Accepted Manuscript

Title: RECENT ADVANCES IN SAMPLE PREPARATION

TECHNIQUES AND METHODS OF SULFONAMIDES
DETECTION - A REVIEW

Author: Stanislava G. Dmitrienko Elena V. Kochuk Vladimir

V. Apyari Veronika V. Tolmacheva Yury A. Zolotov

PII: S0003-2670(14)01006-X
DOI: http://dx.doi.org/doi:10.1016/j.aca.2014.08.023
Reference: ACA 233423

To appear in: Analytica Chimica Acta

Received date: 25-3-2014

Revised date: 7-8-2014
Accepted date: 11-8-2014

Please cite this article as: Stanislava G.Dmitrienko, Elena V.Kochuk, Vladimir
V.Apyari, Veronika V.Tolmacheva, Yury A.Zolotov, RECENT ADVANCES
IN SAMPLE PREPARATION TECHNIQUES AND METHODS OF
SULFONAMIDES DETECTION - A REVIEW, Analytica Chimica Acta
http://dx.doi.org/10.1016/j.aca.2014.08.023

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RECENT ADVANCES IN SAMPLE PREPARATION TECHNIQUES AND
METHODS OF SULFONAMIDES DETECTION - A REVIEW

Stanislava G. Dmitrienko*, Elena V. Kochuk, Vladimir V. Apyari, Veronika V.

Tolmacheva, Yury A. Zolotov

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Lomonosov Moscow State University, Chemistry Department, 119991 Leninskie gory, 1/3,

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Moscow, Russia

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* Corresponding author. Tel.: +7(495)939-46-08; Fax: +7(495)939-46-75; e-mail: <ABS-Highlights ►

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HEAD>dmitrienko@analyt.chem.msu.ru

Graphical abstract

Highlights
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 An overview on recent trends in the sample preparation and determination of
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sulfonamides is given
 A comparison of different methods of real samples preparation is made
 The general chromatographic and other methods of SAs determination are discussed
Examples of SAs determination in different matrices are given.
d


e

ABSTRACT
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Sulfonamides (SAs) have been the most widely used antimicrobial drugs for more than 70 years,
and their residues in foodstuffs and environmental samples pose serious health hazards. For this
reason, sensitive and specific methods for the quantification of these compounds in numerous
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matrices have been developed. This review intends to provide an updated overview of the recent
trends over the past five years in sample preparation techniques and methods for detecting SAs.
Examples of the sample preparation techniques, including liquid-liquid and solid-phase
extraction, dispersive liquid-liquid microextraction and QuEChERS, are given. Different
Ac

methods of detecting the SAs present in food and feed and in environmental, pharmaceutical and
biological samples are discussed.

Keywords: Sulfonamides, Sample preparation, Extraction, Residue determination, Multi-class methods,

Liquid chromatography–tandem mass spectrometry
1

1
Abbreviations: Ac, Acetate; AD, Amperometric detection; ACN, Acetonitrile; CE, Capillary
electrophoresis; DAD, Diode-array detector; DLLME, Dispersive liquid-liquid microextraction; DSPE,
Dispersive solid-phase extraction; EDTA, Ethylenediaminetetraacetic acid; ELISA, Enzyme-linked
immunosorbent assays; EMIS, Electrochemical magnetoimmunosensor; ESI, Electrospray ionization
source; EtOH, Ethanol; F, Formate; FL, Fluorescence detection; HPLC, High-performance liquid
chromatography; HPLC-MS/MS, High-performance liquid chromatography tandem mass spectrometry;
LLE, Liquid–liquid extraction; LLME, Liquid-liquid microextraction; LOD, Limit of detection; LOQ,
Limit of quantification; MeOH, Methanol; MIPs, Molecular imprinted polymers; MRLs, Maximum

Page 1 of 76
Contents
1. Intrroduction
2. Sample preparation
2.1. Food
2.2. Feed
2.3. Environmental samples
2.3.1. Waters
2.3.2. Soils, manure and sediments
2.4. Pharmaceutical and clinical samples
3. Analytical methods
3.1. Chromatographic methods

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3.1.1. Detection with MS and multi-class analysis

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3.1.2. Detection with other techniques
3.2. Electrophoretic methods

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3.3. Microbiological assays
3.4. Immunoassays
3.5. Biosensors

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3.6. Other methods
4. Conclusions and outlook
Refferences

1. Introduction
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The problem of food and environmental sample contamination by veterinary drugs

is of great concern [1 – 3]. There are systematised data on detection of antibiotics in

d

foods of animal origin [4, 5], milk [6], honey [7], fish [8], and feed [9], as well as in
e

environmental samples [10]. According to a previous review [5], sulfonamides (SAs) and
pt

fluoroquinolones are among the most commonly used veterinary antibiotics. For
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example, the frequency of cases in which SAs were detected in food is 20 %, whereas

other antibiotics were detected as follows: fluoroquinolones – 19 %, aminoglycosides –

