foods

Review
Liquid Chromatography Analysis of Common
Nutritional Components, in Feed and Food
Carolina Cortés-Herrera 1 , Graciela Artavia 1 , Astrid Leiva 2 and Fabio Granados-Chinchilla 2, *
1 Centro Nacional de Ciencia y Tecnología de Alimentos (CITA), Universidad de Costa Rica,
Ciudad Universitaria Rodrigo Facio 11501-2060, Costa Rica; carolina.cortesherrera@ucr.ac.cr (C.C.-H.);
graciela.artavia@ucr.ac.cr (G.A.)
2 Centro de Investigación en Nutrición Animal, Universidad de Costa Rica, Ciudad Universitaria Rodrigo 11501-2060,
Costa Rica; astrid.leiva@ucr.ac.cr
* Correspondence: fabio.granados@ucr.ac.cr; Tel.: +506-2511-2028; Fax: +506-2234-2415




Received: 14 September 2018; Accepted: 5 November 2018; Published: 20 December 2018


Abstract: Food and feed laboratories share several similarities when facing the implementation of
liquid-chromatographic analysis. Using the experience acquired over the years, through application
chemistry in food and feed research, selected analytes of relevance for both areas were discussed.
This review focused on the common obstacles and peculiarities that each analyte offers (during the
sample treatment or the chromatographic separation) throughout the implementation of said
methods. A brief description of the techniques which we considered to be more pertinent,
commonly used to assay such analytes is provided, including approaches using commonly
available detectors (especially in starter labs) as well as mass detection. This manuscript
consists of three sections: feed analysis (as the start of the food chain); food destined for
human consumption determinations (the end of the food chain); and finally, assays shared by
either matrices or laboratories. Analytes discussed consist of both those considered undesirable
substances, contaminants, additives, and those related to nutritional quality. Our review is
comprised of the examination of polyphenols, capsaicinoids, theobromine and caffeine, cholesterol,
mycotoxins, antibiotics, amino acids, triphenylmethane dyes, nitrates/nitrites, ethanol soluble
carbohydrates/sugars, organic acids, carotenoids, hydro and liposoluble vitamins. All analytes
are currently assayed in our laboratories.

Keywords: food and feed analysis; liquid chromatography; challenges; nutritional analysis;
additives; contaminants

1. Introduction
Food and feed analysis are paramount to assess both nutritional quality and safety of commodities.
Interconnectivity of food sources [1,2] and new processing techniques [3] make for a more diverse
and complex food supply. Legal thresholds have been stipulated that establish acceptable levels for
individual chemical additives, residues, and contaminants in products [4,5]. Feed is a paramount target
for analysis since it situates at the start of the food chain and poor feed quality can affect the yield on
food-producing animals [6]. Understanding the complexities of food safety is the goal of approaches
such as One Health [7], Farm-to-Fork [6], or MyToolbox [8]. Furthermore, feed contaminants
carryover downstream can reach products such as meat, eggs, and milk (see for example the
transference of aflatoxin M1 from aflatoxin B1 -contaminated feed). Ingredients either destined for
food or feed production (e.g., cereals) are among the fundamental constituents for several staple
commodities. Other regulations require food and feed labeling to list ingredients relating to the
nutritional content [9,10]. All stakeholders involved in the food and feed chain must be able to

Foods 2019, 8, 1; doi:10.3390/foods8010001 www.mdpi.com/journal/foods

Foods 2019, 8, 1 2 of 63

assess product quality and safety. Hence, it is imperative to rely on techniques that meet several
analytical performance parameters. More and more, food and feed analysis methods are based on
LC (liquid-chromatography) [11,12], which has proven to be an optimal technology for screening,
detection, and quantification of a vast variety of analytes (see Table 1). The reason behind this is related
to the molecular affinity between the analyte and also: i. the mobile phase (which is usually a mixture
of solvents) ii. stationary phase (modified silica and polymer scaffolds). Within the LC approach
itself, several alternatives are available for a researcher to resolve a specific task at hand. Each analyte
presents its own unique trials.

Table 1. Typical food and feed analytes assayed using HPLC (High-Performance Liquid-Chromatography).
Additives
Analyte Category Examples Relevance in Feed and Food Quality
Acidulants Used in beverage, food, and feed production, are part of the primary metabolism, are often
Acetic acid, lactic acid, and citric acid [13]. produced by fermentation. Acidic additives serve as buffers to regulate acidity, antioxidants,
preservatives, flavor enhancers, and sequestrants. Related to beneficial effects on animal health and
growth performance as feed additives.
Antioxidants Lipid and protein oxidation can impact meat quality, nutrition, safety, and organoleptic properties.
Gallic, rosemarinic, canosic, and caffeic acids, Antioxidants are added during animal production and meat processing to enhance the nutritional
glabrene, procyanidins, quercetin, catechin α-, β-, γ-, and health benefits of meat and minimize the formation of carcinogens for the chemical safety of
and δ-tocopherols, Eugenol, Carnosine, Tyr-Phe-Glu, cooked and processed meats [14,15]. They can also be used to extend food [16] and feed [17] shelf
and Tyr-Ser-Thr-Ala. life.
Preservatives
Acetates, bacteriocins, benzoates (p-hydroxybenzoic Usually, act as bacteriostatic and bactericidal agents to prevent microbial spoilage, antimicrobials
acid), borates, carbonates, lactates, nitrates/nitrites, not only extend shelf life, but they also enhance the product’s safety [18].
parabens, propionates, sorbates, and sulfites.
Flavors and fragrances
Alcohols, methyl ketones, 2,3-butanedione, lactone, Widely used in food, beverage, feed, cosmetic, detergent, chemical and pharmaceutical formulations
butanoic acid, esters, isovaleric acid, pyrazines, [19].
geosmin, vanillin, benzaldehyde, terpenes.
Sweeteners Non-nutritive sweeteners have become an essential part of daily life and are in increasing demand
Approved as food additives: saccharin, aspartame, as it is used in a wide variety of dietary and medicinal products [20]. They play a role in the
acesulfame potassium, sucralose, neotame, reduction of table sugar [21]. In the case of artificial sweeteners, their use is controversial as they
advantame. Generally regarded as safe (GRAS): have associated with health risks [20,22] and water pollution [23]; currently, the use of natural
Steviosides [28–31]. sweeteners is supported as an alternative [24]. Sweetened products must be subject to verification to
ensure the presence of the sweetener. Furthermore, sweeteners are regulated food additives [25]
unless recognized as safe [26,27].
Natural Components
Analyte Category Examples Relevance in Feed and Food Quality
Inorganic ions
Sulfites, sulfates, phosphate, polyphosphate, nitrate Essential in both raw and processed products, related to food nutritional quality, preservation,
and nitrite, N-nitroso compounds, cyanide, bromide, technological processing, and safety [32].
bromate, chloride, chlorite, fluoride, iodide.
Lipids and fatty acids
C1:0 (formic/methanoic), C2:0 (acetic/ethanoic), C3:0
(propionic/propanoic), C4:0 (butyric/butanoic), C6:0
(caproic/hexanoic), C8:0 (caprylic/octanoic), C10:0
(capric/decanoic), C12:0 (lauric/dodecanoic), C12:0
Major constituents of foods and feeds, of dietary importance as a significant source of energy.
(myristic/tetradecanoic), C16:0
Provide essential fat-soluble nutrients. Are prone to peroxidation. Part of biological membranes.
(palmitic/hexadecanoic), 9c-C16:1
(palmitoleic/(9Z)-hexa-dec-9-enoic), stearic, oleic,
ricinoleic, vaccenic, linoleic, α-linoleic, γ-linoleic,
arachidic, eicosapentaenoic, behenic, erucic,
docosahexaenoic, lignoceric, cholesterol [33–37].
Biogenic amines Nitrogen-based toxic compounds, mainly formed through decarboxylation of amino acids. Relevant
Putrescine, histamine, cadaverine. for quality and safety of diverse foods such as dairy products [38], fermented goods [39] including
wines [40], fishery commodities [41].
Amino acids
The main fermentative amino acids for animal Part of a protein-containing diet, and as supplemented individual products. Amino acids are used
nutrition are L-lysine, L-threonine, and L-tryptophan. in medical (parenteral) nutrition and dietary supplements [42].
DL-Methionine.
Carbohydrates
Glucose is the primary energy source for fetal growth, The most abundant feed energy in diets for some species of animals [43,44].
and lactose is crucial for the development of human
and animal infants alike.
Vitamins
Fat-soluble: retinol (retinol (vitamin A) and retinyl
acetate, and palmitate), tocopherols (α- (vitamin E),
β-, γ-, and δ- and their acetates), ergocalciferol
(vitamin D2 ), cholecalciferol (vitamin D3 ),
Complex unrelated compounds present in minute amounts in natural foodstuffs. Essential to
phylloquinone (vitamin K1 ), menaquinone (vitamin
normal metabolism; their deficiency causes disease.
K2 ), 7-dehydrocholesterol, β-carotene. Hydrosoluble
or B complex vitamins: thiamine (B1 ), Riboflavin (B2 ),
flavin mononucleotide or riboflavin-50 -phosphate,
niacin/nicotinamide riboside/niacinamide (B3 ),
pantothenic acid (B5 ),
pyridoxine/pyridoxamine/pyridoxal (B6 ), biotin (B7 ),
folates (B9 ), cobalamines (B12 ) [45].
Foods 2019, 8, 1 3 of 63

Table 1. Cont.
Additives
Analyte Category Examples Relevance in Feed and Food Quality
Alkaloids
Octopamine, synephrine, tyramine,
N-methyl-tyramine, hordenine in bitter orange
products [47], morphine, codeine, thebaine,
papaverine, and noscapine in poppy straw [48], Alkaloids are natural compounds with a characteristic cyclic structure and a nitrogen atom [46].
caffeine and trigonelline in coffee [49], indole and Alkaloid-containing plants are an essential part of the regular diet, present as natural constituents of
oxindole alkaloids in Uncaria sp. [50], theobromine several food products [46]. The most common use for alkaloid-containing plants is as stimulants
and caffeine in tea [51] and coffee [52], Harman increased concentrations of these compounds can be attained within the food chain as a result of
alkaloids (harmane and harmine) in passion fruit [53], food processing, as food contaminants or as food flavorings [46].
ergot alkaloids in animal feed (ergometrine,
ergotamine, ergocornine, ergocryptine, ergocristine
[54], piperine [55].
Residues and Contaminants
Analyte Category Examples Relevance in Feed and Food Quality
Chemotherapeutics and antiparasitic drugs Antibiotics are extensively utilized in productive animals with therapeutic, prophylactic,
Tetracyclines [56]. metaphylactic, growth promoting, and food effectiveness enhancing ends. These practices that have
been reflected in veterinary residues in products for human consumption (meat, eggs, and milk) and
is also related to directly with allergies and antimicrobial resistance.
Mycotoxins Mycotoxins are practically ubiquitous contaminants, classified as teratogenic, carcinogenic and
Aflatoxins [58]. immunosuppressive, and that affects a great variety of grains, fruits and seeds, as well as eggs, dairy
products, compounds feeds, and other feed ingredients [57].

Pesticides Used for crop protection and to treat infestations in livestock. Their poor use results in
Atrazine, glyphosate, aminomethylphosphonic acid, contamination of the environment and the food itself, impacting human health. Residues usually
phenoxy herbicides. found in vegetables, fruits, honey, fish, eggs, milk, and meat, serving as potential sources of
contamination to consumers [59–61].

To successfully analyze or isolate a compound, a researcher is faced with several questions:

What is the problem to solve, the objective or purpose for the analysis? Is the required data qualitative
or quantitative? Are there two or multiple compounds to be separated? What are the physicochemical
characteristics of the target(s)? What matrix was the analyte recovered from and which interferences
are expected? What is the amount of analyte expected to be recovered? What equipment is accessible
in the laboratory?
Considering the above, a suitable column (Table 2) and detection system must be selected (Table 3).
Sample preparation can aid to solve some of these issues, especially those regarding interferences and
sensitivity but cannot solve issues with poor detector choice. For example, if sensitivity is a problem
using the selected detection system on hand and no other system is available, the initial sample
mass can be increased, or a concentration step (evaporation or solid phase extraction (SPE)) can be
performed. Additionally, the sample injection volume can be expanded to improve sensitivity.

Table 2. General conditions required for each mode of chromatography.

Type of Liquid
Micro Semi-Micro Conventional Semi-Preparative Preparative Process
Chromatography
Column internal diameter, mm 0.3 < x ≤ 1.0 1.0 < x ≤ 3.0 4.0 < x ≤ 8.0 8.0 < x ≤ 20.0 20.0 < x ≤ 50.0 x > 50.0
Eluent flow rate, mL min−1 0.001 < x ≤ 0.1 0.1 < x ≤ 0.4 0.4 < x ≤ 2.0 2.0 < x ≤ 10.0 10.0 < x ≤ 150.0 x > 150.0

Additionally, automation is relevant for conserving resources and reducing turnover times.
An analyst can program an autosampler to increasingly adjust the volume of a standard with a fixed
concentration. For example, to construct a calibration curve between 1000 and 62.5 µg L−1 , one could
use a 1000 µg L−1 standard and instruct the sampler to take from the same vial 20 µL, 10 µL, 5 µL, 2.5 µL,
1.25 µL, consecutively. The sampler will construct a calibration curve without analyst intervention
and this automation will reduce errors. Autosamplers are designed to inject small volumes without
significant loss, with good precision, and adequate reproducibility. They can also inject variable
amounts, dilute the sample prior to injection and perform precolumn derivatization [91]. If a sample
is outside of calibration standard of higher concentration, an analyst can inject a different volume
to ensure it will fit among the calibration curve range. However, injection volume has an impact on
peak shape. The method must be validated to show this is a valid approach. (See for example, [92]).
Reference for one example of the versatility of an LC system and capabilities for its automation. In this
review, we intend to give the reader a thorough background on the common analyses performed,
for quality assurance and safety, in food and feed laboratories. We will include the most recent and
Foods 2019, 8, 1 4 of 63

relevant experience gathered for each test while pointing out the difficulties that each essay presents
and the common ground shared by both types of laboratories.

Table 3. Characteristics of the most common detectors used in liquid chromatography.

Detector Type Range of Applications, Attributes, and Minimal
Applications in Feed and Food Quality
Detectable Quantity Limitations
Non-Destructive Detectors
Photodiode-Array (PDA)/Variable wavelength (VW)/UV-vis
Selective; universal at low wavelengths, 3D spectra comparison can Sulfonated azo dyes in beverages, hard candy and fish roe samples [62], purity of
determine peak purity, can detect nanograms. caffeine reference material [63], sulfamethazine and trimethoprim in liquid feed
Chromophore must be presesent, solvents transparent to the wavelength premixes [64], nitrofurans animal feed [65].
used must be provided.
Fluorescence (FL)
Very selective and specific; monitors two wavelengths simultaneously, 3D
fluorescent spectra, fluorescent fingerprinting/fluorescence pattern analysis
Sulfonamides [67] and fluoroquinolones [68] in animal feed, aflatoxins in agricultural
[66] gradients do not affect baseline significantly, can detect low picograms.
food crops [69] and milk [70].
Fluorophore must be present, derivative formation and quenching are
often needed.
Electrochemical (EC)
Very selective; oxidation or reduction must be possible, can detect from
femtograms to nanograms. Macrolide antibiotics in animal feeds [71], vitamin C in oranges and apples [72].
Conductive mobile phase, susceptible to background noise and
electrode degradation.
Refractive index (RI)
Universal (All compounds affect refractive properties) and versatile; solvent
compatible, relatively simple, can detect micrograms. Inulin in chicory roots [73], total carbohydrates in wine and wine-like beverages [74].
Gradient incompatible, high S/N ratio when the pump is mixing two or
more solvents, susceptible to temperature and flow variation.
Conductivity
Selective; an ionic form of compound necessary can detect low pictograms. Choline, and trimethylamine in feed additives [75], L-carnitine, choline, and metal
Suppression of mobile phase background conductivity, special ions in infant formula [76].
equipment required.
Destructive Detectors
Mass spectrometry Analysis of acrylamide in food [77], tiamulin, trimethoprim, tylosin, sulfadiazine,
Selective and specific; based on a specified mass/charge ratio, ion and sulfamethazine residues in medicated feed [78], multiclass antibiotics in eggs
fractionation, can detect low nanograms. Expensive, expert users are [79], zearalenone and deoxynivalenol metabolites in milk [80], cattle feed analysis of
needed for equipment and data manipulation. Aspergillus clavatus mycotoxins [81], choline chloride in feed and feed premixes [82].
Radioactivity
Selective; Distribution and mass balance wide response range can
detect pictograms. Drug metabolite identification [83].
Large-volume flow cells can produce peak broadening and decreased
the resolution.
Evaporative light scattering (ELS)
Universal; Nonvolatile analyte nebulization, can detect in the range N-acetylglucosamine and N-acetylgalactosamine in dairy foods [84], sucralose and
of nanograms. related compounds [85], spectinomycin and associated substances [86].
Volatile buffers required, poor reproducibility and limited dynamic range.
Corona-Charged aerosol [87]
Universal; can detect non-ultraviolet and weakly ultraviolet active
compounds [88], ionized particles measured by an electrometer, can detect Erythritol, xylitol, sorbitol, mannitol, maltitol, fructose, glucose, sucrose, and maltose
low nanograms. in food products [89], fumonisins in maize [90].
Volatile buffers required.

2. Measurements of Commonly Consumed Food Commodities

2.1. Polyphenols
Polyphenols usually refers to several chemical compounds including flavanols (e.g., catechins and
tannins from tea), flavanones (i.e., hesperidin from citrus fruits), flavonols (e.g., quercetin from tea,
apples and onions), “chlorogenic acids” (including hydroxycinnamic acids caffeic, ferulic, p-coumarinic
acids usually extracted from coffee), anthocyanins (which are partly responsible for imparting color to
plant structures), and stilbenes (e.g., from berries, grape skins and peanuts) (Figure 1) [93].
These compounds, secondary metabolites from plants [94], have, among other functions,
a protective capability within the vegetable tissue, structure, and support [94], and, even,
pollinator attraction [95]. For example, chlorogenic acid (i.e., the esterification product between
caffeic and quinic acid) is an intermediate in lignin biosynthesis [96]. Data suggests that long-term
consumption of such compounds can have beneficial effects [94] as it can improve an organism’s
antioxidant capacity [93] which in turn relates, for example, to cognitive improvement [97] and
reduction in adipogenesis and oxidative stress [98]. Fruits, especially berries, are [97–101] rich in these
bioactive compounds, both extractable [102,103] and non-extractable [104]. From the technological
standpoint, polyphenol safeguard is paramount to achieve functional foods [105] with added value
Foods 2019, 8, 1 5 of 63

(e.g., beverages) and a bioactive capacity of compounds as close as those from the raw material.
Several operation units have been applied to fruits to assess polyphenol retention after processing
including nanofiltration [101], high hydrostatic pressure [106], and drying [107,108]. Method-wise,
the solvent has a profound effect on the number and type of polyphenols extracted. Polyphenol analysis
must first identify the type of matrix to be analyzed, the chemical nature of the polyphenols of
interest, and different solvents and solvent systems should be examined. The most appropriate
solvent for the case in hand (i.e., maximizing compound diversity and yield) should be the one
selected [109]. For example, Flores and coworkers resuspended the methanolic extract in hexane,
chloroform,
Foods 2018, 7, x ethyl acetate,
FOR PEER and n-butanol and reanalyzed each fraction. Ethyl acetate fraction exhibited
REVIEW 6 of 62
the best results [110]. Finally, though polyphenols are usually related to health applications [111,112],
Finally, thougheffects
antinutritional polyphenols
should beare usually related
considered to health
[109]. Some applications
examples [111,112],
of polyphenol antinutritional
analysis are included
effects
in Tableshould
4. be considered [109]. Some examples of polyphenol analysis are included in Table 4.

Figure
Figure 1.
1. Polyphenols
Polyphenols structure
structure and
and classification
classification [97].
[97]. Highly
Highly functionalized
functionalized structures
structures account
account for
for
the
the molecules radical scavenging, metal ion chelating, and enzyme inhibition. Hydrogen bonding can
molecules radical scavenging, metal ion chelating, and enzyme inhibition. Hydrogen bonding can
stabilize
stabilize phenoxyl
phenoxyl radicals.
radicals.

Table 4. Polyphenol analysis in different matrices, based on liquid chromatography, and varied
approaches to determine them.

Measurement Method,
Matrix Analytes Identified Extraction Method Reference
Chromatographic Column
Preparative LC: 250 × 20
mm Eurospher 100–5 C18
Anthocyanins (68.6%),
H2O, membrane Identification: was
Berries hydroxycinnamic acids [98]
ultrafiltration HPLC/DAD/ESI±-MSn, 150 ×
(23.9%), flavonols (4.4%)
2.1 mm, 5 µm. λ 280, 325,
360 and 520 nm
MeOH/H2O (70 mL/100 LC-TOF-ESI± (m/z range
Costa Rican Ellagic acid, myricetin,
mL). Freeze-dryed pulp, 100–1000), Synergi Hydro [110]
guava quercitrin, and quercetin
mechanical dispersion RP 80A 250 × 4.6 mm, 4 µm.
Acetone/H2O/HCOOH
Brazilian guava, Hydrolyzable and HPLC-DAD-ESI−-MSn,
(70:29:1). Freeze-dryed
jambolan, nance, condensed tannins, Aqua RP18 150 × 2.0 mm, 3 [113]
pulp, accelerated solvent
and lúcuma flavonols, and flavanols µm.
extraction
Foods 2019, 8, 1 6 of 63

Table 4. Polyphenol analysis in different matrices, based on liquid chromatography, and varied
approaches to determine them.
Matrix Analytes Identified Extraction Method Measurement Method, Chromatographic Column Reference
Anthocyanins (68.6%), Preparative LC: 250 × 20 mm Eurospher 100–5 C18
Berries hydroxycinnamic acids (23.9%), H2 O, membrane ultrafiltration Identification: was HPLC/DAD/ESI± -MSn , [98]
flavonols (4.4%) 150 × 2.1 mm, 5 µm. λ 280, 325, 360 and 520 nm
MeOH/H2 O (70 mL/100 mL).
Ellagic acid, myricetin, quercitrin, LC-TOF-ESI± (m/z range 100–1000), Synergi Hydro
Costa Rican guava Freeze-dryed pulp, mechanical [110]
and quercetin RP 80A 250 × 4.6 mm, 4 µm.
dispersion
Brazilian guava, Acetone/H2 O/HCOOH
Hydrolyzable and condensed tannins, HPLC-DAD-ESI− -MSn , Aqua RP18
jambolan, nance, and (70:29:1). Freeze-dryed pulp, [113]
flavonols, and flavanols 150 × 2.0 mm, 3 µm.
lúcuma accelerated solvent extraction
Rosmarinic acid (12.7–85.3%),
scutellarein-7-O-glucuronide Ethanol (EtOH)/H2 O (75
Perilla frutescents (L.) UPLC-PDA-ESI− -TOF-MS, Kintex XB C18 column
(6.5–45.1%), caffeic acid, mL/100 mL). Accelerate solvent [114]
Britton 150 × 2.1 mm, 1.7 µm
apigenin-7-O-diglucuronide, and extraction (N2 1200 psi 70 ◦ C)
apigenin-7-O-glucuronide
e.g., caffeic acid hexosides,
homovanillic acid hexoside, and Methanol (MeOH)/H2 O HPLC-DAD-ESI− -MS/MS, Zorbax 300SB-C18
Solanum lycopersicum L. [115]
dicaffeoylquinic acid (increasing (80 mL/100 mL) column (2.1 × 150 mm; 5 µm)
trend)
Rubus fruticosus L., Gallic acid (138.0–443.5 mg kg−1 fresh
HCOOH/MeOH/H2 O LC-FLD λex 280, 320, 322 nm λem 360 nm.
Prunus spinos L. and weight), rutin (13.9–22.8 mg kg−1 [116]
(0.1/70/29.9) Eclipse XDB C18 150 × 4.6 mm
Cornus mas L. fresh weight)
Gallic acid, caffeic acid (+)-catechin,
Green, herbal and (–)-epicatechin, (–)-epigallocatechin, LC-PDA/FLD scan 260–400 nm absorbance matching
95 ◦ C for 10 min [117]
fruit teas procyanidin B1 , and procyanidin B2 Zorbax Eclipse XDB-C18 , 150 × 4.6 mm, 5 µm
contribute to 43.6–99.9%
Vanillic, ellagic, gallic, p-coumaric,
chlorogenic, caffeic, ferulic,
MeOH/H2 O (62.5 mL/100 mL). HPLC-DAD at 260, 280, 329, and 520 nm.
Dried and candied fruit rosmarinic acids, and myricetin, [118]
Sonication Zorbax Eclipse Plus C18 column 150 × 4.6 mm, 3.5 µm
quercetin, kaempferol, delphinidin,
cyanidin, and pelargonidin
Ellagitannins, flavones, flavonols,
flavanols, proanthocyanidins,
dihydrochalcones, and
Freeze dried pulp, MeOH/H2 O UHPLC-DAD-ESI+ -MS/MS, BHE Shield RP18
Pink guava anthocyanidins, and non-flavonoids [119]
(90:10), sonication 150 × 2.1 mm, 1.7 µm.
such as phenolic acid derivatives,
stilbenes, acetophenones, and
benzophenones
Microfiltrate (tubular ceramic HPLC-DAD-ESI+ -IT-MS/MS Lichrosrb ODS-2
Blackberry juice [120]
membrane) 250 × 4.6 mm, 5 µm

Gordon and coworkers used accelerated solvent extraction (ASE) to characterize polyphenolic
compounds in Psidium guineense Sw., Syzygium cumini (L.) Skeels, Byrsomina crassifolia (L.) Kunth,
and Pouteria macrophylla (Lam.) Eyma. [113]. ASE techniques allow for multiple extractions
simultaneously. Swifter assays are obtained which, in turn, expedite research results and minimize
solvent waste [114] when compared to common extraction methods (e.g., Soxhlet, sonication).
Anton and coworkers investigated the effect of ripening in tomato polyphenols content and
antioxidant capability. A differential mass spectrometry approach allowed the authors to conclude
that cultivar-dependent patterns are observed during ripening (e.g., maximum concentrations of
polyphenols achieved half-ripe stage) [115]. Radovanović and coworkers, associated polyphenols
from berries to antibacterial activity [116]. Veljković and coworkers analyzed phenolic compounds in
different types of tea. Nettle/pineapple, and bearberry/raspberry teas showed the lowest and highest
phenolic contents, respectively [117]. Miletić assessed polyphenols in dried and candied fruit. In this
particular case, acid hydrolysis was applied to the previously dispersed methanolic extracts to free
matrix-bound polyphenols [118]. One g tert-butyl hydroquinone/100 mL was added during extraction
as a radical sink to protect polyphenols. Kowalska and coworkers used preparative chromatography
to remove non-phenolics [98].
Tentative screening for Psidium friedrichsthalianum (Berg) Niedenzu pulp showed 1,5-dimethyl
citrate, 1-trans-cinnamoyl-β-D-glucopyranoside, sinapic aldehyde-4-O-β-D-glucopyranoside,
1,3-O-diferuloylglycerol, and 3,30 ,4-tri-O-methylellagic acid-40 -O-D-glucopyranoside [110].
Phenolic compounds from pink guava from Costa Rica have been recently reported, n = 60
phenolic compounds were characterized. The authors report for the first time in P. guajava n = 42
compounds in the fruit’s peel and flesh, and n = 24 new compounds, e.g., phlorizin, nothofagin,
astringin, chrysin-C-glucoside, valoneic acid bilactone, cinnamoyl-glucoside, and two dimethoxy
cinnamoyl-hexosides [119]. During polyphenol analysis, HLB® SPE (Hydrophilic-Lipophilic,
Foods 2019, 8, 1 7 of 63

Balance Solid Phase Extraction) cartridges are used routinely for clean-up. At least one research
group has applied this approach to assay polyphenols and vitamin C in plant-derived materials [121].
Interestingly, when using the Folin–Ciocalteu spectrophotometric approach, ascorbate is considered
interference
Foods 2018, and
7, x FORmust be eliminated from the eluate (usually taking advantage of 8ascorbate
PEER REVIEW of 62
thermolability) or else the measurements are overestimated. However, simultaneous retention of both
assay polyphenols and vitamin C in plant-derived materials [121]. Interestingly, when using the
analytes in the SPE cartridge can be exploited, if HPLC methods are used instead. We recommend that
Folin–Ciocalteu spectrophotometric approach, ascorbate is considered interference and must be
in countries in which fruits with high polyphenol content are readily available (and in considerable
eliminated from the eluate (usually taking advantage of ascorbate thermolability) or else the
quantities), preparative
measurements separation However,
are overestimated. of polyphenol fractions
simultaneous is a possibility
retention for obtaining
of both analytes in the SPE pure
compounds (See for example, [122]). Finally, vanillic acid was reported in cocoa pod
cartridge can be exploited, if HPLC methods are used instead. We recommend that in countries in polyphenol-rich
extracts. Interestingly, the application −1 of this cocoa extract to a vegetable oil improved
which fruits with high polyphenol of 2000 mg
content are Lreadily available (and in considerable quantities),
its oxidative
preparativestability and shelf-life
separation [123].
of polyphenol fractions is a possibility for obtaining pure compounds (See for
example, [122]). Finally, vanillic acid was reported in cocoa pod polyphenol-rich extracts.
Method Application
Interestingly, the Experience
application of 2000 mg L−1 of this cocoa extract to a vegetable oil improved its
oxidative stability and shelf-life [123].
In our laboratory, ultrasound-assisted extraction is preferred for reducing processing time
and avoiding degradation
Method Application of the compounds. Additionally, polyphenols are quite light sensitive,
Experience
hence yellow lights are used during the extraction using acetone-water or methanol-water solutions.
In our laboratory, ultrasound-assisted extraction is preferred for reducing processing time and
As the polyphenol family is extensive and chemically diverse, a surface response design is always
avoiding degradation of the compounds. Additionally, polyphenols are quite light sensitive, hence
recommended
yellow lights to assess
are usedthe appropriateness
during the extractionofusing
the solvent systemor(i.e.,
acetone-water selecting a solvent
methanol-water solutions.that
Asprovides
the
the highest
polyphenol family is extensive and chemically diverse, a surface response design is alwayscause
yields). Samples with a high lipid content (i.e., > 5 g total fat/100 g) usually
significant interferences
recommended andthe
to assess must be defatted previous
appropriateness to polyphenol
of the solvent system (i.e.,extraction.
selecting aItsolvent
is usual to add
that
additional
providesantioxidants (e.g., ascorbic
the highest yields). acid)a to
Samples with polyphenol
high lipid contentextracts
(i.e., > 5gtototal
protect them
fat/100 from cause
g) usually oxidation.
significant
Finally, interferences
it is common to findand must be
natural defatted
existing previous to polyphenol
polyphenols as adductsextraction.
with proteinIt is usual to add
or carbohydrate
additional antioxidants (e.g., ascorbic acid) to polyphenol extracts to protect
moieties. These adducts are usually formed by non-covalent interactions (e.g., salt bridges); them from oxidation.
Finally,
therefore, byit adjusting
is commonthe to find natural
extract existing
ionic polyphenols
strength, one can as remove
adducts with theseprotein or carbohydrate
artifacts. Sugar adducts
moieties. These adducts are usually formed by non-covalent interactions (e.g., salt bridges); therefore,
are considerably more difficult to analyze since only a few compounds are commercially available
by adjusting the extract ionic strength, one can remove these artifacts. Sugar adducts are considerably
(e.g., cyanidin 3-O-glucoside chloride). Hydrolysis (mild acidic, basic or enzymatic) is the usual
more difficult to analyze since only a few compounds are commercially available (e.g., cyanidin 3-O-
approach to circumvent
glucoside the lack of(mild
chloride). Hydrolysis theseacidic,
commercial standards.isAvailability
basic or enzymatic) of masstospectrometry
the usual approach circumvent or
nuclear
the magnetic
lack of theseresonance (NMR)
commercial can helpAvailability
standards. elucidate unknown compoundsor
of mass spectrometry and adducts.
nuclear magnetic
resonance (NMR) can help elucidate unknown compounds and adducts.
2.2. Capsaicinoids
2.2. Capsaicinoids
Capsaicinoids are plant metabolites from the Capsicum genus which give pungency to chili
Capsaicinoids
peppers [124]. Scoville are plant
scale metabolites
which measures from
thethe Capsicum
spiciness of genus which
the fruits give pungency
(originally, testedto
bychili
sensory
peppers
assays) [124]. Scoville
is reported scale which
in function measures the
of capsaicin spiciness of the
concentration fruits
(i.e., mg (originally,
capsaicin kg−by
tested 1 ×
sensory
16 [125]).
assays)
Today, is reported
the most in function
reliable, of capsaicin
rapid, and efficientconcentration (i.e., mg capsaicin
method to identify kg−1 × 16
and quantify [125]). Today,isthe
capsaicinoids HPLC.
most reliable, rapid, and efficient method to identify and quantify capsaicinoids is HPLC. Measurement
Measurement of this molecules is significant as a quality measure of chili pepper (22 domesticated
of this molecules is significant as a quality measure of chili pepper (22 domesticated varieties consumed
varieties consumed regularly worldwide), a crop which is of significant cultural and global trade market
regularly worldwide), a crop which is of significant cultural and global trade market value [126]. More
valuethan
[126]. More than 20 different capsaicinoids have been described; the foremost capsaicinoids
20 different capsaicinoids have been described; the foremost capsaicinoids found in these plant
foundstructures
in these include
plant structures include
capsaicin and capsaicin and
dihydrocapsaicin [127]dihydrocapsaicin
(Figure 2). [127] (Figure 2).

Figure
Figure 2. Chemical
2. Chemical structures
structures forfor (A)capsaicin
(A) capsaicin (8-methyl-N-vanillylamide)
(8-methyl-N-vanillylamide) andand
(B) (B)
dihydrocapsaicin
dihydrocapsaicin
(8-methyl-N-vanillylnonamide), the aromatic vanillyl radical is shown
(8-methyl-N-vanillylnonamide), the aromatic vanillyl radical is shown in red.in red.

2.2.1.2.2.1. Measurement
Measurement of Capsaicin
of Capsaicin andDehydrocapsaicin
and Dehydrocapsaicin in
inReal
RealSamples
Samples
Research
Research reports
reports havehave described
described capsaicinoidanalysis;
capsaicinoid analysis; the
themost
mostrecent areare
recent summarized in Table
summarized 5.
in Table 5.
Garcés-Claver and coworkers determined capsaicin and dihydrocapsaicin in two different scenarios, i.e.,
Garcés-Claver and coworkers determined capsaicin and dihydrocapsaicin in two different scenarios,
fruits grown in summer and then in spring [128]. The authors concluded that capsaicinoids varied largely
Foods 2019, 8, 1 8 of 63

i.e., fruits grown in summer and then in spring [128]. The authors concluded that capsaicinoids varied
largely among fruit families and that these families did not respond similarly to producing these
capsaicinoids when their fruits were grown in the two seasons tested [128].

Table 5. Common chromatographic conditions used for capsaicinoid analysis.

Measurement Method, Sensitivity, mg L−1 or mg
Matrix Extraction Method Reference
Chromatographic Column kg−1 Fruit Dry Weight
RP-LC/MS-TOF/ESI− ,
pseudo-molecular ions [M-H]−
Peppers Capsicum annuum L. ACN, mechanical shaking 304.2 and 306.2 m/z. IS 0.06 [128]
4,5-dimethoxybenzyl)-4-methyloctamide,
250 × 4.6 mm, 5 µm
1. Sequential centrifugal partition
Natural capsaicinoid mixture C7 H16 /EtAOc/MeOH/H2 O chromatography.
Preparative chemistry [129]
(capsaicin/dihydrocapsaicin 67:33) (1:1:1:1) 2. Nucleosil 100-5 C18 column
(125 × 3 mm, 5 µm, UV 280 nm
HPLC-UV using a wavelength of
Hot chilies, green peppers, red
EtOH 222 nm and a Betasil C18 150 × 0.10 [130]
peppers, and yellow peppers
4.6 mm, 3 µm column
Immunoaffinity column, SPE
loading solvent, 5 mL LC-ESI+ -MS/MS, Hypersil Gold,
Vegetable and waste oils 0.03 [131]
MeOH/H2 O (5:95), washing 100 × 2.1 mm, 3.0 µm
solvent PBS, MeOH for elution
IS capsaicin-d3 , and
dihydrocapsaicin-d3 .
Edible and crude vegetable oils SPE C18 , MeOH RP-UPLC-ESI-MS/MS, ZORBAX 0.5 [132]
Eclipse Plus C18 50 × 2.1 mm,
1.8 µm)
UV and FLD λex 280 and λem
Austrian chili peppers ACN/H2 O (35:65) 310 nm, UPLCTM BEH C18 50 × 2.1 0.136 [133]
mm, 1.7 µm
UHPLC–DAD–APCI-MS/MS,
Brazilian Capsicum chinense Jacq. MeOH sonication Hypersil Gold C18 100 × 3 mm, 0.0027 [134]
1.9 µm
FLD λex 280 and λem 325 nm,
South Korean red peppers MeOH/H2 O (95:5), 80 ◦ C 2 h Zorbax Eclipse XDB-C18 75 × 3 mm, 0.06 [135]
3.5 µm)

SPE: Solid phase extraction. UPLC: Ultra-Performance Liquid-Chromatography.