Ac

residue limits; MRM, Multiple reaction monitoring; MS, Mass spectrometry; MS/MS, Tandem mass
spectrometry; MSPD, Matrix solid-phase dispersion; MWCNTs, multi-walled carbon nanotubes; PLE,
Pressurised liquid extraction; PSA, Primary-secondary amine; QqLIT, Quadrupole linear ion-trap; QTOF-
MS, Quadrupole time of flight mass spectrometry; SAA, Sulfacetamide; SAM, Sulfanilamide; SAs,
Sulfonamids; SCP, Sulfachloropyridazine; SDD, Sulfadimidine; SDM, Sulfadimethoxine; SDO,
Sulfadoxine; SDZ, Sulfadiazine; SML, Sulfametrole; SMP, Sulfamethoxypyridazine; SMR,
Sulfamerazine; SMT, Sulfamethizole; SMX, Sulfamethoxazole; SMZ, Sulfamethazine; SPE, Solid-phase
extraction; SPME, Solid-phase microextraction; SPY, Sulfapyridine; SQX, Sulfaquinoxaline; SSA,
Sulfisoxazole; SSZ, Sulfasalazine; STZ, Sulfathiazole; ToF-MS, Time-of-flight mass spectrometry;
UHPLC-MS/MS, Ultra-high-performance liquid chromatography tandem mass spectrometry; UV,
Ultraviolet detection.

Page 2 of 76
15 %, phenicols–15 %, β-lactams – 15 %, oxazolidinones – 8 % and tetracyclines – 8 %

[5].

SAs are derivatives of sulfanilic acid (p-aminobenzenesulfonic acid) and are one

of the oldest classes of antimicrobial drugs, which have been used for the treatment of

humans and animals from the middle of the twentieth century. SAs act as bacteriostatic

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agents and possess chemotherapeutic activity against infections caused by gram-positive

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and gram-negative bacteria and some protozoa (causative agents of malaria,

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toxoplasmosis, etc.).

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The precursor of SAs is sulfanilamide (p-aminobenzenesulfonamide), better

known as streptocid, which was first synthesised in 1908 and was widely used as an
an
intermediate in the production of dyes. SAs were discovered to have antibacterial

properties in 1935 by G. Domagk. The basis for their bacteriostatic action is the structural
M
similarity between the sulfanilamide moiety and p-aminobenzoic acid (PABA), which is
d

involved in the biosynthesis of dihydrofolic and folic acids and other substances utilised
e

by the microorganisms. SAs act as antimicrobials by blocking the synthesis of

pt

dihydrofolic acid by preventing the formation of dihydropteroic acid from

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dihydropteridine and PABA, which involves dihydropteroate synthetase. SAs compete

with PABA, resulting in no formation of dihydrofolic acid but instead the formation of its
Ac

analogue [11]. These features of the mechanism confer high sensitivity to SAs only to

microorganisms that synthesise their own dihydrofolic acid, and microorganisms and

cells that utilise dihydrofolic acid as a finished product are not sensitive to SAs.

To date, more than 10000 sulfanilamide derivatives have been synthesised, and 40

of these are applied in medical and veterinary practices. The structures of the most

commonly used SAs are represented in Fig. 1. Some patterns that illustrate the

Page 3 of 76
relationship between the chemical structure of these chemotherapeutic agents and their

action are known, and the physiological activity of SAs has been found to be conditioned

by the presence of an SO2NH group in the chemical structure that can be changed with

the addition of various radicals at the R position:

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H2N S NH R

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O

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The most active SA derivatives contain heterocyclic radicals. Many SAs are based

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on pyrimidine, pyridazine and other heterocycles, and it turns out that the displacement of

the para-amino group to the meta- or ortho-position deprives the compound of its
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bacteriostatic action. If the hydrogen atom in the amino group is replaced with different

radicals, the compound loses its activity, but if these radicals are detached in the human
M
body, the activity of the compound will be retained. Introduction of some additional
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substituents into the aromatic ring results in the decrease or complete loss of the
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physiological activity of the SA.

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All SAs are white or slightly yellowish, odourless powders, and some have a bitter
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taste. Most of these substances are poorly soluble or practically insoluble in water. The

solubility of SAs in acids and alkalis is conditioned by their amphoteric properties, which
Ac

are due to the presence of the basic aromatic amino group (pKa1 2–2.5) and the amide

group, which contains a labile hydrogen atom with acidic properties (pKa2 5–8). The

acidic properties of SAs are more pronounced than their basic properties, and thus SAs

are positively charged in acidic medium at pH < 2, neutral at pH 3 – 5, and negatively

charged at pH > 5 [2].

Page 4 of 76
 The high efficiency of SAs and their relatively low cost have stimulated their

ubiquitous use in veterinary practices. SAs are used in veterinary medicine in pure

formulations or in combination with other antibiotics not only to combat infectious

diseases, but as additives to animal feed in order to promote growth and to increase the

productivity of livestock and poultry despite the fact that the use of antibiotics for this

t
purpose is forbidden in several parts of the world. For example, according to the data

ip
given in a previous review [11], the 2009 consumption of SAs in mg per kilogram of

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meat produced in Denmark was 4.82 (pork), 17.2 (cattle), 0.033 (broilers) and 58.5 (fish).

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As a result of misconduct in the prophylaxis and treatment of animals or due to

non-compliance with holding times before slaughter, traces of SA drugs can enter foods
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of animal origin. Strictly speaking, every human is a passive consumer of these drugs,

which are obtained from meat and dairy products, eggs and honey that inherit SAs from
M
the treatment of bacterial diseases in animals, poultry or bees. The systematic human
d

intake of SAs through foods is dangerous, as SAs can have adverse effects that most
e

commonly manifest in the form of allergic reactions, disbacteriosis, suppression of

pt

enzyme activity, alteration of the intestinal microflora, and promotion of sustainable

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forms of pathogens. In addition, there is evidence of hemotoxicity and carcinogenic

effects of some SAs, particularly SMZ [12]. It should be noted that certain decomposition
Ac

products of SAs can be active and potentially more toxic than the parent compounds [13].

The long-term use of SAs has resulted in a large number of SA-resistant bacterial strains,

which has stimulated the production of combined SA preparations containing

trimethoprim (Bactrim, Septrin, Biseptol, etc.) aimed at delaying proliferation of SA-

resistant microorganisms and enhancing the antibacterial effect of the SAs.

Page 5 of 76
 SAs enter the soil and water from food chains that involve human and animal

waste. A considerable portion of SAs enter the environment from flushing water from

pharmaceutical companies as well as poultry and pig farms. Every year more than 20000

tons of SAs enter the environment worldwide, with maximum amounts of SAs being

found in pig manure and soils. The SA content in other samples is as follows: sea water <

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groundwater < surface water < treated wastewater < untreated municipal wastewater <

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hospital wastewater < silt < soil < agricultural runoff < landfill leakage < manure. Of the

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SAs detected in the environment, SMZ is the most common and has been detected in

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50 % of the all analysed samples [11].

The widespread and often uncontrolled use of SAs in veterinary practices

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contributes to the potential presence of SA residues in livestock production, posing a

potential threat to human health. In most countries, the maximum residue limits (MRLs)
M
for total SAs in food are as follows: 100 mg kg-1 for meat and honey and 100 mg l-1 for
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milk [12]. There is also the problem of detecting the SAs present in environmental
e

samples, such as pharmaceutical and agricultural wastewater, surface and groundwaters,

pt

and soils.
ce

The data on the detection of SAs in foods [14 – 18], drugs and biological fluids

[19] using immunochemical methods [14 – 16], capillary electrophoresis [17] and HPLC
Ac

[18, 19] have been summarised. The steady increase in the number of research

publications related to SAs demonstrates the relevance of the issues associated with these

substances (Fig. 2). Fig. 2 shows that since 1995, there has been a four-fold increase in

the annual production of scientific publications with the title, abstract or keywords

containing the term “sulfonamide”. Moreover, since 2005, the percent of the publications

containing the term “sulfonamide” in the title (the studies where SAs are of prime

Page 6 of 76
importance) has also grown by approximately 1.5-fold after a period of stagnation from

2000 – 2005 (Fig. 2). This indicates a positive shift in the focus of scientists toward SAs

as an important research subject.

The present review outlines the most recently published methods of the sample

preparation and detection of SAs in food, feed, environmental samples, pharmaceuticals

t
and biological fluids that have been published mainly in the last five years.

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2. Sample preparation

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Sample preparation is a key step prior to the detection of SAs present in various

matrices. Sample preparation must be performed in order to extract SAs from different
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matrices, to eliminate interfering effects of associated components and to reduce the

detection limit. An important problem that is often faced while isolating and
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preconcentrating SAs arises due to their decreased hydrophobicity and the low degrees of
d

extraction. It is also important to extract SAs from different matrices without altering the
e

nature of the SAs, which imposes stringent requirements on the choice of the
pt

preconcentration conditions.
ce

According to previous reviews [20, 21], the current trends in food and

environmental sample preparation include the following: the miniaturization of the

Ac

equipment for sampling and sample preparation; the decreasing of the mass of a sample

to be analysed; the reduction in amounts or exclusion of organic solvents used; the

development of schemes for simultaneous extraction and preconcentration of the greatest

number of analytes from a single sample; the reduction of the number of stages of

analysis by combining the steps of sampling and sample preparation; and the

Page 7 of 76
development of automated methods for the preconcentration, thereby increasing the

performance of the analysis.