Goll and coworkers optimized a cyclic solid support free liquid–liquid partition to separate
a capsaicin and dehydrocapsaicin mixture into two sequentially collected product streams.
This approach may serve as a base for compound purification before chemical characterization.
With this optimization, the authors demonstrated theoretical and predictive tools are useful in
preparative chemistry and process design [129].
The pretreatment of capsaicinoid determination (i.e., extraction steps) is usually straightforward,
and the majority of methods are based on methanol-based extraction. However, Lu and coworkers
reviewed several techniques that can be used to extract capsaicinoids successfully [136]. Ma and
coworkers [131] used capsaicin and dihydrocapsaicin, and nonivamide [132] were selected as
adulteration markers to authenticate vegetable oils. No capsaicinoid compounds were found in
edible vegetable oils, thereby ruling out a possible adulteration source. The authors prepared
immunosorbents by covalently coupling highly specific capsaicinoid polyclonal antibodies with
CNBr-activated Sepharose 4B and packed into a polyethylene column [131]. This research is
interesting, from the clean-up standpoint, since the authors adjusted the major parameters affecting
the immunoaffinity column extraction efficiency (i.e., loading, washing, and eluting conditions) [131].
Schmidt and coworkers compared different chili peppers available in Austria and compared their
contents of capsaicin and dihydrocapsaicin [133]. The authors used UPLC (Ultra-Performance
Liquid-Chromatography) and hence obtained a reduced resolved chromatogram for both compounds
of just 1.7 min. [133]. The authors also corroborated that the highest capsaicinoids content was in the
fruits’ placenta and the seeds. Similarly, Sganzerla and coworkers obtained a complete separation
under 4 min [134]. The above examples correspond to high-throughput methods of analysis.
Finally, ingested capsaicinoids can persist in the bloodstream and can be determined in plasma
using LC coupled with tandem mass spectrometry [137]. Intestinal absorption and metabolisms
(via capsaicinoid glucuronides) have also been reported for a mammal [138]. At the same time,
using LC coupled with tandem mass spectrometry [137]. Intestinal absorption and metabolisms (via
capsaicinoid glucuronides) have also been reported for a mammal [138]. At the same time, dietary
capsaicin has been linked to the browning of adipose tissue, which in turn, promotes energy
expenditure [139].
Foods 2019, 8, 1 9 of 63

2.2.2. Method Application Experience

dietary capsaicin has been linked to the browning of adipose tissue, which in turn, promotes energy
As expenditure [139].
shown, capsaicinoids can very well be measured by using a wavelength in the 200–400 nm
UV range. However,
2.2.2. fluorescence
Method Application analysis can be performed (λex 280 nm λem 338 nm) improving
Experience
sensitivity dramatically [134], an approach preferred by our laboratory for routine analysis. A short
As shown, capsaicinoids can very well be measured by using a wavelength in the 200–400 nm
column UVwith a smaller
range. particle
However, size seems
fluorescence to improve
analysis both resolution
can be performed and
(λex 280 nm λemsensitivity.
338 nm) improving
sensitivity dramatically [134], an approach preferred by our laboratory for routine analysis. A short
2.3. Caffeine andwith
column Theobromine
a smaller particle size seems to improve both resolution and sensitivity.

Caffeine and theobromine

2.3. Caffeine and Theobromineare naturally occurring methylxanthines with antioxidant potential [140]
(Figure 3). There are some
Caffeine misconceptions
and theobromine regardingoccurring
are naturally health effects caused by with
methylxanthines caffeine ingestion [140].
antioxidant
On the contrary, theobromine
potential [140] (andarecocoa)
(Figure 3). There consumptionregarding
some misconceptions has demonstrated beneficial
health effects caused effects [141].
by caffeine
ingestion
Coffee, cocoa, [140].
tea, and Oncaffeine-containing
the contrary, theobromine (and cocoa)
beverages consumption
(e.g., soft andhas demonstrated
energy drinks)beneficial
are widespread
and relevant food commodities. For example, caffeine intake has been calculated at 25 andare
effects [141]. Coffee, cocoa, tea, and caffeine-containing beverages (e.g., soft and energy drinks) 50 mg per
widespread and relevant food commodities. For example, caffeine intake has been calculated at 25 and
day for children and adolescents aged 2–11 and 12–17 years, respectively. The more relevant caffeine
50 mg per day for children and adolescents aged 2–11 and 12–17 years, respectively. The more relevant
sources were soda
caffeine andwere
sources tea as
sodawell
andas teaflavored
as well asdairy
flavored(for children
dairy aged aged
(for children < 12 years) andand
< 12 years) coffee
coffee(for those
aged 12 (for
years and
those agedabove). Similarly,
12 years and above). caffeine consumption
Similarly, has beenhas
caffeine consumption between 2.5–3 2.5–3
been between and 400
and mg kg−1
400weight) −
mg kg day1 bw−1(body weight) day − 1 for children and adults,[142,143].
respectivelyThe[142,143]. The evidence
bw (body for children and adults, respectively evidence is suggesting an
is suggesting an alimentary impact as some nutrients are poorly
alimentary impact as some nutrients are poorly absorbed when combined with alkaloids absorbed when combined with [140].
alkaloids [140]. Caffeine analysis is common in the food industry (e.g., quality control in beverages)
Caffeineand analysis is common in the food industry (e.g., quality control in beverages) and research
research (e.g., alkaloid carrying plants); it has also been incorporated in academia and student
(e.g., alkaloid carrying
curricula [144]. plants); it has also been incorporated in academia and student curricula [144].

Figure 3.Figure 3.
Chemical Chemical
structures structures
for (A)forcaffeine
(A) caffeine (1,3,7-trimethylxanthine), (B)
(1,3,7-trimethylxanthine), (B) theobromine
theobromine (3,7-
(3,7-dimethylxanthine), (C) theophylline (1,3-dimethylxanthine), (D) paraxanthine (1,7-dimethylxanthine),
dimethylxanthine), (C) theophylline (1,3-dimethylxanthine), (D) paraxanthine (1,7-
and (E) antipyrine (2,3-Dimethyl-1-phenyl-3-pyrazoline-5-one or phenazone). (F) Caffeine
dimethylxanthine), and (E)
biotransformation antipyrine
pathway (2,3-Dimethyl-1-phenyl-3-pyrazoline-5-one
is dependent on the CYP1A2 and CYP2A6 enzyme or phenazone).
system. 1. (F)
Caffeine1,3,7-trimethylxanthine
biotransformation pathway
2. is dependent on
1,7-dimethylxanthine 3. the CYP1A2 and 4.CYP2A6
7-methylxanthine enzyme
7-methyluric system. 1.
acid
5. 1-mthyluric acid 6. 5-acetylamino-6-formylamino-3-methyluracil
1,3,7-trimethylxanthine 2. 1,7-dimethylxanthine 3. 7-methylxanthine 4. 7-methyluric acid7. 1,7-dimethyluric acid 8. 5. 1-
5-acetylamino-6-amino-3-methyluracil [145].
mthyluric acid 6. 5-acetylamino-6-formylamino-3-methyluracil 7. 1,7-dimethyluric acid 8. 5-
acetylamino-6-amino-3-methyluracil [145].
Foods 2019, 8, 1 10 of 63

2.3.1. Alkaloid Analysis and Reported Application to Real Samples

Several methods have been developed for alkaloid analysis in food samples. Also, methods for
studying the fate of these alkaloids have been documented (Table 6). For example, Grujić-Letić and
coworkers, analyzed 12 commercial tea and coffee products, non-alcoholic energy drinks and foods
(including mate, green tea, and black tea), 5 combined preparations of over the counter non-steroid
anti-inflammatories and water samples collected from 7 representative locations of the Danube
River [146]. This paper represents a clear example of method versatility, as a single analyte was
recovered, from variable matrices, and assessed using a similar procedure. This analysis was not
only used for characterization, but also demonstrated a potential for quality control in commercial
products (e.g., compliance of the nutritional label) and water. In water samples, the highest caffeine
concentration found was 306.120 ± 0.082 ng L−1 during springtime. Gonçalves and coworkers recently
demonstrated that caffeine might be a suitable chemical marker of domestic wastewater contamination
in surface waters [147].

Table 6. Summary of conditions regarding alkaloid analysis.

Measurement Method, Sensitivity, mg

Matrix Extraction Method Reference
Chromatographic Column L−1 or mg kg−1
Food Samples
DAD 270 nm (caffeine) Nova-Pak C18
150 × 3.9 mm, 5 µm, mobile phase:
Energy drinks Sonication for degassing 0.023 [148]
MeOH, NaH2 PO4 /hexanesulfonic
acid (C6 H13 SO3 H)
25 mmol L−1 NaAOc/HAOC buffer,
Sonication for degassing.
Energy drinks pH 6.0, an inertsil OctaDecylSilane-3V 0.19 [149]
“Dilute and shoot”
250 × 4.6 mm, 5 µm, UV 230 nm
Defat with C6 H14 , 1. HPLC 250 × 4.60 mm, 5 µm
Cocoa Acetone/H2 O/HAOc 2. UPLC Acquity HSS T3 0.001 for both LCs [150]
(70/29.5/0.5) 100 × 2.1 mm, 1.8 µm
UHPLC-Q-Orbitrap-MS/MS
Cocoa-based Defat by mechanical dispersion polyphenols (n = 35, ESI− ) and Theobromine 0.03,
[151]
products with C6 H14 , MeOH/H2 O (80:20) alkaloids (n = 2, ESI+ ) Kinetex caffeine 0.04
biphenyl 100 × 2.1 mm, 1.7 µm
HP-TLC LiChrospher silica gel plates,
Mate beer and fluorescence indicator and mobile
Sonication for degassing, ACN. 0.4 [152]
mate soft drinks phase acetone/toluene/chloroform
(4:3:3) UV 274 nm
Biological Samples
Waters Atlantis C18 150 × 4.6 mm,
Human and 5 µm. Mobile phase: 15 mmol L−1
Ultracentrifugation, 12,000 rpm 0.02 [153]
synthetic plasma PBS (pH 3.5)/ACN (83:17). PDA 274
nm, IS: antipyrine
Mobile phase:
H2 O/HAOc/MeOH/ACN (79:1:20:2),
Human saliva Chloroform/isopropanol (85:15) 0.032 [154]
Kromasil 100 C18 250 × 4.6 mm, 5 µm,
30 ◦ C, UV 273 nm
10 mmol L−1 PBS (pH 6.8)/ACN
SPE polymeric 96-well plates
Human and (gradient mode). Zorbax® SB-Aq
Strata-X™. Elution: 0.1 [155]
neonate plasma narrow bore RR 100 × 2.1 mm,
MeOH/H2 O/HAOc (70:29:1)
3.5 µm), 40 ◦ C, UV 273 nm

Shrestha and coworkers developed a method for use as quality control. Concentrations of
Nepalese tea and coffee ranged from 1.10 to 4.30 mg caffeine kg−1 dry basis [156]. Fajara and Susanti
also determined caffeine in coffee beverages; they found 109.7–147.7 mg caffeine kg−1 per serving [157].
Gliszczyńska-Świgło and Rybicka used both a photodiode and fluorescence detector to monitor both
caffeine and water-soluble vitamins, simultaneously, in energy drinks [148]. Aşçı and coworkers
analyzed caffeine in soft drinks [158]. The authors used Behnken response surface design to optimize
HPLC conditions. Optimized variables included pH, 6.0, flow rate, 1.0 mL min−1 and a mobile
phase ratio, 95% [158]. Similarly, preservatives sorbate and benzoate also can be determined with
caffeine simultaneously in sports drinks [149]. Ortega and coworkers compared data from HPLC- and
UPLC-MS/MS (MS/MS also known as tandem mass spectrometry). The authors analyzed procyanidin
Foods 2019, 8, 1 11 of 63

oligomers (mono to nonamers) and catechin, epicatechin, caffeine, theobromine. The analysis was
performed under 12.5 min [150]. Recently, Rodríguez-Carrasco and coworkers used to analyze
polyphenols and alkaloids in cocoa-based products. Mainly, they compared three different coffee
varieties including “Forastero”, “Trinitario”, and “Criollo”. Mostly, theobromine was found in major
quantities relative to caffeine except Criollo 70 and 75% where the theobromine/caffeine ratio is
ca. 1:1. Of all samples examined, Criollo varieties showed the highest quantities of alkaloids. [151].
Interestingly, a positive association has been described between cacao polyphenol absorption and
theobromine [159]. Other identifying markers, such as fatty acids, have also been reported as tools for
discrimination among coffee varieties. The authors were able to discern Coffea arabica (Arabica) and
Coffea canephora (Robusta) using ∑MUFA, 18:3n3, ∑MUFA/∑SFA [160].

2.3.2. Alkaloid Bioavailability and Transference to Biological Samples

Caffeine is rapidly absorbed following oral consumption; maximum blood (plasma) levels are
usually reached within 30 min [140]. Caffeine bioavailability studies have been performed in human
plasma, for example, Alvi and Hummami monitored caffeine and antipyrine (Figure 3). Caffeine in
human plasma was stable for at least 24 h at room temperature or 12 weeks at –20 ◦ C [153]. Caffeine is
a demonstrated therapeutic agent for apnea of prematurity. Hence, López-Sánchez developed
a method to monitor caffeine in serum to demonstrate that the drug had achieved its therapeutic
levels (i.e., 30 or 35 µg mL−1 ) [161]. Cleanup using SPE adapted in multiple well plates, as the one
used in the former study, is an easy way to process several samples simultaneously, instead of the
one-on-one cartridge approach. Only in 85% and 78% of the cases studied, maternal and newborn
absorption of caffeine was demonstrated, respectively. Another research group investigated caffeine
metabolism based on CYP1A2 enzyme activity. The presence and ratio of theophylline, paraxanthine,
theobromine, and caffeine (Figure 3) was evaluated in human saliva [154]. The authors collected
saliva of healthy subjects after consumption of a caffeinated beverage and obtained data of compared
chromatographic profiles from the saliva of smoking (active xenobiotic hepatic metabolism) and
non-smoking subjects [154]. Saliva, plasma, and urine already have been demonstrated valuable
to intervention studies for cocoa [155,162]. Kobayashi used differential chromatogram analysis to
narrow the signal width for caffeine, in urine samples, to improve separation demonstrating that
peak enhancing posterior to injection is possible [163]. Finally, Ramdani and co-workers incorporated
green and black tea powder into bovine diets demonstrating that alkaloids, catechins, and theaflavins
diminished ammonia and methane productions without any detrimental effect on rumen functions
in vitro [51].
Although theobromine is not a usual analyte for feed analysis, is noteworthy that the 2002/EC/32
regulation sets limits for the analyte at 300 mg kg−1 for compound feed, except for adult cattle feed,
where the threshold is laxer (i.e., 700 mg kg−1 ).

2.3.3. Method Application Experience

Tea and coffee sample extracts are rich in tannins and other non-desired compounds that may
generate matrix effects and reduce the shelf life of an analytical column. We have successfully used
MgO to remove said interferences while increasing the extract pH. An alkaline medium ensures
positively charged alkaloid molecules. Furthermore, defatting is vital for an adequate recovery when
a lipid-rich sample is treated (e.g., cacao seeds, >30 g total fat/100 g), especially, if aqueous extracting is
employed. We suggest the use of efficient organic solvents; n-hexane, petroleum benzine, for example,
have been exploited. Minimal amounts possible should be used, as this otherwise generates waste.
Chlorinated solvents and ethyl ether should be avoided, as alkaloids exhibit some degree of solubility
in these solvents which, in turn, may affect recovery.
Foods 2019, 8, 1 12 of 63

2.4. Cholesterol
Cholesterol ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]2,3,4,7,8,
9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol), is a waxy steroid metabolite
found in the cell membranes and transported in the blood plasma of all animals [164]. This sterol
plays a role in metabolic (e.g., precursor for bile acids and steroid hormones) and structural
processes (e.g., regulates biological membrane fluidity) [165,166]. Cholesterol can be introduced
to the metabolism through de novo synthesis or diet [162]. In plant structures, similar compounds are
found such as phytosterols and stanols [167]. However, when analyzing cholesterol, one must consider
that the amount of cholesterol made by many plants is not negligible [168]. Nutritional information
regarding cholesterol content in food and intake through dietary sources is relevant, as overload
can drastically increase plasma cholesterol levels and, hence, health risks. From a methodological
standpoint, a considerable advantage in using the LC approach is that lipid oxidation is negligible,
as measurements can be performed at relatively low temperatures. Herein are detailed some examples
of cholesterol analysis in food samples (Table 7).
Albuquerque and coworkers compared both HPLC and UPLC for the analysis of eggs, egg yolks,
sour cream, and chicken nuggets. The latter approach rendered a method with 8-fold less solvent
waste and ca. 4-fold more sensitivity, with a decreased analysis time (i.e., 4 min) [166]. The initial
sample mass used from the assay was optimized; 0.25 g and 1 g for samples with relative lower
(e.g., sour cream) and higher (e.g., egg yolk) cholesterol contents. The authors also compared different
cooking methods for the chicken nuggets (baked vs. deep frying). They found that cholesterol content
was higher in the oven baked goods. This is a result of the processing as the meat loses water during
baking. Meanwhile, water/oil exchange occurs during frying. Although several solvents were tested,
the authors concluded that an acetonitrile/2-propanol solvent system was the most successful in
eluting the cholesterol molecule [166]. Cholesterol analysis usually renders clean chromatograms since
most interferences are eliminated by saponification. Saponification segregates the molecule of interest
from the saponifiable lipid fraction (e.g., acylglycerols) and hydrolyzes cholesterol esters. This step
has been considered critical for cholesterol analysis in food matrices [166]. Furthermore, Cruz and
coworkers, quantitatively, compared several extraction methods on freeze dried and thawed seafood
samples [169]. In this regard, the direct saponification and extraction considerably reduce solvent waste,
while the Smedes method used non-chlorinated solvents (is a greener approach). Better recoveries
for vitamin E are obtained when the analysis is performed before saponification step (e.g., modified
Folch, Smedes). The authors were able to analyze α-tocopherol, cholesterol, and fatty acids all
from the same extract and applied the optimized method to octopus, squid, mackerel, and sardine
successfully. From the assayed samples, squid and sardine showed higher values of cholesterol and
vitamin E, respectively. Interestingly, normal phase chromatography was used to assess vitamin
E [169]. Saldanha and Bragagnolo also used normal phase chromatography. The authors used
very mild conditions during saponification, which are paramount to avoid cholesterol oxidation.
Also, they monitored cholesterol contents after heat treatment and demonstrated that it decreased
significantly, with a simultaneous increase of the cholesterol oxides contents (i.e., 19-hydroxycholesterol,
24(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-hydroxycholesterol,
and 7-ketocholesterol) [170]. Bauer and coworkers analyzed cholesterol and cholesterol oxides in
milk samples using reversed-phase chromatography. [171]. The presence of cholesterol oxides can
indicate the source and nature of the food, as well as the storage and processing conditions suffered
by a commodity. The authors conclude that milk has physicochemical characteristics that make
it more resistant to oxidation of cholesterol compared to other products of animal origin. In this
regard, several sample preparation methods for cholesterol oxides have been detailed elsewhere [173].
Daneshfar and coworkers used dispersive liquid–liquid microextraction as an alternative to the
extraction and clean-up steps in sample preparation [172]. In this case, ethanol was used as a disperser
solvent and carbon tetrachloride as an extraction solvent [172]. This work is a fine example of
parameter optimization during method validation; different dispersion (i.e., EtOH, acetone, and ACN)
Foods 2019, 8, 1 13 of 63

and extraction (i.e., CS2 , CH2 Cl2 , CHCl3 , and CCl4 ) solvents were tested, as well as variables such as
pH, volume and time. However, the authors fail to explain how they obtain total cholesterol from
a complex matrix (for example, a method must be able to free cholesterol from its esterified form) when
no hydrolysis is performed (i.e., ensuring not just the mere quantification of unbound/free cholesterol).

Table 7. Measurement techniques meant for cholesterol in food samples.

Measurement Method, Sensitivity, mg L−1 or
Matrix Extraction Method Reference
Chromatographic Column mg kg−1
Egg-, dairy-and 1. SupelcosilTM LC-18-DB 150 × 4.6 mm, 3 µm 3
ACN/2-propanol [166]
meat-based
products 2. Acquity UPLC® BEH C18 50 × 2.1 mm,
0.7
1.7 µm, UV 210 nm
1. In situ: KOH 2 mol L−1 /MeOH, 80 ◦ C, Vitamin E: FLD λex 290 λem 330). Cholesterol:
N2 , C6 H14 UV 210 nm, Supelcosil™ LCSI 75 × 3.0 mm, Vitamin E: 0.05
Seafood
2. Modified Folch: MeOH/CH2 Cl2 (1:2), 3 µm, mobile phase: n-hexane and 1,4-dioxane Cholesterol: 10 [169]
saponification (97.5:2.5) IS: tocol
3. Smedes: 2-propanol/cyclohexane
(1:1.25), saponification
1. Nova Pack CN HP 300 × 3.9 mm, 4 µm,
n-hexane/2-propanol (97:3), UV 210 nm
KOH 50 g/100 g/EtOH, 25 ◦ C, 22 h, in the
Seafood (cholesterol oxides), RID (cholesterol 0.01 [170]
dark, C6 H14
and epoxides)
2. Confirmation: HPLC-APCI-MS QTRAP®
Restek C18 150 × 6 mm, 5µm, mobile phase:
KOH 50 g/100 g/EtOH, 25 ◦ C, 22 h, in the ACN/2-propanol (95:5), UV 202 nm
Dairy product 11.10 [171]
dark, C6 H14 25-hydroxy and cholesterol, 227 nm
7-ketocholesterol

Egg and dairy 1. Egg yolk and milk: pretreatment with

CLC-ODS-C8 150 × 6 mm, 5µm. Mobile phase:
product and ACN 0.01 [172]
ACN/EtOH (50:50), HPLC-UV 210 nm,
vegetable oil 2. Liquid–liquid dispersion (DLLME) EtOH
(800 µL)/CCl4 (35 µL).

It should be pointed out that though the chlorinated solvents are used in very small quantities,
they are still classified by the IARC (International Agency for Research on Cancer) as possible human
carcinogens (group 2B). Finally, Robinet and coworkers used a cholesterol esterase in an unrelated
matrix to avoid chemical saponification [174]. In this regard, cholesterol esterases (most active at pH
7.0, 37 ◦ C, and in the presence of taurocholate) and lipases (most active at pH 7.7, and 37 ◦ C [175]) are
commercially available.

Method Application Experience

We suggest two major points: i. that it is recommendable to perform the saponification first and
then the solvent-aided extraction ii. a response surface design may be useful to optimize the length of
the saponification treatment.

3. Determinations Designed for Feed and Feed Ingredients

3.1. Mycotoxins

3.1.1. Recent Approaches for the Determination of Mycoxotins in Feeds

Mycotoxins are secondary metabolites mainly by fungi Aspergillus, Penicillium, Fusarium and
Alternaria species, in stress situations, which involve changes in temperature, moisture or pH
in plants [58,176,177]. Currently there are more than 400 types of mycotoxins as ubiquitous
contaminants in a wide variety of foods [178,179], such as, corn, cocoa, sorghum, wheat, oats,
rye, cotton, peanuts, coffee, dairy products, eggs, among others [180]. Among the best known
are ochratoxin (OTA), zearalenone (ZEA), trichothecenes, aflatoxin B1 (AFB1 ), fumonisin B1 (FB1 ) and
their metabolites. The last two are listed as carcinogenic by the IARC [181]. Mycotoxins, in general,
are teratogenic, mutagenic, carcinogenic, and can possess an immunosuppressive effect in both animals
and humans [178,182], which can be aggravated by factors such as the animal species, the concentration
of the toxin and synergism existing among them, in addition to the health and nutritional status of the
animal [182,183]. Also, the direct effects on health, including decreased weight gain, feed conversion
Foods 2019, 8, 1 14 of 63

inefficiency, reduced production, and a decrease of the food system profitability, the increase in feedstuff
costs, medical treatments, and ineffectiveness when exploiting the genetic potential of animals [183].
At an organ level, in the liver, AFB1 can generate several metabolites, which include aflatoxin
M1 (AFM1 ), which is transferred to milk, a complete food nutritionally, and which is vital in the
development of the first years of life [184,185]. Also, the AFM1 is a compound declared as a carcinogen
that is very resistant to pasteurization and freezing [180,183]. Therefore, being trawl compounds in the
trophic chain, which involve the adverse effects on livestock production, with an obvious risk to the
health of consumers, it stresses the need for laboratories to possess the ability to analyze a large number
of analytes in a single sample. In this way, the amount of information can be increased, and a wider
diagnosis can be made about the safety of the food and feed industry.
In this regard, Table 8 shows a summary of methods developed for the identification and
quantification of mycotoxins, by different research groups, focused mainly on animal feed. For example,
Njumbe Ediage and coworkers developed a technique capable of determining 25 mycotoxins in cassava
meal, peanut cakes, cornmeal, and different sorghum varieties. The most exciting thing, in this case,
is how the researchers solved the affinity fact of fumonisin and ochratoxin with the amino groups
(due to the presence of carboxylic acid moiety, Figure 4) [177,186]. The researcher divided their extract
into two portions, one to which formic acid and dichloromethane were added. After cleanup, the two
independent shares were remixed evaporated at 40 ◦ C, reconstituted with MeOH/H2 O/CH3 COOH,
and 5 mmol L−1 CH3 COO− NH4 + . During MS-based mycotoxin separations, flows are usually
kept low, so solvent nebulization and evaporation are performed swiftly. The mobile phase is
generally accompanied by an acetic or formic acid buffer to improve ionization especially for those
compounds without readily ionizable functional groups (e.g., aflatoxins). Also, the formate ion is
added in both solvents as one solvent depletes during the gradient separation and the buffer must
always be present in a similar proportion [177,186]. Dzuman and coworkers and Rasmussen and
coworkers, used, as an extraction method, a modification of the QuEChERS method, (Quick, Easy,
Cheap, Effective Rugged, and Safe usually used for pesticide analysis). Both research groups coincide
that QuEChERS adaptations for mycotoxin analysis open the possibility toward the simultaneous
assay of several and distinct groups of contaminants (e.g., pesticides and mycotoxins) [179,187].

Table 8. Measurement techniques meant for mycotoxins in feed samples.

Number of
Measurement Method,
Matrix Analytes/Execution Extraction Method Reference
Chromatographic Column
Time (min)
Cassava meal, peanut
MeOH/CH3 CO2 CH2 CH3 /H2 O
cakes, cornmeal, LC: Symmetry RP-18 150 × 2.1 mm,
25/28 (70:20:10), cleanup was performed [177,186]
and different sorghum 5 µm, Identification: MS/MS/ESI+
using amino SPE cartridges
varieties
Cereals, compound feed LC: Acquity UP3 HSS T3 100 × 2.1 mm,
56/50 Modified QuEChERS method [179]
and silages 1.8 µm, Identification: MS/MS/ESI±
Acid acidified ACN and sodium
acetate was used to separate the LC: Ascentis Express C18 , 150 × 2.1 mm,
Bovine milk 10/30 [185]
aqueous from the hydrophilic 2.7 µm, Identification: MS/MS/ESI+
phase from milk
LC: Gemini® C6 -Phenyl 100 × 2.0 mm,
Silage 27/44 Modified QuEChERS method [187]
3 µm, Identification: MS/MS/ESI±
84 and 62
ACN/H2 O/CH3 COOH (79:20:1) LC: Gemini® C18 , 150 × 4.6 mm, 5 µm,
Millet and Sorghum respectively/Not [188,189]
mixture Identification: MS/MS/ESI±
Indicated
 Acid acidified ACN and
LC: Ascentis Express C18, 150
sodium acetate was used to
Bovine milk 10/30 × 2.1 mm, 2.7 µm, [185]
separate the aqueous from the
Identification: MS/MS/ESI+
hydrophilic phase from milk
LC: Gemini® C6-Phenyl 100 ×
Silage 27/44 Modified QuEChERS method 2.0 mm, 3 µm, Identification: [187]
MS/MS/ESI±
Foods 2019, 8, 1Millet and 84 and 62 LC: Gemini® C18, 150 × 4.6 15 of 63
ACN/H2O/CH3COOH
respectively/Not mm, 5 µm, Identification: [188,189]
Sorghum (79:20:1) mixture
Indicated MS/MS/ESI±

Figure 4. Chemical structures for (A) ochratoxin A, (B) ochratoxin B, (C) ochratoxin C, blue colored
Figure 4. Chemical structures for (A) ochratoxin A, (B) ochratoxin B, (C) ochratoxin C, blue colored
circles represent changes in the structure between ochratoxins, loss of Cl and OH in ochratoxin B and
circles represent changes in the structure between ochratoxins, loss of Cl and OH in ochratoxin B and
C respectively render a more lipophilic molecule. Et = C2H5, and (D) are the general backbone of
C respectively
Fumonisins. FB1 = 721.83 lipophilic
render a more g mol−1 R1: H molecule.
R2: OH R3: OH; EtFB=2 C 2 H5 , and
= 705.84 g mol(D)
−1 R1: are
OH Rthe
2: H general
R3: OH; FBbackbone
3 = of
Fumonisins. FB1g =
705.84 721.83
mol g mol−3:1OH;
−1 R1: H R2: H R
R1 :FB
H4 =R689.84
2 : OH g R
mol
3 : −1 R1: H R2: H R3: H. Functional−groups
OH; FB 2 = 705.84 g mol 1 R : OH
1 R
colored2 :
inH R3 : OH;
FB3 = 705.84 green and−
g mol red
1R represent −1 R :respectively.
a positively and negatively ionizable moiety,
1 : H R2 : H R3 : OH; FB4 = 689.84 g mol 1 H R2 : H R3 : H. Functional groups
colored in green and red represent a positively and negatively ionizable moiety, respectively.
3.1.2. Agricultural by-Products as Feed Ingredients
Agricultural
3.1.2. Agricultural and food-industry
by-Products as Feed residues are valuable to animal nutrition as they are rich in many
Ingredients
bioactive and nutraceutical compounds, such as polyphenolics, carotenoids and dietary fiber among
Agricultural and
others [190]. food-industry
Agro-byproducts, residues
used in animal are
feed,valuable to animal
originate from perishablenutrition
crops and, asassuch,
theyareare rich in
susceptible
many bioactive andtonutraceutical
fungal infection compounds,
[191]. Hence, mycotoxin
such assurveillance of these carotenoids
polyphenolics, materials contemplating
and dietary fiber
the most common contaminants present in such matrices, but also considering emerging
among others [190]. Agro-byproducts, used in animal feed, originate from perishable crops and,
contaminants (e.g., beauvericin, enniantins, and fusaproliferin) [191,192] is paramount. The food
as such, are susceptible
industry generally toincludes
fungal practices
infection that[191]. Hence,
guarantee mycotoxin
the safety surveillance
of the product meant forof human
these materials
contemplating
consumption. Residues destined for animal production may not be subject to the same scrutiny. For emerging
the most common contaminants present in such matrices, but also considering
contaminantsexample, thebeauvericin,
(e.g., wine industry with a production
enniantins, and estimated at 27 million[191,192]
fusaproliferin) liters worldwide. Presence of The food
is paramount.
OTA in wine has been widely investigated [193]. However, with the development of new methods,
industry generally includes practices that guarantee the safety of the product meant for human
it has been possible to find up to 36 different mycotoxins. (See for example, [194]).
consumption. Countries
Residues destined
where for animal
the production production
of wine may notcompared
is the predominant, be subject to the
to other same
types of scrutiny.
For example, the wine
industry, industryamount
a considerable with a ofproduction
waste must estimated
be repurposed.at 27
Asmillion
such, thisliters
mightworldwide.
be of use as Presence
a of
OTA in wine ruminant (such widely
has been as cows and goats) feed ingredient,
investigated where the pulp,
[193]. However, withhusks, and seeds, mightof
the development offer
new to methods,
it has beenthe animal diet: fiber, energy, fatty acids, and antioxidant compounds which improve ruminal health,
possible to find up to 36 different mycotoxins. (See for example, [194]).
echoing in the quality of meat and milk [195–197]. As yet another benefit from this waste processing,
Countries where
the use of grape theseeds
production
as mycotoxinof wine is thehas
adsorbents predominant,
been investigatedcompared to [198]
both in vitro otherand
types of industry,
in vivo
a considerable amount
(e.g., pigs [199]). of waste must be repurposed. As such, this might be of use as a ruminant
(such as cows and goats) feed ingredient, where the pulp, husks, and seeds, might offer to the
3.2. Antibiotics
animal diet: fiber, energy, fatty acids, and antioxidant compounds which improve ruminal health,
echoing in3.2.1.
the quality of meat and
Recent Multiresidue and milk [195–197].
Multi-Class Analysis Asof yet another
Antibiotics benefit from this waste processing,
in Feeds
the use of grape seeds as mycotoxin adsorbents has been investigated
Antibiotics are bioactive substances used against bacteria as a therapeutic, both in vitro [198]orand in vivo
metaphylaxis
(e.g., pigs [199]).
prophylactic agent both in humans and animals [200–202]. In livestock, some antibiotics are included

3.2. Antibiotics

3.2.1. Recent Multiresidue and Multi-Class Analysis of Antibiotics in Feeds

Antibiotics are bioactive substances used against bacteria as a therapeutic, metaphylaxis or
prophylactic agent both in humans and animals [200–202]. In livestock, some antibiotics are included
in animal diets as growth promoter (e.g., monensin, narasin, ractopamine), decrease feed conversion,
improve feed efficiency, and overall cost-effectiveness of animal production systems [203,204].
Overuse of veterinary pharmaceuticals in livestock, aquaculture, and the feed industry is reflected
in the incidence of residues found in animal-derived food products (e.g., meat, eggs, milk,
and honey) [201,205–207]. Antibiotic biotransference through the food chain may contribute to allergic
reactions, mutanogenic and cancerogenic effects, found in humans and animals; additional to the
growing rates of antimicrobial resistance [208,209]. Considering these issues, organizations worldwide
(e.g., European Commission, United States Food and Drug Administration, World Health Organization)
have generated protocols that help control, regulate and surveil the use of antibiotics in food-producing
Foods 2019, 8, 1 16 of 63

animals [208,210–212]. Hence, similar to mycotoxins, development of analytic methods that allow for
identifying and quantitating a broad spectrum of compounds from a sample, directly contributes to
surveillance programs for feedstuff manufacturing (raw materials or feed ingredients, compound feed,
and premixes) and, similarly, those commodities derived from food-producing animals.
Table 9 shows a summary of the different characteristics of validated methods for the identification
and quantification of veterinary antibiotics in different types of matrices. Molognoni and coworkers,
optimized a method for the determination of spectinomycin, halquinol y zilpaterol in compound
feed demonstrating once again the capabilities of mass spectrometry to assess two or more
families of seemingly unrelated compounds. The authors tried both hydrophilic interaction and
reverse-phase chromatography. Though HILIC (Hydrophilic Interaction Liquid Chromatography)
offered good results, it requires a longer analysis time (i.e., up to 5 additional min), and is pH
sensitive. Reverse-phase chromatography requires a relatively inexpensive column that is usually
available in laboratories and which analytical instrumentation providers generally keep in stock.
Additionally, a more effective separation was archived using heptafluorobutyric acid in the mobile
phase [202].