2.1. Food

Over the past five years, SAs have been mostly detected in meat [22 – 56] and

milk [37, 38, 46, 47, 53, 56 – 91], and less often in fish [37, 92 – 106], eggs [27, 46, 47,

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53, 57, 100, 107 – 112], honey [37, 47, 53, 61, 113 – 129] and baby food [130 – 132]

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(Fig. 3). The problem of detection of these compounds in complex matrices such as meat,

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fish, eggs, honey, and milk at or below the MRLs has been successfully solved through

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the use of powerful methods of chemical analysis such as high-performance liquid

chromatography (HPLC) coupled to mass spectrometry (MS) or tandem mass

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spectrometry (MS/MS) and through rational choice of a sample preparation technique

and by combining preconcentration techniques with the subsequent detection of SAs.

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One of the first steps in the preparation of solid food samples is the extraction of
d

SAs from the homogenised samples by using either a polar or non-polar organic solvent
e

with subsequent evaporation and change of the solvent. Solvent extraction from the solid
pt

matrices is carried out as follows: a weighed portion of a finely ground solid sample is
ce

placed in a vial, the selected solvent is added, and the vial is stirred for a certain period of

time (from a few minutes to several hours). The phases are separated by filtration. The
Ac

complex composition of the matrices, which often contain large amounts of fats and

proteins, makes the development of such methods a non-trivial task.

Acetonitrile is often used to extract SAs from meat [22 – 25, 38 – 43], fish [92,

93], honey [113, 114, 129] or baby food [130]. Other organic solvents are also used such

as methanol [56, 129], ethylacetate [53, 107], or dichloromethane [34, 95]. A

supramolecular solvent based on reverse micelles derived from decanoic acid dissolved

Page 8 of 76
in tetrahydrofuran [28] as well as mixtures of acetonitrile with chloroform [25], water

[94, 111] and phosphoric acid [27], or mixtures of methanol with dimethylsulfoxide [55]

and water [106] have also been used. To intensify the sample preparation, solvent

extraction from the solid matrices is carried out in an ultrasonic bath [24, 25, 36, 42, 92]

or under a microwave field [41, 111]. As an example, an extraction of SAs from different

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fish samples [92, 93] showed that acetonitrile provides the highest degree of extraction

ip
compared to methanol, acetone, and sodium acetate buffer. Additional purification of the

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samples to remove fats is carried out by extracting with hexane and/or by solid-phase

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extraction (SPE).

Apart from the classical techniques, many alternative extraction procedures have
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been developed. These are used prior to the detection of SAs and are summarised in

Table 1.
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A new procedure for the isolation of SAs from meat, the so-called liquid-liquid
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extraction with fast partition at very low temperature (LLE-FPVLT), has been proposed
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[29, 44]. The sample preparation includes the following operations:

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Mixing of the
Freezing by Separation of the
finely ground
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Homogenization, placing into a

The procedure
sample (~ 2 g) described analyte-rich
is simple, solvent-saving and free of additional purification
centrifugation container with
with acetonitrile acetonitrile phase
liquid nitrogen
(4steps.
mL)
Ac

In recent years, there has been remarkable interest in the development of food

sample preparation for the simultaneous isolation of the greatest possible number of

analytes from a single sample for their subsequent detection using multiresidue methods

[133]. Examples of the simultaneous extraction of SAs with other antibiotics and their

detection by HPLC-MS are given in Table 2. Other methods for the isolation of SAs from

Page 9 of 76
food include pressurised liquid extraction (PLE), matrix solid-phase dispersion (MSPD)

and QuEChERS (Table 1).

The main advantages of PLE include high performance and its ease of automation.

Sample preparation is carried out using commercially available automated systems for

accelerated solvent extraction under pressure. Both organic solvents and water in the

t
subcritical state are used as the extractants in this method. Performing liquid extraction at

ip
high temperature and pressure results in quick and efficient isolation of substances with a

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minimum consumption of the solvent. The increased temperature accelerates extraction

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and increases the degree of extraction, whereas the high pressure keeps the solvent in the

liquid state. Acetonitrile was used for the PLE-based isolation of SAs from meat [32],

fish [99] and eggs [108].

an
MSPD, first proposed in 1989, allows for the preparation of samples from solid
M
and viscous food products and raw materials with both high and low fat contents. Food
d

sample preparation using this method includes the following operations:

e

Thorough
Mixing of the Purification by
pt

homogenization Elution of the

sample (~ 0.5 g)
The
with an MSPD method
adsorbent
in awas
mortar,
used to extract SAs from
target meat [30, 31, extraction
analytes of fats
48] and fish [97],
transferring into a with hexane
(~ 1.5 g)
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and a comparison of thecartridge

effectiveness of different sorbents and eluents has been given

[30]. Silica gel, diatomaceous earth and neutral alumina were used as the sorbents, and
Ac

methanol, hexane, dichloromethane, acetonitrile and acetone were the eluents.

Diatomite/acetone was recognised the best sorbent/eluent combination. Furthermore,

adsorption by C18 followed by the extraction of SAs with a mixture of acetonitrile and

dichloromethane (1:1, v/v) [48, 97] or adsorption by nanotubes and extraction with a

mixture of acetonitrile and 50 mmol L-1 ammonium acetate solution (95:5, v/v) [31] have

also been used.

Page 10 of 76
 The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) sample

preparation method was initially developed to analyse pesticides in fruits and vegetables

(2003) [134]. The QuEChERS methodology involves two steps: the first step is extraction

based on partitioning via salting-out, which involves the equilibrium between aqueous

and organic layers; the second step is dispersive SPE, which involves further clean-up

t
using MgSO4 and different sorbents such as C18 or a primary-secondary amine (PSA) to

ip
remove interfering substances. An integrated approach to the combined use of extraction,

cr
separation and purification of the extract allows for sample preparation with the minimal

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loss of an analyte on laboratory ware, filters, a rotary evaporator, sorbents or dehydrating

reagents. By simplifying and/or merging some classical sample preparation methods, this
an
method both reduces the amount of sample used and provides significant saving of

reagents, materials, energy and time required for the analysis. To date, many modified
M
schemes have been developed using this approach. Unlike the original method, they also
d

employ buffers. The QuEChERS method has established itself as a universal method for
e

the preparation of a wide variety of samples, including food, plants, vegetables, fruits,
pt

grains, biological samples, soils, and waters. The method has been standardised by
ce

normative document EN 15662-2007.

The QuEChERS procedure was used to isolate SAs from milk [70, 71] and for the
Ac

simultaneous extraction of SAs and other veterinary drugs from different types of foods

(Table 3). A comparative study of the effectiveness of food sample preparation using

QuEChERS and other extraction methods was made [70, 71, 109], demonstrating that

this method allows for the extraction of SAs along with other antibiotics from milk [70]

and eggs [109] more efficiently (with degrees of separation > 85%) than SPE, liquid

extraction and MSPD. Furthermore, the sample preparation using QuEChERS was very

Page 11 of 76
fast and cheap due to the absence of the solvent evaporation and purification steps which

are present in the other three extraction methods. Using this method, the preparation of

15 samples was completed in less than 1 h while the other extraction methods required at

least 3 – 4 hours. The QuEChERS method meets requirements for today’s chemical

analysis methodologies, as it allows for the quick and efficient extraction of SAs as well

t
as other veterinary antibiotics from foods. For simplifying the conventional QuEChERS-

ip
like pretreatment, two straightforward extraction procedures named “STEMIT” (single-

cr
tube extraction with multisorbent impurity trapping) and “SEP/MAC” (single-tube

us
extraction/partitioning multifunction adsorption clean-up) were evaluated [37].

In recent years, the isolation of SAs from milk and honey has been performed
an
using traditional liquid-liquid extraction (LLE) with ethyl acetate [53, 58, 86],

chloroform:acetone mixture (65:35, v/v) [59], acetonitrile [79, 81, 82], hexane [83] along
M
with cloud point extraction [63, 90] and different types of liquid-liquid microextraction
d

(LLME) [115, 120, 121]. Among these methods, dispersive LLME (DLLME, developed
e

in 2006) has attracted the greatest interest [33, 60, 61, 71, 78, 128]. The main idea of this
pt

method is to obtain a sub-micron emulsion of an extractant in the solution under analysis,

ce

which increases the surface of mass-exchange and decreases the time required for

analysis. Equilibrium in such systems is usually achieved in less than 1 min. A poorly
Ac

water-miscible or water-immiscible organic solvent is used in this method as the

extractant and an organic polar solvent that is well miscible with water and capable of

dissolving the extractant is used as a dispersant (dispersing solvent). The sample solution,

the extractant and the dispersing solvent are mixed in a centrifuge tube, shaken, and the

resulting emulsion is centrifuged. The use of ultrasound [78] or ionic liquids [60, 61, 101,

130] simplifies this method remarkably as it avoids the addition of a dispersant.

Page 12 of 76
 As mentioned above, the solvent-based extraction of SAs from solid foodstuffs is

often combined with SPE, which was used to preconcentrate SAs from meat [27, 39 − 42,

46], fish [96, 104], eggs [46, 107], honey [33, 116 – 118, 126] and milk [46, 65, 67, 72,

73, 75]. Commercially available cartridges filled with different polymeric sorbents are

often used for this purpose and include the following: Oasis MCX [39, 116], Oasis HLB

t
[46, 65, 73, 75, 107, 117], Nexus Abselul [27], BondElut SCX [33], Cleanert PEP [40],

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Strata SCX [41], LiChrolut C18 [42], HySphere C18 HD [96] and Sep-Pak C18 [126]. For

cr
the selective isolation of SAs from food, carbon nanomaterials [43] and molecular

us
imprinted polymers (MIPs) [66, 67, 72, 104] have been used successfully. Acetonitrile