Table 9. Measurement techniques meant for veterinary antibiotics in food and feed samples.
Number of
Measurement Method, Chromatographic
Matrix Analytes/Execution Extraction Method Reference
Column
Time
Recent Multiresidue and Multi-Class Analysis of Antibiotics in Feeds
During extraction, fat was removed and
clean up performed using an SPE PRiME
BEH C18 column
Rendering products 40/Not Indicated HLB cartridge, eluate evaporated to [201]
Identification: HPLC-MS/MS/ESI+
dryness and reconstituted with ACN and
formic acid
Hypersil Gold HILIC (150 × 3.0 mm, 5 µm)
Compound feed 3/Not Indicated Formic acid/H2 O (80:10) and C18 (50 × 2.1 mm, 3.5 µm). [202]
Identification: HPLC-MS/MS/ESI+
Pig, poultry, ACN/H2 O (90:10) acidified with C18 Vensusil XBP (50 × 2.1 mm, 3.0 µm,
62/13 [209]
and cattle feed CH3 COOH. 100 Å). Identification: HPLC-MS/MS/ESI+
Acidic extraction with hydrochloric acid Acquity UPLC HSS
Feed 10/Not Indicated (0.5 mol L−1 aqueous solution), and T3 (150 × 2.1 mm, 1.7 µm). Identification: [213]
purified by SPE cartridge HPLC-MS/MS/ESI±
Multiresidue Analysis of Antibiotics in Foods
Extraction with ammonium formate and X-SELECT C18 (150 × 2.1 mm, 3.5 µm)
Fish muscle 41/20 [205]
ACN/H2 O (80:20) Identification: HPLC-MS/MS/ESI±
Extraction with formic acid in water XBridge BEH C18 (100 × 2.1 mm, 2.5 µm).
Shrimp 24/8 [206]
and ACN Identification HPLC-MS/MS/ESI+
Poroshell 120 ECC18 (50 × 3.0 mm, 2.7 µm)
Poultry muscle tissue ACN extraction
14/14 Identification: HPLC-MS/MS/ESI± [207]
and eggs Centrifugation at 0 ◦ C 45 min
(quadrupole linear ion trap)
Modified QuEChERS method
ZORBAX Eclipse XDB C-18 (150 × 4.6 mm,
Honey 6/Not Indicated Extraction was performed using ACN [214]
5 µm). Identification: HPLC-MS/MS/ESI+
and MgSO4 and NaCl

ACN: Acetonitrile.

3.2.2. Multiresidue Analysis of Antibiotics in Foods

Barreto and coworkers developed a method to assay n = 14 different coccidiostats (i.e., lasalocid A,
maduramicin, monensin, narasin, salinomycin, semduramicin, robenidine, diclazuril, toltrazuril,
trimethoprim, chlopidol, amprolium, diaveridin y nicarbazin) in poultry muscle tissue and eggs;
after testing several chromatographic columns, they selected the one that completed the separation
under less time (i.e., 14 min). The authors used low temperature clean-up as an alternative to SPE,
reducing costs, time and ion suppression. Internal standards where used to compensate intense
matrix effects [207]. Regarding aquaculture, Kang and coworkers analyzed n = 41 antibiotics in fish
muscle [205]. Similarly, Kumar Saxena and coworkers developed and validated n = 24 antibiotics
(including quinolones, sulfonamides, and tetracyclines) in shrimp, and they preferred to use methanolic
separation [206]. Finally, Shendy and coworkers identified n = 6 different classes of antibiotics in honey
with a modified QuEChERs procedure simultaneously. Extraction was performed using ACN and
MgSO4 and NaCl [214].
Foods 2019, 8, 1 17 of 63

For both mycotoxins and antibiotics, a review was made of the wide variety of methods used
in the food industry for the simultaneous, extraction of multiple analytes. For the identification
and quantification of each chemical, a sensitive and selective tool is required. It is here that mass
spectrometry has been useful, by reducing costs and response time. [185,202,209].

3.2.3. Method Application Experience (Mycotoxins and Antibiotics)

A multitoxin (n = 26) analysis was applied to feedingstuffs using, as a reference, a method
previously described by Wang and coworkers in cornmeal. ACN/CH3 COOH/H2 O (74:1:25) was used
for extraction and cleanup we exploited the versatility of HLB cartridges (which allow the retention of
a wide array of analytes with the least of interferences) [215]. When compared with immunoaffinity
columns, this sorbent is less prone to fracturing and do not require low temperatures for storage.
Later, the recovered extract was evaporated to dryness using vacuum at 60 ◦ C and reconstituted
with MeOH. The method relies on the 12.5-fold concentration of the original analyte to improve
sensitivity. In the case of antibiotics (n = 23), we based our procedure on that described by Duelge and
coworkers [216]. We extracted and eluted analytes using an ACN/MeOH solution. Again, we trusted
the versatility of HLB SPE cartridges during cleanup. Both assays were single quadrupole equipped LC
system using ESI+ and relied on a reverse phase separation (Zorbax Eclipse Plus, 100 × 3 mm, 3.5 µm).
For mycotoxin separation, the mobile phase consisted of a gradient using acidified (0.1 mL/100 mL
formic acid) ACN and H2 O. For antibiotics, the gradient consisted of three different acidifed solvents
ACN, H2 O, and MeOH. In our experience, the first two-solvent gradient (starting with water) can
separate most antibiotic families (β-lactams, tetracyclines, macrolides, streptogramins, lincosamides,
aminoglycosides). Our gradient finishes with MeOH which is the only solvent capable of eluting
coccidiostats (e.g., monensin and narasin). Efficient chromatographic separation was achieved under
35 min.

3.3. Amino Acids

Protein building blocks (i.e., amino acids), biologically, can be separated into two main groups.
Exogenic/essential amino acids (i.e., Arg, Phe, His, Ile, Leu, Lys, Met, Thr, Trp, and Val), are not
synthesized by the organism and must be provided in the diet to cover the requirement. The remaining
amino acids are endogenic (i.e., Ala, Cys, Asp, Glu, Pro, Ser, Tyr, and Gly). Several of these amino
acids (e.g., Lys, Met, Thr, and Trp) are prepared synthetically and are commercially available to use
as feed additives. The purity of these additives must be routinely checked and adequately verified.
Hence, methodological development is paramount for quality control for determination of amino
acids in feed materials and feed mixtures. However, few reports have focused on feed. As a result;
we intend to give an overview of the methods available in related matrixes.

3.3.1. Fish Tissue

In a comprehensive research article, Mohanty and coworkers reported the complete
amino acid profile (except tryptophan which was assessed spectrophotometrically and
basic hydrolysis) for 27 different food fishes. [217]. Derivatization was performed using
6-aminoquinolyl-N-hydroxysucciminidyl carbamate (AQC), this specific reagent requires neutral
pH to work. Adduct formation has the advantage of being stable and reacting with secondary amines.
No variability among profiles was found in fishes of the same species from different locations. They also
related the concentration of the amino acid found in the fish with the environment in which they
live (e.g., marine and cold-water fishes showed relatively higher amounts of Met). At the same time,
they recommend the consumption of certain fish species for several amino acids dietary deficiency
in humans [217]. Example of methods suitable to analyze amino acids in diverse matrixes is shown
in Table 10.
Foods 2019, 8, 1 18 of 63

Table 10. Sample pretreatment, derivatization and measurement conditions for amino acids in feeds.
Measurement Method,
Matrix Hydrolysis Derivatization Reference
Chromatographic Column
Applications in Feed and Related Matrices
Licrospher 100 RP 18 125 × 4 mm,
Spirulina sp. Various physical methods 2-mercaptoethanol [218]
FLD λex 360 λem 460 nm
1. Total AA: HClO4 8 mol
Supelcosil LC18 -DB 250 × 4.6 mm,
L−1 , 150 ◦ C for 2 h, 140 ◦ C
5 µm. Gradient 0.7 mol L−1
for 4 h, 120 ◦ C for 8 h, and Triethylendiamine (TEA), phenylisothiocianate
Spirulina sp. acetate buffer pH 6.4/TEA, H2 O [219]
110 ◦ C for 22 h. 2. Free AA: (separation of protonated species)
and ACN/H2 O (80:20). UV λ
CCl3 COOH, sodium
254 nm
deoxycholate
Zorbax AAA at 40 ◦ C
Pyrogallol, HCl 8.3 mol L−1
40 mmol L−1 NaH2 PO4 pH 7.8,
Spirulina sp. 70–80 ◦ C 2 h, IS o-phtaldialdehyde (OPA) [220]
ACN/MeOH/H2 O (45:45:10),
triundecanoin
2.0 mL min−1 , UV λ 338 nm
UHPLC EZ:faastTM 4u AAA-MS,
Soncation, EZ:faastTM Free 250 × 2.0 mm, 3 µm. IS:
Plants propyl chloroformate [221]
Amino Acid Kit homoarginine, methionine-d3 ,
and homophenylalanine
Chamomile Shimpack column (250 × 4.6 mm,
Free amino acids: Sonication AccQ Fluor, 55 ◦ C [222]
flowers 5 µm) FLD λex 250 λem 395 nm
Ion exchange chromatography,
Rapeseed meal HCl 6 mol L−1 , 110 ◦ C 23 h Ninhydrin Vis 570 nm (Pro 440 nm), [223]
IS: Norleucine
HCl 6 mol L−1 0.1 g/100 mL
Feed Borate buffer pH 10, OPA (primary-) and FMOC Zorbax Eclipse-AAA 40 ◦ C, λex
phenol, 150 ◦ C 6 h, [224]
ingredients (secondary amines) 262 λem 338 nm
Reacti-ThermTM
AccQ-Tag Ultra C-18 100 × 2.1
6 mol L−1 HCl 110 ◦ C 16–23
Feed AQC, borate buffer mm, 1.7 µm). UPLC PDA 260 nm, [225]
h, peformic acid, HBr,
IS: DL-2-aminobutyric acid
6 mol L−1 HCl 110 ◦ C closed AccQ-Fluor Reagent (AQC in 0.2 mol L−1 borate
Fish tissue FLD λex 250 λem 395 nm. RP C18 [217]
vessel 24 h buffer pH 8.8)
Selected Applications
Matrix Hydrolysis and treatment Measurement method, chromatographic column Reference
UPLC BEH C18 50 × 2.1 mm, 1.7 µm, 130 Å, UV 202–208 nm.
Sodium dodecyl sulfate (SDS), enzymatic
Lipoprotein Phosphate buffer 50 mmol L−1 pH 4.35/sodium azide and Phosphate [226]
digestion (e.g., pronase E, muramidase)
buffer 75 mmol L−1 pH 4.95/MeOH (85:15)
SDS, sonication, DNAse, RNAse, and trypsin.
CF3 COOH/MeOH, UPLC-TOF/MS-ESI+ CSH C18 100 × 2.1 mm,
Peptidoglycan HCl for teichoic acids. Hydrolases [227]
1.7 µm, UV 210 nm
(mutanolysin)
Fermentation, HCl 0.1 mol L−1 , mechanical UPLC-ESI+ -MS Acquity UPLC BEH C18 150 × 2.1 mm, 1.7 µm and
Cocoa beans [228]
dispersion, ethyl ether LC/ESI+ -MS/MS Aeris Peptide XB-C18 150 × 2.1 mm, 3µm
n-hexane defat, Tris/HCl pH 7.5, SDS, RP-HPLC Jupiter Proteo 250 × 10 mm, 4 µm FLD λex 280 λem = 360 nm
Olive seeds dithiothreitol, high-intensity focus ultrasound, RP-HPLC-ESI+ -QTOF-MS, Ascentis Express Peptide ES-C18 [229]
acetone precipitation, alcalase hydrolysis 100 × 2.1 mm, 2.7 µm

3.3.2. Filamentous Cyanobacteria, Spirulina sp.

Spirulina sp. is a filamentous cyanobacterium that have been recognized for its nutritional
value as a feed ingredient and supplement, and has been related to health benefits in humans [230].
Its nutrient profile has been reported previously, and it has even exhibited a higher amino acid
value (except for Lys, Glu, Pro, His) when compared with that of soybean meal (a staple feed
ingredient) [218]. Additionally, based on this profile, they calculated energy for a broiler diet.
Nurcahya Dewi and coworkers applied different physical treatments (i.e., drying, sonication 30/60 min,
reflux 60/90 ◦ C, maceration in MeOH) to determine their effects on Spirulina sp. amino acid profile,
which they concluded is rich in amino acids related to umami flavor (i.e., Asp and Glu). Drying and
methanol maceration showed to be the treatment that delivered the highest (8.37 g/100 g) and lowest
(2.34 g/100 g) contents of Glu, respectively [231].
Campanella and coworkers assayed total and free amino acids from Spirulina sp.; they found
that freshwater Spirulina contained relatively high concentrations of non-essential amino acids.
The authors indicate that the samples tested were lysine-rich and limited in sulfur-containing
amino acids. Free amino acids constitute as high as 2% of the amino acid input. Method-wise,
the authors used an oxidation-capable acid, this is chancy as it may contribute to analytes deterioration.
Additionally, the mobile phase already included the derivatization agent [219].
Foods 2019, 8, 1 19 of 63

Al-Dhabi and Arasu quantified polyunsaturated fatty acids, sugars, polyphenol and total and
free amino acids in Spirulina sp. In contrast to the authors mentioned above, this research group used
pre-column derivatization and a dedicated column for analysis. Total amino acids contents ranged
from 11.49 to 56.14 mg/100 g; from which essential amino acids accounted for 17.00 to 39.18%. [220].

3.3.3. Compound Feedstuff

For the specific case of feed, a time-reduced (i.e., complete separation in an eight-minute
chromatographic run) analysis has been recently developed [225]. AOAC OMASM includes two
different assays to determine amino acids based on LC; 992.12 design for pet foods using fluorescence
and 999.13 include ninhydrin/Orto-phthalaldehyde (OPA) fluorescence or pulsed coulometric
detection. Finally, a report made by Wang and coworkers described a successful simultaneous analysis
of 20 amino acids without using derivatization using an evaporative light scattering detector [232].
More recently, underivatized amino acids have also been monitored using hydrophilic interaction
liquid chromatography coupled with tandem mass spectrometry [233]. Herein, we included some
examples of derivatization agents. However, we suggest the reader access a paper written by Masuda
and Dohmae, which not only cites the four most commonly used reagents for amino acid derivatization,
but also identifies their strengths and weaknesses [234].

3.3.4. Bacterial Cell Walls, Peptidoglycan, and Food-Extracted Peptides

A less common application for LC, is to monitor the products from the hydrolysis of
bacterial cell walls (using enzymatic physical, and chemical approaches) and posterior fragment
analysis. Desmarais and coworkers design a method that included the digestion of Braun’s
lipoprotein. Muropeptide fragments (monomers-trimers), 3,3-diaminopimelic acid among others [226].
Kühner and coworkers developed a similar application; complete muropeptide hydrolysis was
accomplished within 24 h. UPLC/MS was used to monitor fragments. After BH4 − reduction,
both Gram-positive to Gram-negative bacteria can be evaluated after gradient modification [227].
In this regard, MSD (Mass Spectrometry Detectors) serve as a good reference for additional mass
information, which will ease the peptidoglycan in silico reconstitution. This application has not found
accommodation in food or feed, but it can correctly be adapted for bioreactions/fermentations or
lactic bacteria.
Other applications for LC include, for example, the work by Marseglia and coworkers.
They identified n = 44 different peptides from cocoa beans. The peptide fragmentation pattern
in fermented cocoa samples was used to describe the geographic origin, different fermentation levels,
and roasting. Vicilin, a storage protein, was identified in cocoa bean samples, information that
can be useful to understand the biological activity of cocoa and to determine the aroma relevant
peptides [228]. MS assisted analysis is advantageous as amino acids lack any distinctive chromophores
and already have readily ionizable moieties. Prados and coworkers recently have described a method
to isolate, characterize and identify peptides that can downregulate adipogenesis. The authors also
used semipreparative fractionation to achieve the initial peptide separation [229].

3.3.5. Method Application Experience

When facing fresh feed products (e.g., wet pet food, forages) additional operation units such as
lyophilization is necessary before sample treatment (see, for example, [235]). To obtain individual
amino acids, most applications require acid or alkaline hydrolysis. However, amino acids are extremely
susceptible to oxygen during hydrolysis, to prevent quantitative losses, we recommend the sample
hydrolysis steps suggested elsewhere for furosine [236]. Additionally, pyrogallol in 1 g/100 mL is also
used as a radical receptor (i.e., a radical sink) to avoid amino acid degradation. Particularly, Trp, Thr,
and Tyr are usually lost during acid hydrolysis, cysteine is oxidized to cysteic acid, and asparagine
and glutamine (if present) will transform to their respective acids. Hydrolysis may be performed using
a feed of known concentration in parallel as a reference [237].
Foods 2019, 8, 1 20 of 63

From the sample preparation standpoint, we have applied a Supelco ENVI-Carb SPE cartridge
for cleanup as hydrolysate retain undesired particulates. A translucent extract is obtained after
SPE that will be suitable for both FLD (Fluorescence Detector) and UV-Vis (Ultraviolet-Visible)
detection-based analysis. Also, cleaner chromatograms are obtained as interferences are significantly
reduced. This cartridge will adsorb (including those responsible for coloration) a great range of
molecules, while the (charged) amino acids will not be retained. Sodium azide is applied routinely for
extract storage. However, best results are obtained when measurements are performed immediately
after preparation steps.
We have used a method based on OPA pre-column derivatization adapted from an established
method for biopharmaceuticals [238]. We also recommend the strict use of a C18 guard column
to increase column lifespan. When applied to feed samples and feed ingredients, essential amino
acids covered include Arg, His, Ile, Leu, Lys, Met, Phe Val, and non-essential Ala, Asp/Asn, Glx,
Cys/CY2, Gly, and Ser for a total of 14 amino acids. OPA derivatization is only effective under alkaline
conditions (usually performed using borate buffer pH 8–10). Therefore, the feed hydrolysate must be
neutralized (to pH 7.0) before injection, as the buffer will not be able to compensate for the [H3 O+ ]
that results from the acid treatment. Furthermore, 9-fluorenylmethyl chloroformate (FMOC) must be
included during derivatization (additional to OPA) to obtain proline and hydroxyproline amino acids
(see, for example, [224]).
Method automatization (when an automatic sampler is available) can concede an advantage
since the reaction occurs in situ within the needle. Automated precolumn derivatization is also
useful for unstable adducts (e.g., OPA derivatives). A benefit of amino acid derivatization is
that most adducts can be monitored using a UV/VWD (Ultraviolet/Variable Wavelength Detector)
or DAD/PDA (Diode-Array Detection/Photodiode-Array Detection), so even if the fluorescence
detector is not available, analysis can still be performed. Though, the fluorescent detector can filter
interferences, begetting cleaner chromatographs. We have also used the same method to assess the
purity of feed grade amino acids, and taurine. The technique can also be applied to energy drinks to
evaluate taurine in as a very simple “dilute and shoot” method after sonication for sample degassing.
Interestingly, ninhydrin and OPA can detect complementary analytes to methods based in ninhydrin
(see, for example, [223]).

3.4. Triphenylmethane Dyes

Malachite green is a dye usually used in aquaculture as a fungicide and antiparasitic due to its
low cost and effectiveness [239]. The widespread use of this substance is not without downsides,
though, including residue accumulation in fish tissue and contamination of sediments and water
bodies, which can affect non-target organisms downstream (see, for example, [240,241]).
Recent and improved methods have found acceptance to monitor these kinds of dyes in fish
tissue. For example, Hidaya and coworkers already conducted a short review on techniques available
for detection of malachite and leucomalachite green in the fish industry [242].
Within, this paper, several LC-based techniques are mentioned. Triphenylmethane dyes
suffer from reversible redox reactions; each form can be oxidized or reduced to one another
(see, for example, [243]; Figure 5).
Foods 2019, 8, 1 21 of 63
Foods 2018, 7, x FOR PEER REVIEW 23 of 62

Figure
Figure 5.5. Chemical
Chemical structures
structures for
for three
three triphenylmethane
triphenylmethane dyes
dyes which
which are
are sharing
sharing aa common
common phenyl
phenyl
backbone sharing a methylidyne. Each molecule
backbone sharing a methylidyne. Each molecule has extended π-delocalized electrons justifying their
π-delocalized electrons justifying their
crystal
crystal coloration
coloration and
andvisible
visiblelight
lightabsorption
absorption(ca.
(ca. 621
621nm
nmfor
formalachite
malachitegreen).
green).

Table
Table11 11shows
showsaasummary
summaryof of various
various methods
methods for
for the
the extraction
extraction and and identification
identification of of malachite
malachite
green
green and its metabolites. Although it is a common contaminant in aquaculture production, and
and its metabolites. Although it is a common contaminant in aquaculture production, and
research
researchfocuses
focuses onon fresh
fresh residues
residues from
from aquaculture
aquaculture production
production animals
animals (fish,
(fish, shrimp,
shrimp, lobster,
lobster, among
among
others),
others), thethe development
development of of methods
methods should
should also
also be
be extended
extended to to the
the analysis
analysis ofof feed
feed [244]
[244] asas fish
fish and
and
shrimp
shrimp feed are made from marine by-products. Doses on fish or shrimp range from 0.05–0.2 mgmg
feed are made from marine by-products. Doses on fish or shrimp range from 0.05–0.2 L−1
Las−1anasactive
an active ingredient have been used. Treatments for fish eggs include dosages
ingredient have been used. Treatments for fish eggs include dosages of 5 mg L−1 is usually of 5 mg L −1 is

usually
suggested for fish for
suggested fishLaboratories
tanks. tanks. Laboratories
usuallyusually
measure measure
malachite malachite greenequipment
green with with equipment
able toable to
detect
detect − 1
tissue tissue
residuesresidues
belowbelow
2 µg2 kg
µg−1kg (maximum
(maximum permitted
permitted residue
residue limit
limit ininfish
fishtissue;
tissue;[250]).
[250]). A A very
very
interesting approach was made by Furusawa who developed a green chemistry
interesting approach was made by Furusawa who developed a green chemistry method for malachite method for malachite
green
green andand its
its metabolite
metabolite[251].
[251].
As previouslyTable mentioned, Wang and coworkers used solid-phase microextraction with the
11. Measurement techniques meant for triphenylmethane dyes.
excellent result to assess malachite green, crystal violet and their respective metabolites using a
Measurement Method,
monolithic fiber [245]. Bae LeeExtraction
Matrix and coworkers
Method homogenized fish tissue samples, and the
Chromatographic Column
extracted
Reference
residues were partitioned into dichloromethane
− 1
Extraction with 0.1 mol L NH4 O2 C2 H3
and an in situ oxidation with 2,3-dichloro-5,6-
LC: Cloversil-C18 250 × 4.6 mm, 5 µm.
dicyano-1,4-benzoquinone. Afterward, 0.25 g mL−1 , was performed
cleaned-up Identification:on neutral+ alumina and
Fresh fish muscles buffer, pH 4.5, HAH solution [243]propyl
MS/MS/ESI
1 mol L−1 p-TSA solution and can
sulfonic acid cation-exchange solid-phase extraction cartridges. Malachite green and crystal violet
Extraction with McIlvaine buffer, TSA, and LC: Prodigy ODS-3 C18 150 × 4.6 mm,
Channel
were Catfish muscle
determined at 618 and 588 nm using HPLC-Vis detector common+approach[244]
[246]. A MS/MS/ESI included
TMPD. Oasis MCX SPE columns 3 µm. Identification:
analyzing dyes using traditional detectors and adding a step that
LC: Phenomenex C18 included
140, 250 × 4.6confirmation
mm, 5 by MS.
µm. Identification: UV 558 nm (malachite
Chengyun
Aquacultureand coworkers relied
water Noton Oasis® MCX (a green
indicated strong cation exchange-based
and crystal violet), FLD λex 265, λem
adsorbent)
[245] to
perform clean-up. After a two-step, QuEChERS extraction, dispersive 360 nm (leucosolid phase extraction coupled
forms)

with, both,fisha products

reverse phase and
Extraction strong
with anion
ammonium exchange
acetate buffer, (as250
LC: × 4.6as
well mm,a mixed mode
5 µm Capcell PAK adsorbent)
C18 cleanup
Processed [246]
HAH solution, p-TsOH solution, and can Identification: MS/MS/ESI+
was tested. Residues of the dyes were evaluated in codfish [247]. However, we do not see how anion
Modified QuEChERS Extraction:
Fish tissue LC: XCharge C18 column
exchange favors dye-stationary NHphase
4 O2 CH interaction,
and can since all parent compounds are positively[247] charged.
−
Noteworthy,Salmonusually reverse phase
Extraction C2 H3 O2 columns
buffer, p-TSAcan resolve YMC
solution, these types
phenyl of50dyes
3-4-5 with
× 4.0 mm, ease, even[248]
3 µm if several
hydroxylamine and can Identification: LC-MS/ESI+ /APCI
analytes are to be evaluated simultaneously. Croatia and Iran are specific examples of countries
Chromolith® Performance RP-18e
which have stated have
Fish feed foundwith
Extraction residues
ACN/CHof this dye in fish tissue
3 OH/CH3 COOH (100 ×[252,253]. Both cases demonstrate
4.6 mm) Identification: [249] the
MS/MS/ESI +
need to assess these compounds in food items. However, both research groups used immunoassays
to evaluate the contaminant. AOAC method OMASM 2012.25 is a reference based on LC-MS/MS to
Astriphenylmethane
assess previously mentioned, dyes andWang and coworkers
their metabolites used solid-phase
in aquaculture products. microextraction with
the excellent
Additionally, resultUStoFDA assess malachite
reference methodgreen, crystalon
is based violet and theirof respective
the isolation malachite greenmetabolites
using
using a monolithic
alumina/propyl fibersolid
sulfonic [245].
phaseBae Lee and
extraction coworkers
cartridges previoushomogenized fish tissue
to Non-Discharge samples,
Atmospheric
and the extracted
Pressure Chemicalresidues
Ionization were
and partitioned
an LC-MSn; into dichloromethane
quantification was performed and an in insalmon
situ oxidation with
[248]. Finally,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone.
since fish and shrimp compound feed can also Afterward,
be based cleaned-up
in aquaculture was performed
by-product onmeal,
neutral
as alumina
a source
and propyl sulfonic acid cation-exchange solid-phase extraction
of protein, contaminated tissue can reach the final product. Hence, the need for feed analysis cartridges. Malachite green andis
crystal violet were determined at
evident, as it shows, Abro and coworkers [249].618 and 588 nm using HPLC-Vis detector [246]. A common approach
included analyzing dyes using traditional detectors and adding a step that included confirmation by
Foods 2019, 8, 1 22 of 63

MS. Chengyun and coworkers relied on Oasis® MCX (a strong cation exchange-based adsorbent) to
perform clean-up. After a two-step, QuEChERS extraction, dispersive solid phase extraction coupled
with, both, a reverse phase and strong anion exchange (as well as a mixed mode adsorbent) cleanup
was tested. Residues of the dyes were evaluated in codfish [247]. However, we do not see how anion
exchange favors dye-stationary phase interaction, since all parent compounds are positively charged.
Noteworthy, usually reverse phase columns can resolve these types of dyes with ease, even if several
analytes are to be evaluated simultaneously. Croatia and Iran are specific examples of countries which
have stated have found residues of this dye in fish tissue [252,253]. Both cases demonstrate the need
to assess these compounds in food items. However, both research groups used immunoassays to
evaluate the contaminant. AOAC method OMASM 2012.25 is a reference based on LC-MS/MS to
assess triphenylmethane dyes and their metabolites in aquaculture products.
Additionally, US FDA reference method is based on the isolation of malachite green using
alumina/propyl sulfonic solid phase extraction cartridges previous to Non-Discharge Atmospheric
Pressure Chemical Ionization and an LC-MSn ; quantification was performed in salmon [248].
Finally, since fish and shrimp compound feed can also be based in aquaculture by-product meal,
as a source of protein, contaminated tissue can reach the final product. Hence, the need for feed
analysis is evident, as it shows, Abro and coworkers [249].

4. The Common Ground among Measurements Performed in Food and Feed Laboratories

4.1. Nitrates and Nitrites

Nitrates and nitrites are natural compounds that are part of the nitrogen cycle, but especially high
dosages of these ions are registered because of anthropological activities [254,255]; they enter human
diets by means of drinking water, leafy vegetables, and cured meats. Noteworthy, these ions have been
authorized as additives in several countries including the European Community [256,257].
Though there is evidence that both ions have a relevant biological and physiological function,
special attention has been paid to nitrates and nitrites and their metabolites such as N-nitrosamines and
nitrous oxide as all these molecules may pose a health hazard [256,257]. For example, these compounds
have been related to colorectal cancer [256–261]. Hence, risk management and assessment in food have
been proved necessary [258]. Regarding the quantification of NO2 − and NO3 − using HPLC, there are
two main approaches used i.e., ion exchange and reverse phase columns (Table 12).

4.1.1. Ion Exchange Chromatography

When analyzing crops, one must consider that cultivar, and harvest date can affect the nitrate
levels of selected vegetables. Hence, maximum levels have been set by European legislation
accordingly [262]. For example, Brkić and coworkers analyzed several leafy greens (n = 200) in
two different seasons, in order to evaluate differences in ion content and encountered considerable
differences among vegetable and sampling season [263]. Pardo-Marin and coworkers assessed
vegetable-based baby foods, considering the levels found within these types of foods. They calculated
ion ingestion between 13–18% of the acceptable daily intake for an infant. [264]. Quijano and coworkers
assessed the nitrate content of vegetables (n = 533); they obtained values up to 3509 mg kg−1 in chard
samples. They calculated an intake of 490 mg kg−1 bw day−1 for a young population, values which
tend to increase the risk of exceeding acceptable intake values [265]. The main advantages in using ion
exchange columns are that the separation can be accomplished using aqueous buffers which are made
up from relatively cheap salts, making the methods apt for green chemistry and avoid mobile phase
drift [263].

4.1.2. Ion Pairing and Reverse Phase Chromatography

Tetrabutylammonium salt has also been used as an ion-pairing agent coupled with reverse
phase columns (Table 12). For example, Hsu and coworkers used a reverse phase approach to
Foods 2019, 8, 1 23 of 63

assess both ions in cured meats and vegetables. The authors found the highest values of NO3 −
in spinach (4849.6 mg kg−1 ) and for NO2 − in hot dogs (78.6 mg kg−1 ) [266]. Nitrite tends to
oxidate to NO3 − , the authors cite several factors affecting nitrate and nitrite recovery in foods
(e.g., temperature, pH, metals). Usually, non-desired compounds found in greens differ from those
found in meat products, for which proteins interfere significantly. Meat sample extracts will need pH
adjustments and higher temperatures are needed to improve recovery. Some of these parameters must
be monitored during analysis, especially when vegetables are subject of study [266]. Croituru used
a similar approach to assess human, rabbit, rat urine as well as vegetables. However, they roduced
adducts (an azo dye, HO3 SC6 H4 –N=N–C10 H6 NH2 ) based on Greiss reaction (sulfanilic acid form
a diazonium cation (HO3 SC6 H4 –N≡N+ ) with NO2 − and then with 1-naphthylamine) for NO2 −
that was measured at 520 nm [267,268]. Interestingly, the authors followed the reaction with
mass spectrometry. We encourage the reader to pay special attention to this paper as highlights
difficulties during method development. The author concluded that while useful, the use of Greiss
reaction, spectrophotometrically, is unadvisable as several samples may exhibit additional confounding
compounds that may behave similarly as the NO2 − ion adduct. However, is quite valuable as
a derivatizing agent when coupled with HPLC; the method can work with samples of different
origins without the need for further modifications [267]. Samples were decolorized with carbon and
ZnSO4 was applied for protein precipitation to overcoming this matrix interference and enhance the
sensitivity. Croituru and coworkers used a validated method to assess NO2 − and NO3 − in vegetables
for self-consumption; toxicologically speaking, the NO2 − content found in the samples was deemed
too low to represent a hazard [269].
Stationary phases containing only alkyl chains have been used, but it is also possible to find mixed
stationary phases, for example, Abdulkair and coworkers assayed NO2 − and NO3 − using a stationary
phase containing both alkyl groups and phenyl groups (Table 12) to separate both ions successfully
after sonication [270].
Chou and coworkers assessed both ions in vegetables and observed a high concentration
variability was observed which reflect differences in environmental conditions [271]. The authors also
optimized critical chromatographic parameters such as pH, organic solvent fraction, and flow [271].
In this regard, the methanol fraction optimization was demonstrated to be paramount to improve
octylammonium solubility and achieve an optimal resolution between both ions. In contrast, pH and
flow variations tend to have an effect only on chromatographic run times and not so much in resolution.
Foods 2019, 8, 1 24 of 63

Table 12. Common chromatographic approaches for the determination of nitrate and nitrite ion.

Measurement Method,
Matrix Mobile Phase Composition Reference
Chromatographic Column
Ion Exchange Chromatography
Waters IC-PAK HC anion exchanger
Leafy greens 10 g L−1 of KH2 PO4 , pH 3.0 [263]
(150 × 4.6 mm), UV λ 214 nm
Waters IC-PAK HC anion exchanger
Baby foods Phosphate 5 mmol L−1 (pH 6.5) [264]
(150 × 4.6 mm), UV λ 214 nm
Waters IC-PAK HC anion exchanger
Vegetables Phosphate 5 mmol L−1 (pH 6.5) [265]
150 × 4.6 mm, 10 µm, UV λ 214 nm
Reverse Phase Chromatography
Measurement method,
Matrix Ion pair reagent Mobile phase composition Reference
chromatographic column
Cured meat and Tetrabutyl Phenomenex C18 110 Å Gemini
MeOH:H2 O (75:25) [266]
vegetables ammonium (TBA) 250 × 4.6 mm, 5 µm. PDA λ 214 nm
X Bridge C18 , 50 × 2.1 mm, 2.5 µm.
TBA, Greiss Gradient
Vegetables UV-Vis λ 222 (nitrate) and 520 nm [267]
reagent MeOH/ACN/H2 O
(nitrite)
ACN/2 mmol L−1 RP-thermophenyl hexyl,
Cured meats 3 mmol L−1 TBA [270]
HPO4 2− pH 4 150 × 4.6 mm, 3 µm, UV λ 205 nm
0.1 mol L−1
octyl OA buffer pH 7.0/MeOH Phenomenex Luna C18 250 × 4.6 mm,
Vegetables [271]
ammonium salt (70:30) 5 µm, UV λ 213 nm
Dried vegetables Triethylamine C6 H13 SO3 H, H2 PO4 − , C13 250 × 4.6 mm, 5 µm, UV λ
[272]
and water (TEA) TEA pH 3.0/MeOH (80:20) 222 nm.
0.01 mol L−1 AcclaimTM Polar Advantage and C18
Ham n-octylamine/TBA n-octylamine/5 mmol L−1 Thermo Scientific™, HyPURITY™, [273]
TBA pH 6.5 250 × 4.6 mm, 5 µm

4.1.3. Miscellaneous Methods for Nitrates and Nitrites

In contrast with ion pairing approaches, dos Santos and coworkers developed a method based
on the reaction of the NO2 − with 2,3-diaminonaphthalene to yield a highly specific fluorescent
2,3-naphthotriazole adduct (λex 375 λem 415 nm), under acidic conditions, to assess the ions in
beetroot [274]. Cassanova and coworkers have developed an application for HPLC derivatization
based on VCl3 , 4-nitroaniline, methanesulfonic acid, and N-(1-naphthyl)-ethylenediamine. Under these
conditions, a post-column reduction of nitrate to nitrite can be accomplished [275].

4.1.4. Method Application Experience

The preferred methodology used in our laboratories is based on the chromatographic
determination of NO2 − and NO3 − anions simultaneously. Reverse phase (using a C18 column, i.e.,
Zorbax Eclipse 5.0 µm, 4.6 mm × 150 mm, set at 30 ◦ C and 0.6 mL min−1 ) HPLC-PDA or -VWD
(213 nm as the absorption spectra maximum) is sufficient to perform the assay [266,271]. It is important
to emphasize that for the detection and separation of inorganic anions, in this case NO3 − and NO2 − ,
the mobile phase must contain a complementary counter ion that interacts with it and with the bonded
stationary phase of the column concurrently. In the absence of the counter ion, no interaction with the
column is achieved and, as a result, no retention will be obtained at all, as the ions would come out in
the void. In this scenario, a tetrabutylammonium salt (e.g., tetrabutylammonium hydrogen sulfate,
TBAHS, 155837 Sigma-Aldrich) is a possibility (Figure 6B). In this case, the four alkyl chains from the
reagent interact with the eighteen-carbon alkyl chains of the stationary phase and, at the same time,
with the NO2 − /NO3 − . The elution order may be explained by considering a more delocalized negative
charge (among three oxygen atoms) in NO3 − and the bent geometry of NO2 − due to the nitrogen
atom-containing an electron lone pair. Interestingly, NO2 − is a larger anion (0.192 nm), when hydrated,
than NO3 − (0.179 nm) [276]. Now, depending on the length of the column, the affinity of the this will
not be sufficient to resolve peaks from the solvent front (specially the first peak; NO2 − ), this issue is
easily solved including acetonitrile in the mobile phase, using slower flows, a longer column or even
an ion pair agent with longer alkyl chains (e.g., octylamine). The mobile phase used is 20% acetonitrile,
from the reagent interact with the eighteen-carbon alkyl chains of the stationary phase and, at the
same time, with the NO2−/NO3−. The elution order may be explained by considering a more
same time, with the NO2−/NO3−. The elution order may be −explained by considering a more
delocalized negative charge (among three oxygen atoms) in NO3 and the bent geometry of NO2− due
delocalized negative charge (among three oxygen atoms) in NO3− and the− bent geometry of NO2− due
to the nitrogen atom-containing an electron lone pair. Interestingly, NO2− is a larger anion (0.192 nm),
to the nitrogen atom-containing an electron lone pair. Interestingly, NO2 is a larger anion (0.192 nm),
when hydrated, than NO3−− (0.179 nm) [276]. Now, depending on the length of the column, the affinity
when hydrated, than NO3 (0.179 nm) [276]. Now, depending on the length of the column, the affinity
of the
Foods this8, will
2019, 1 not be sufficient to resolve peaks from the solvent front (specially the first peak; 25 NO −),
of263
of the this will not be sufficient to resolve peaks from the solvent front (specially the first peak; NO2−),
this issue is easily solved including acetonitrile in the mobile phase, using slower flows, a longer
this issue is easily solved including acetonitrile in the mobile phase, using slower flows, a longer
column or even an ion pair agent with longer alkyl chains (e.g., octylamine). The mobile phase used
column
80% or even
TBAHS an ion
5 mmol L−pair
1 , atagent withInterestingly,
a 6.55pH. longer alkyl chainsinjecting
(e.g., octylamine). The
withmobile phase used
is 20% acetonitrile, 80% TBAHS mmol L−1−1, at a 6.5 when
pH. Interestingly,a solution
when injectingboth ions present
a solution with
is
and20% acetonitrile, 80% TBAHSthe 5 mmol L , (the
at a 6.5 pH. obtained),
Interestingly, when injecting a solution with
bothations
the present
same concentration,
and at the same response
concentration, signals
the response (theissignals
very similar in area/height
obtained), and,
is very similar
both
as ions present and at the same concentration, the response (the signals obtained), is very similar
in such, sensitivity
area/height and,isasvery
such, close for bothisanions.
sensitivity very close for both anions.
in area/height and, as such, sensitivity is very close for both anions.

Figure6.6.Schematic
Schematic representation for interaction
the interaction of nitrite ion with (A) a cation stationary
exchange
Figure
Figure 6. Schematicrepresentation forfor
representation the of nitrite
the interaction ion with
of nitrite ion(A)with
a cation
(A) exchange
a cation exchange
stationary
phase or (B)phase or (B)
interaction interaction
with TBAHS with TBAHS
present present
in present
the mobilein the
phasemobile phase
and phase and
stationary phase C18 .phase CC1818..
stationary phase
stationary phase or (B) interaction with TBAHS in the mobile and stationary

The same
The same methodology
methodology has has been
been used
used inin feed
feed toto assay
assay hayhay samples
samples (Figure
(Figure 7A,B)
7A,B) thatthat were
were
The same methodology has been used in feed to assay hay samples (Figure 7A,B) that were
presumed as the
presumed the source
sourceofofintoxication
intoxicationininhorseshorses [277]. In this
[277]. case,case,
In this fromfrom
ten samples assayed,
ten samples three
assayed,
presumed as the source of intoxication in horses [277]. In this
−1 ) case, from ten samples assayed, three
(average
three concentrations
(average of 92.77of
concentrations ± 60.88
92.77mg ± kg −1) and six (average
60.88 mg kg and concentrations
six (average of 92.13 ±
concentrations47.55 mg
of
(average concentrations
−1) samples tested−positive
1
of 92.77 ± 60.88 mg kg−1) and six− (average concentrations
− of 92.13 ± 47.55 mg
kg for NO − and NO3−, respectively (unpublished
92.13 ± 47.55 mg kg ) samples tested positive for NO2 and NO3 , respectively (unpublished data).
2 data). Forage and swine
kg−1) samples tested positive for NO2− and NO3−, respectively (unpublished data). Forage and swine
compound
Forage feed samples
and swine compound (n =feed
10) have
samplesalso(nbeen
= 10)assayed
have also with
beenthis method
assayed obtaining
with values
this method from <5
obtaining
compound feed samples (n = 10) have also been assayed with this method obtaining values from <5
to 23.69
values and<52.30
from to 4.96
to 23.69 andand
2.30925.15
to 4.96toand1135.10
925.15and 989.51 and
to 1135.10 to 1479.71
989.51 to mg kg−1−1 for
1479.71 mgNO kg−2−−1 and
for NONO2 3−−,
to 23.69 and 2.30 to 4.96 and 925.15 to 1135.10 and 989.51 to 1479.71 mg kg for NO 2 and NO3−,
and NO3 − , respectively
respectively on both accounts.
on both In accounts.
the case ofInfeeds and of
the case fishfeeds
meals,andwhich suffer from
fish meals, whichsevere
suffermatrix
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respectively on both accounts. In the case of feeds and fish meals, which suffer from severe matrix
effects,matrix
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from the cartridgeusing the mobileusing
is performed phase.the mobile phase.
the elution from the cartridge is performed using the mobile phase.

Figure 7. Chromatograph of (A) an aqueous 10 mg L−1−1 nitrite (4.95 min) and nitrate (6.26 min) standard
Figure 7. Chromatograph
Chromatograph of
of (A)
(A) an aqueous
anwith
aqueous 10 mg
10 mg LL−1nitrite (4.95 min) and
andnitrate (6.26
(6.26min) standard
Figure
(B) hay7.sample after extraction hot water, SPE nitrite (4.95
cleanup, min)
and nitrate
micropore min)
filtration standard
presence of
(B)
(B) hay
hay sample
sample after
after extraction
extraction with
with hothot water,
water, SPESPE cleanup,
cleanup, and and micropore
micropore filtration
filtration presence
presence of
of nitrite
nitrite (4.91 min) and nitrate (6.23 min) is evident.
nitrite (4.91and
(4.91 min) min) and nitrate
nitrate (6.23ismin)
(6.23 min) is evident.
evident.

4.1.5. Legislation
Regulation 2002/EC/32 sets limits for NO2 − in fish meal (i.e., 60 mg NaNO2 kg−1 ) and complete
feedingstuffs (i.e., 15 mg NaNO2 kg−1 ) excluding those intended for pets except birds and aquarium
fish. We refer the reader to two thorough reviews that tackle regulatory as well as methodological
topics [278,279].

4.2. Carotenoids
Chemically, carotenoids are conjugated hydrocarbons that may be further classified as
carotenes (without any oxygen molecules) and xanthophylls (with one or more oxygen molecules).
Carotenoids are widespread natural pigments, are recognizable from the bright colors (yellow, orange,
Foods 2019, 8, 1 26 of 63

red, or purple) that they often confer on plant and animal organ. The molecules responsible for
producing said coloration must be attained from dietary sources. For example, lutein and zeaxanthin
are carotenoid pigments that impart yellow or orange color to various common foods such as
cantaloupe, pasta, corn, carrots, orange/yellow peppers, fish, salmon and eggs, β-carotenoid and
isomer are found in sweet potatoes, dark leafy greens, butternut squash, lettuce, red bell peppers,
apricots, broccoli, and peas, and lycopene are in tomato. As molecules with a conjugated double
bond system, carotenoids serve several physiological functions (e.g., antioxidants, immunostimulants,
photoprotection, visual tuning, among others). This electron delocation causes them to be particularly
unstable compounds, especially sensitive to light, heat, oxygen, and acids. Hence, several precautions
have been taken while extracting carotenoids. For example, must be carried out in dim lighting;
use rotary evaporation at low temperature and reduced pressure also it has to be carried out under
a stream of nitrogen. Finally, samples should be stored in the dark, at about −20 ◦ C [280,281].
Carotenoids are fat soluble but, because of the high moisture content of plant tissues, a preliminary
extraction solvent miscible with water (e.g., methanol or ethanol) is generally necessary to allow for
penetration of the extraction solvent.
Saponification is required to remove interference as neutral fats, chlorophylls, and chlorophyll
derivatives. Usually, this procedure is carried out with potassium hydroxide in methanol.
Then, it is necessary to perform liquid–liquid extraction using a water-immiscible solvent
(e.g., ethyl acetate, ethyl ether, hexane) to obtain the unsaponifiable fraction, where carotenoids
should be present. [280–298]. The identification and quantification require high-resolution techniques;
the reversed-phase high-performance liquid chromatography has been used routinely to determine
carotenoids because of its satisfactory separation efficiency. So, several factors must be evaluated
to employ HPLC technique such as column type, mobile phase, chromatographic conditions.
Several methods for carotenoid analysis are summarized in Table 13.
Regarding column type, the analysis can be performed using a C18 column. However, YMC C30
Carotenoid dedicated column provides excellent results, had better resolution than a C18 column
for separation of carotenoids and their geometric isomers. The thirty-carbon alkyl chains interaction
with the carotenoid lipophilic profile guarantee less peak distortion and better resolution [280,281].
Compounds such as α/β/γ/δ/ε-carotene, lutein, zeaxanthin, β-cryptoxanthin, dehydrolutein,
anhydrolutein, astaxanthin, galloxanthin, α-doradexanthin, adonirubin, and canthaxanthin can all be
separated using the aforementioned chromatographic column.
According to Huck and coworkers, the flow rate did not significantly influence the resolution,
but it is essential to use an adequate flow to generate acceptable column back pressure.
Also, they studied the effect of column temperature on the separation of lutein, zeaxanthin,
β-cryptoxanthin, and β-carotene. The column temperature was varied between 21 and 80 ◦ C; the best
selectivity being achieved at 21 ◦ C, at a temperature of 34 ◦ C, zeaxanthin could not be easily separated
from lutein. The authors concluded that maintaining a constant temperature during carotenoid
analysis is critical as small changes in the ambient temperature can cause significant changes in the
chromatographic selectivity of the carotenoids and at temperatures higher than 60 ◦ C, the investigated
carotenoids unstable.
In the case of the mobile phase, the same authors indicated that carotenoid selectivity was better
using tetrahydrofuran, rather than ethyl acetate, and also better than MeOH and ACN. Carotenoids are
sensitive to degradation on the stationary phase of the column by the presence of silanol groups.
Foods 2019, 8, 1 27 of 63

Table 13. Common chromatographic approaches for the determination of carotenoids.

Measurement Method,
Matrix Extraction Method Reference
Chromatographic Column
HPLC-PDA
Quantitative: C18 Nova-Pak ODS 300 × 3.9 mm,
Extracted from the crushed peel with acetone
Camu–camu (Myrciaria dubia 4 µm set at 29 ◦ C, mobile phase ACN/H2 O/ethyl
transferred to petroleum ether/diethyl ether and [282]
(Kunth) Macvaugh) acetate For Qualitative: C30 YMC Carotenoid 250
saponified with 10% KOH methanolic
× 4.6 mm, 3 µm at 33 ◦ C. Mobile phase
MeOH/MTBE (methyl tert-butyl ether)
Extraction with n-hexane–EtOH–acetone–toluene PDA, YMC Carotenoid (250 × 4.6 mm, 5 µm,
Algae species, Chlorella vulgaris,
(10:6:7:7) 1 h, Saponification: 40 g/100 mL MeOH/ACN/H2 O (84:14:2) and CH2 Cl2 gradient [284]
and Scenedesmus regularis
methanolic KOH at 25 ◦ C in the dark for 16 h UV λ 450 nm
Plasma and liver extract: n-hexane:MTBE (1:1)
Tissues of a species of colored bird Adipose tissue, retina, beak, legs: Saponification PDA, YMC Carotenoid 250 × 4.6 mm, 5 µm,
[285]
(Taeniopygia guttata) 0.02 mol L−1 methanolic MeOH:ACN:CH2 Cl2 linear gradient)
KOH for 6 h, organic solvent extraction
Extraction with hexane/acetone/EtOH (2/1/1)
containing MgCO3 and BHT (butylated PDA, YMC Carotenoid 250 × 4.6 mm, 5 µm,
Taiwanese sweet potatoes (Ipomoea
hydroxytoluene) by 0.5 h, Saponification with at 25 ◦ C, MeOH–ACN–H2 O (84:14:2) and CH2 Cl2 , [287]
batatas (L.) Lam.)
40 g/100 mL methanolic KOH for 3 h under UV λ 450 nm
nitrogen gas at 25 ◦ C
PDA Phenomenex LUNA C18 ODS, 250 × 4.6 mm,
Mashed orange-fleshed sweet
Extraction with acetone, THF, and THF:MeOH (1:1) 5 µm, ACN:THF:MeOH: 1 g/100 mL NH4 C2 H3 O2 [288]
potato
(68:22:7:3) at room temperature and 450 nm
HPLC–APCI± –MS, Phenomenex Luna Si C18
column (250 × 2 mm, 5 µm), ACN
Extraction with THF and MeOH (1:1), petroleum
0.1 g/100 mL/MeOH (0.05 mol L−1 NH4 C2 H3 O2 ,
Selected vegetables ether containing 0.1 g/100 mL BHT and 50 mL [289]
0.05 mL/100 mL TEA)/CHCl3 (0.1 g/100 mL
10 g/100 mL NaCl
BHT)/n-heptane (0.1 g/100 mL BHT), ambient
temperature
Extraction under subdued yellow light. 50:50
Fresh and Processed Fruits and acetone/hexane UV, YMC Carotenoid 250 × 4.6 mm, 5 µm, mobile
[290]
Vegetables Saponification: saturated methanolic KOH phase 89:11 MeOH/MTBE
SPE Alumina N Sep Pak
Freeze-dried papaya homogenized in hexane:
HPLC-DAD-MS/MS-ESI+ , C30 YMC Carotenoid
Papaya (Carica papaya L., cv. CH2 Cl2 (1:1). Organic phase was separated and
250 × 4.6 mm, 3 µm, at 15 ◦ C. The mobile phase [291]
Maradol) saponified with methanolic KOH 40 g/100 mL (1:1)
MeOH and MTBE
for 1 h at 50 ◦ C
MeOH ethyl acetate, and light petroleum (bp DAD, YMC Carotenoid 250 × 3.0 mm, 3 µm at
Papaya (Carica papaya L.) 40−60 ◦ C) containing 0.1 g/L of both BHT and 25 ◦ C. Mobile phase MeOH/MTBE/H2 O (91:5:4) [292]
BHA (butylated hydroxyanisole) and MeOH/MTBE/H2 O (6:90:4)
Sample with CaCO3 , NaCl solution (30 g/100 mL, HPLC-PDA-MS/ESI+ , C30 YMC Carotenoid
were extracted 250 × 3.0 mm, 3 µm, MeOH/H2 O/aqueous
Yellow and red nance fruits
MeOH/ethyl acetate/light petroleum 1:1:1 NH4 C2 H3 O2 1 mol L−1 (90:8:2) and [295]
(Byrsonima crassifolia (L.) Kunth)
Saponification: methanolic KOH 30 g/100 mL MTBE/methanol/aqueous NH4 C2 H3 O2 1 mol L−1
stirring for 23 h (78:20:2), gradient, at 40 ◦ C. UV λ 450 nm
HPLC-PDA-MS/ESI± , C30 YMC Carotenoid
Red and Yellow Physalis (Physalis Extraction: CaCO3 , MeOH/ethyl (250 × 4.6 mm, 3 µm, mobile phase
alkekengi L. and P. pubescens L.) acetate/petroleum ether (1:1:1), containing MeOH/MTBE/H2 O (80:18:2) and [297]
Fruits and Calyces 0.1 g L−1 BHA and BHT., sonication MeOH/MTBE/H2 O (8:90:2), both 0.4 g L−1
NH4 C2 H3 O2 .Gradient.

Solvent modifiers could be added to the mobile phase, for example triethylamine (TEA).
Free silanol groups on the surface of silica deprotonate in the presence of the basic molecules,
preventing the analyte from interacting with the medium. The TEA generates a positive impact
on peak symmetry, reducing the peak tailing effect, reducing the retention time, and improving the
recovery. The addition of triethylamine to the mobile phase can also have negative consequences,
such as changes in the pH of the mobile phase; therefore, it is recommended that TEA be used in low
concentration (less than 0.05 mL/100 mL) [299].
When using chlorinated solvents, the addition of ammonium acetate to the MeOH provides
sufficient buffer capacity to prevent losses due to acid degradation of carotenoids. Some papers use
MTBE as part of the mobile phase. The advantage in using this solvent, instead of chlorinated
solvents, lies in the MTBE is less volatile (55.2 vs. 39.6 ◦ C, respectively) and less toxigenic.
Depending on the solvent system, a good compound separation may require a longer run time
and poorer resolution compared with MeOH/ACN/H2 O/CH2 Cl2 . Carotenoid content in tropical
pigment-bearing fruits [281,295,300–302], and fish [302] have also been described.

Method Application Experience

The preferred methodology used in our laboratories is based on the work by Gayosso and
coworkers with some modifications [282]. We use MTBE/MeOH as the mobile phase with a gradient
Foods 2019, 8, 1 28 of 63

system for 45 min with YMC C30 (150 × 3.0 mm, 3 µm) at 0.6 mL min−1 and 30 ◦ C. These conditions
were applied to identify and quantify carotenoids in food matrices such as palm oil, peach palm,
sweet potatoes, papaya, and guava. We extracted the carotenoids from these matrices using
a saponification procedure, followed by extraction with ethyl ether. This solvent evaporates at 40 ◦ C
and the residue is reconstituted in CHCl3 . Undesired coextractants (e.g., waxes, sterol and tocophenol
esters) are usually better solubilized with this solvent than MTBE saving from additional filtration
steps and within-system precipitation. Optimization of injection volumes and initial composition of the
mobile phase can somewhat mitigate the effect that injecting in a different solvent [303]. Analogous to
polyphenols, carotenoid extraction methods must contemplate ester hydrolysis or other treatments to
ensure the quantitation of overall amounts of carotenoids. For example, it is common to find carotenoid
esters in food matrices, and these adducts present several intrinsic difficulties during carotenoid
determination [295]. However, mass spectrometry-based LC is a powerful tool able to discriminate both
parent compounds and their esters [295]. Recently, Wen and coworkers identified n = 69 carotenoids
esters in Physalis alkekengi L. and P. pubescens L. fruits [297]. Additionally, BHA and BHT are common
organic-solvent-soluble antioxidants to preserve carotenoid integrity [298]. Finally, our laboratory has
also assessed carotenoid content in plasma from colored tropical frogs (Agalychnis callidryas).

4.3. Carbohydrates and Sugars Soluble in Ethanol

Animal feeds are, by definition, based on vegetable/plant sources that use carbohydrates as
storage compounds, structure elements, and energy sources [10]. Then, carbohydrates form the most
substantial portion of the organic matter in feeds; they can be divided into two main categories
non-structural and structural carbohydrates. We encourage the reader to examine an excellent review
of carbohydrate and organic acid in food commodities intended for human consumption by da Costa
and Conte-Junior [304]. A great starting point for reviewing different approaches for carbohydrate
analysis is the thesis written by de Goeij [305].

4.3.1. Carbohydrate Measurement Using Amino-Based Columns

Xu and coworkers compared two methods for sample cleanup and extraction. A macroporous
resin was compared to a solid phase sorbent based on alkyl chain. From the two approaches,
SPE showed less analyte loss (11.32 vs. 0.69%). However, the discoloration ratio was similar for
both methods. Sugar profile from molasses samples was obtained [305] after pigments, nitrogen
compounds, and inorganic ions were removed. The analysis was performed using two NH2 -based
columns. Under the same conditions, it was concluded that the Zorbax Carbohydrate column showed
better performance. Agius and coworkers recently developed a method to determine organic acid and
sugars in tomato fruits [306]; the authors used ACN to improve peak shape. RID (Refractive Index
Detector) is used for carbohydrate analysis since sugars do not have chromophores and alternative
detectors (e.g., MS) are expensive. RID is the detector of choice in many labs for sugar profiling
(Table 14) despite its relative lack of sensitivity. However, usual concentrations found in fruits
counteract the issue.
Foods 2019, 8, 1 29 of 63

Table 14. Different methods and stationary phases to assess carbohydrates in food matrixes.

Mobile Phase Measurement Method,

Matrix Sample Pretreatment, Extraction Reference
Composition Chromatographic Column
Amine-Based Columns
RID, IS: maltose
1. SPE Sep-Pak C18
1. Zorbax Carbohydrate
Molasses 2. Microporous resin discoloration ACN:H2 O (75:25) [307]
2. UltimateTM XB-NH2 ; both
(Seplite LX), 30 ◦ C
250 × 4.6 mm, 5 µm
Filtration, ACN/H2 O (45:55), SPE Nucleodur 100-5 NH2
Tomato ACN:H2 O (80:20) [306]
Chromabond NH2 125 × 4 mm, RID, IS: lactose
Amide-Based Columns
gradient
Confectionery, Defat (when applicable), ACN/H2 O + 0.05 UPLC-ELSD, Acquity BEH Amide
chocolate products, H2 O 80 ◦ C (+EtOH for mL/100 mL (50, 100, 150) × 2.1 mm, 1.7 µm, [308]
snacks chocolate products) ethanol- and 85 ◦ C
triethyl- amine
RID, Sugar PakTM 300 × 6.5 mm,
Apple Juice Filtration H2 O [309]
10 µm, 80 ◦ C
Ligand-Based Columns
1. H2 O 92 ◦ C
2. Reflux MeOH/H2 O (50:50)
10 mmol L−1 RID, UHPLC Aminex HPX 87H
Tubers 3. Activated Charcoal/MeOH [310]
H2 SO4 300 × 7.8 mm, 9 µm, 18 ◦ C
4. 2x·MeOH/H2 0 50:50 SPE
Bond-Elut C18
Liquids: H2 O/EtOH (50:50) 1. HPLC-RID Ultron PS-80P
Solids: H2 O 65 ◦ C, sonication, 1. H2 O 300 × 6.5 mm, 10 µm, 50 ◦ C
Foods [311]
+EtOH Fat-/Protein-rich: 2. ACN:H2 O (9:1) 2. LC-MS-ESI± Unison
H2 O/EtOH (20:80), sonication UK-Amino 150 × 2.0 mm, 3 µm
Derivatization-Based Approaches
MeOH extraction, benzyl
Gradient PDA λ 228 and 248 nm, Shim-pack
Fruit tree buds alcohol/NaOH 8 mol L−1 , SPE [312]
ACN/H2 O C18 250 × 4.6 mm, 5 µm
C18
NH4 O2 CH3 50
RID (sucrose/fructose)/UV λ 310,
2,3-naphtalenediamine, iodine, mmol L−1 pH 5.0
Foods FLD λex 320 λem 360 nm, C18 [313]
HAOc in ACN/MeOH
250 × 4.6 mm
(70:30)
Normal Phase
Glycine max (L.) ◦ C, H2 O/ACN + ELSD, PrevailTM Carbohydrate ES
H2 O 55 ACN [314]
Merr Acetone (75:25) 250 × 4.6 mm, 5 µm
Complex Carbohydrates (e.g., Inulin and Fructans)
1. Knauer Eurokat Pb
2. Nucleosil CHO 620
Plants and feed H2 O or H2 SO4 0.01
H2 O 3. Nucleosil CHO 682 (Pb) [315]
materials mol L−1
4. Biorad Aminex HPX-87C. All
columns 300 × 7.8 mm, RID
HCl 60 mmol L−1 70 ◦ C, 90 mmol L−1 Carbopac-PA-100
Wheat [316]
quenching Na2 CO3 NaOH 250 × 4.0 mm, PAD
Fungus sucrose ACN/0.04 g/100 RID, Knauer Eurospher 100-5
Filtration [317]
fermentation mL NH4 OH (70:30) NH2 Vertex 25 × 4.6 mm
heat stable amylase and RID, Zorbax Carbohydrate
Starch from feeds ACN/H2 O (80:20) [318]
amyloglucosidase 150 × 4.6 mm, 5 µm
Diluted 1:9 with EtOH 70 mL/100 ACN/0.1 mol L−1
mL and g/100 mL UV λ 245 nm, Eclipse XDB-C18
Wine [319]
1-phenyl-3-methyl-5-pyrazolone NH4 C2 H3 O2 250 × 4.6 mm, 5 µm
derivatization (70:30)
Microplate polysaccharide
5 mmol L−1
Bacterial hydrolysis 4 mol L−1 CF3 COOH, Gravity C18 , 100 × 2 mm, 1.8 µm,
NH4 C2 H3 O2 pH [320]
Exopolysaccharide 90 min at 121 ◦ C. Derivatization HPLC-UV-Ion trap/ESI+ -MS
5.6/ACN gradient
1-phenyl-3-methyl-5-pyrazolone

4.3.2. Carbohydrate Measurement Using Amide-Based Columns

Koh and coworkers developed a method using an amide-based column, which is designed
to retain polar molecules [308]. Contrary to their amino counterparts, these columns can retain
Foods 2019, 8, 1 30 of 63

analytes wide range of mobile phase pH. Thirteen sugars were separated including monosaccharides,
disaccharides, sugar alcohols. This separation is impressive since it includes several molecules
commonly used as sugar substitutes or replacement sweeteners. Organic amines within the
mobile phase are used as stationary phase modifiers [308]. The authors recommended the use of
a 150 mm column as the reduction of time of analysis using shorter lengths, compromise resolution.
However, peaks obtained on longer columns are typically wider peaks resulting in lower sensitivity
due to increased diffusion.

4.3.3. Carbohydrate Measurement Using Ligand Exchange-Based Columns

Duarte-Delgado and coworkers assayed four different extraction methods for sucrose, glucose,
and fructose, and demonstrated that a double aqueous MeOH extraction was the more efficient
approach for the determination of these sugars [310]. The authors used SPE and guaranteed the
removal less polar compounds and avoid possible co-elution with sugars during HPLC analysis.
Extraction method seems to be more critical for mono than disaccharides, and starch gelification
appears to be an interference when extraction is performed with hot water. Zielinski and coworkers
a cation exchange gel in calcium form column to determine sucrose, D-glucose, fructose, and sorbitol
in different ripe stages and during senescence of Malus domestica (Suckow) Borkh [309].
Senescent apple juice showed higher sugar concentration; a stage in which fruit is better suited
for fermentation. Shindo and coworkers used recovered sugars from samples such as orange juice,
yogurt, chewing gum, milk, and biscuits (this last matrix needed a triple extraction to obtain adequate
recoveries). Additionally, the authors optimized column temperature and flow rate [311].

4.3.4. Reverse Phase Columns and Sugar Derivatization Techniques

Several detection systems are used to detect carbohydrate after chromatographic separation,
an approach commonly used is the pulsed electrochemical detection. A thorough review of this
technique has been already written by Corradini and coworkers [321]. Evaporative light scattering
detector has also been used to assay sugars. Dvořáčkova and coworkers wrote a comprehensive
review of this technique [322]. The most common detector for chromatographic analysis of sugars is
refractive index. UV detection is usually inconvenient as the wavelength 210 nm (low range of the
UV) has the disadvantage of exhibiting interferences. An easy way to circumvent this to derivatize
using pyrazolones (e.g., 1-phenyl-3-methyl-5-pyrazolone) to form Schiff bases with reducing sugars
and monitor using 248 nm. This approach only works for reducing sugars. Hence, sucrose will not
be detectable. Additionally, a C18 column (usually readily available) can be used to separate the
adducts. Canesin and coworkers analyzed sorbitol from lateral buds of fruit trees (e.g., black mulberry,
peach, avocado, and pear) as a way to monitor primary photosynthesis products [312]. In this case,
traditional detection systems are not useful as levels of sorbitol are in the µg per mg.
Hung and coworkers were able to add a fluorophore to aldol sugars assisting in their detection
and mass fragmentation [313]. Naphthylimidazole fluorescent derivatives were obtained successfully
for sugars (only for reducing aldoses) extracted from beverages such as fruit juice, yogurt, coffee drink,
milk tea, and flavored milk. Additionally, oligosaccharides from a Solanaceae were identified using
the approach above and NMR as an additional confirmatory tool. Recently, special attention has been
drawn toward added sugars in food commodities; sterner regulations have been set in different
countries due to population health concerns such as obesity, diabetes, and heart disease [323].
Hung and coworkers also used their approach to assess added sugar in the food items tested [313].
Carbohydrates analysis in food should contemplate, systematically, added sugars during chemical
determinations [324].

4.3.5. Aqueous Normal Phase Chromatography for Sugars

Interestingly, Valliyodan and coworkers used an aqueous normal phase approach based on
a hydrophilic polymeric gel) to assess sugars from soybean. The addition of just 20–30 mL/100 mL
Foods 2019, 8, 1 31 of 63

of acetone to acetonitrile, in the mobile phase, permitted the successful separation of galactose from
glucose [314].

4.3.6. Complex Carbohydrates and Conjugates

Hydrolysis of complex (mainly structural) carbohydrates has been used previously to assess
them [325,326] by indirect determination of their basic units and building blocks. Several approaches
can be used to achieve this [325]. However, HPLC can be an attractive one since it provides high
specificity and selectivity. As hydrolysis usually produces considerable concentrations of the monomer,
usually sensitivity is not an issue. For example, we have used endo-1,4-β-mannanase (EC 3.2.1.78)
to break down and indirectly determine mannan, monitoring mannose. Similarly, hydrolysis can
be used to assess the quality of commercial mannanase. Mannanase is commonly used as a feed
ingredient to improve nutrient absorption [10]. Here, an enzyme of known activity (a standard, see for
example E-BMANN from Megazyme) is directly compared to the commercial one (the feed additive);
a galactomannan polysaccharide (like guar gum) can be used as the substrate.
Weiß and Alt described an exhaustive method to assay sugars in plant materials and feeds.
Separation of the following was achieved: inulin, verbascose, stachyose, raffinose, cellobiose,
sucrose, isomaltose, maltose, lactose, glucose, xylose, galactose, rhamnose, arabinose, fructose,
mannose, ribose, and mannitol [315]. Flow rate, temperature, mobile phase composition, and
injection volume were optimized. From the series of columns tested, the Nucleosil® Sugar 682 Pb
(Macherey-Nagel GmbH & Co. KG, Düren, Germany) was finally used at 85 ◦ C, H2 O at 0.4 mL
min−1 , and using 20 µL. Recent data show that inulin-rich diets can benefit gut microbiome,
notwithstanding, routine inulin analysis in feeds is uncommon [327]. It was not until very recently
that the minimal performance requirements were established for fructans analysis in feed, pet food,
and their ingredients [328]. However, excess dietary fructans have demonstrated adverse health
effects in equines [329]. AOAC® (Rockville, Maryland, USA) Official MethodSM 997.08 is available
to assess fructans in food products using ion exchange chromatography with pulsed amperometric
detection. The method is based on two-step hydrolysis using amyloglucosidase (to remove starch)
and inulinase. Measurement of simple sugars in different food-derived extract fractions is performed
using glucoheptose as an internal standard. The same principle has been used to assess fructose
derived from fructans in pet food [330]. Verspreet and coworkers analyzed fructan from wheat
grains after acid hydrolysis. Mild conditions used during hydrolysis avoid the release from other
naturally occurring saccharides in wheat that would otherwise interfere during the fructan estimation
(e.g., raffinose oligosaccharides) [316]. Correia and coworkers have developed a method to analyze
fructooligosaccharides [317]. These sugars are dietary and are used as food ingredients (incorporated
as dietary fibers in commodities). The authors monitored sucrose pathway fermentation products
from Aspergillus aculeatus as a potential source of fructooligosaccharides; fructose, glucose, sucrose,
1-kestose, nystose, and 1F -Fructofuranosylnystose were monitored.
We have used enzymatic hydrolysis to obtain glucose from starch molecules present in feed
and feed ingredients. Total and resistant starch was measured in several matrices including
(e.g., silages) [318]. Bai and coworkers analyzed mono- and oligosaccharides from Hakka rice [319]
as a measure of quality for sugars such as isomaltotriose, isomaltose, panose, maltose, and glucose.
Finally, the determination of bacterial exopolysaccharides has also been reported [320,331].

4.3.7. Method Application Experience

Amine-based columns (e.g., Zorbax® Carbohydrate (Agilent technologies, Santa Clara, USA),
Ultisil® XB-NH2 (Welch Materials, Inc, Texas, USA)) are successful in separating mono and
disaccharides in foods especially those containing lactose (such as dairy products). However, this type
of stationary phase suffers easily from poisoning as amine functional groups form covalent bonding
with several compounds (e.g., Schiff bases). As the amine functional group is sensitive to pH changes,
extracts must be adjusted to avoid changes in the chemical form of the stationary phase functional
Foods 2019, 8, 1 32 of 63

group as this may affect repeatability/reproducibility or even obliterate the column capacity for
retention. Hence, the elimination of interferences is paramount. Additionally, when retention capacity
is lost, it is possible to apply changes in the mobile phase composition and flow (e.g., to increase
acetonitrile concentration and reduce flow).
A particular case is that of coffee samples. Amine-based columns especially suffer when
analyzing coffee extracts as they contain phenolic acids (e.g., chlorogenic, syringic, ferulic,
protocatechuic and hydroxybenzoic acid) and alkaloids (e.g., caffeine, caffeic acid, theophylline,
trigonelline) [332]. Costa Rican regulations accept not more than 10 g/100 g sucrose in roasted coffee.
Hence, monitoring sugar levels, as a quality standard, in these products is paramount. When routine
quality control in coffee samples is necessary, we recommend to use stationary phases more resistant
to pH changes (e.g., amide-based), include mobile phase modifiers (e.g., triethylamine), or intensive
extract clean up.
In the case of animal compound feed, for example, suckling pigs feed usually contain lactose.
Contrary to the amine-based column (Figure 8A,B and Figure 9A), ion exclusion (e.g., Agilent Hi-Plex
Ca, Phenomenex® RezexTM RCU-USP Ca2+ (Torrance, California, USA)) is better equipped to deal
with a larger range of samples and is less prone to deteriorate.

Figure 8. Chromatographs of (A) 2 g/100 mL standard mixture of four sugars including fructose
(5.24 min), glucose (6.26 min), sucrose (9.12 min), and lactose (13.09 min) separated using amino
column (Zorbax Carbohydrate, 0.7 mL min−1 , 80 ACN: 20 H2 O). (B) Sugar content of a molasses
sample after hot water extraction, fructose (5.18 min) and glucose (6.31 min) signals are evident.
(C) 1 g/100 mL standard solution for arabinose (3.89 min) (D) 1 g/100 mL standard solution for xylose
(4.30 min) (E) 1 g/100 mL standard solution for ribose (4.76 min), and (F) 1 g/100 mL standard solution
for mannose (5.42 min). Signal at ca. 1.80 min corresponds to the solvent front; constant in all injections.
 min), glucose (6.26 min), sucrose (9.12 min), and lactose (13.09 min) separated using amino column
(Zorbax Carbohydrate, 0.7 mL min−1, 80 ACN: 20 H2O). (B) Sugar content of a molasses sample after
hot water extraction, fructose (5.18 min) and glucose (6.31 min) signals are evident. (C) 1 g/100 mL
standard solution for arabinose (3.89 min) (D) 1 g/100 mL standard solution for xylose (4.30 min) (E)
Foods 12019,
g/1008, 1mL standard solution for ribose (4.76 min), and (F) 1 g/100 mL standard solution for mannose33 of 63
(5.42 min). Signal at ca. 1.80 min corresponds to the solvent front; constant in all injections.

Figure 9.
Figure Schematicrepresentation
9.Schematic representation of
ofsugar
sugarinteraction
interaction mechanism
mechanism using
using (A)
(A) amine
amine based
based (B)
(B)calcium
calcium
ion-based ligand
ion-based ligand exchange
exchange column.
column.

types of columns are able to separate H+ (organic acids/monosaccharides), Na+ ,

These Acids
4.4. Organic
Ca (sugars/alcohols), Ag+ and Pb2+ (oligosaccharides). Sugars and alcohols are separated using
2+
Organic acids are common substances found naturally in several foods and result from
ligand exchange (Figure 9B) and organic acids by ion exchange. During ligand exchange, the more
fermentation processes [334]. As such, they are responsible for the particular flavor and aroma of
complex sugars elute first whereas simple ones, such as fructose, elute last, opposite to the elution order
commercially relevant commodities such as wine, vinegar, fermented meats, and yogurt, to name
found in amine-based columns. Another advantage is that ion exchange columns need ultra-high
just a few. These substances have found widespread use in the food and feed industry as
purity water (type I) to segregate analytes while amino-based columns require acetonitrile in the
preservatives (increasing shelf-life, [335]) and antimicrobials (due to their bacteriostatic properties)
mobile phase to perform. However, amino-based columns have the inherent advantage that complete
chromatographic runs can be achieved under 12 min (while setting the column at 30 ◦ C). Meanwhile,
a good separation using exchange columns can be extended up to 30 min at 60 ◦ C. As LC-MS usually
use low flow rates to aid in solvent nebulization, it is harder to develop methods using this type of
column due to their dimensions. When using ion exchange based-columns, EDTA can be used to
sequestrate ions present on complex sample extracts reducing interferences. Peak tailing or fronting is
also common when there is already wear of the chromatographic column. Finally, mathematic designs
can be used to optimize method critical parameters and attributes, at least one research group has used
this approach (i.e., Monte-Carlo simulation) to analyze sugars in herbs [333].