[27], methanol [40], ammonium – acetonitrile [39] and ammonium – methanol solutions
an
[41] as well as mixtures of acetonitrile or methanol with acetic acid [104, 118] are used

as the eluents. Monolithic capillary columns provide an alternative to the use of SPE
M
cartridges [76, 105]. The advantages of monolithic capillary columns are their high
d

efficiency and stability combined with the convenience of their introduction into on-line
e

flow preconcentration systems.

pt

The routine monitoring of food products for residual amounts of SAs requires
ce

rapid methods of analysis. Recently, solid-phase microextraction (SPME) has been

increasingly used for this purpose [57, 77, 80]. Compared with the conventional methods
Ac

of extraction such as LLE or SPE, SPME allows pure extracts to be obtained with less

solvent consumption, which significantly improves the analytical signals. The adsorbents

used in this method are different polymeric materials (polydimethylsiloxane, polyacrylate

and carbowax/divinylbenzene, etc.) supported on a solid matrix. To improve the

efficiency and selectivity by which analytes are isolated, the adsorbents are further

modified, for instance, by molecular imprinted polymers (MIPs). Thus, Chen et al. [80]

Page 13 of 76
have developed a fast, selective and efficient method of SPME in which the adsorbent

consists of MIP microbeads. The method was applied to the separation and

preconcentration of SMZ from milk before its detection by capillary electrophoresis with

a UV detector. The recoveries of SMZ from milk were 89 – 110 %. To isolate SAs from

milk and meat, an SPME technique on a 1x1-cm polypropylene membrane containing a

t
polymeric sorbent based on methacrylic acid and ethyleneglycoldimethacrylate was

ip
developed [57]. Using the porous membrane, the authors managed to avoid

cr
contamination of the sorbent by solid particles. For the detection of SAs in milk, a

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miniature device based on a medical syringe filled with 30 mg of water-compatible

poly(hydroxyethyl methacrylate) was designed [74].

an
One of the newest techniques of food sample preparation is stir bar sorptive

extraction in which glass magnetic stirrer bars coated with polydimethylsiloxane are
M
used. The stir bar is placed in a vessel containing a sample that is placed on a magnetic
d

stirrer and mixed for a certain time. Then, the bar is recovered, dried, and desorption of
e

the compounds is performed. In several studies, this method of sample preparation was
pt

used for the isolation of SAs from meat [34, 35] and milk [64, 69]. To increase the
ce

efficiency of SAs sorption, the polydimethylsiloxane coating was further modified with

MIPs [34], monolithic polymeric materials [35, 64] or C18 [69].

Ac

Among the other SPE methods, there is an interesting approach based on magnetic

particles coated with MIPs synthesised by grafting [26, 68, 119]. Particles of Fe 3O4 were

treated with modifiers (ethylene glycol, polyvinyl alcohol or oleic acid), dipped into a

solution containing all components necessary for the synthesis of MIP, and the

polymerization was carried out. Composite materials based on Fe3O4 and MIPs retain all

of the properties needed to recognise template molecules and can be easily separated

Page 14 of 76
from the solution by applying magnetic field. These were used for the selective extraction

of SAs from meat [26], milk [68] and honey [119].

2.2. Feed

The practical use of SAs and other antibiotics as farm animal feed additives first

became widespread in the 1950s. SAs are added to feed for therapeutic or prophylactic

t
purposes and despite the fact that the antimicrobial effect of SAs is less than that of

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antibiotics, they are cheaper and more widely available in dealing with infectious

cr
livestock and poultry diseases. The concentration of SAs in feed ranges from 70 to 800

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mg kg-1 [135], leading to their accumulation in the bodies of animals and contamination

of animal-derived foods. SMZ, SQX and SDM are the most frequently detected in feed.
an
A survey of the literature [53, 94, 135 – 148] has shown that the researchers have

mainly focused on the search for express methods of feed sample preparation and their
M
combination with the subsequent SA detection, which mostly has been performed by
d

HPLC, HPLC-MS or HPLC-MS/MS. In recent years, many different extractants have

e

been suggested for extracting SAs from feed, including acetonitrile [138, 142, 143],
pt

mixtures of acetonitrile and water (95:5, v/v) [53, 94, 135, 141, 142], mixtures of
ce

chloroform and acetone (50:50, v/v) [144] or ethylacetate and water (99:1, v/v) [145] as

well as ternary mixtures methanol:acetonitrile:McIlvaine buffer (37.5:37.5:25, v/v/v)

Ac

[137, 146]; and acetonirile:methanol:1%-formic acid (65:25:10, v/v/v [139] and

80:10:10, v/v/v [140]). In several papers, the sample preparation was carried out using an

ultrasonic bath [139, 140, 146]. PLE [136, 148] and modified QuEChERS [147, 148]

were also used. Further purification and preconcentration of the extracts was carried out

using SPE with Oasis HLB [135, 148] and Strata SCX [144] cartridges or magnetic

nanoparticles coated with SMZ-imprinted polymers (Fe3O4@MIPs) [142]. In most works

Page 15 of 76
[136, 137, 138 – 140, 146, 148], SAs were extracted from feed along with other

antibiotics and medicines.