4.4. Organic Acids

Organic acids are common substances found naturally in several foods and result from
fermentation processes [334]. As such, they are responsible for the particular flavor and aroma
of commercially relevant commodities such as wine, vinegar, fermented meats, and yogurt, to name
just a few. These substances have found widespread use in the food and feed industry as preservatives
(increasing shelf-life, [335]) and antimicrobials (due to their bacteriostatic properties) (large-scale use
of benzoic acid in beverages is a clear example, [335]). Organic acids can be determined by HPLC
using ion exchange columns. Several anion exchange columns have already been mentioned in the
previous section. Usually, though, measurement is commonly made using a UV detector at 210 nm
(detection of absorption of carboxyl groups). Solvents and columns may vary slightly, but an isocratic
method is sufficient to separate the analytes. The sample preparation for organic acid determination
in beverages can be straightforward. In some cases, depending on the clarification, process suffered
by the final product, it may consist of centrifugation and microfiltration. For an excellent primer for
organic acids, we recommend the book edited by Vargas [336].

4.4.1. Reverse Phase Chromatography Analysis in Foods

Neffe-Skocińska and coworkers analyzed sugars, ethyl alcohol and organic acids in Kombucha tea
beverages (a fermented brew) with an emphasis on glucuronic acid (which has been associated with
Foods 2019, 8, 1 34 of 63

health benefits) [337]. The fermentative profile was evaluated for 10 days at 3 different temperatures.
Nour and coworkers use low temperature so they can separate 6 compounds (oxalic, tartaric, malic,
lactic, citric, and ascorbic) in 13 min. The applied temperatures (i.e., 10 ◦ C) in a 250 mm column using
0.7 mL min−1 flow rates ensures optimal resolution of the compounds while obtaining adequate peak
shapes within a reasonable time. A buffer adjusted at 2.8 pH guarantees that the compounds of interest
are maintained during chromatography as protonated species [338].
Different citrus juices were tested finding concentrations of citric acid ranging from 7.39 × 104
to 6.89 × 104 mg L−1 . Reverse phase separation of acids uses buffers (e.g., the H2 PO4 − /HPO4 2−
pair) or salts (e.g., Na2 SO4 ) to accomplish separation. The advantage of this approach is that usually
C8 /C18 columns are readily available and are relatively inexpensive. The downside resides in that
the use of this kind of mobile phases increase the possibility of crystal precipitation in the pump and
capillaries. The buffer has to be prepared daily (to circumvent microbial growth), and pH values
strictly supervised (to avoid retention time shifts). Lobo Roriz and coworkers determined organic
acids in three different medicinal plants which are widely consumed as infusions. Gomphrena globosa L.
showed the highest levels or organic acids (mainly malic and oxalic) [339].
Pterospartum tridentatum (L.) Willk. and Cymbopogon citratus (DC.) Stapf showed higher levels of
citric and succinic acids, respectively. Acid content depends on inherent plant genetic characteristics
and edaphoclimatic conditions. The authors also analyzed sugars (using HPLC-RID Eurospher
100–5 NH2 column and melezitose as internal standard) and, interestingly, tocopherols (α, γ,
and δ-tocopherol, normal phase YMC Polyamide II column and fluorescence at λex and λem 290 nm
and 330 nm). Scherer and coworkers used a reverse phase column to assess ascorbic acid stability in
apple, orange and lemon juices. They also compared nutritional analysis reported within the food
labels for ascorbic acid with that obtained experimentally [340].

4.4.2. Ion Exchange Chromatography Analysis in Foods

Llano and coworkers analyzed sugars, acids, and furfural in pulp mill residue. The authors used
a resin-based cross-linked gel column for low molecular-weight chain acids, alcohols, and furfurals.
This method is particularly interesting since a comparison between 2 sets of columns for each
application was tested (Table 15). The authors also included specific details for each column and
optimize temperature, injection volume, and flow rate. Size exclusion also seem to have a role in
sugar separation using ion exchange columns [341]. Though this application is not specifically for
food, xylooligosaccharides (from revalorization alternatives for materials derived from the pulp
mill enterprise) have found applications in the food industry and have been even linked to health
benefits [342]. Saleh Zaky and coworkers reported a simultaneous analysis of chlorides, sugars,
and acids [343]. However, the paper states that several inorganic ions are retained with the same
strength within the column (all tested ions have different physicochemical properties; i.e., hydration
spheres, charge among others). We have not been able to repeat this procedure. The authors did
analyze sugars and acids (i.e., citric, lactic, acetic) and ethanol in a grand variety of food products
including energy drinks, sodas, tomato juice and sauce, brine, milk, whey, cheese, and hummus.
An interesting paper focused on the determination of organic acids from olive fruits. Different organic
acid profiles were found for unique fruit varieties. They found oxalic, malic, succinic, and citric as
main organic acids [344].
Foods 2019, 8, 1 35 of 63

Table 15. Determination of organic acids and in foods and silage.

Sample Pretreatment,
Matrix Mobile Phase Composition Measurement Method, Chromatographic Column Reference
Extraction
Reverse Phase-Based Columns
20 mmol H2 PO4 − pH 2.4/MeOH
Kombucha H2 O 70–80 ◦ C, filtration Luna C18 250 × 4.6 mm, 5 µm 30 ◦ C UV λ 210 nm [337]
(97:3)
Juice extraction, depulping, Hypersil Gold aQ 250 × 4.6 mm, 5 µm 10 ◦ C UV λ 214 nm
Fresh fruits 50 mmol L−1 H2 PO4 − pH 2.8 [338]
centrifugation (254 ascorbic acid)
Medicinal plants Sphere-Clone C18 250 × 4.6 mm, 5 µm, 35 ◦ C UV λ 215 nm
(HPO3 )n extraction 3.6 mmol L−1 H2 SO4 [339]
infusions (254 ascorbic acid)
Fruit juices Filtration 0.01 mol L−1 KH2 PO4 pH 2.6 RP-C18 150 × 4.6 mm, 3 µm, UV λ 210 nm [340]
SPE C18
0.005 mol L−1 H3 PO4 pH 2.1,
Wine Elution: mobile phase (for Lichrosorb RP-C18 150 × 4.0 mm, 5 µm, UV λ 210 nm [345]
1 mL/100 mL CAN
acid protonation)
Ion Exchange-Based Columns
RID for all cases,
1. Ultrapure water 79 ◦ C
1. Aminex HPX-87P Pb2+ 300 × 7.8 mm, 9 µm
2. Ultrapure water 68 ◦ C
Sulfite pulp mill Filtration 2. Transgenomic® CHO-782 Pb2+ 300 × 7.8 mm, 7 µm [341]
3. H2 SO4 0.005 mol L−1 30 ◦ C
3. Bio-rad Aminex HPX-87H 300 × 7.8 mm, 9 µm
4. H2 SO4 0.005 mol L−1 60 ◦ C
4. Shodex SH-1011 H+ 300 × 8.0 mm, 6 µm
Liquid samples: filtration
Food samples 0.005 mol L−1 H2 SO4 35 ◦ C Hi-Plex H 300 × 8.0 mm, 6 µm, RID [343]
Solid samples: H2 O 85 ◦ C
Maceration in H2 O MeOH
Olive fruits 0.1 g/100 mL H3 PO4 Shodex RSpak KC-118 300 × 8.0 mm, UV λ 214 nm [344]
(75:25)
Filtration, SPE strong anion ◦ C,
Wines 0.065 mL/100 mL H3 PO4 Aminex HPX-87H 300 × 7.8 mm, 9 µm, 65 UV λ 210 nm [346]
exchange
Centrifugation, sonication,
Fermented shrimp waste (HPO3 )n pH 2.1 SS Exil ODS 250 × 4.0 mm, 5 µm, UV λ 210 nm [347]
filtration
Silage H2 O 100 ◦ C 6 mmol L−1 HClO4 Shodex KC 811 300 × 8.0 mm, 7 µm 50 ◦ C, UV λ 210 [348]
Ion Exclusion-Based Analysis
Precondition: 10 mmol L−1 SDS 3
Filtration, heat-aided h 0.3 mL min−1
Drinks Kinetex XB-C18 150 × 4.6 mm, 2.6 µm, UPLC-UV λ 210 nm [349]
degassing Elution: 1.84 mmol L−1 H2 SO4
pH 2.43

Mihaljević and coworkers separated organic acids in wine. Organic acid profile
(especially glucuronic and galacturonic acids levels) was able to distinguish among Traminer vs.
Welsch produced Croatian wines. Mobile phase rate was reduced during chromatography when target
acids were glucuronic, gluconic, galactaric, and galacturonic [346]. Diacids and citric acid considerably
differ structurally (e.g., number of carbons). Meanwhile, the reduction of flow rate responds to the
subtle differences among these intimately related structures, making them more difficult to resolve.
Sánchez-Machado and coworkers preserved shrimp tissue through fermentation with lactic acid
bacteria. A complete separation of lactic, citric and acetic acid was accomplished. Sonication time and
initial sample mass were optimized during the assay [347]. Finally, though most of the tests regarding
organic acids extraction-wise are straightforward, even in brightly colored samples (e.g., fruits [350]),
still SPE cleanup has been applied, with adequate recoveries, to these extracts to remove interferences
as anthocyanins and carbohydrates that may co-elute during acid analysis (especially relevant if
a non-selective detector is used) [345].

4.4.3. Ion Exclusion Chromatography Analysis in Foods

Fasciano and coworkers modified a reverse phase column, a C18 column was dynamically
modified by running a solution of SDS through the column (Table 15). They separated organic
acids after optimizing sulfuric acid concentration, flow rate, and pH; an example of how a reverse
column can be made more versatile. A wide array of compounds in juices and sodas were analyzed
using ion exclusion chromatography [349]. For a detailed description of ion exclusion chromatography,
we encourage the reader to pay special attention to this paper introduction.

4.4.4. Silages
The maturity of the crop governs silage quality at harvest. However, fermentation in the silo
further influences the nutritive value of silage. Coblentz and Akins recently published a detailed
discussion of silages [351]. Similarly, Khan and coworkers wrote a more specific review based
on maize silages [352]. In both papers, references to silage quality based on organic acids are
Foods 2019, 8, 1 36 of 63

mentioned. Since silage is the result of this fermentation process, the organic acid analysis is used
to monitor its quality. Concentrations of fermentation acids do not seem closely related to silage
intake; however, they are decisive in the balance of volatile fatty acids produced in the rumen.
In turn, affecting gluconeogenic metabolism and influencing milk and body composition in productive
livestock. Several researchers have dedicated efforts to not only assess organic acid concentrations
from silages but also have studied the effect that organic acid has on silage fermentation. For example,
Ke and coworkers included malic or citric acid at concentrations of 0.1 to 0.5 g/100 g during
alfalfa ensiling of alfalfa and concluded that these levels improved silage fermentation quality [348].
Additionally, both acids can be further used as feed additives that have proven to promote animal
performance. Silva and coworkers determined the fermentation profile of alfalfa silages treated with
microbial inoculants at different fermentation periods under tropical conditions [353]. From the strains
tested P. pentosaceus showed the most efficiency suggesting its use as a silage inoculant. The sample
pretreatment just consisted of extract acidification with metaphosphoric acid, gravity-aided filtration,
and centrifugation.

4.4.5. Method Application Experience

We have used ligand exchange-based analysis to routinely screen silage quality (Figure 10).
Sample pretreatment consists of metaphosphoric acid extraction. We also have taken advantage
of sample extraction for ammoniacal nitrogen (a modified version of method 941.04). In this type
of columns, poly and diacids are eluted first. Monocarboxylic acids will elute later on during the
chromatographic run in order of increasing alkyl chain length (i.e., formic, acetic, propionic, butyric).
Finally, we have used liquid chromatography coupled with a variable wavelength detector set at
210 nm, a Hi-Plex H (300 × 7.7 mm and 8 µm particle size) column kept at 60 ◦ C, and a 50 mmol
L−1 H2 SO4 solution with a flow rate of 0.6 mL min−1 to monitor ammonium propionate, added as
a preservative, in dry dog foods (see for example, [354]). Average concentrations of (693.12 ± 75.63)
mg kg−1 have been obtained for local products. Sieved (at 0.5 mm particle size) dog food was treated
with hot water to extract the propionate quantitatively. Both RID and UV detectors can be used for
both organic acid and sugar (and alcohol) analysis. Using RID will enable the user to monitor all
the compounds above simultaneously, but RID detectors suffer from low sensitivity when compared
to others.
Foods 2018, 7, x FOR PEER REVIEW 38 of 62

Figure
Figure 10.
10. Chromatographs
Chromatographs of of (A)
(A) Mix
Mix of
of organic
organic acid
acid standards
standards malic
malic acid
acid (9.24
(9.24 min)
min) methanoic
methanoic acid
acid
(formic acid,10.92
(formic acid, 10.92min),
min),ethanoic
ethanoicacid
acid (acetic
(acetic acid,
acid, 11.65
11.65 min),
min), propanoic
propanoic acidacid (propionic
(propionic acid, acid,
12.62 12.62
min),
min), lactic(14.92
lactic acid acidmin),
(14.92 min), 2-methylpropanoic
2-methylpropanoic acid (isobutyric
acid (isobutyric acid, 17.22 acid,
min), 17.22
butanoicmin),
acidbutanoic acid
(butyric acid,
(butyric acid, 18.52 min). (B) A silage sample after extraction −with
1 acid 0.01 mol
18.52 min). (B) A silage sample after extraction with acid 0.01 mol L H2 SO4 . Fermentation products L−1 H2SO4.

Fermentation products
identified at 18.499 min,identified at 18.499
14.903 min, 12.606 min,
min.14.903
The min, 12.606
signal at ca.min. The
5.70 minsignal at ca. 5.70
corresponds tomin
the
corresponds
solvent front.to the solvent front.

4.5. Vitamins
Vitamins are essential micronutrients
micronutrients that
that humans
humans andand animals
animals need
need for
for normal
normal metabolism.
metabolism.
The lack of these nutrients in dietary sources can cause serious disease; trace amounts of these
compounds are required for growth and reproduction. Based on their solubility, vitamins have been
divided into two groups: those soluble in organic non-polar solvents and water-soluble vitamins.
Thus, the vitamins from the B-complex and vitamin C are classified water soluble while the fat-
soluble vitamins are isoprenoid compounds, namely vitamins A, D, E and K. This last group is found
in small amounts on foodstuffs, associated with lipids; stored in the liver and fatty tissues, and are
Foods 2019, 8, 1 37 of 63

compounds are required for growth and reproduction. Based on their solubility, vitamins have been
divided into two groups: those soluble in organic non-polar solvents and water-soluble vitamins.
Thus, the vitamins from the B-complex and vitamin C are classified water soluble while the fat-soluble
vitamins are isoprenoid compounds, namely vitamins A, D, E and K. This last group is found in small
amounts on foodstuffs, associated with lipids; stored in the liver and fatty tissues, and are eliminated
slower than water-soluble vitamins [45].

4.5.1. Fat-Soluble Vitamins

Vitamin A is commonly expressed as retinol equivalents but can occur in different chemical
forms, i.e., retinal, retinoic acid and retinyl esters. In foods, it is very common to find this vitamin as
retinyl esters, more specifically, as acetate, propionate, or palmitate [355]. This vitamin is involved in
immune function, vision, reproduction, and cellular communication [356]. Vitamin E is a term used
to designate some related compounds as tocopherols and tocotrienols, it is found in fat products of
vegetal origin, mainly oils. The most common tocopherols that can be found in food and feed are
α/β/δ/γ-tocopherol in different proportion. Mixed tocopherols are considered the most effective
lipid-soluble antioxidants [357]. Vitamin D is naturally present in very few foods like sea products,
eggs, meat, and dairy products, the most commonly found members are vitamin D2 and D3 , but it is
also produced endogenously when ultraviolet rays from sunlight strike the skin and trigger vitamin
D synthesis from 7-dehydrocholesterol [358]. It promotes the absorption of calcium, regulates bone
growth and plays a role in immune function [359]. Lastly, vitamin K is an essential nutrient for animals
and humans because it is required for functioning of the blood clotting cascade [360], just as vitamin
D, vitamin K can be found in two forms i.e., phylloquinone (vitamin K1 , found in green plant leaves
e.g., spinach, collards, lettuce, and broccoli [361]) and menaquinone (vitamin K2 , bacterium residing in
the vertebrate intestine [362]).
Since these compounds are involved in metabolic pathways, and are paramount in health
promotion in animals and humans, it is crucial to determine their content in food and feed to
comply with daily requirements and quality control. That is why several studies have been
conducted regarding the extraction and quantitative analysis of these vitamins, either individually or
simultaneously [359,363–382].

Sample Preparation
Most of the analytical methods involve previous steps of sample preparation like saponification,
solid-liquid or liquid–liquid extractions, followed by a concentration step before HPLC analysis
(Table 16). The sample pre-treatment is critical for an accurate method. That is why there are many
aspects that need to be controlled, Qian and Sheng have studied seven different variables to take
into account for simultaneous analysis of vitamins in animal feed. These variables were related
to the extraction procedure: (1) sample particle size, (2) solvent, (3) the ratio of sample to solvent,
(4) extraction with and without N2 protection, (5) extraction time, (6) equipment and (7) the use of SPE
for cleanup [367]. They evaluated how each of the variables affected both the coefficient of variation
and the recovery of each of the vitamins in order to obtain extraction conditions that would allow
them to satisfy each of the vitamins in a satisfying way.
Foods 2019, 8, 1 38 of 63

Table 16. Determination of fat vitamins and in foods and feeds.

Measurement Method,
Matrix Vitamin Extraction Method Reference
Chromatographic Column
LC-MS/MS-ESI+ C18 50 × 2.1 mm, 2.6 µm 40 ◦ C.
Lipase at 37 ◦ C 2 h in PBS
Infant and nutritional Mobile phase: H2 O/ACN 50:50 with
K1 Extraction with hexane and concentrate [377]
formulas 0.1 mL/100 mL HCOOH and ACN/MeOH 75:25,
with N2 , reconstituted with MeOH
with 2.5 mmol L−1 NH4 CO2 H, gradient system
Extraction with 0.2 g/100 mL NH3 and
Additives, premixes, HPLC-PDA C18 150 × 4.6 mm, 5 µm. Mobile
A, E, K3 , D3 EtOH, sonication 40–50 ◦ C for 20–30 min in [366]
complete feed phase MeOH/H2 O (98:2), UV λ 230 nm
the dark, SPE (OASIS HLB)
Vitamin B Analyses.
Agilent ZORBAX Eclipse Plus C18 250 × 4.6 mm,
Vitamin B Analyses. 5 µm. Mobile phase MeOH/0.023 mol L−1 H3 PO4
0.1 mol L−1 H2 SO4 incubation 30 min at pH 3.54 (33:67) UV λ 270 nm at room temperature
121 ◦ C, pH 4.5, 2.5 mol L−1 NaC2 H3 O2 , Ascorbic Acid Analyses
Takadiastase enzyme PDA, 0.1 mol L−1 KC2 H3 O2 pH 4.9 and
B2 , B3 , B6 , B12 ,
Okra (Abelmoschus Ascorbic Acid Analyses ACN/H2 O (33:67). UV λ 254 nm at room
C, E, K, D, A, [365]
esculentus) Extraction 0.3 mol L−1 metaphosphoric temperature
β-Carotene
acid and 1.4 mol L−1 HAOc β-carotene:
Saponification KOH 50 g/10 g in EtOH in Agilent TC-C18 250 × 4.6 mm, 5 µm,
reflux at 50 ◦ C for 40 min. Extraction ACN/MeOH/ethyl acetate (88:10:2) at 453 nm.
with ether Fat-soluble vitamins
Eclipse XDB-C18 150 × 4.6 mm, 5 µm, MeOH at:
325, 265, 290, 244 nm
PDA, TSKGel ODS-100V, 150 × 4.6 mm, 5 µm at
A, D3 , E, B1 , Extraction with Carrez I and Carrez II
Fruits, juices, and 30 ◦ C. Gradient 0.01 mL/100 mL TFA in H2 O and
B2 , B3 , B5 , B6 , solutions [364]
supplements MeOH at 320, 275, 253, 290, 258, 218, 289, 360,
B9 , B12 C Cleaned by SPE (RP18 Bakerbond)
and 262 nm
DAD, at 228 and 266. C18 200 × 4.6 mm, 5 µm
Saponification with KOH (60% g/100 mL
Cheeses D3 ambient temperature Mobile phase: [378]
in H2 O). Extraction: MeOH/CHCl3
MeOH/ACN/H2 O (49.5:49.5:1)
PDA, C18 250 × 4.6 mm, 5 µm at 30 ◦ C, 3.0 g/100
Saponification: KOH/EtOH + ascorbic acid.
mL SDS and 0.02 mol L−1 PBS at pH 7, with 15.0
Mechanical shaking. Extraction: hexane,
Dairy and soybean oil K1 , D3 , E, A mL/100 mL butyl alcohol (organic solvent [379]
evaporated at 40 ◦ C and re-dissolved in
modifier). 230 and 300 nm (K1 , D3 and E vitamins),
MeOH
280 nm (A, E, D3 , and K1 )
HPLC-UV-Vis 325, 264, and 280 nm (A, D, K and E),
K1 , D3 , D2 , E, Extraction: hexane, concentrate with N2
Milk sample C18 DBS 150 × 3 mm, 3 µm at room temperature; [380]
A and reconstituted with MeOH
MeOH/H2 O (99:1) and MeOH/THF (70:30)
HPLC-EC Vydac 201 TP54 150 × 4.6 mm, 5 µm.
Vegetables, fruits, and Extraction with 2-propanol/hexane and
K1 Mobile phase: MeOH/0.05 mol L−1 NaC2 H3 O2 , [381]
berries CHCl3 /MeOH
pH 3 (96:4)
Saponification KOH (600 g L−1 ) in EtOH
FLD, COSMOSIL π-NAP 250 × 4.6 mm, 5 µm at
with pyrogallol for 45 min at 70 ◦ C,
Rice E 25 ◦ C. Mobile phase: MeOH/H2 O/ACN (80:13:7). [382]
mechanical shaking. Microextraction:
Vitamin E isomers λex 290 and λem 330 nm
CCl4 /ACN (1:10)

Regarding the first variable in meals and flours, subsample variability and homogeneity is closely
linked to particle size. Qian and Sheng observed that large sample particle size causes an incomplete
vitamin A extraction with high variability [367]. We have noted that for fresh products, with a total
fat content greater than 10 g/100 g and high moisture content (i.e., greater than 85%) (e.g., avocado
(Persea americana Mill.) and peach palm (Bactris gasipaes Kunth)), it is advisable to freeze dry the sample,
before analysis, to promote homogenization and eliminate water that interferes with fat-soluble
compound extraction.
Qian and Sheng developed the assay procedure without saponification, but nevertheless it is
a widespread procedure used in the analysis of vitamins since it is an efficient way of removing
interferences of lipid origin that can be found in the matrix [363,365,368,374,378,379,382]. Since it is
based on an alkaline digestion (i.e., heated KOH or NaOH aqueous or alcoholic solutions) there is
a disadvantage, the saponification could generate oxidation of the vitamins, which translate into a loss
for vitamin degradation and low recovery percentage [366,368]. Some researchers have made use of
the antioxidant ability of some compounds such as BHT, BHA, TBHQ, ascorbic acid or pyrogallol
to reduce oxidation losses [363,365,367,368,371,374,379,380]. Nevertheless, saponification procedures
take time, and the extractions procedures are not always straightforward, because emulsions are
generated, as Lim and co-worker mentioned in their comparison of extraction methods for determining
tocopherols in soybeans [369], these inconveniences introducing considerable variation, low recovery,
and reproducibility, situations that we have also seen in the development of this type of methodologies.
Foods 2019, 8, 1 39 of 63

Recently, alternatives to saponification process for the extraction of vitamins have been used,
some papers use enzyme-catalyzed hydrolysis and alcoholysis of ester bonds in vitamin A and
E esters to facilitate their determination in milk powder and infant formula. They assayed six lipase
preparations and one esterase preparation using diisopropyl ether, hexanes/ethanol and supercritical
CO2 containing ethanol. Three of the lipases’ preparations from Candida antarctica (Novozyme 435),
Rhizomucor miehei (Lipozyme IM) and Pseudomonas cepacia, showed considerably higher activity toward
retinyl palmitate but there was no observed activity with α-tocopheryl acetate [372]. In feed, Xue and
co-workers applied enzymolysis instead of saponification with a basic proteinase named Savinase
in 30 min of incubation time at 40 ◦ C getting good results in the determination of four fat-soluble
vitamins (K3 , A, D3 , E) [366].
With respect to the solvent type and ratio solvent: sample, there is a wide variety of solvents
available for the fat-soluble extraction, most of the methods use solvents such as hexane, heptane,
chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, ethyl ether, and the choice will depend
on the type of matrix to work with (Table 16). For example, in the case of animal feed [367], a poor
resolution was observed using hexane and chloroform, generating an overestimation of vitamin D,
such mixture does not allow a good separation during centrifugation which produced a high %RSD.
If a mixture of acetone/CHCl3 (30:70) is used, the results in terms of variability and recovery
of vitamins are outstanding, mainly for vitamin A. In low-fat matrices (less than 0.1 g/100 g,
e.g., fruit juices), this solvent system has the disadvantage of generating emulsions and, hence,
low recoveries. In the case of dairy and infant formulas where the presence of milk proteins is
a hindrance, the extraction of the lipid part has been reported using saponification and extraction with
hexane, leading to vitamin degradation in fat. It is also an extensive process [373,374]. For this reason,
a group of researchers developed a fat extraction methodology using a mixture of CH2 Cl2 :EtOH 2:1
and separation at 4 ◦ C with a centrifuge, giving satisfactory results for analysis of FAMES so it could
be applied in the extraction of vitamins in these matrices [375].
Regarding extraction time and equipment, Qian and Sheng used vortex mixer for several minutes,
rotatory mixer and supersonic mixer, these last two methods were not as effective for extraction of
vitamin A and other vitamins due to low recoveries [367]. Hung used a rotatory mixer for extraction
of vitamins D2 and D3 during one hour [376]. We have found, that for foodstuffs, the most efficient
sample treatment is to rely on the combination of a vortex mixer for one minute, a rotatory mixer for
30 min or supersonic mixer for 15 min.
Effective extraction can be aided if the solvent contains a percentage of an appropriate antioxidant.
N2 has been used in some protocols to the protection [377,380], of extracted vitamins from degradation
because the solvent vapor that replaces air over the surface of extraction mixture has a protective
antioxidant effect [367]. Qian and Sheng showed evidence that this protection did not influence in the
mean values of vitamins A, D, and E and pro-vitamin D, but decreased the variation coefficient [367].

Chromatographic Analysis
The analytical method for the determination of vitamins in food and feed, is liquid
chromatography (HPLC or UPLC) due to its, selectivity, short time of analysis, and high resolution.
Methods based on chromatography can be easily automated and can determine several compounds at
the same time.
Methods range from using normal phase chromatography with silica columns to reverse phase
chromatography with C8 , C18, and C30 columns (Table 16). Lee and coworkers studied three different
columns to separate vitamin A and E: an NH2 column, C30, and C18. Concerning the resolution,
they observed the β-tocopherol and γ-tocopherol peaks of vitamin E were not separated and appeared
as a single overlapping peak when using a C18, but it could be separated using an NH2 column.
Regarding detection and quantification limits the NH2 column presented values lower than C8 column
but higher than C18 .
Foods 2019, 8, 1 40 of 63

The solvent systems to use as mobile phase vary depending on the selected approach, in the case
of normal phase chromatography the solvents systems mostly used are 2-propanol/hexane in different
proportion, but also can be use methanol/hexane/THF (97.25:2.5:0.25), or hexane/MTBE (96:4) [374].
In reverse phase the most common are MeOH-H2 O, MeOH-ACN, both techniques can be used in
gradient o isocratic mode.
As mentioned before, the liquid chromatography technique has a wide variety of monitoring
techniques including PDA, FLD, ECD, ELSD or MSD. The most commonly used detector for vitamins
is FLD, which is considerably more sensitive and selective than UV. Therefore, it is possible to carry
out a simultaneous determination of vitamin A and E, for which a programming of the equipment
is required so that at certain time intervals it uses the excitation wavelength (λex ) and emission
wavelength (λem ) specifies for each vitamin, for vitamin E, λex = 285 and λem = 310 nm, for vitamin A
the configuration at λex = 325 and λem = 470 nm, but no other vitamins could be detected such as K
or D. Alternatively, PDA can work with multiple UV wavelengths and determine the four vitamins
at the same time. Mass spectrometry coupled chromatography is usually the most versatile option.
However, it requires that the laboratory has the resources for its acquisition. We have successfully
applied mass spectrometry to assess tocopherols in feed supplements and animal biological samples
(Figure 11A–F).

Figure 11. Single quadrupole LC/MS ESI+ chromatographs of (A) Total ion chromatogram
α-tocopherol (a 1 mg L−1 solution in butanol) signal positively identified at 11.75 min (B) Mass
spectra for α-tocopherol (a 1 mg L−1 solution in butanol) using a cone energy of 120 V extracted from
a signal with a retention time of 11.71 min (C) α-tocopherol (retention time 11.77 min) identified in
a chicken plasma sample after extraction with chloroform and butanol (D) α-tocopherol in selected ion
monitoring (SIM) mode using a cone energy of 120 V extracted from signal with a retention time of
11.82 min (E). α-tocopherol acetate in an injectable vitamin E solution for veterinary use using a “dilute
and shoot” approach (16.32 min), and (F) α-tocopherol acetate in SIM mode using a cone energy of
60 V extracted from signal with a retention time of 16.34 min.
 with a retention time of 11.71 min (C) α-tocopherol (retention time 11.77 min) identified in a chicken
plasma sample after extraction with chloroform and butanol (D) α-tocopherol in selected ion
monitoring (SIM) mode using a cone energy of 120 V extracted from signal with a retention time of
11.82 min (E). α-tocopherol acetate in an injectable vitamin E solution for veterinary use using a
“dilute
Foods 2019, 8, 1and shoot” approach (16.32 min), and (F) α-tocopherol acetate in SIM mode using a cone 41 of 63
energy of 60 V extracted from signal with a retention time of 16.34 min.

4.5.2. Hydrosoluble
4.5.2. Hydrosoluble Vitamins
Vitamins
One of
One of the
the main
main issues
issuesthat
thatthe
thehydrosoluble
hydrosolublevitamins
vitaminsanalysis
analysisexhibit
exhibitis is that
that each
each molecule
molecule is
is structurally different. Hence, to assess each vitamin, different conditions must
structurally different. Hence, to assess each vitamin, different conditions must be applied to be applied to the
the
HPLC system
HPLC system toto assess
assess each
each vitamin.
vitamin. Kim
Kim published
published aa paper
paper in
in which
which ion
ion pairing chromatography
pairing chromatography
was used to monitor six different vitamins (nicotinic acid, nicotinamide, folic
was used to monitor six different vitamins (nicotinic acid, nicotinamide, folic acid, andacid, and pyridoxine)
pyridoxine)
in the feed [383]. Sodium hexanosulfate was used to with this approach; all
in the feed [383]. Sodium hexanosulfate was used to with this approach; all six vitamins cansix vitamins can be
be
quantified using
quantified using the
the same
same chromatographic
chromatographic run run (using
(using the
the same
same wavelength
wavelength and and column
column for
for all
all
species) (Figure 12).
species) (Figure 12).

Figure
Figure 12.12. Hydrosoluble
Hydrosolublevitamin
vitaminanalysis
analysisbased
basedononion
ionpairing
pairing[383].
[383].(A)
(A)Successful
Successfulseparation ofof
separation 7
7 complex B vitamins including niacin (nicotinic acid, B3 , 6.67 min), FMN (B2 , 14.12 min), pyridoxal,
complex B vitamins including niacin (nicotinic acid, B3, 6.67 min), FMN (B 2, 14.12 min), pyridoxal (B 6

17.007 min),
(B6 , 17.007 pyridoxamine
min), pyridoxamine (B6,(B18.607 min), pyridoxine (B6, 19.963 min), folic acid (B9, 20.630 min),
6 , 18.607 min), pyridoxine (B6, 19.963 min), folic acid (B9, 20.630 min),
and
and thiamine
thiamine(B(B
1, 25.074 min). (B) Analysis of a vitamin premix destined for feed formulation. Another
1 , 25.074 min). (B) Analysis of a vitamin premix destined for feed formulation.
advantage presented
Another advantage is that the
presented separation
is that can be performed
the separation using ausing
can be performed reverse phasephase
a reverse C18 column.
C18 column.

A usual problem that arises with the analysis of this compounds is the fact that pH changes
affect their chemical behavior drastically
drastically both
both during
during extraction
extraction and
and chromatographic
chromatographic separation.
separation.
The author circumvented this issue using a mobile phase and sample extraction solution spiked
with 0.1 mL/100 mL acetic acid, maintaining all species protonated. Midttun and coworkers
described an extensive two-phase analysis based on an LC-MS/MS (to determine fat soluble
and water soluble). In the chloroform/isooctane phase all-trans retinol, 25-hydroxyvitamin D2 ,
25-hydroxyvitamin D3 , α-tocopherol, γ-tocopherol, and phylloquinone were retained. The hydrophilic
phase (in which water-soluble vitamins were found), was mixed with ethanol, water, pyridine,
and methyl chloroformate as a derivatizing agent. In this assay there can be a third phase
(i.e., the methyl chloroformate fraction) that it is reserved for gas chromatography analysis of amino
acids [384]. As an excellent example of hydrosoluble vitamins analysis using LC-MS/MS in the food
industry, is the determination of 15 compounds in beverages using a multi-mode column (SM-C18
column, 150 × 2.0 mm, 3 µm; Imtakt Co., Kyoto, Japan), which provided reverse-phase, anion- and
cation-exchange capacities, and therefore improved the retention of highly polar analytes such as
water-soluble vitamins. The use of this column removes the need for an ion pair reagent in the mobile
phase [385]. Finally, we encourage the reader toward a recent and ample review regarding fat- and
hydrosoluble vitamins, respectively [386,387].

4.5.3. Method Application Experience

In our experience in the development of a methodology for the determination of fat-soluble
vitamins in food matrices, the most challenging part has been the sample pretreatment. As mentioned
above, several factors have to be considered. For the species retinyl acetate and palmitate, we chose
to use a direct extraction to avoid decomposition by the saponification process. Extraction can be
performed with hexane, ethyl acetate or chloroform; as the last solvent is far easier to eliminate,
during concentration steps, is considered the most suitable option. Isopropanol is a useful solvent
for reconstitution.
Foods 2019, 8, 1 42 of 63

This method applies to dry matrices as flour, bakery products, freeze-dried pulps or fortified sugar.
In the case of dairy products, we highly recommend the use of dichloromethane/ethanol. Separation is
carried out using an HPLC-DAD set at 325 nm and a Zorbax Eclipse XDB-C8 (150 × 4.6 mm, 5 µm)
column at 50 ◦ C. Shifting the solvent system form a MeOH/H2 O (90:10) to MeOH/2-propanol/ACN
(95:1.5:3.5) saves up to 5 min of chromatographic run time and better peak shape, for the palmitate,
is obtained (Figure 13A,B).

Figure 13. Chromatographs for vitamin A standards mixtures separated with C8 column at 325 nm and
50 ◦ C, of (A) retinyl acetate (3.11 min) and retinyl palmitate (17.63 min) using MeOH/H2 O (90:10) and
(B) retinyl acetate (2.89 min) and retinyl palmitate (13.30 min) using MeOH/2-propanol/acetonitrile
(95:1.5:3.5).

Monitoring the analytes at 295 nm, using MeOH/H2 O (90:10) as solvents at a flow rate of
0.5 mL min−1 , tocopherols can be successfully separated (i.e., retention times for δ/γ/α-tocopherol
5.35, 6.03 and 8.59 min, respectively; Rs 1.97). In samples with relatively high-fat content, saponification
is necessary to eliminate interferences that usually share similar retention times that those for
α-tocopherol. Food and feed samples can be fortified or can naturally contain both, vitamin D2
and D3 . Therefore, for a method to be suitable, simultaneous detection of both analytes is a must.
Under the conditions above, C8 stationary phases are incapable of resolving both species.
A C18 column heated at 30 ◦ C and a MeOH/2-propanol/ACN (90:3:7) solvent system, with a flow
set at 0.3 mL min−1 , can achieve a resolution of 1.25 (Figure 14A,B). Though a mobile phase composed
MeOH and H2 O is highly desirable, a drawback using this solvent system in complex matrices is
that α-tocopherol can be interference for the identification of vitamin D3 and vice versa (Figure 14C).
A solvent gradient, a column with longer alkyl chains (e.g., C30 ) or the use an MS detector may be
employed to solve this issue.
Foods 2019, 8, 1 43 of 63

Figure 14. Separation for vitamin D2 +D3 standards at 264 nm using (A) a C8 column and (B) a C18
column D2 (16.47 min) y D3 (17.24 min). Analysis performed at 30 ◦ C using MeOH/2-propanol/ACN
(90:3:7) (C) Superposed chromatograms for vitamin D2 + D3 (blue line) and δ/γ/α-tocopherol standards
using a C18 column and MeOH/H2 O (90:10), 30 ◦ C.

5. Conclusions
LC is a powerful and versatile tool for food and feeds analysis, food and feed matrices are
complex mixtures that on occasion present to the researcher difficulties as analytes of interest must
be extracted and purified before injecting into the LC system. Advantages that chromatography
provides when applied to food or feed analysis include sensitivity (determination of trace amounts,
especially important in the case of contaminants, residues, controlled or undesired substances),
automation and high throughput (reducing time and user dependence in laboratories with
considerable workloads), simultaneous determination of multiple analytes. Food and feed chemists
must make an effort to develop methods that provide a faster response and with few possible
numbers of steps. Several current methods that are based on other step-full or less automated,
or specific techniques can be reinvented, transformed and transplanted to LC analysis to improve
sensitivity, specificity, and selectivity (For example, exchanging a spectrophotometric-based piperine
analysis for a chromatography one). Within the myriad of alternatives, the LC approach delivers,
each on its own seldom can solve a research problem, and each technique (i.e., each detector,
each chromatographic column or sample treatment) has its shortcomings and limits as to which
data is to provide. Usually, a multiphasic methodology is desirable to reach an appropriate conclusion.
Hence, LC methods are more useful when are tailored to fit a purpose. Nowadays, mass spectrometry
coupled liquid chromatography is an almost widespread technique that can provide molecule
confirmation, ease trace analysis and allows the assay of multiple analytes simultaneously. Not only
is a versatile tool for routine analysis but, research-wise, it provides more information about the
target molecules and opens a valuable doorway toward a myriad of applications in food analysis
including metabolomics, proteomics, and parvomics. Notwithstanding, as demonstrated above,
traditional detectors are still the most commonly available in most laboratories. With a proper
Foods 2019, 8, 1 44 of 63

sample pretreatment, some traditional-detector-based methods are, regarding analytical performance,

comparable to those based on MS.

Author Contributions: Conceptualization: C.C.-H and F.G.-C.; Methodology: C.C.-H, G.A., F.G.-C., A.L.;
Software: F.G.-C.; Investigation: C.C.-H, G.A., F.G.-C., A.L.; Resources: C.C.-H, G.A., F.G.-C., A.L.; Data Curation:
F.G.-C.; Writing—original draft preparation: C.C.-H, G.A., F.G.-C., A.L., C.C.-H, G.A., F.G.-C., A.L.; Visualization:
F.G.-C..; Supervision: F.G.-C, G.A; Funding Acquisition: C.C.-H.
Funding: This research was funded by the University of Costa Rica grants number A2502, B2062, B2066, B2659,
B8042, B5084. B3097, ED-427 and ED-428 and the APC was funded by the Office of the Vice Provost for Research
of the University of Costa Rica.
Acknowledgments: Marelyn Rojas Lezama is acknowledged for her help doing the experiments regarding nitrate
and nitrite in the hay. Special thanks to Guy Lamoureux Lamontagne for his suggestions.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Ercsey-Ravasz, M.; Toroczkai, Z.; Lakner, Z.; Baranyu, J. Complexity of the international agro-food trade
network and its impact on food safety. PLoS ONE 2012, 7, e37810. [CrossRef]
2. Fair, K.R.; Bauch, C.T.; Anand, M. Dynamics of the Global Wheat Trade Network and Resilience to Shocks.
Sci. Rep. 2017, 7, 7177. [CrossRef] [PubMed]
3. Keding, G.B.; Schneider, K.; Jordan, I. Production and processing of foods as core aspects of nutrition-sensitive
agriculture and sustainable diets. Food Secur. 2013, 5, 825–846. [CrossRef]
4. Canady, R.; Lane, R.; Paoli, G.; Wilson, M.; Bialk, H.; Hermansky, S.; Kobielush, B.; Li, J.-E.; Llewellyn, C.;
Scimeca, J. Determining the applicability of threshold of toxicological concern approaches to substances
found in foods. Crit. Rev. Food Sci. Nutr. 2013, 53, 1239–1249. [CrossRef] [PubMed]
5. Van der Fels-Klerx, H.J.; Adamse, P.; de Jong, J.; Hoogenboom, R.; de Nijs, M.; Bikker, P. A model for
risk-based monitoring of contaminants in feed ingredients. Food Control 2017, 72, 211–218. [CrossRef]
6. Allard, D.G. The ‘farm to plate’ approach to food safety—Everyone’s business. Can. J. Infect. Dis. 2002, 13, 185–190.
[CrossRef]
7. Kniel, K.E.; Kumar, D.; Thakur, S. Understanding the complexities of food safety using
a “One Health” approach. Microbiol. Spectr. 2018, 6. [CrossRef]
8. Krska, R.; de Nijs, M.; McNerney, O.; Pichler, M.; Gilbert, J.; Edwards, S.; Suman, M.; Magan, N.; Rossi, V.;
van der Fels-Klerx, H.J.; et al. Safe food and feed through an integrated toolbox for mycotoxin management:
The MyToolBox approach. World Mycotoxin J. 2016, 9, 487–495. [CrossRef]
9. Wartella, W.A.; Lichtenstein, A.H.; Boon, C.S. Front-of-Package Nutrition Rating Systems and Symbols; Institute
of Medicine of the National Academies Press: Washington, DC, USA, 2010; ISBN 978-0-309-15827-5.
10. Association of American Feed Control Officials (AAFCO). Pet feed regulation. In AAFCO Official Publication;
AAFCO: Atlanta, GA, USA, 2018.
11. Yashin, Y.I.; Yashin, A.Y. Analysis of food products and beverages using high-performance liquid
chromatography and ion chromatography with electrochemical detectors. J. Anal. Chem. 2004, 59, 1237–1243.
[CrossRef]
12. Nollet, L.M.L. Liquid chromatography in food analysis. In Encyclopedia of Analytical Chemistry; Meyers, R.A.,
McGorrin, R.J., Eds.; John Wiley and Sons: Hoboken, NJ, USA, 2006. [CrossRef]
13. Quitmann, H.; Fan, R.; Czermak, P. Acidic organic compounds in beverage, food, and feed production.
In Biotechnology of Food and Feed Additives; Zorn, H., Czermak, P., Eds.; Springer: Berlin/Heidelberg, Germany,
2014; ISBN 978-3-662-43760-5.
14. Jiang, J.; Xiong, Y.L. Natural antioxidants as food and feed additives to promote health benefits and quality
of meat products: A review. Meat Sci. 2016, 120, 107–117. [CrossRef] [PubMed]
15. Candan, T.; Bağdatl, A. Use of natural antioxidants in poultry meat. CBU J. Sci. 2017, 13, 279–291.
16. Chanadang, S.; Koppel, K.; Aldrich, G. The impact of rendered protein meal oxidation level on shelf-life,
sensory characteristics, and acceptability in extruded pet food. Animals 2016, 6, 44. [CrossRef] [PubMed]
17. Sanches-Silva, A.; Costa, D.; Alburquerque, T.G.; Buonocore, G.G.; Ramos, F.; Castilho, M.C.; Machado, A.V.;
Costa, H.S. Trends in the use of natural antioxidants in active food packaging: A review. Food Addit. Contam.
Part A 2014, 31, 374–395. [CrossRef] [PubMed]
Foods 2019, 8, 1 45 of 63

18. Erickson, M.C.; Doyle, M.P. The challenges of eliminating or substituting antimicrobial preservatives in
foods. Annu. Rev. Food Sci. Technol. 2017, 8, 371–390. [CrossRef]
19. Gupta, C.; Prakash, D.; Gupta, S. A biotechnological approach to microbial based perfumes and flavours.
J. Microbiol. Exp. 2015, 2. [CrossRef]
20. Das, A.; Chakraborty, R. An introduction to sweeteners. In Sweeteners, Reference Series in Phytochemistry;
Mérillon, J.-M., Ramawat, K.G., Eds.; Springer International Publishing: Basel, Switzerland, 2018;
ISBN 978-3-319-27026-5.
21. Mooradian, A.D.; Smith, M.; Tokuda, M. The role of artificial and natural sweeteners in reducing the
consumption of table sugar: A narrative review. Clin. Nutr. ESPEN 2017, 18, 1–8. [CrossRef]
22. Durán, S.; Dávila, L.A.; Contreras, M.C.E.; Rojas, D.; Costa, J. Noncaloric sweeteners in children:
A controversial theme. BioMed Res. Int. 2018, 2018, 4806534.
23. Subedi, B.; Kannan, K. Fate of Artificial Sweeteners in Wastewater Treatment Plants in New York State, USA.
Environ. Sci. Technol. 2014, 48, 13668–13674. [CrossRef]
24. Khan, S.A. Artificial sweeteners: Safe or unsafe? J. Pak. Med. Assoc. 2015, 25, 225–227.
25. García-Almeida, J.M.; Conejo-Pareja, I.M.; Muñoz-Garach, A.; Gómez-Pérez, A.; García-Alemán, J.
Sweeteners: Regulatory Aspects. In Sweeteners, Reference Series in Phytochemistry; Mérillon, J.-M.,
Ramawat, K.G., Eds.; Springer International Publishing: Basel, Switzerland, 2016; ISBN 978-3-319-27026-5.
26. Shah, R.; de Jager, L.S. Recent analytical methods for the analysis of sweeteners in food: A
regulatory perspective. Food Drug Adm. Papers 2017, 5, 13–31.
27. Sharma, A.; Amarnath, S.; Thulasimani, M.; Ramaswamy, S. Artificial sweeteners as a sugar substitute:
Are they really safe? Indian J. Pharmacol. 2016, 48, 237–240. [CrossRef] [PubMed]
28. Vanĕk, T.; Nepovím, A.; Valíček, P. Determination of stevioside in plant material and fruit teas. J. Food
Compos. Anal. 2001, 14, 383–388. [CrossRef]
29. Chaturvedula, V.S.P.; Zamora, J. Reversed-Phase HPLC analysis of steviol glycosides isolated from
Stevia rebaudiana Bertoni. Food Nutr. Sci. 2014, 5, 1711–1716. [CrossRef]
30. Wang, T.-H.; Avula, B.; Tang, W.; Wang, M.; Elsohly, M.A.; Khan, I.A. Ultra-HPLC method for quality and
adulterant assessment of steviol glycosides sweeteners—Stevia rebaudiana and stevia products. Food Addit.
Contam. Part A 2015, 32, 674–685.
31. Martono, Y.; Riyanto, S.; Rohman, A.; Martono, S. Improvement method of fast and isocratic RP-HPLC
analysis of major diterpene glycoside from Stevia rebaudiana leaves. AIP Conf. Proc. 2016, 1755, 080001–080008.
32. Sádecka, J.; Polonský, J. Determination of inorganic ions in food and beverages by capillary electrophoresis.
J. Chromatogr. 1999, 834, 401–417. [CrossRef]
33. Rios, R.V.; Pessanha, M.D.F.; de Almeida, P.F.; Viana, C.L.; Lannes, S.C. Application of fats in some
food products. Food Sci. Technol. 2014, 34, 3–15. [CrossRef]
34. Li, H.; Zhou, X.; Gao, P.; Li, Q.; Li, H.; Huang, R.; Wu, M. Inhibition of lipid oxidation in foods and feeds
and hydroxyl radical treated fish erythrocytes: A comparative study of Ginkgo biloba leaves extracts and
synthetic antioxidants. Anim. Nutr. 2016, 2, 234–241. [CrossRef]
35. Kerr, B.J.; Kellner, T.A.; Shurson, G.C. Characteristics of lipids and their feeding value in swine diets. J. Anim.
Sci. Biotechnol. 2015, 6, 30. [CrossRef]
36. Shurson, G.C.; Kerr, B.J.; Hanson, A.R. Evaluating the quality of feed fats and oils and their effects on pig
growth performance. J. Anim. Sci. Biotechnol. 2015, 6, 10. [CrossRef]
37. Pickova, J. Importance of knowledge on lipid composition of foods to support development towards
consumption of higher levels of n-3 fatty acids via freshwater fish. Psychol. Res. 2009, 58, S39–S45.
38. Benkerroum, N. Biogenic amines in dairy products: Origin, incidence, and control means. Compr. Rev. Food
Sci. Food Saf. 2016, 15, 801–826. [CrossRef]
39. Spano, G.; Russo, P.; Lonvaud-Funel, A.; Lucas, P.; Alexandre, H.; Grandvalet, C.; Coton, E.; Coton, M.;
Barnavon, L.; Bach, B.; et al. Biogenic amines in fermented foods. Eur. J. Clin. Nutr. 2010, 64, S95–S100.
[CrossRef] [PubMed]
40. Landete, J.M.; Ferrer, S.; Polo, L.; Pardo, I. Biogenic amines in wines from three Spanish regions. J. Agric.
Food Chem. 2005, 53, 1119–1124. [CrossRef]
41. Biji, K.B.; Ravishankar, C.N.; Venkateswarlu, R.; Mohna, C.O.; Srinivasa, T.K. Biogenic amines in seafood:
A review. J. Food Sci. Technol. 2016, 53, 2210–2218. [CrossRef] [PubMed]
Foods 2019, 8, 1 46 of 63

42. Karau, A.; Grayson, I. Amino acids in human and animal nutrition. Adv. Biochem. Eng. Biotechnol. 2014, 143, 189–228.
[PubMed]
43. Hardy, K.; Brand-Miller, J.; Brown, K.D.; Thomas, M.G.; Copeland, L. The importance of dietary carbohydrate
in human evolution. Q. Rev. Biol. 2015, 90, 251–268. [CrossRef] [PubMed]
44. Bach Knudsen, K.E.; Lærke, H.N.; Ingerslev, A.K.; Hedemann, M.S.; Nielsen, T.S.; Theil, P.K. Carbohydrates
in pig nutrition—Recent advances. J. Anim. Sci. 2016, 94, 1–11. [CrossRef]
45. McDowell, L.R. Vitamins in Animal and Human Nutrition; Iowa State University Press: Ames, IA, USA, 2000;
pp. 15–217. ISBN 0-8138-2630-6.
46. Koleva, I.I.; van Beek, T.A.; Soffers, A.E.M.F.; Dusemund, B.; Rietjens, I.M.C.M. Alkaloids in the human food
chain—Natural occurrence and possible adverse effects. Mol. Nutr. Food Res. 2012, 56, 30–52. [CrossRef]
47. Di Lorenzo, C.; Dos Santos, A.; Colombo, F.; Moro, E.; Dell’Agli, M.; Restani, P. Development and validation
of HPLC method to measure active amines in plant food supplements containing Citrus aurantium L.
Food Control 2014, 46, 136–142. [CrossRef]
48. Endlová, L.; Laryšová, A.; Vrbovsky, V.; Navrátilová, Z. Analysis of alkaloids in poppy straw by
high-performance liquid chromatography. IOSR J. Eng. 2015, 3, 1–7.
49. Arai, K.; Terashima, H.; Aizaewa, S.; Taga, A.; Yamamoto, A.; Tsutsumiuchi, K.; Kodama, S. Simultaneous
Determination of Trigonelline, Caffeine, Chlorogenic Acid and Their Related Compounds in Instant Coffee
Samples by HPLC Using an Acidic Mobile Phase Containing Octanesulfonate. Anal. Sci. 2015, 31, 831–835.
[CrossRef] [PubMed]
50. Wang, H.-B.; Qi, W.; Zhang, L.; Yuan, D. Qualitative and quantitative analyses of alkaloids in Uncaria species
by UPLC-ESI-Q-TOF/MS. Chem. Pharm. Bull. 2014, 62, 1100–1109. [CrossRef] [PubMed]
51. Ramdani, D.; Chaudhry, A.S.; Seal, C.J. Alkaloid and polyphenol analysis by HPLC in green and black tea
powders and their potential use as additives in ruminant diets. AIP Conf. Proc. 2018, 1927, 0300081–0300086.
52. Yashin, A.; Yashin, Y.; Xia, X.; Nemzer, B. Chromatographic Methods for Coffee Analysis: A Review.
J. Food Res. 2017, 6, 60–82. [CrossRef]
53. Pereira, C.A.M.; Rodrigues, T.R.; Yariwake, J.H. Quantification of Harman alkaloids in sour passion fruit
pulp and seeds by a novel dual SBSE-LC/Flu (stir bar sorptive extraction-liquid chromatography with
fluorescence detector) method. J. Braz. Chem. Soc. 2014, 25, 1472–1483. [CrossRef]
54. Kowalczyk, E.; Patyra, E.; Grelik, A.; Kwiatek, K. Development and validation of an analytical
method for determination of ergot alkaloids in animal feedingstuffs with high performance liquid
chromatography-fluorescence detection. Pol. J. Vet. Sci. 2016, 19, 559–565. [CrossRef]
55. Upadhyay, V.; Sharma, N.; Joshi, H.M.; Malik, A.; Mishra, M.; Singh, B.P.; Tripathi, S. Development and validation of
rapid RP-HPLC method for estimation of piperine in Piper nigrum L. Int. J. Herb. Med. 2013, 1, 6–9.
56. Granados-Chinchilla, F.; Rodriguez, C. Tetracyclines in food and feedingstuffs: From regulation to analytical methods,
bacterial resistance, and environmental and health implications. J. Anal. Methods Chem. 2017, 2017, 1315497. [CrossRef]
57. Alshannaq, A.; Yu, J.-H. Occurrence, toxicity, and analysis of major mycotoxins in food. Int. J. Environ. Res.
Public Health 2017, 14, 632. [CrossRef]
58. Granados-Chinchilla, F.; Molina, A.; Chavarría, G.; Alfaro-Cascante, M.; Bogantes-Ledezma, D.;
Murillo-Williams, A. Aflatoxins occurrence through the food chain in Costa Rica: Applying the One Health
approach to mycotoxin surveillance. Food Control 2017, 82, 217–226. [CrossRef]
59. Oliveira, F.A.; Pereira, E.N.C.; Gobbi, J.M.; Soto-Blanco, B.; Melo, M.M. Multiresidue method for detection of
pesticides in beef meat using liquid chromatography coupled to mass spectrometry detection (LC-MS) after
QuEChERS extraction. Food Addit. Contam. Part A 2018, 35, 94–109. [CrossRef] [PubMed]
60. Schnabel, K.; Schmitz, R.; von Soosten, D.; Frahm, J.; Kersten, S.; Meyer, U.; Breves, G.; Hackenberg, R.;
Spitzke, M.; Dänicke, S. Effects of glyphosate residues and different concentrate feed proportions on
performance, energy metabolism and health characteristics in lactating dairy cows. Arch. Anim. Nutr.
2017, 71, 413–427. [CrossRef] [PubMed]
61. Velkoska-Markovska, L.; Petanovska-Ilievska, B.; Markovski, A. Application of high performance liquid
chromatography to the analysis of pesticide residues in apple juice. Contemp. Agric. 2018, 67, 93–102.
[CrossRef]
62. Recjczak, T.; Tuzimski, T. Application of high-performance liquid chromatography with diode array detector
for simultaneous determination of 11 synthetic dyes in selected beverages and foodstuffs. Food Anal. Methods
2017, 10, 3572–3588. [CrossRef]
Foods 2019, 8, 1 47 of 63

63. Shehata, A.B.; Rizk, M.S.; Rend, E.A. Certification of caffeine reference material purity by ultraviolet/visible
spectrophotometry and high-performance liquid chromatography with diode-array detection as two
independent analytical methods. J. Food Drug Anal. 2016, 24, 703–715. [CrossRef] [PubMed]
64. Cancho Grande, B.; Falcón, M.S.G.; Comesaña, M.R.; Gándara, J.S. Determination of sulfamethazine and
trimethoprim in liquid feed premixes by HPLC and diode array detection, with an analysis of the uncertainty
of the analytical results. J. Agric. Food Chem. 2001, 49, 3145–3150. [CrossRef]
65. Barbosa, J.; Moura, S.; Barbosa, R.; Ramos, F.; Silveira, M.I. Determination of nitrofurans in animal feeds by
liquid chromatography-UV photodiode array detection and liquid chromatography-ionspray tandem mass
spectrometry. Anal. Chim. Acta 2007, 586, 359–365. [CrossRef]
66. Horigome, J.; Kozuma, M.; Shirasaki, T. Fluorescence pattern analysis to assist food safety—-Food analysis
technology driven by fluorescence fingerprints. Hitachi Rev. 2016, 65, 248–253.
67. Borràs, S.; Companyó, R.; Guiteras, J. Analysis of sulfonamides in animal feeds by liquid chromatography
with fluorescence detection. J. Agric. Food Chem. 2011, 59, 5240–5247. [CrossRef]
68. Patyra, E.; Kwiatek, K. Determination of fluoroquinolones in animal feed by ion pair high-performance
liquid chromatography with fluorescence detection. Anal. Lett. 2017, 50, 1711–1720. [CrossRef]
69. Wacoo, A.P.; Wendiro, D.; Vuzi, P.C.; Hawumba, J.F. Methods for Detection of Aflatoxins in Agricultural
Food Crops. J. Appl. Chem. 2014, 2014, 70629. [CrossRef]
70. Shuib, N.S.; Makahleh, A.; Salhimi, S.M.; Saad, B. Determination of aflatoxin M1 in milk and dairy products
using highperformance liquid chromatography-fluorescence with post column photochemical derivatization.
J. Chromatogr. A 2017, 1510, 51–56. [CrossRef] [PubMed]
71. González de la Huebra, M.J.; Vincent, U.; von Holst, C. Sample preparation strategy for the
simultaneous determination of macrolide antibiotics in animal feedingstuffs by liquid chromatography with
electrochemical detection (HPLC-ECD). J. Pharm. Biomed. Anal. 2007, 43, 1628–1637. [CrossRef] [PubMed]
72. Gazdik, Z.; Ztka, O.; Petrlova, J.; Adam, V.; Zehnalek, J.; Horna, A.; Reznicek, V.; Beklova, M.; Kizek, R.
Determination of vitamin C (ascorbic acid) using high performance liquid chromatography coupled with
electrochemical detection. Sensors 2008, 8, 7097–7112. [CrossRef]
73. Başaran, U.; Akkbik, M.; Mut, H.; Gülümser, E.; Doğrusöz, M.C.; Koçoğlu, S. High-Performance liquid
chromatography with refractive index detection for the determination of inulin in chicory roots. Anal. Lett.
2018, 51, 83–95. [CrossRef]
74. Kupina, S.; Roman, M. Determination of total carbohydrates in wine and wine-like beverages by HPLC with
a refractive index detector: First action 2013.12. J. AOAC Int. 2014, 97, 498–505. [CrossRef]
75. Zhang, J.; Zhu, Y. Determination of betaine, choline and trimethylamine in feed additive by ion-exchange
liquid chromatography/non-suppressed conductivity detection. J. Chromatogr. A 2007, 1170, 114–117.
[CrossRef]
76. Wei, D.; Xang, X.; Wang, N.; Zhu, Y. A rapid ion chromatography column-switching method for online
sample pretreatment and determination of L-carnitine, choline and mineral ions in milk and powdered
infant formula. RSC Adv. 2017, 7, 5920–5927. [CrossRef]
77. Troise, A.D.; Fiore, A.; Fogliano, V. Quantitation of acrylamide in foods by high-resolution mass spectrometry.
J. Agric. Food Chem. 2014, 62, 74–79. [CrossRef]
78. Patyra, E.; Nebot, C.; Gavilán, R.E.; Cepeda, A.; Kwiatek, K. Development and validation of an LC-MS/MS
method for the quantification of tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine in
medicated feed. Food Addit. Contam. Part A 2018, 35, 882–891. [CrossRef] [PubMed]
79. Wang, K.; Lin, K.; Huang, X.; Chen, M. A simple and fast extraction method for the determination of
multiclass antibiotics in eggs using LC-MS/MS. Journal of Agricultural and Food Chemistry. J. Agric.
Food Chem. 2017, 65, 5064–5073. [CrossRef] [PubMed]
80. Winkler, J.; Kersten, S.; Valenta, H.; Meyer, U.; Engelhardt, U.H.; Dänicke, S. Development of a multi-toxin
method for investigating the carryover of zearalenone, deoxynivalenol and their metabolites into milk of
dairy cows. Food Addit. Contam. Part A 2015, 32, 371–380.
81. Botha, C.J.; Legg, M.J.; Truter, M.; Sulyok, M. Multitoxin analysis of Aspergillus clavatus-infected feed samples
implicated in two outbreaks of neuromycotoxicosis in cattle in South Africa. Onderstepoort J. Vet. Res. 2014, 81, e1–e6.
[CrossRef] [PubMed]
Foods 2019, 8, 1 48 of 63

82. Granados-Chinchilla, F.; Artavia, G. A straightforward LC approach using an amine column and single
quad mass detector to determine choline chloride in feed additives and feeds. MethodsX 2017, 4, 297–304.
[CrossRef] [PubMed]
83. Nassar, A.-E.F.; Bjorge, S.M. On-Line liquid chromatography-accurate radioisotope counting coupled
with a radioactivity detector and mass spectrometer for metabolite identification in drug discovery
and development. Anal. Chem. 2003, 75, 785–790. [CrossRef] [PubMed]
84. Kim, H.J.; Bae, I.K.; Jeong, M.H.; Park, H.J.; Jung, J.S.; Kim, J.E. A new HPLC-ELSD method for simultaneous
determination of N-acetylglucosamine and N-acetylgalactosamine in dairy foods. Int. J. Anal. Chem.
2015, 2015, 892486. [CrossRef]
85. Yan, W.; Wang, N.; Zhang, P.; Zhang, J.; Wu, S.; Zhu, Y. Simultaneous determination of sucralose and
related compounds by high-performance liquid chromatography with evaporative light scattering detection.
Food Chem. 2016, 204, 358–364. [CrossRef]
86. Wang, Y.; Wang, M.J.; Li, J.; Yao, S.C.; Xue, J.; Zou, W.B.; Hu, C.Q. determination of spectinomycin and related
substances by HPLC coupled with evaporative light scattering detection. Acta Chromatogr. 2015, 207, 93–109.
[CrossRef]
87. Ligor, M.; Studzińska, S.; Horna, A.; Buszewski, B. Corona-charged aerosol detection: An analytical approach.
Crit. Rev. Anal. Chem. 2013, 43, 64–78. [CrossRef]
88. Vehovec, T.; Obreza, A. Review of operating principle and applications of the charged aerosol detector.
J. Chromatogr. A 2010, 1217, 1549–1556. [CrossRef]
89. Grembecka, M.; Lebiedzińska, A.; Szefer, P. Simultaneous separation and determination of erythritol, xylitol,
sorbitol, mannitol, maltitol, fructose, glucose, sucrose and maltose in food products by high performance
liquid chromatography coupled to charged aerosol detector. Microchem. J. 2014, 117, 77–82. [CrossRef]
90. Szekeres, A.; Budai, A.; Bencsik, O.; Németh, L.; Bartók, T.; Szécsi, Á.; Mesterházy, Á.; Vágvölgyi, C.
Fumonisin measurement from maize samples by high-performance liquid chromatography coupled with
corona charged aerosol detector. J. Chromatogr. Sci. 2014, 52, 1181–1185. [CrossRef]
91. Enri, F.; Steuer, W.; Bosshart, H. Automation and validation of HPLC-Systems. Chromatographia 1987, 24, 201–207.
92. Oláh, E.; Tarnai, M.; Fekete, J. Possibility of large volume injection and band focusing in UHPLC.
J. Chromatogr. Sci. 2013, 51, 839–844. [CrossRef] [PubMed]
93. Williamson, G. The role of polyphenols in modern nutrition. Nutr. Bull. 2017, 42, 226–235. [CrossRef]
94. Pandey, K.B.; Rizvi, S.I. Plant polyphenols as dietary antioxidants in human health and disease. Oxidaive Med.
Cell. Longev. 2009, 2, 270–278. [CrossRef] [PubMed]
95. Beaulieu, M.; Franke, K.; Fischer, K. Feeding on ripening and over-ripening fruit: Interactions between sugar,
ethanol and polyphenol contents in a tropical butterfly. J. Exp. Biol. 2017, 220, 3127–3134. [CrossRef]
96. Vanholme, R.; Demedts, B.; Morreel, K.; Ralph, J.; Boerjan, W. Lignin Biosynthesis and Structure. Plant Physiol.
2010, 153, 895–905. [CrossRef]
97. Bensalem, J.; Dal-Pen, A.; Gillard, E.; Fréderic, C.; Pallet, V. Protective effects of berry polyphenols against
age-related cognitive impairment. Nutr. Aging 2015, 3, 89–106. [CrossRef]
98. Kowalska, K.; Olejnik, A.; Szwajgier, D.; Olkowicz, M. Inhibitory activity of chokeberry, bilberry, raspberry
and cranberry polyphenol-rich extract towards adipogenesis and oxidative stress in differentiated 3T3-L1
adipose cells. PLoS ONE 2017, 12, e0188583. [CrossRef] [PubMed]
99. Khalifa, I.; Zhu, W.; Li, K.-K.; Li, C.-M. Polyphenols of mulberry fruits as multifaceted compounds:
Compositions, metabolism, health benefits, and stability—A structural review. J. Funct. Foods 2018, 40, 28–43.
[CrossRef]
100. Pérez-Jiménez, J.; Neveu, V.; Vos, F.; Sclbert, A. Identification of the 100 richest dietary sources of polyphenols:
An application of the Phenol-Explorer database. Eur. J. Clin. Nutr. 2010, 64, S112–S120. [CrossRef] [PubMed]
101. Acosta, O.; Vaillant, F.; Pérez, A.M.; Dornier, M. Concentration of polyphenolic compounds in blackberry
(Rubus adenotrichos Schltdl) juice by nanofiltration. J. Food Process Eng. 2017, 40, e12343. [CrossRef]
102. Haminiuk, C.W.; Maciel, G.M.; Plata-Oviedo, M.S.V.; Peralta, R.M. Phenolic compounds in
fruits—An overview. Int. J. Food Sci. Technol. 2012, 47, 2023–2044. [CrossRef]
103. Karasawa, M.M.G.; Mohan, C. Fruits as Prospective Reserves of bioactive Compounds: A Review.
Nat. Prod. Bioprospect. 2018, 8, 335–346. [CrossRef]
104. Reynoso-Camacho, R.; Rufino, M.S.M.; Amaya-Cruz, D.M.; Pérez, A.M. Non-extractable polyphenols in
tropical fruits: Occurrence and health-related properties. In Non-extractable Polyphenols and Carotenoids:
Foods 2019, 8, 1 49 of 63

Importance in Human Nutrition and Health; Saura-Calixto, F., Pérez-Jiménez, J., Eds.; Royal Society
of Chemistry: Cambridge, UK, 2018; ISBN 978-1-78801-106-8.
105. Abbas, M.; Saeed, F.; Anjum, F.M.; Afzaal, M.; Tufail, T.; Bashir, M.S.; Ishtiaq, A.; Hussain, S.; Suleria, H.A.
Natural polyphenols: An overview. Int. J. Food Prop. 2016, 20, 1689–1699. [CrossRef]
106. Kaşikci, M.B.; Bağdatlioğlu, N. High hydrostatic pressure treatment of fruit, fruit products and fruit juices:
A review on phenolic compounds. J. Food Health Sci. 2016, 2, 27–39.
107. Miletić, N.; Mitrović, O.; Popović, B.; Nedović, V.; Zlatković, B.; Kandić, M. Polyphenolic content and
antioxidant capacity in fruits of plum (Prunus domestica L.) cultivars “Valjevka” and “Mildora” as influenced
by air drying. J. Food Qual. 2013, 36, 229–237. [CrossRef]
108. McSweeny, M.; Seetharaman, K. State of polyphenols in the drying process of fruits and vegetables. Crit. Rev.
Food Sci. Nutr. 2015, 55, 660–669. [CrossRef]
109. Koffi, E.; Sea, T.; Dodehe, Y.; Soro, S. Effect of solvent type on extraction of polyphenols from twenty three
Ivorian plants. J. Anim. Plant Sci. 2010, 5, 550–558.
110. Flores, G.; Dastmalchi, K.; Wu, S.-B.; Whalen, K.; Dabo, A.J.; Reynertson, K.A.; Foronjy, R.F.; D’Armiento, J.M.;
Kennelly, E.J. Phenolic-rich extract from the Costa Rican Guava (Psidium friedrichsthalianum) pulp with
antioxidant and anti-inflammatory activity. Potential for COPD therapy. Food Chem. 2013, 141, 889–895.
[CrossRef] [PubMed]
111. Tresserra-Rimbau, E.; Arranz, S.; Vallverdu-Queralt, A. New Insights into the Benefits of Polyphenols in
Chronic Diseases. Oxidative Med. Cell. Longev. 2017, 2017, 1432071. [CrossRef] [PubMed]
112. Yang, J.; Dwyer, J. Exploring Possible Health Effects of Polyphenols in Foods. Nutr. Today 2017, 52, 62–72.
[CrossRef]
113. Gordon, A.; Jungfer, E.; da Silva, B.A.; Maia, J.G.S.; Marx, F. Phenolic constituents and antioxidant capacity
of four underutilized fruits from the Amazon region. J. Agric. Food Chem. 2011, 59, 7688–7699. [CrossRef]
[PubMed]
114. Assefa, A.D.; Jeong, Y.-J.; Kim, D.-J.; Jeon, Y.-A.; Ok, H.-C.; Baek, H.-J.; Sung, J.-S. Characterization, identification,
and quantification of phenolic compounds using UPLC-Q-TOF-MS and evaluation of antioxidant activity of
73 Perilla frutescens accessions. Food Res. Int. 2018, 111, 153–167. [CrossRef]
115. Anton, D.; Bender, I.; Kaart, T.; Roasto, M.; Heinonen, M. Changes in polyphenols contents and
antioxidant capacities of organically and conventionally cultivated tomato (Solanum lycopersicum L.) fruits
during ripening. Int. J. Anal. Chem. 2017, 2017, 2367453. [CrossRef]
116. Radovanović, B.C.; Anđelković, A.S.M.; Radovanović, A.B.; Anđelković, M.Z. Antioxidant and antimicrobial activity
of polyphenol extracts from wild berry fruits grown in southeast Serbia. Trop. J. Pharm. Res. 2013, 12, 813–819.
[CrossRef]
117. Veljković, J.N.; Pavlović, A.N.; Mitić, S.S.; Tošić, S.B.; Stojanović, G.S.; Kaličanin, B.M.; Stanović, D.M.;
Stojković, M.B.; Mitić, M.N.; Brcanović, J.M. Evaluation of individual phenolic compounds and antioxidant
properties of black, green, herbal and fruit tea infusions consumed in Serbia: Spectrophotometrical and
electrochemical approaches. J. Food Nutr. Res. 2013, 52, 12–24.
118. Miletić, N.; Popović, B.; Mitrović, O.; Kandić, M.; Leposavić, A. Phenolic compounds and antioxidant
capacity of dried and candied fruits commonly consumed in Serbia. Czech J. Food Sci. 2014, 32, 360–368.
[CrossRef]
119. Rojas-Garbanzo, C.; Zimmermann, B.F.; Schulze-Kaysers, N.; Schieber, A. Characterization of phenolic
and other polar compounds in peel and flesh of pink guava (Psidium guajava L. cv. ‘Criolla’) by ultra-high
performance liquid chromatography with diode array and mass spectrometric detection. Food Res. Int.
2017, 100, 445–453. [CrossRef] [PubMed]
120. Azofeifa, G.; Quesada, S.; Pérez, A.M.; Vaillant, F.; Michel, A. Effect of an in vitro digestion on the antioxidant
capacity of a microfiltrated blackberry juice (Rubus adenotrichos). Beverages 2018, 4, 30. [CrossRef]
121. Georgé, S.; Brat, P.; Alter, P.; Amiot, M.J. Rapid determination of polyphenols and vitamin C in plant derived
products. J. Agric. Food Chem. 2005, 53, 1370–1373. [CrossRef] [PubMed]
122. Gouvêa, A.; de Araujo, M.C.; Schulz, D.F.; Pacheco, S.; Godoy, R.; Cabral, L. Anthocyanins
standards (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) isolation from freeze-dried açaí
(Euterpe oleraceae Mart.) by HPLC. Ciênc. Tecnol. Aliment. 2012, 32, 43–46. [CrossRef]
123. Teboukeu, G.B.; Djikeng, F.T.; Klang, M.J.; Ndomou, S.H.; Karuna, M.S.L.; Womeni, H.M.
Polyphenol antioxidants from cocoa pods: Extraction optimization, effect of the optimized extract, and
Foods 2019, 8, 1 50 of 63

storage time on the stability on palm olein during thermoxidation. J. Food Process. Preserv. 2018, 42, e13592.
[CrossRef]
124. Ryu, W.-K.; Kim, H.-W.; Kim, G.-D.; Rhee, H.-I. Rapid determination of capsaicinoids by colorimetric method.
J. Food Drug Anal. 2017, 25, 798–803. [CrossRef]
125. Collins, M.D.; Wasmund, L.M.; Bosland, P.W. Improved method for quantifying capsaicinoids in Capsicum
using high-performance liquid chromatography. Hortic. Sci. 1995, 30, 137–139.
126. Guzmán, I.; Bosland, P.W. Sensory properties of chile pepper heat e and its importance to food quality and
cultural preference. Appetite 2017, 117, 186–190. [CrossRef]
127. Kobata, K.; Sugawara, M.; Mimura, M.; Yawaza, S.; Watanabe, T. Potent Production of Capsaicinoids and
Capsinoids by Capsicum Peppers. J. Agric. Food Chem. 2013, 61, 11127–11132. [CrossRef]
128. Garcés-Claver, A.; Gil-Ortega, R.; Álvarez-Fernández, A.; Arnedo-Andrés, M.S. Inheritance of capsaicin and
dihydrocapsaicin, determined by HPLC-ESI/MS, in an intraspecific cross of Capsicum annuum L. J. Agric.
Food Chem. 2007, 55, 6951–6957. [CrossRef]
129. Goll, J.; Frey, A.; Minceva, M. Study of the separation limits of continuous solid support free liquid-liquid
chromatography: Separation of capsaicin and dihydrocapsaicin by centrifugal partition chromatography.
J. Chromatogr. A 2013, 1284, 59–68. [CrossRef] [PubMed]
130. Othman, Z.A.A.; Ahmed, Y.B.H.; Habila, M.A.; Ghafar, A.A. Determination of capsaicin and dihydrocapsaicin
in Capsicum fruit samples using high performance liquid chromatography. Molecules 2011, 16, 8919–8929.
[CrossRef] [PubMed]
131. Ma, F.; Yang, Q.; Matthäus, B.; Li, P.; Zhang, Q.; Zhang, L. Simultaneous determination of capsaicin and
dihydrocapsaicin for vegetable oil adulteration by immunoaffinity chromatography cleanup coupled with
LC-MS/MS. J. Chromatogr. B 2016, 1021, 137–144. [CrossRef] [PubMed]
132. Zhou, C.; Ma, D.; Cao, W.; Shi, H.; Jiang, Y. Fast simultaneous determination of capsaicin, dihydrocapsaicin
and nonivamide for detecting adulteration in edible and crude vegetable oils by UPLC-MS/MS. Food Addit.
Contam. Part A 2018, 35, 1447–1452. [CrossRef] [PubMed]
133. Schmidt, A.; Fiechter, G.; Fritz, E.-M.; Mayer, H.K. Quantitation of capsaicinoids in different chilies from
Austria by a novel UPLC method. J. Food Compos. Anal. 2017, 60, 32–37. [CrossRef]
134. Sganzerla, M.; Coutinho, J.P.; Tavares de Melo, A.M.; Godoy, E.T. Fast method of capsaicinoids analysis from
Capsicum chinense fruits. Food Res. Int. 2014, 64, 718–725. [CrossRef]
135. Dang, Y.M.; Hong, Y.S.; Lee, C.L.; Khan, N.; Park, S.; Jeong, S.-W.; Kim, K.S. Determination of Capsaicinoids
in Red Pepper Products from South Korea by High-Performance Liquid Chromatography with Fluorescence
Detection. Anal. Lett. 2018, 51, 1291–1303. [CrossRef]
136. Lu, M.; Ho, C.-T.; Huang, Q. Extraction, bioavailability, and bioefficacy of capsaicinoids. J. Food Drug Anal.
2017, 25, 27–36. [CrossRef] [PubMed]
137. Zhang, Q.; Hu, J.; Sheng, L.; Li, Y. Simultaneous quantification of capsaicin and dihydrocapsaicin in rat
plasma using HPLC coupled with tandem mass spectrometry. J. Chromatogr. B 2010, 878, 2292–2297.
[CrossRef]
138. Kuzma, M.; Fodor, K.; Maász, G.; Avar, P.; Mózsik, G.; Past, T.; Fischer, E.; Perjési, P. A validated HPLC-FLD
method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat.
J. Pharm. Biomed. Anal. 2015, 103, 59–66. [CrossRef]
139. Baskaran, P.; Krishnan, V.; Ren, J.; Thyagarjan, B. Capsaicin induces browning of white adipose tissue and
counters obesity by activating TRPV1 channel-dependent mechanisms. Br. J. Pharmacol. 2016, 173, 2369–2389.
[CrossRef]
140. Wolde, T. Effects of caffeine on health and nutrition: A Review. Food Sci. Qual. Manag. 2014, 30, 59–65.
141. Martínez-Pinilla, E.; Oñatibia-Asibia, A.; Franco, R. The relevance of theobromine for the beneficial effects of
cocoa consumption. Front. Pharmacol. 2015, 6, 1–5. [CrossRef]
142. Ahluwalia, N.; Herrick, K. Caffeine intake from food and beverage sources and trends among children
and adolescents in the United States: Review of national quantitative studies from 1999 to 2011. Adv. Nutr.
2015, 6, 102–111. [CrossRef] [PubMed]
143. Verster, J.C.; Koenig, J. Caffeine intake and its sources: A review of national representative studies. Crit. Rev.
Food Sci. Nutr. 2018, 58, 1250–1259. [CrossRef] [PubMed]
Foods 2019, 8, 1 51 of 63