2.3. Environmental samples

In the past decade, the interest in detecting SAs in the environment has

significantly increased. These substances and their metabolites, along with surfactants,

t
personal hygiene products and other compounds associated with the human life, enter the

ip
environment as complex mixtures and achieve penetrance primarily through the crude

cr
and treated city wastewater [149]. A considerable portion of SAs enter the environment

us
through flushed water from pharmaceutical enterprises and poultry and pig farms.

According to the data systematised in the reviews [11, 150], the content of SAs in
an
untreated wastewaters ranges from 0.01 to 19.2 μg L-1, and in treated wastewaters ranges

from 0.004 to 6.0 μg L-1. SMX has been the most frequently detected SA in the ground
M
water in many countries [150].
d

2.3.1. Waters
e

SAs have been detected in underground [151], surface [33, 61, 101, 152, 155, 157,
pt

159, 160, 162 – 171] and wastewater [153, 154, 156 – 158, 161]. The preparation of these
ce

samples was often carried out by SPE using commercially available cartridges, such as

Oasis HLB [151 – 159], Strata-X [160], or Bond Elut-ENV [161]. In some cases, to
Ac

remove negatively charged humic and fulvic acids, a subsidiary anion exchange cartridge

(SAX) has been used [155, 158].

The Oasis HLB, Hysphere C18 EC, and PRLP cartridges were compared for their

efficiency in extracting SAs [152]. The best cartridge was Hysphere C18 EC, while the

best eluents were methanol, acetone or a methanol – acetone mixture (1:1, v/v) in

comparison to acetonitrile. The good sorption ability toward SAs was attributed to hyper-

Page 16 of 76
crosslinked polystyrene, which sorbed by 98–100% of SMZ, SMX, SMP and SCP using

a microcolumn filled with 30 mg of this sorbent [162, 163]. Carbon nanotubes [164],

magnetic iron oxide nanoparticles coated with octadecyltrimethylammonium bromide

[165] and composites based on Fe3O4 and either carbon nanotubes [164] or graphene

[166] were also suggested as the sorbents for isolating SAs from waters through SPE. A

t
portable device based on a micropipette filled with 1 mg of graphene was developed for

ip
flow micro-SPE of SAs [167].

cr
Alternative miniaturised methods of extracting SAs from natural water before their

us
chromatographic determination are single-drop liquid-phase microextraction into a drop

of extractant formed at the tip of a syringe needle [168], membrane microextraction [169,
an
170] and dispersive liquid-liquid microextraction [61, 101, 171]. Ionic liquids are

increasingly being used as the extractants [33, 61, 101, 168, 169].
M
Other drug substances such as tetracyclines [154 – 157], quinolones [154, 171],
d

macrolides [155, 158], trimethoprim [154, 160], chloramphenicol [154, 161] and
e

hormones [155] have been found along with SAs in water.

pt

2.3.2. Soils, manure and sediments

ce

In recent years, interest in the determination of SAs in soils, manure and sediments

has grown remarkably [172 − 183]. The preparation of these environmental samples
Ac

includes SA extraction with acetonitrile [172 – 175], methanol [176 – 178], nonionic

surfactant Triton X-114 [179] and mixtures of these extractants with different buffers

[175, 177, 178]. SAs were extracted from solid samples using ultrasonic extraction [174,

175, 177], microwave-assisted extraction [175, 179], PLE [175, 180 – 182] and MSPD

[183].

Page 17 of 76
 The extraction of six SAs (SDZ, SDD, STZ, SCP, SDM, and SQX) from soils was

investigated by different extraction techniques, such as conventional mechanical shaking,

microwave-assisted extraction, ultrasonic extraction and PLE [175]. The recoveries of

SAs in an acetonitrile : pH 9 buffer (20:80, v/v) solution from overnight spiked soils

ranged from 40 to 80%, depending on the SA and the soil, and did not depend on the

t
extraction technique. Recoveries obtained from aged SA residues were lower and

ip
depended on the extraction technique applied, with the microwave-assisted extraction

cr
being the most efficient.

us
Additional clean-up of the samples is often carried out by SPE using anion-

exchange and polymer cartridges (Oasis HLB, Strata X) [172, 177, 178, 180].

2.4. Pharmaceutical and clinical samples

an
The preparation of sulfonamide-containing pharmaceuticals, in which the SA
M
content is usually approximately 0.1 – 1 g, usually involves powdering a sample and
d

dissolving a weighed portion of it in methanol [184 − 189]. The concentration levels of

e

SAs in clinical samples vary within a quite broad range. SAs are usually detected at a rate
pt

of approximately 0.1 – 100 μg mL-1 in blood plasma and 0.001 – 1 μg mL-1 in urine.
ce

Liquid-liquid extraction is used for isolation of SAs from blood serum and plasma [61,

185, 190, 191] or urine [190, 192] and still remains the main method for preparation of
Ac

biological samples prior to SA determination. Acetonitrile [185], ethylacetate [192], 1%

phosphoric acid, [191] and a mixture of acetonitrile with phosphoric acid [190] are used

as the extractants. Sedimentation of proteins in urine was performed using 2%

trichloroacetic acid [192]. In some papers, liquid extraction was combined with SPE

[190, 191]. For example, the aqueous phase was additionally purified on a Nexus Abselut

SPE cartridge [190]. SAs were eluted with acetonitrile, preconcentrated by evaporation,

Page 18 of 76
derivatised by putrescine in sodium acetate buffer (pH 3.4) and analysed by HPLC-FL.