144. Li, X.; Simmons, R.; Mwongela, S.M. Two-stage course-embedded determination of caffeine and related
compounds by HPLC in caffeine containing food, beverages and (or) related products. J. Lab. Chem. Educ.
2017, 5, 19–25.
145. Lowry, J.A.; Pearce, R.E.; Gaedigk, A.; Venneman, M.; Talib, N.; Shaw, P.; Leeder, J.S.; Kearns, G.L. Lead and
its effects on cytochromes P450. J. Drug Metab. Toxicol. 2012, S5, S004. [CrossRef]
146. Grujić-Letić, N.; Rakić, B.; Šefer, E.; Milanović, M.; Nikšić, M.; Vujić, I.; Milić, N. Quantitative determination
of caffeine in different matrices. Maced. Pharm. Bull. 2016, 62, 77–84.
147. Gonçalves, E.S.; Rodrigues, S.V.; Vieira da Silva-Filho, E. The use of caffeine as a chemical marker of
domestic wastewater contamination in surface waters: Seasonal and spatial variations in Teresópolis, Brazil.
Rev. Ambient. Agua 2017, 12, 192–202. [CrossRef]
148. Gliszczyńska-Świglo, A.; Rybicka, I. Simultaneous determination of caffeine and water-soluble vitamins in energy
drinks by HPLC with photodiode array and fluorescence detection. Food Anal. Methods 2015, 8, 139–146. [CrossRef]
149. Mazdeh, F.Z.; Moradi, Z.; Moghaddam, G.; Moradi-Khatoonabadi, Z.; Aftabdari, F.E.; Badaei, P.;
Hajimahmoodi, M. Determination of synthetic food colors, caffeine, sodium benzoate and potassium
sorbate in sports drinks. Trop. J. Pharm. Res. 2016, 15, 183–188. [CrossRef]
150. Ortega, N.; Romero, M.-P.; Macià, A.; Reguant, J.; Anglès, N.; Morelló, J.-R.; Motilva, M.-J. Comparative
study of UPLC-MS/MS and HPLC-MS/MS to determine procyanidins and alkaloids in cocoa samples.
J. Food Compos. Anal. 2010, 23, 298–305. [CrossRef]
151. Rodríguez-Carrasco, Y.; Gaspari, A.; Graziani, G.; Santini, A.; Ritieni, A. Fast analysis of polyphenols and
alkaloids in cocoa-based products by ultra-high performance liquid chromatography and orbitral high
resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS). Food Res. Int. 2018, 111, 229–236. [CrossRef]
[PubMed]
152. Oellig, C.; Schunck, J.; Schwack, W. Determination of caffeine, theobromine and theophylline in Mate beer
and Mate soft drinks by high-performance thin-layer chromatography. J. Chromatogr. A 2018, 1533, 208–212.
[CrossRef]
153. Alvi, S.N.; Hammami, M.M. Validated HPLC method for determination of caffeine level in human plasma
using synthetic plasma: Application to bioavailability studies. J. Chromatogr. Sci. 2011, 49, 292–296. [CrossRef]
154. Begas, E.; Kouvaras, E.; Tsakalof, A.K.; Bounitsi, M.; Asprodini, E.K. Development and validation of
a reverse-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.
Biomed. Chromatogr. 2015, 29, 1657–1663. [CrossRef]
155. Rodríguez, A.; Costa-Bauza, A.; Saenz-Torres, C.; Rodrigo, D.; Grases, F. HPLC method for urinary
theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion
in children. Clin. Biochem. 2015, 48, 1138–1143. [CrossRef]
156. Shrestha, S.; Rijal, S.K.; Pokhrel, P.; Rai, K.P. A simple HPLC method for determination of caffeine content in
tea and coffee. J. Food Sci. Technol. 2016, 9, 74–78.
157. Fajara, B.E.P.; Susanti, H. HPLC determination of caffeine in coffee beverage. IOP Conf. Ser. Mater. Sci. Eng.
2017, 259, 01211. [CrossRef]
158. Aşçi, B.; Zor, Ş.D.; Dönmez, Ö.A. Development and validation of HPLC method for the simultaneous
determination of five food additives and caffeine in soft drinks. Int. J. Anal. Chem. 2016, 2016, 2879406.
[CrossRef]
159. Yamamoto, T.; Takahashi, H.; Suzuki, K.; Hirano, A.; Kamei, M.; Goto, T.; Takahashi, N.; Kawada, T. Theobromine
enhances absorption of cacao polyphenol in rats. Biosci. Biotechnol. Biochem. 2014, 78, 2059–2063. [CrossRef] [PubMed]
160. Romano, R.; Santini, A.; Le Grottaglie, L.; Manzo, N.; Visconti, A.; Ritieni, A. Identification markers based on
fatty acid composition to differentiate between roasted Arabica and Canephora (Robusta) coffee varieties in
mixtures. J. Food Compos. Anal. 2014, 35, 1–9. [CrossRef]
161. López-Sánchez, R.C.; Lara-Díaz, V.J.; Aranda-Gutiérrez, A.; Martínez-Cardona, J.A.; Hernández, J.A. HPLC
method for quantification of caffeine and its three major metabolites in human plasma using fetal bovine
serum matrix to evaluate prenatal drug exposure. J. Anal. Methods Chem. 2018, 2018, 2085059. [CrossRef]
[PubMed]
162. Ptolemy, A.S.; Tzioumis, E.; Thomke, A.; Rifai, S.; Kellogg, M. Quantification of theobromine and caffeine in
saliva, plasma and urine via liquid chromatography-tandem mass spectrometry: A single analytical protocol
applicable to cocoa intervention studies. J. Chromatogr. A 2010, 878, 409–416. [CrossRef] [PubMed]
Foods 2019, 8, 1 52 of 63

163. Kobayashi, J.; Ikeda, K.; Terada, H.; Mochizuki, M.; Sugiyama, H. HPLC determination of caffeine using
a photodiode array detector and applying a derivative processing to chromatograms. Bull. Nippon Vet. Life
Sci. Univ. 2014, 63, 48–57.
164. Naviglio, D.; Gallo, M.; Le Grottaglie, L.; Scala, C.; Ferrara, L.; Santini, A. Determination of cholesterol in
Italian chicken eggs. Food Chem. 2012, 132, 701–708. [CrossRef]
165. Ribeiro, S.M.L.; Luz, S.S.; Aquino, R.S. The role of nutrition and physical activity in cholesterol and aging.
Clin. Geriatr. Med. 2015, 31, 401–416. [CrossRef]
166. Albuquerque, T.G.; Oliveira, M.B.P.P.; Sanches-Silva, A.; Costa, H.S. Cholesterol determination in foods:
Comparison between high performance and ultra-high performance liquid chromatography. Food Chem.
2016, 193, 18–25. [CrossRef] [PubMed]
167. Gylling, H.; Simonen, P. Are plant sterols and plant stenols are a viable future treatment for dyslipidemia?
Expert Rev. Cardiovasc. Ther. 2016, 4, 549–551. [CrossRef] [PubMed]
168. Sonawane, P.D.; Pollier, J.; Panda, S.; Szymanski, J.; Massalha, H.; Yona, M.; Unger, T.; Malitsky, S.; Arendt, P.;
Powels, L.; et al. Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism. Nat. Plants
2016, 3, 16205. [CrossRef] [PubMed]
169. Cruz, R.; Casal, S.; Mendes, E.; Costa, A.; Santos, C.; Morais, S. Validation of a single-extraction procedure
for sequential analysis of vitamin E, cholesterol, fatty acids, and total fat in seafood. Food Anal. Methods
2012, 6, 1196–1204. [CrossRef]
170. Saldanha, T.; Bragagnolo, N. Effect of grilling on cholesterol oxide formation and fatty acids alterations
in fish. Ciênc. Tecnol. Aliment. 2010, 30, 385–390. [CrossRef]
171. Bauer, L.C.; Santana, D.A.; Macedo, M.S.; Torres, A.G.; de Souza, N.E.; Simionato, J.I. Method validation
for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD. J. Braz.
Chem. Soc. 2014, 25, 161–168. [CrossRef]
172. Daneshfar, A.; Khezeli, T.; Lotfi, H.J. Determination of cholesterol in food samples using dispersive
liquid-liquid micro extraction followed by HPLC-UV. J. Chromatogr. B 2009, 877, 456–460. [CrossRef]
173. Georgiou, C.A.; Constantinou, M.S.; Kapnissi-Christodoulou, C.P. Sample preparation: A critical step in the
analysis of cholesterol oxidation products. Food Chem. 2014, 145, 1918–1926. [CrossRef]
174. Robinet, P.; Wang, Z.; Hazen, S.L.; Smith, J.D. A simple and sensitive enzymatic method for cholesterol
quantification in macrophages and foam cells. J. Lipid Res. 2010, 51, 3364–3369. [CrossRef]
175. Carvalho, P.O.; Campos, P.R.B.; Noffs, M.D.; Fegolente, P.B.L.; Fegolente, L.V. Enzymatic hydrolysis of
salmon oil by native lipases: Optimization of process parameters. J. Braz. Chem. Soc. 2009, 20, 117–124.
[CrossRef]
176. Codex Alimentarius. Code of Practice for the Prevention and Reduction of Mycotoxin Contamination in
Cereals (CAC/RCP 51-2003). 2003. Available online: http://www.fao.org/fao-who-codexalimentarius/
codex-texts/codes-of-practice/es/ (accessed on 20 June 2018).
177. Njumbe Ediage, E.; Van Poucke, C.; De Saeger, S. A multi-analyte LC-MS/MS method for the analysis of
23 mycotoxins in different sorghum varieties: The forgotten sample matrix. Food Chem. 2015, 117, 397–404.
[CrossRef]
178. Binder, E. Managing the risk of mycotoxins in modern feed production. Anim. Feed Sci. Technol. 2007, 133, 149–166.
[CrossRef]
179. Dzuman, Z.; Zachariasova, M.; Lacina, O.; Veprokova, Z.; Slavokova, P.; Hajslova, J. A rugged
high-throughput analytical approach for the determination and quantification of multiple mycotoxins
in complex feed matrices. Talanta 2014, 121, 263–272. [CrossRef]
180. Chavarría, G.; Molina, A.; Leiva, A.; Méndez, G.; Wong-González, E.; Cortés-Muñoz, M.; Rodríguez, C.;
Granados-Chinchilla, F. Distribution, stability, and protein interactions of Aflatoxin M1 in fresh cheese.
Food Control 2017, 73, 581–586. [CrossRef]
181. Ostry, V.; Malir, F.; Toman, J.; Grosse, Y. Mycotoxins as human carcinogens, the IARC Monographs
classification. Mycotoxins Res. 2016, 33, 65–73. [CrossRef]
182. CAST. Mycotoxins: Risks in Plant, Animal, and Human Systems; Council for Agricultural Science and
Technology: Ames, IA, USA, 2003; pp. 13–48. ISBN 1-887383-22-0.
183. Molina Alvarado, A.; Zamora-Sanabria, R.; Granados-Chinchilla, F. A Focus on Aflatoxins in Feedstuffs: Levels
of Contamination, Prevalence, Control Strategies, and Impacts on Animal Health. In Aflatoxin Control, Analysis,
Detection and Health Risks; Lukman Bola Abdulra’uf, Ed.; IntechOpen: London, UK, 2017; pp. 115–152.
Foods 2019, 8, 1 53 of 63

184. Codex Alimentarius. Code of Practice for the Reduction of Aflatoxin B1 in Raw Materials and Supplemental
Feedingstuffs for Milk Producing Animals (CAC/RCP 45-1997). 1997. Available online: http://www.fao.
org/fao-who-codexalimentarius/codex-texts/codes-of-practice/es/ (accessed on 20 June 2018).
185. Flores-Flores, M.; González-Peñas, E. Development and validation of a high performance liquid
chromatographic-mass spectrometry method for the simultaneous quantification of 10 trichothecenes in
ultra-high temperature processed cow milk. J. Chromatogr. A 2015, 1419, 37–44. [CrossRef]
186. Njumbe Ediage, E.; Diana Di Mavungu, J.; Monbaliu, S.; Van Peteghem, C.; De Saeger, S. A Validated
Multianalyte LC–MS/MS Method for Quantification of 25 Mycotoxins in Cassava Flour, Peanut Cake and
Maize Samples. J. Agric. Food Chem. 2011, 59, 5173–5180. [CrossRef] [PubMed]
187. Rasmussen, R.; Storm, I.; Rasmussen, P.; Smedsgaard, J.; Nielsen, K. Multi-mycotoxin analysis of maize
silage by LC-MS/MS. Anal. Bioanal. Chem. 2010, 397, 765–776. [CrossRef]
188. Chala, A.; Taye, W.; Ayalew, A.; Krska, R.; Sulyok, M.; Logrieco, A. Multimycotoxin analysis of sorghum
(Sorghum bicolor L. Moench) and finger millet (Eleusine coracana L. Gaten) from Ethiopia. Food Control
2014, 45, 29–35. [CrossRef]
189. Vishwanath, V.; Sulyok, M.; Labuda, R.; Bicker, W.; Krska, R. Simultaneous determination of 186 fungal
and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry.
Anal. Bioanal. Chem. 2009, 395, 1355–1372. [CrossRef]
190. Alija, C.M.; Brar, S.K.; Verma, M.; Tyagi, R.D.; Godbout, S.; Valéro, J.R. Bio-processing of agro-byproducts to
animal feed. Crit. Rev. Biotechnol. 2012, 32, 382–400.
191. Mikušová, P.; Ritieni, A.; Santini, A.; Juhasová, G.; Šrobárová, A. Contamination by mould of grape berries
in Slovakia. Food Addit. Contam. Part A 2010, 27, 738–747. [CrossRef]
192. Santini, A.; Ferracane, R.; Meca, G.; Ritieni, A. Overview of analytical methods for beauvericin and
fusaproliferin in food matrices. Anal. Bioanal. Chem. 2009, 395, 1253–1260. [CrossRef]
193. Gil-Serna, J.; Vázquez, C.; González-Jaén, M.T.; Patiño, B. Wine contamination with ochratoxins: A Review.
Beverages 2018, 4, 6. [CrossRef]
194. Pizzutti, I.R.; de Kok, A.; Scholten, J.; Righi, L.W.; Cardoso, C.D.; Rohers, G.N.; da Silva, R.C. Development,
optimization and validation of a multimethod for the determination of 36 mycotoxins in wines by liquid
chromatography–tandem mass spectrometry. Talanta 2014, 129, 352–363. [CrossRef] [PubMed]
195. Nistor, E.; Dobre, A.; Dobre, A.; Bampidis, V.; Ciola, V. Grape pomace in sheep and dairy cows feeding.
J. Hortic. For. Biotechnol. 2014, 18, 146–150.
196. Guerra-Rivas, C.; Gallardo, B.; Mantecón, Á.R.; del Álamo-Sanza, M.; Manso, T. Evaluation of grape pomace
from red winw by-products as feed for sheep. J. Sci. Food Agric. 2017, 97, 1885–1893. [CrossRef] [PubMed]
197. Kerasioti, E.; Terzopoulou, Z.; Komini, O.; Kafantaris, I.; Makri, S.; Stagos, D.; Gerasopoulos, K.;
Anisimov, N.Y.; Tsatsakis, A.M.; Kouretas, D. Tissue specific effects of feeds supplemented with grape
pomace or olive oil mill wastewater on detoxification enzymes in sheep. Toxicol. Rep. 2017, 4, 364–372.
[CrossRef] [PubMed]
198. Avantaggiato, G.; Greco, D.; Damascelli, A.; Solfrizzo, M.; Visconti, A. Assessment of Multi-mycotoxin
Adsorption Efficacy of Grape Pomace. J. Agric. Food Chem. 2014, 62, 497–507. [CrossRef] [PubMed]
199. Gambacorta, L.; Pinton, P.; Avantaggiato, G.; Oswald, I.P.; Solfrizzo, M. Grape Pomace, an Agricultural
Byproduct Reducing Mycotoxin Absorption: In Vivo Assessment in Pig Using Urinary Biomarkers. J. Agric.
Food Chem. 2016, 64, 6762–6771. [CrossRef]
200. Anadón, A.; Martínez-Larrañaga, M.; Ares, I.; Martínez, M. Chapter 7—Regulatory Aspects for the Drugs
and Chemicals Used in Food-Producing Animals in the European Union. In Veterinary Toxicology: Basic and
Clinical Principles, 3rd ed.; Gupta, R.C., Ed.; Academic Press: Cambridge, MA, USA, 2018; pp. 103–131.
201. Decheng, S.; Peilong, W.; Yang, L.; Ruiguo, W.; Shulin, W.; Zhiming, X.; Su, Z. Simultaneous determination
of antibiotics and amantadines in animal-derived feedstuffs by ultraperformance liquid
chromatographic-tandem mass spectrometry. J. Chromatogr. B 2018, 1095, 183–190. [CrossRef]
202. Molognoni, L.; Coelho de Souza, N.; Antunes de Sá Ploêncio, L.; Amadeu Micke, G.; Daguer, H.
Simultaneous analysis of spectinomycin, halquizol, zilpaterol, and melamine in feedingstuffs by ion-pair
liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2018, 1569, 110–117. [CrossRef]
203. Cancho Grande, B.; García Falcón, M.S.; Simal Gándara, J. El uso de los antibióticos en la alimentación
animal: Perspectiva actual. Cienc. Tecnol. Aliment. 2000, 3, 39–47.
Foods 2019, 8, 1 54 of 63

204. Rojek-Podgórska, B. EU Legislation in Progress: Review of Medicated feed Legislation. European Parliamentary
Research Service (EPRS). 2016. Available online: http://www.europarl.europa.eu/RegData/etudes/BRIE/2016/
583843/EPRS_BRI%282016%29583843_EN.pdf (accessed on 10 July 2018).
205. Kang, H.; Lee, S.; Shin, D.; Jeong, J.; Hong, J.; Rhee, G. Occurrence of veterinary drug residues in farmed
fishery products in South Korea. Food Control 2018, 85, 57–65. [CrossRef]
206. Kumar Saxena, S.; Rangasamy, R.; Krishnan, A.; Singh, D.; Uke, S.; Kumar Malekadi, P.; Sengar, A.;
Peer Mohamed, D. Simultaneous determination of multi-residue and multi-class antibiotics in aquaculture
shrimps by UPLC-MS/MS. Food Chem. 2018, 260, 336–343. [CrossRef] [PubMed]
207. Barreto, F.; Ribeiro, C.; Barcellos Hoft, R.; Dalla Costa, T. A simple and high-throughput
method for determination and confirmation of 14 coccidiostats in poultry muscle eggs using
liquid chromatography-quadrupole linear ion trap–tandem mass spectrometry (HPLC-QqLIT-MS/MS):
Validation according to European Union 2002/657/EC. Talanta 2017, 168, 43–51. [PubMed]
208. World Health Organization. Global Action Plan on Antimicrobial Resistance. WHO Library Cataloguing
2015. Available online: http://www.wpro.who.int/entity/drug_resistance/resources/global_action_plan_eng.pdf
(accessed on 18 July 2018).
209. Camargo Valese, A.; Molognoni, L.; Coelho de Souza, N.; Antunes de Sá Ploêncio, L.; Oliveira Costa, A.;
Barreto, F. Development, validation and different approaches for the measurement uncertainty of
a multi-class veterinary drugs residues LC-MS method for feeds. J. Chromatogr. B 2017, 1053, 48–59.
[CrossRef] [PubMed]
210. European Commission. Directive 2001/82/EC of the European Parliament and of the Council of 6 November
2001 on the Community code relating to veterinary medicinal products. Off. J. Eur. Communities 2001, L 311, 1–66.
Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32001L0082&from=ES
(accessed on 10 July 2018).
211. European Commission. Regulation (EC) No 726/2004 of the European Parliament and of the Council of
31 March 2004 laying down Community procedures for the authorization and supervision of medicinal
products for human and veterinary use and establishing a European Medicines Agency. Off. J.
Eur. Communities 2004, L 136, 1–33. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/
PDF/?uri=CELEX:32004R0726&from=ES (accessed on 10 July 2018).
212. US Food and Drug Administration. Medicated Feeds. Available online: https://www.fda.gov/AnimalVeterinary/
Products/AnimalFoodFeeds/MedicatedFeed/default.htm#license (accessed on 19 July 2018).
213. Robert, C.; Basseur, P.-Y.; Dubois, M.; Delahaut, P.; Gillard, N. Development and validation of rapid
multiresidue and multi-class analysis for antibiotics and anthelmintics in feed by ultra high performance
liquid chromatography coupled to tandem mass spectrometry. Food Addit. Contam. Part A 2016, 33, 1312–1323.
[CrossRef]
214. Shendy, A.; Al-Ghobashy, M.; Gad Alla, S.; Lotfy, H. Development and validation of a modified QuEChERS
protocol coupled to LC-MS/MS for simultaneous determination of multi-class antibiotic residues in honey.
Food Chem. 2016, 190, 982–989. [CrossRef] [PubMed]
215. Wang, Y.; Xiao, C.; Guo, J.; Yuan, Y.; Wang, J.; Liu, L.; Yue, T. Development and application of method for the
analysis of 9 mycotoxins in maize by HPLC-MS/MS. J. Food Sci. 2013, 78, 1752–1756. [CrossRef]
216. Duelge, K.; Nishshanka, U.; De Alwis, H. An LC-MS/MS method for the determination of antibiotic residues
in distillers grains. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2017, 1053, 81–86. [CrossRef]
217. Mohanty, B.; Mahanty, A.; Ganguly, S.; Sankar, T.V.; Chakraborty, K.; Rangasamy, A.; Paul, B.; Sarma, D.;
Mathew, S.; Kunnath Asha, K.; et al. Amino acid compositions of 27 food fishes and their importance in
clinical nutrition. J. Amino Acids 2014, 2014, 269797. [CrossRef]
218. 218Ribeiro Alvarenga, R.; Borges Rodrigues, P.; de Souza Cantarelli, V.; Gilberto Zangeronimo, M.; da Silva
Júnior, J.; da Silva, L.; Moreira dos Santos, L.; Pereira, L. Energy values and chemical composition of spirulina
(Spirulina platensis) evaluated with broilers. Rev. Bras. Zootec. 2011, 40, 992–996. [CrossRef]
219. Campanella, L.; Russo, M.V.; Avino, P. Free and total amino acid composition in blue-green algae. Ann. Chim.
2002, 92, 343–352. [PubMed]
220. Abdullah Al-Dhabi, N.; Valan Arasu, M. Quantification of Phytochemical from Commercial Spirulina
Products and Their Antioxidant Activities. Evid. Based Complement. Alternat. Med. 2016, 2016, 7631864.
Foods 2019, 8, 1 55 of 63

221. Dziagwa-Becker,
˛ M.M.; Ramos, J.M.M.; Topolski, J.K.; Oleszek, W.A. Determination of free amino acids in plants
by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). BioSci. Trends 2011, 5, 231–238.
[CrossRef]
222. Ma, X.; Zhao, D.; Li, X.; Meng, L. Chromatographic method for determination of the free amino acid content
of chamomile flowers. Pharmacogn. Mag. 2015, 11, 176–179. [PubMed]
223. Salazar-Villanea, S.; Bruininx, E.M.A.M.; Gruppen, H.; Hendriks, W.H.; Carré, P.; Quinsac, A.;
van der Poel, A.F.B. Physical and chemical changes of rapessed meal proteins during toasting and their
effects on in vitro digestibility. J. Anim. Sci. Biotechnol. 2016, 7, 62. [CrossRef]
224. Jajić, I.; Krstović, S.; Glamočić, D.; Jakšić, S.; Abramović, B. Validation of an HPLC method for the
determination of amino acids in feed. J. Serbian Chem. Soc. 2013, 78, 839–850. [CrossRef]
225. Szkudzińska, K.; Smutniak, H.; Rubaj, J.; Korol, W.; Bielecka, G. Method validation for determination of
amino acids in feed by UPLC. Accredit. Qual. Assur. 2018, 22, 247–252. [CrossRef]
226. Desmarais, S.M.; Cava, F.; de Pedro, M.A.; Casey Huang, K. Isolation and Preparation of Bacterial Cell Walls
for Compositional Analysis by Ultra Performance Liquid Chromatography. J. Vis. Exp. 2014, 83, e51183.
[CrossRef]
227. Kühner, D.; Stahl, M.; Demircioglu, D.D.; Bertsche, U. From cells to muropeptide structures in 24 h:
Peptidoglycan mapping by UPLC-MS. Sci. Rep. 2014, 4, 7494. [CrossRef]
228. Marseglia, A.; Sforza, S.; Faccini, A.; Bencivenni, M.; Palla, G.; Caligian, A. Extraction, identification and
semi-quantification of oligopeptides in cocoa beans. Food Res. Int. 2014, 63, 382–389. [CrossRef]
229. Prados, I.M.; Marina, M.L.; García, M.C. Isolation and identification by high resolution liquid
chromatography tandem mass spectrometry of novel peptides with multifunctional lipid lowering capacity.
Food Res. Int. 2018, 111, 77–86. [CrossRef] [PubMed]
230. Holman, B.W.B.; Malau-Aduli, A.E.O. Spirulina as livestock supplement and animal feed. J. Anim. Physiol.
Anim. Nutr. 2013, 97, 615–623. [CrossRef] [PubMed]
231. Nurcahya Dewi, E.; Amalia, U.; Mel, M. The effect of Different Treatments to the Amino Acid Contents of
Micro Algae Spirulina sp. Aquatic Procedia 2016, 7, 59–65. [CrossRef]
232. Wang, Y.; Shen, K.; Li, P.; Zhou, J.; Chao, Y. Simultaneous determination of 20 underivatized amino acids by
high performance liquid chromatography-evaporative light-scattering detection. Se Pu 2011, 29, 908–911.
[PubMed]
233. Prinsen, H.C.M.T.; Schiebergen–Bronkhorst, B.G.M.; Roeleveld, M.N.; Jans, J.J.M.; de Sain-van der
Velden, M.G.M.; Visser, G.; van Hasselt, P.M.; Verhoeven-Duif, N.M. Rapid quantification of underivatized
amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tándem
mass-spectrometry. J. Inherit. Metab. Dis. 2016, 39, 651–660. [CrossRef]
234. Masuda, A.; Dohmae, N. Amino acid analysis of sub-picomolar amounts of proteins by pre-column fluorescence
derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Biosc. Trends 2011, 5, 231–238. [CrossRef]
235. Dhillon, M.K.; Kumar, S.; Gujar, G.T. A common HPLC-PDA method for amino acid analysis in insects
and plants. Indian J. Exp. Biol. 2014, 52, 73–79.
236. Artavia, G.; Rojas-Bogantes, L.; Granados-Chinchilla, F. Two alternative chromatography methods assisted
by the sulfonic acid moiety for the determination of furosine in milk. MethodsX 2018, 5, 639–647. [CrossRef]
237. Dolowy, M.; Pyka, A. Application of TLC, HPLC and GC methods to the study of amino acid and peptide
enantiomers: A review. Biomed. Chromatogr. 2013. [CrossRef]
238. Bartolomeo, M.; Maisano, F. Validation of a Reversed-Phase HPLC Method for Quantitative Amino
Acid Analysis. J. Biomol. Tech. 2006, 17, 131–137.
239. Zhou, X.; Zhang, J.; Pan, Z.; Li, D. Review of Methods for the Detection and Determination of Malachite
Green and Leuco-Malachite Green in Aquaculture. Crit. Rev. Anal. Chem. 2018, 14, 1–20. [CrossRef]
[PubMed]
240. Adel, M.; Dadar, M.; Oliveri Conti, G. Antibiotics and malachite green in farmed rainbow trout
(Oncorhynchus mykiss) from Iranian markets: A risk assessment. Int. J. Food Prop. 2017, 20, 402–408.
[CrossRef]
241. Sudova, E.; Machova, J.; Svobodova, Z.; Vesely, T. Negative effects of malachite green and possibilities of its
replacement in the treatment of fish eggs and fish: A review. Vet. Med. 2007, 52, 527–539. [CrossRef]
242. Hidayah, N.; Abu Bakar, F.; Mahyudin, N.A.; Faridah, S.; Nur-Azura, M.S.; Zaman, M.Z. Detection of
malachite green and leuco-malachite green in fishery industry. Int. Food Res. J. 2013, 20, 1511–1519.
Foods 2019, 8, 1 56 of 63

243. Xie, J.; Peng, T.; Chen, D.; Zhang, Q.; Wang, G.; Wang, X.; Guo, Q.; Jiang, F.; Chen, D.; Deng, J.
Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC-VIS detection
after immunoaffinity column clean-up. J. Chromatogr. B 2013, 913–914, 123–128. [CrossRef] [PubMed]
244. Chen, G.; Miao, S. HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their
Leuco Metabolite Residues in Channel Catfish Muscle. J. Agric. Food Chem. 2010, 58, 7109–7114. [CrossRef]
[PubMed]
245. Wang, Y.; Liao, K.; Huang, X.; Yuan, D. Simultaneous determination of malachite green, crystal violet
and their leuco-metabolites in aquaculture water samples using monolithic fiber-based solid-phase
microextraction coupled with high performance liquid chromatography. Anal. Methods 2015, 7, 8138.
[CrossRef]
246. Bae Lee, J.; Yun Kim, H.; Mi Jang, Y.; Young Song, J.; Min Woo, S.; Sun Park, M.; Sook Lee, H.; Kyu Lee, S.;
Kim, M. Determination of malachite green and crystal violet in processed fish products. Food Addit. Contam.
Part A 2010, 27, 953–961. [CrossRef]
247. Chengyun, Z.; Jie, W.; Xuefang, D.; Zhimou, G.; Mingyang, L.; Xinmiao, L. Fast analysis of malachite
green, leucomalachite green, crystal violet and leucocrystal violet in fish tissue based on a modified
QuEChERS procedure. Chin. J. Chromatogr. 2014, 4, 419–425.
248. Turnipseed, S.B.; Andersen, W.C.; Roybal, J.E. Determination and Confirmation of Leucomalachite Green in Salmon
using No-Discharge Atmospheric Pressure Chemical Ionization LC-MS. J. AOAC Int. 2005, 88, 1312–1317.
249. Abro, K.; Mahesar, S.A.; Iqbal, S.; Perveen, S. Quantification of malachite green in fish feed utilizing liquid
chromatography-tandem mass spectrometry with a monolithic column. Food Addit. Contam. Part A 2014, 31, 827–832.
[CrossRef] [PubMed]
250. EFSA. Malachite green in food, EFSA Panel on Contaminants in the Food Chain (CONTAM). EFSA J. 2016, 14, 4530.
[CrossRef]
251. Furusawa, N. An isocratic Toxic Chemical-Free Mobile Phase HPLC-PDA analysis of Malachite Green and
Leuco-Malachite Green. Chromatography 2014, 1, 75–81. [CrossRef]
252. Bilandžić, N.; Varenina, I.; Solomun Kolanović, B.; Oraić, D.; Zrnčić, S. Malachite green residues in farmed
fish in Croatia. Food Control 2012, 26, 393–396. [CrossRef]
253. Barani, A.; Tajik, H. Malachite green residue in farmed fish in north-west part of Iran. Int. J. Food Prop.
2017, 20, S580–S585. [CrossRef]
254. Bedale, W.; Sindelar, J.J.; Milkowski, A.L. Dietary nitrate and nitrite: Benefits, risks, and evolving perceptions.
Meat Sci. 2016, 120, 85–92. [CrossRef] [PubMed]
255. Wakida, F.T.; Lerner, D.N. Non-agricultural sources of groundwater nitrate: Review and case study. Water Res.
2005, 39, 3–16. [CrossRef]
256. Deutsche Forschungsgemeinschaft. Nitrate and Nitrite in Diet: An Approach to Assess Benefit and Risk for
Human Health; Institut für Lebensmitteltoxikol: Hannover, Germany, 2014; p. 42.
257. Iammarino, M.; Di Taranto, A.; Cristino, M. Monitoring of nitrites and nitrates levels in leafy vegetables
(spinach and lettuce): A contribution to risk assessment. J. Sci. Food Agric. 2014, 94, 773–778. [CrossRef]
258. Lundberg, J.O.; Gladwin, M.T.; Ahluwalia, A.; Benjamin, N.; Bryan, N.S.; Butler, A.; Cabrales, P.; Fago, A.;
Feelisch, M.; Ford, P.C.; et al. Nitrate and nitrite in biology, nutrition and therapeutics. Nat. Chem. Biol.
2009, 5, 865–869. [CrossRef]
259. Shiva, S. Nitrite: A physiological store of nitric oxide and modulator of mitochondrial function. Redox Biol.
2013, 1, 40–44. [CrossRef]
260. Espejo-Herrera, N.; Gràcia-Lavedan, E.; Boldo, E.; Aragonés, N.; Pérez-Gómez, B.; Pollán, M.; Molina, A.J.;
Fernández, T.; Martín, V.; La Vecchia, C.; et al. Colorectal cancer risk and nitrate exposure through drinking
water and diet. Int. J. Cancer 2016, 139, 334–346. [CrossRef] [PubMed]
261. Ward, M.H. Too Much of Good Thing? Nitrate from Nitrogen Fertilizers and Cancer. Rev. Environ. Health
2009, 24, 357–363. [CrossRef] [PubMed]
262. European Commission. Regulation (EC) No 1881/2006 of 19 December 2006 setting levels for certain
contaminants in foodstuff. Off. J. Eur. Communities 2006, 016.011, 1–35.
263. Brkić, D.; Bošnir, J.; Bevardi, M.; Gross Bošković, A.; Miloš, S.; Lasić, D.; Krivohlavek, A.; Racz, A.;
Mojsović-Ćuić, A.; Uršulin Trstenjak, N. Nitrate in leafy green vegetables and estimated intake. Afr. J.
Tradit. Complement. Altern. Med. 2017, 14, 31–41. [PubMed]
Foods 2019, 8, 1 57 of 63

264. Pardo, O.; Yusà, V.; Villalba, P.; Perez, J.A. Monitoring programme on nitrate in vegetables and
vegetable-based baby foods marketed in the Region of Valencia: Levels and estimated daily intake.
Food Addit. Contam. 2010, 27, 478–486. [CrossRef] [PubMed]
265. Quijano, L.; Yusà, V.; Font, G.; McAllister, C.; Torres, C.; Pardo, O. Risk assessment and monitoring programme
of nitrates through vegetables in the Region of Valencia (Spain). Food Chem. Toxicol. 2017, 100, 42–49. [CrossRef]
[PubMed]
266. Hsu, J.; Arcot, J.; Lee, N.A. Nitrate and nitrite quantification from cured meat and vegetables and their
estimated dietary intake in Australians. Food Chem. 2009, 115, 334–339. [CrossRef]
267. Dumitru Croitoru, M. Nitrite and nitrate can be accurately measured in samples of vegetal and animal origin
using an HPLC-UV/VIS technique. J. Chromatogr. B 2012, 911, 154–161. [CrossRef]
268. Tsikas, D. Analysis of nitrite and nitrate I biological fluids by assays based on the Griess reaction: Appraisal
of the Griess reaction in the L-arginine/nitric oxide area of research. J. Chromatogr. B 2007, 851, 51–70.
[CrossRef]
269. Dumitru Croitoru, M.; Fülöp, I.; Miklos, A.; Hosszú, B.; Tátar, V.; Muntean, D. Presence of nitrate and nitrite
in vegetables grown for self-consumption. Farmacia 2015, 63, 530–533.
270. Yagoub Abdulkair, B.; Elzupir, A.O.; Alamer, A.S. An Ultrasound Assessed Extraction Combined with
Ion-Pair HPLC Method and Risk Assessment of Nitrite and Nitrate in Cured Meat. J. Anal. Methods Chem.
2018, 2018, 1907151. [CrossRef] [PubMed]
271. Chou, S.; Chung, J.; Hwang, D. A High Performance Liquid Chromatography Method for Determining
Nitrate and Nitrite Levels in Vegetables. J. Food Drug Anal. 2003, 11, 233–238.
272. Nemade, K.; Fegade, U.; Ingle, S.; Attarde, S. High Performance Liquid Chromatography Method for Determination
of Nitrite and Nitrate in Vegetable and Water samples. Int. J. Adv. Sci. Tech. Res. 2014, 4, 238–250.
273. Scheeren, M.B.; Arul, J.; Gariépy, C. Comparison of different method for nitrite and nitrate determination
in meat products. In Proceedings of the 59th International Congress of Meat Science and Technology,
Izmir, Turkey, 18–23 August 2013.
274. Dos Santos Baião, D.; Conte-Junior, C.; Flosi Paschoalin, V.; Silveira Alvares, T. Quantitative and
Comparative Contents of Nitrate and Nitrite in Beta vulgaris L. by Reversed. Phase High-Performance
Liquid Chromatography-Fluorescence. Food Anal. Methods 2016, 9, 1002–1008. [CrossRef]
275. Casanova, J.A.; Gross, L.K.; McMullen, S.E.; Schenck, F.J. Use of Greiss reagent containing Vanadium(III) fro
post-column derivatization and simultaneous determination of nitrite and nitrate in baby food. J. AOAC Int.
2006, 89, 447–451. [PubMed]
276. Marcus, Y. Thermodynamics of Solvation of Ions. J. Chem. Soc. Faraday Trans. 1991, 87, 2995–2999. [CrossRef]
277. Oruc, H.H.; Akkoc, A.; Uzunoglu, I.; Kennerman, E. Nitrate Poisoning in Horses Associated with Ingestion
of Forage and Alfalfa. J. Equine Vet. Sci. 2010, 30, 159–162. [CrossRef]
278. Merino, L.; Örnemark, U.; Toldrá, F. Chapter Three—Analysis of Nitrite and Nitrate in Foods: Overview of
Chemical, Regulatory and Analytical Aspects. Adv. Food Nutr. Res. 2017, 81, 65–107. [PubMed]
279. Wang, Q.; Yu, L.; Liu, Y.; Lin, L.; Lu, R.; Zhu, J.; He, L.; Lu, Z. Methods for the detection and determination of
nitrite and nitrate: A review. Talanta 2017, 165, 709–720. [CrossRef]
280. O’Neil, C.A.; Schwartz, S.J. Chromatographic analysis of cis/trans carotenoid isomers. J. Chromatogr.
1992, 624, 235–252. [CrossRef]
281. Wilberg, V.C.; Rodriguez-Amaya, D.B. HPLC Quantitation of Major Carotenoids of Fresh and Processed
Guava, Mango and Papaya. LWT-Food Sci. Technol. 1995, 28, 474–480. [CrossRef]
282. Zanatta, C.F.; Mercadante, A.Z. Carotenoid composition from the Brazilian tropical fruit camu–camu
(Myrciaria dubia). Food Chem. 2007, 101, 1526–1532. [CrossRef]
283. Chen, J.P.; Tai, C.Y.; Chen, B.H. Improved liquid chromatographic method for determination of carotenoids
in Taiwanese mango (Mangifera indica L.). J. Chromatogr. A 2004, 29, 261–268. [CrossRef]
284. Inbaraj, B.S.; Chien, J.T.; Chen, B.H. Improved High Performance Liquid Chromatographic Method for
Determination of Carotenoids in the Microalga Chlorella pyrenoidosa. J. Chromatogr. A 1102, 1102, 193–199.
[CrossRef] [PubMed]
285. McGraw, K.J.; Toomey, M.B. Carotenoid accumulation in the tissues of zebra finches: Predictors of
integumentary pigmentation and implications for carotenoid allocation strategies. Physiol. Biochem. Zool.
2010, 83, 97–109. [CrossRef] [PubMed]
Foods 2019, 8, 1 58 of 63

286. Aluç, Y.; Kankılıç, G.B.; Tüzün, I. Determination of carotenoids in two algae species from the saline water of
Kapulukaya reservoir by HPLC. J. Liquid Chromatogr. Relat. Technol. 2018, 41, 1–8. [CrossRef]
287. Shih-Chuan, L.; Jau-Tien, L.; Deng-Jye, Y. Determination of cis- and trans- α- and β-carotenoids in Taiwanese
sweet potatoes (Ipomoea batatas (L.) Lam.) harvested at various times. Food Chem. 2009, 116, 605–610.
288. Van Jaarsveld, P.J.; Marais, D.W.; Harmse, E.; Nestel, P.; Rodriguez-Amaya, D.B. Retention of β-carotene in
boiled, mashed orange-fleshed sweet potato. J. Food Compos. Anal. 2006, 19, 321–329. [CrossRef]
289. Huck, C.; Popp, M.; Scherz, H.; Bonn, G.K. Development and Evaluation of a New Method for
the Determination of the Carotenoid Content in Selected Vegetables by HPLC and HPLC–MS–MS.
J. Chromatogr. Sci. 2000, 38, 441–449. [CrossRef]
290. Lessin, W.J.; Catigani, G.L.; Schwartz, S.J. Quantification of cis-trans Isomers of Provitamin A Carotenoids in
Fresh and Processed Fruits and Vegetables. J. Agric. Food Chem. 1997, 45, 3728–3732. [CrossRef]
291. Gayosso-García, L.E.; Yahia, E.M.; González-Aguila, G.A. Identification and quantification of phenols,
carotenoids, and vitamin C from papaya (Carica papaya L., cv. Maradol) fruit determined by
HPLC-DAD-MS/MS-ESI. Food Res. Int. 2011, 44, 1284–1291. [CrossRef]
292. Schweiggert RFSteingass, C.B.; Esquivel, P.; Carle, R. Chemical and Morphological Characterization
of Costa Rican Papaya (Carica papaya L.) Hybrids and Lines with Particular Focus on Their Genuine
Carotenoid Profiles. J. Agric. Food Chem. 2012, 60, 2577–2585.
293. Chacón-Ordóñez, T.; Schweiggert, R.M.; Bosy-Westphal, A.; Jiménez, V.M.; Carle, R.; Esquivel, P.
Carotenoids and carotenoid esters of orange- and yellow-fleshed mamey sapote (Pouteria sapota (Jacq.)
H.E. Moore & Stearn) fruit and their postprandial absorption in humans. Food Chem. 2017, 221, 673–682.
[PubMed]
294. Rojas-Garbanzo, C.; Gleichenhagen, M.; Heller, A.; Esquivel, P.; Schulze-Kaysers, N.; Schieber, A.
Carotenoid profile, antioxidant capacity, and chromoplasts of pink guava (Psidium guajava L. Cv. ‘Criolla’)
during fruit ripening. J. Agric. Food Chem. 2017, 65, 3737–3747. [CrossRef] [PubMed]
295. Irias-Mata, A.; Jiménez, V.M.; Steingass, C.B.; Schweiggert, R.M.; Carle, R.; Esquivel, P. Carotenoids
and xanthophyll esters of yellow and red nance fruits (Byrsonima crassifolia (L.) Kunth) from Costa Rica.
Food Res. Int. 2018, 111, 708–714. [CrossRef] [PubMed]
296. Marutti, L.R.B.; Mercadante, A.Z. Carotenoid esters analysis and occurrence: What do we know so far?
Arch. Biochem. Biophys. 2018, 648, 36–43. [CrossRef] [PubMed]
297. Wen, X.; Hempel, J.; Schweiggert, R.M.; Ni, Y.; Carle, R. Carotenoids and carotenoid esters of red and yellow
Physalis (Physalis alkekengi L. and P. pubescens L.) fruits and calyces. J. Agric. Food Chem. 2017, 65, 6140–6151.
[CrossRef] [PubMed]
298. De Rosso, V.; Mercadante, A. Identification and Quantification of Carotenoids, by HPLC-PDA-MS/MS,
from Amazonian Fruits. J. Agric. Food Chem. 2007, 55, 5062–5072. [CrossRef]
299. Ligor, M.; Kováčová, J.; Gadzała-Kopciuch, R.; Studzińska, S.; Bocian, S.; Lehotay, J.; Buszewski, B. Study of
RP HPLC Retention Behaviours in Analysis of Carotenoids. Chromatographia 2014, 77, 1047–1057. [CrossRef]
300. Schex, R.; Lieb, V.; Jiménez, V.M.; Esquivel, P.; Schweiggert, R.; Carle, R.; Steingrass, C.B.
HPLC-DAD-APCI/ESI-MSn analysis of carotenoids and α-tocopherol in Costa Rican Acrocomia aculeata
fruits of varying maturity stages. Food Res. Int. 2018, 105, 645–653. [CrossRef]
301. Schweiggert, R.M.; Vargas, E.; Conrad, J.; Hempel, J.; Gras, C.C.; Ziegler, J.U.; Mayer, A.; Jiménez, V.;
Esquivel, P.; Carle, R. Carotenoids, carotenoid esters, and anthocyanins of yellow-, orange-, and red-peeled
cashew apples (Anacardium occidentale L.). Food Chem. 2016, 200, 274–282. [CrossRef] [PubMed]
302. Chacón-Ordoñez, T.; Esquivel, P.; Jiménez, V.M.; Carle, R.; Schweiggert, R.M. Deposition Form
and Bioaccessibility of Keto-Carotenoids from Mamey Sapote (Pouteria sapota), Red Bell Pepper
(Capsicum annuum), and Sockeye Salmon (Oncorhynchus nerka) Filet. J. Agric. Food Chem. 2016, 64, 1989–1998.
[CrossRef] [PubMed]
303. Dolan, J.W. How Much Can I Inject? Part II: Injecting in Solvents Other than Mobile Phase. LCGC N. Am.
2014, 32, 854–859.
304. Pereira da Costa, M.; Conte-Junior, C.A. Chromatographic methods for the determination of carbohydrates
and organic acids in foods of animal origin. Compr. Rev. Food Sci. Food Saf. 2015, 14, 586–600. [CrossRef]
305. De Goeij, S. Quantitative Analysis Methods for Sugars. Master’s Thesis, Universiteit van Amsterdam,
Amsterdam, The Netherlands, August 2013.
Foods 2019, 8, 1 59 of 63

306. Agius, C.; von Tucher, S.; Poppenberger, B.; Rozhon, W. Quantification of sugars and organic acids in
tomato fruits. MethodsX 2018, 5, 2537–2550. [CrossRef] [PubMed]
307. Xu, W.; Liang, L.; Zhu, M. Determination of Sugars in Molasses by HPLC Following Solid-Phase Extraction.
Int. J. Food Prop. 2015, 18, 547–557. [CrossRef]
308. Koh, D.-W.; Park, J.-W.; Lim, J.-H.; Yea, M.-J.; Bang, D.-Y. A rapid method for simultaneous quantification of
13 sugars and sugar alcohols in food products by UPLC-ELSD. Food Chem. 2018, 240, 694–700. [CrossRef]
309. Zielinkski, A.A.F.; Braga, C.M.; Demiate, I.M.; Beltrame, F.L.; Nogueira, A.; Wosiaki, G. Development and
optimization of a HPLC-RI method for the determination of major sugars in apple juice and evaluation of
the effect of the ripening stage. Food Sci. Technol. 2014, 34, 38–43. [CrossRef]
310. Duarte-Delgado, D.; Narváez-Cuenca, C.E.; Restrepo-Sánchez, L.P.; Kushalappa, A.; Mosquera-Vásquez, T.
Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose
in tubers of Solanum tuberosum Group Phureja. J. Chromatogr. B 2015, 975, 18–23. [CrossRef]
311. Shindo, T.; Sadamasu, Y.; Suzuki, K.; Tanaka, Y.; Togawa, A.; Uematsu, Y. Method of quantitative analysis by
HPLC and confirmation by LC-MS of sugar alcohols in foods. Shokuhin Eiseigaku Zasshi 2013, 54, 358–363.
[CrossRef]
312. Canesin, R.C.F.S.; Isique, W.D.; Buzetti, S.; Aparecida de Souza, J. Derivation method for determining sorbitol
in fruit trees. Am. J. Plant Sci. 2014, 5, 3457–3463. [CrossRef]
313. Hung, W.-T.; Chen, Y.-T.; Wang, S.-H.; Liu, Y.-C.; Yang, W.-B. A new method for aldo-sugar analysis in
beverages and dietary foods. Funct. Food Health Dis. 2016, 6, 234–245.
314. Valliydan, B.; Shi, H.; Nguyen, H.T. A simple analytical method for high-throughput screening of major
sugars from soybean by normal-phase hplc with evaporative light scattering detection. Chromatogr. Res. Int.
2015, 2015, 757649. [CrossRef]
315. Wieß, K.; Alt, M. Determination of single sugars, including inulin, in plants and feed materials by
high-performance liquid chromatography and refraction index detection. Fermentation 2017, 3, 36.
316. Verspreet, J.; Pollet, A.; Cuyvers, S.; Vergauwen, R.; Van den Ende, W.; Delcour, J.A.; Courtin, C.M. A simple
and accurate method for determining wheat grain fructan content and average degree of polymerization.
J. Agric. Food Chem. 2012, 60, 2102–2107. [CrossRef] [PubMed]
317. Correia, D.M.; Dias, L.G.; Veloso, A.C.A.; Dias, T.; Rocha, I.; Rodrigues, L.R.; Peres, A.M. Dietary sugars
analysis: Quantification of fructooligosaccharides during fermentation by HPLC-RI method. Front. Nutr.
2014, 1, 11. [CrossRef]
318. Salazar Murillo, M.M.; Granados-Chinchilla, F. Total starch in animal feeds and silages based on the
chromatographic determination of glucose. MethodsX 2018, 5, 83–89. [CrossRef]
319. Bai, W.; Fang, X.; Zhao, W.; Huang, S.; Zhang, H.; Qian, M. Determination of oligosaccharides and
monosaccharides in Hakka rice wine by precolumn derivation high-performance liquid chromatography.
J. Food Drug Anal. 2015, 23, 645–651. [CrossRef]
320. Madhuri, K.V.; Prabhakar, K.V. Recent trends in the characterization of microbial exopolysaccharides.
Orient. J. Chem. 2014, 30, 895–904. [CrossRef]
321. Corradini, C.; Cavazza, A.; Bignardi, C. High-performance anion-exchange chromatography coupled
with pulsed electrochemical detection as a powerful tool to evaluate carbohydrates of food interest:
Principles and applications. Int. J. Carbohydr. Chem. 2012, 2012, 487564. [CrossRef]
322. Dvořáčková, E.; Šnóblová, M.; Hrdlička, P. Carbohydrate analysis: From sample preparation to HPLC on
different stationary phases coupled with evaporative light-scattering detection. J. Sep. Sci. 2014, 37, 323–337.
[CrossRef] [PubMed]
323. Rippe, J.M.; Angelopoulos, T.J. Added sugars and risk factors for obesity, diabetes and heart disease.
Int. J. Obes. 2016, 40, S22–S27. [CrossRef] [PubMed]
324. Louie, J.C.Y.; Moshtaghian, H.; Boylan, S.; Flood, V.M.; Rangan, A.M.; Barclay, A.W.; Brand-Miller, J.C.; Gill, T.P. A
systematic methodology to estimate added sugar content of foods. Eur. J. Clin. Nutr. 2015, 69, 154–161. [CrossRef]
[PubMed]
325. Uçar, G.; Balaban, M. Hydrolysis of polysaccharides with 77% sulfuric acid for quantitative saccharification.
Turk. J. Agric. For. 2003, 27, 361–365.
326. Yan, X. High performance liquid chromatography for carbohydrate analysis. In High-Performance Liquid
Chromatography (HPLC): Principles, Practices and Procedures; Zhuo, Y., Ed.; Nova Science Publishers:
Hauppauge, NY, USA, 2014; ISBN 978-1-62948-854-7.
Foods 2019, 8, 1 60 of 63

327. Thøgersen, R.; Castro-Mejía, J.L.; Sundekilde, U.K.; Hansen, L.H.; Hansen, A.K.; Nielsen, D.S.; Bertram, H.C.
Ingestion of an inulin-enriched pork sausage product positively modulates the gut microbiome and
metabolome of healthy rats. Mol. Nutr. Food Res. 2018, 13, e1800608. [CrossRef]
328. Szpylka, J.; Thiex, N.; Acevedo, B.; Albizu, A.; Angrish, P.; Austin, S.; Bach Knudsen, K.E.; Barber, C.A.;
Berg, D.; Bhandari, S.D.; et al. Standard Method Performance Requirements (SMPRs® ) 2018.002: Fructans
in Animal Food (Animal Feed, Pet Food, and Ingredients). J. AOAC Int. 2018, 101, 1283–1284. [CrossRef]
[PubMed]
329. Longland, A.C.; Byrd, B.M. Pasture nonstructural carbohydrates and equine laminitis. J. Nut. 2006, 136, 2099S–2102S.
[CrossRef]
330. Stöber, P.; Bénet, S.; Hischenhuber, C. Simplified Enzymatic High-Performance Anion Exchange
Chromatographic Determination of Total Fructans in Food and Pet Foods-Limitations and Measurement
Uncertainty. J. Agric. Food Chem. 2004, 52, 2137–2146. [CrossRef]
331. Rühmann, B.; Schmid, J.; Sieber, V. High throughput exopolysaccharide screening platform: From strain
cultivation to monosaccharide composition and carbohydrate fingerprinting in one day. Carbohydr. Polym.
2015, 122, 212–220. [CrossRef]
332. Affonso, R.C.L.; Voytena, A.P.L.; Fanan, S.; Pitz, H.; Coelho, D.S.; Hortmann, A.L.; Pereira, A.; Uarrota, V.G.;
Hillmann, M.C.; Varela, L.A.C.; et al. Phytochemical composition, antioxidant activity, and the effect of the
aqueous extract of coffee (Coffea arabica L.) bean residual press cake on the skin wound healing. Oxidative Med.
Cell. Longev. 2016, 2016, 1923754. [CrossRef]
333. Shao, J.; Cao, W.; Qu, H.; Pan, J.; Gong, X. A novel quality by design approach for developing an HPLC
method to analyze herbal extracts: A case study of sugar content analysis. PLoS ONE 2018, 13, e019515.
[CrossRef] [PubMed]
334. Swetwiwathana, A.; Visessanguan, W. Potential of bacteriocin-producing lactic acid bacteria for safety
improvements of traditional Thai fermented meat and human health. Meat Sci. 2015, 109, 101–105. [CrossRef]
[PubMed]
335. Gurtler, J.B.; Mai, T.L. Traditional preservatives: Organic acids. Encycl. Food Microbiol. 2014, 3, 119–130.
336. Anyasi, T.A.; Jideani, A.I.O.; Edokpayi, J.N.; Anokwuru, C.P. Application of Organic Acids in Food
Preservation. In Organic Acids, Characteristics, Properties and Synthesis; Vargas, C., Ed.; Nova Sciences
Publishers, Inc.: New York, NY, USA, 2016; pp. 1–47. ISBN 9781634859523.
337. Neffe-Skocińska, K.; Sionek, B.; Ścibisz, I.; Kotoźyn-Krajewska, D. Acid contents and the effect of
fermentation condition of Kombucha tea beverages on physicochemical, microbiological and sensory
properties. CyTA-J. Food 2017, 15. [CrossRef]
338. Nour, V.; Trandafir, I.; Ionica, M.E. HPLC Organic Acid Analysis in Different Citrus Juices under Reversed
Phase Conditions. Not. Bot. Hort. Agrobot. Cluj 2010, 38, 44–48.
339. Lobo Roriz, C.; Barros, L.; Carvalho, A.M.; Ferreira, I.C.F.R. HPLC-Profiles of Tocopherols, Sugars, and
Organic Acids in Three Medicinal Plants Consumed as Infusions. Int. J. Food Sci. 2014, 2014, 241481.
[CrossRef]
340. Scherer, R.; Rybka, A.C.P.; Ballus, C.A.; Dillenburg Meinhart, A.; Texeira Filho, J.; Texeira Godoy, H. Validation
of HPLC method for simultaneous determination of main organic acids in fruits and juices. Food Chem.
2012, 135, 150–154. [CrossRef]
341. Llano, T.; Quijorna, N.; Andrés, A.; Coz, A. Sugar, acid and furfural quantification in a sulphite pulp mill:
Feedstock, product and hydrolysate analysis by HPLC/RID. Biotechnol. Rep. 2017, 15, 75–83. [CrossRef]
342. Jain, I.; Kumar, V.; Satyanarayana, T. Xylooligosaccharides: An economical prebiotic from agroresidues and
their health benefits. Indian J. Exp. Biol. 2015, 53, 131–142.
343. Saleh Zaky, A.; Pensupa, N.; Andrade-Eiroa, Á.; Tucker, G.A.; Du, C. A new HPLC method for simultaneously
measuring chloride, sugars, organic acids and alcohols in food samples. J. Food Compos. Anal. 2017, 56, 25–33.
[CrossRef]
344. Ergönül, P.G.; Nergiz, C. Determination of Organic Acids in Olive Fruit by HPLC. Czech J. Food Sci. 2010, 28, 202–205.
[CrossRef]
345. Tašev, K.; Stefova, M.; Ivanova-Petropulos, V. HPLC method validation and application for organic acid
analysis in wine after solid-phase extraction. Maced. J. Chem. Chem. Eng. 2016, 35. [CrossRef]
346. Mihaljević Žulj, M.; Puhelek, I.; Jagatić Korenika, A.M.; Maslov Bandić, L.; Pavlešić, T.; Jeromel, A. Organic
Acid Composition in Croatian Predicate Wines. Agric. Conspec. Sci. 2015, 80, 113–117.
Foods 2019, 8, 1 61 of 63

347. Sánchez-Machado, D.I.; López-Cervantes, J.; Martínez-Cruz, O. Quantification of Organic Acids in Fermented
Shrimp Waste by HPLC. Food Technol. Biotechnol. 2008, 46, 456–460.
348. Ke, W.C.; Ding, W.R.; Xu, D.M.; Ding, L.M.; Zhang, P.; Li, F.D.; Guo, X.S. Effects of addition of malic or
citric acids on fermentation quality and chemical characteristics of alfalfa silage. J. Dairy Sci. 2017, 100, 1–9.
[CrossRef] [PubMed]
349. Fasciano, J.M.; Mansour, F.R.; Danielson, N.D. Ion-Exclusion High-Performance Liquid Chromatography
of Aliphatic Organic Acids Using a Surfactant-Modified C18 Column. J. Chromatogr. Sci. 2016, 54, 958–970.
[CrossRef]
350. Wang, Y.; Wang, J.; Cheng, W.; Zhao, Z.; Cao, J. HPLC method for the simultaneous quantification of the
major organic acids in Angeleno plum fruit. IOP Conf. Ser. Mater. Sci. Eng. 2014, 62, 012035. [CrossRef]
351. Coblentz, W.K.; Akins, M.S. Silage review: Recent advances and future technologies for baled silages.
J. Dairy Sci. 2018, 101, 4075–4092. [CrossRef] [PubMed]
352. Khan, N.A.; Yu, P.; Ali, M.; Cone, J.W.; Hendriks, W.H. Nutritive value of maize silage in relation to dairy
cow performance and milk quality. J. Sci. Food Agric. 2015, 95, 238–258. [CrossRef]
353. Silva, V.P.; Pereira, O.G.; Leandro, E.S.; Da Silva, K.G.; Ribeiro, K.G.; Mantovani, H.C.; Santos, S.A. Effects of
lactic acid bacteria with bacteriocinogenic potential on the fermentation profile and chemical composition of
alfalfa silage in tropical conditions. J. Dairy Sci. 2016, 99, 1–8. [CrossRef] [PubMed]
354. Kim, H.J.; Lee, M.J.; Kim, H.J.; Cho, S.K. Development of HPLC-UV method for detection and quantification
of eight organic acids in animal feed. J. Chromatogr. Sep. Tech. 2017, 8, 385. [CrossRef]
355. Grune, T.; Lietz, G.; Palou, A.; Ross, A.C.; Stahl, W.; Tang, G.; Thurnham, D.; Yin, S.A.; Biesalski, H.K.
β-Carotene Is an Important Vitamin A Source for Humans. J. Nutr. 2010, 140, 2268S–2285S. [CrossRef]
[PubMed]
356. Gilbert, C. What is vitamin A and why do we need it? Community Eye Health 2013, 26, 65. [PubMed]
357. Ding, Z.; Saldeen, T.G.P.; Mathur, P.; Mehta, J.L. Mixed tocopherols are better than alpha-tocopherol as
antioxidant as good as statins. Curr. Res. Cardiol. 2016, 3, 128–129. [CrossRef]
358. Alshahrani, F.; Aljohani, N. Vitamin D: Deficiency, Sufficiency and Toxicity. Nutrients 2013, 5, 3605–3616.
[CrossRef] [PubMed]
Foods 2019, 8, 1 62 of 63

359. Ortiz-Boyer, F.; Fernandez-Romero, J.M.; Luque de Castro, M.D.; Quesada, J.M. Determination of
vitamins D2 , D3 , K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous
clean-up–preconcentration procedure coupled on-line with liquid chromatography–UV detection. Analyst
1999, 124, 401–406. [CrossRef] [PubMed]
360. Schwalfenberg, G.K. Vitamins K1 and K2: The emerging group of vitamins required for human health.
J. Nutr. Metab. 2017, 345, 229–234. [CrossRef] [PubMed]
361. Harshman, S.G.; Finnan, E.G.; Barger, K.J.; Bailey, R.L.; Haytowitz, D.B.; Gilhooly, C.H.; Booth, S.L.
Vegetables and Mixed Dishes Are Top Contributors to Phylloquinone Intake in US Adults: Data from
the 2011–2012 NHANES. J. Nutr. 2017, 96, 149–154. [CrossRef]
362. Beulens, J.; Booth, S.; van den Heuvel, E.; Stoecklin, E.; Baka, A.; Vermeer, C. The role of menaquinones
(vitamin K2) in human health. Br. J. Nutr. 2013, 110, 1357–1368. [CrossRef]
363. Chavez-Servín, J.; Castellote, A.; Lopez-Sabater, M.C. Simultaneous analysis of Vitamins A and E in infant
milk-based formulae by normal-phase high-performance liquid chromatography-diode array detection
using a short narrow-bore column. J. Chromatogr. A 2003, 1122, 138–143. [CrossRef]
364. Płonka, J.; Toczek, A.; Tomczyk, V. Multivitamin Analysis of Fruits, Fruit–Vegetable Juices,
and Diet Supplements. Food Anal. Methods 2012, 5, 1167–1176. [CrossRef]
365. Sami, R.; Li, Y.; Qi, B.; Wang, S.; Zhang, Q.; Han, F.; Ma, Y.; Jing, J.; Jiang, L. HPLC Analysis of
Water-Soluble Vitamins (B2, B3, B6, B12, and C) and Fat-Soluble Vitamins (E, K, D, A, and β-Carotene) of
Okra (Abelmoschus esculentus). J. Chem. 2014, 2014, 831357. [CrossRef]
366. Xue, X.; You, J.; He, P. Simultaneous Determination of Five Fat-Soluble Vitamins in Feed by High-Performance
Liquid Chromatography Following Solid-Phase Extraction. J. Chromatogr. Sci. 2012, 46, 345–350. [CrossRef]
367. Qian, H.; Sheng, M. Simultaneous determination of fat-soluble vitamins A, D and E and pro-vitamin D2 in
animal feeds by one-step extraction and high-performance liquid chromatography analysis. J. Chromatogr. A
1998, 825, 127–133. [CrossRef]
368. Lee, H.; Kwak, B.; Ahn, J.; Jeong, S.; Shim, S.; Kim, K.; Yoon, T.; Leem, D.; Jeong, J. Simultaneous
Determination of Vitamin A and E in Infant Formula by HPLC with Photodiode Array Detection. Korean J.
Food Sci. Anim. Resour. 2011, 31, 191–199. [CrossRef]
369. Lim, H.; Woo, S.; Sig Kim, H.; Jong, S.; Lee, J. Comparison of extraction methods for determining tocopherols
in soybeans. Eur. J. Lipid Sci. Technol. 2007, 109, 1124–1127. [CrossRef]
370. Odes, S.; Hisil, Y. Analysis of vitamin A in eggs by high pressure liquid chromatography. Die Nahr. 1991, 35, 391–394.
371. Reynolds, S.; Judd, H. Rapid procedure for the determination of vitamins A and D in fortified skimmed milk
powder using high-performance liquid chromatography. Analyst 1989, 109, 489–492. [CrossRef]
372. Turner, C.; Persson, M.; Mathiasson, L.; Adlercreutz, P.; King, J.W. Lipase-catalyzed reactions in organic and
supercritical solvents: Application to fat-soluble vitamin determination in milk powder and infant formula.
Enzym. Microb. Technol. 2001, 29, 111–121. [CrossRef]
373. Jedlička, A.; Klimeš, J. Determination of Water- and Fat-Soluble Vitamins in Different Matrices Using
High-Performance Liquid Chromatography. Chem. Papers 2005, 59, 202–222. [CrossRef]
374. Rupérez, F.J.; Martín, D.; Herrera, E.; Barbas, C. Chromatographic analysis of alpha-tocopherol and related
compounds in various matrices. J. Chromatogr. A 2001, 935, 45–69. [CrossRef]
375. Stefanov, I.; Vlaeminck, B.; Fievez, V. A novel procedure for routine milk fat extraction based on
dichloromethane. J. Food Compos. Anal. 2010, 23, 852–855. [CrossRef]
376. Hung, G.W.C. Determination of Vitamins D2 and D3 in Feedingstuffs by High Performance Liquid
Chromatography. J. Liquid Chromatogr. 1988, 11, 953–969. [CrossRef]
377. Lee, S.; Morrisa, Y.; Grossa, L.; Portera, F.; Ortiz-Colona, F.; Farrowa, J.; Waqara, A.; Phifera, E.; Kerdahia, K.
Development and Single-Lab Validation of an UHPLC-APCI-MS/MS Method for Vitamin K1 in Infant
Formulas and Other Nutritional Formulas. J. Regul. Sci. 2015, 2, 27–35.
378. Wagner, D.; Rousseau, D.; Sidhom, G.; Pouliot MAudet, T.; Vieth, R. Vitamin D3 Fortification, Quantification,
and Long-Term Stability in Cheddar and Low-Fat Cheeses. J. Agric. Food Chem. 2008, 56, 7964–7969.
[CrossRef]
379. Kienen, V.; Costa, W.; Visentainer, J.; Souza, N.; Oliveira, C. Development of a green chromatographic method for
determination of fat-soluble vitamins in food and pharmaceutical supplement. Talanta 2008, 75, 141–146. [CrossRef]
[PubMed]
Foods 2019, 8, 1 63 of 63

380. Gomis, D.B.; Fernández, M.P.; Gutiérrez, M.D. Simultaneous determination of fat-soluble vitamins and
provitamins in milk by microcolumn liquid chromatography. J. Chromatogr. A 2000, 891, 109–114. [CrossRef]
381. Koivu, T.; Piironen, V.; Henttonen, S.; Mattila, P. Determination of Phylloquinone in Vegetables, Fruits, and
Berries by High-Performance Liquid Chromatography with Electrochemical Detection. J. Chromatogr. A
1997, 45, 4644–4649. [CrossRef]
382. Shammugasamy, B.; Ramakrishnan, Y.; Ghazali, H.; Muhammad, K. Tocopherol and tocotrienol contents of
different varieties of rice in Malaysia. J. Sci. Food Agric. 2015, 95, 672–678. [CrossRef]
383. Kim, H.O. Development of an ion-pairing reagent and HPLC-UV method for the detection and quantification
of six water-soluble vitamins in animal feed. Int. J. Anal. Chem. 2016, 2016, 8357358. [CrossRef]
384. Midttun, Ø.; McCann, A.; Aarseth, O.; Kokeide, M.; Kvalheim, G.; Meyer, K.; Ueland, P.M. Combined
measurement of 6 fat-soluble vitamins and 26 water-soluble functional vitamin markers and amino acids
in 50 µL of serum or plasma by high-throughput mass spectrometry. Anal. Chem. 2016, 88, 10427–10436.
[CrossRef] [PubMed]
385. Kakitani, A.; Inoue, T.; Matsumoto, K.; Watanabe, J.; Nagatomi, Y.; Mochizuki, N. Simultaneous
determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS. Food Addit.
Contam. Part A 2014, 31, 1939–1948. [CrossRef] [PubMed]
386. Zhang, Y.; Zhou, W.-E.; Yan, J.-Q.; Liu, M.; Zhou, Y.; Shen, X.; Ma, Y.-L.; Feng, X.-S.; Yang, J.; Li, G.-H. A
Review of the Extraction and Determination Methods of Thirteen Essential Vitamins to the Human Body:
An Update from 2010. Molecules 2018, 23, 1484. [CrossRef]
387. Abano, E.E.; Dadzie, R.G. Simultaneous detection of water-soluble vitamins using the High Performance
Liquid Chromatography (HPLC)—A review. Croat. J. Food Sci. Technol. 2014, 6, 116–123. [CrossRef]

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