The recoveries from serum and urine were 91.2 – 119.0 % and 91.3 – 117.0 %,

respectively. For the simultaneous determination of SMX and trimethoprim in plasma by

HPLC-MS, the isolation of the analytes was performed by SPE on Orpheous DVB-HL

cartridges [191].

t
ip
3. Analytical methods

cr
Analytical methods for the detection and determination of SAs residues can be

us
classified into three groups: quantitative, confirmatory and screening. Quantitative

methods are mostly based on chromatography and capillary electrophoresis and allow for
an
the quantification of SAs. However, these methods usually require complex sample-
M
preparation procedures, multi-step clean-ups, complex laboratory equipment and trained

operators. They are time-consuming and expensive. The main feature of confirmatory
d

methods is their ability to reliably identify a compound. Screening methods can detect an
e

analyte at the level of interest and usually provide semi-quantitative results. They are
pt

specifically designed to avoid false compliant results [193]. These methods must allow
ce

for the reliable checking of samples, and only those samples indicating the presence of

the analyte should be selected for a thorough analysis. The ideal characteristics of a
Ac

screening method are a low percentage of false compliant samples, short analysis time

and high throughput, ease of use, good selectivity and low cost.

In general, the reported methods of SA determination can be grouped depending

on the type of analytical technique applied. The corresponding percentages are depicted

in Fig. 4. As it can be seen, HPLC-MS(/MS) is the most employed analytical method (38

%), followed by HPLC with other detectors (22%) and electrophoresis (15 %).

Page 19 of 76
3.1. Chromatographic methods

HPLC has been widely used for the quantification of SAs due to its high

sensitivity and broad linear range. Currently, the most widely used analytical methods for

SAs are based on a reversed-phase HPLC (RP-HPLC) separation. Among these methods,

HPLC–MS/MS, which can also be considered to be a confirmatory method, has become

t
the main analytical technique for the identification of SAs due to its higher selectivity

ip
and sensitivity than other instrumental methods.

cr
In addition to HPLC, ultra high-performance liquid chromatography (UHPLC) has

us
increasingly been used for the rapid separation of SАs. This technique became possible

due to the relatively new HPLC systems that tolerate ultra-high pressures and use
an
columns with sub-2-μm-particles, improving chromatographic performance (e.g., speed,

sensitivity and resolution) and reducing co-elution of interferences, thereby diminishing

M
matrix effects when compared to conventional HPLC. Rapid multi-residue screening of
d

veterinary antibiotics including SAs in food and environmental samples is one of the
e

most promising applications of UHPLC technology.

pt

3.1.1. Detection with MS and multi-class analysis

ce

Table 4 represents a selection of analytical methods for the detection of SAs by

RP-HPLC and UHPLC with MS detection reported over the past 5 years.
Ac

Being a confirmatory method, MS detection is used to identify and quantify a

substance and can be used to confirm a compound’s molecular structure. The basic

principle of this detection technique is measurement of the mass-to-charge (m/z) ratio of

ionised molecules. HPLC–MS/MS is often applied using a triple quadrupole analyser and

a selected reaction monitoring mode. This mode allows for the confirmation of the

composition of the compound and provides its structural information. In MS/MS, the

Page 20 of 76
most intensive ionic fragment from a precursor ion is used for the quantification. A less

sensitive secondary transition is used as the second criterion in confirmation purposes.

This mode also improves the precision and sensitivity of the analysis but does not collect

the full scan data. This can limit the availability of the full scan data which could

otherwise be used to both identify target analytes and detect additional unknown

t
compounds. The choice of one or another MS approach to monitor certain substances and

ip
residues in live animals and animal products can be referred to the European Union

cr
Commission Decision 2002/657/EC, which established performance criteria and other

us
requirements for analytical methods with different type detection, including MS

[193].The first step in the tandem MS detection is the selection of a precursor ion.
an
HPLC–MS/MS analysis of SAs is usually performed with the electrospray ionization

(ESI) source operating in positive-ionization mode. The protonated molecule [M+H]+

M
was chosen as a precursor ion for quantitation in all developed methods. The common
d

fragment ion m/z 156 represents the sulfanyl ring and is used for the quantification of the
e

majority of SAs [10].

pt

One of the advantages of MS/MS is the fact that complete HPLC separation of the
ce

target analytes is not necessary for selective detection. However, it is always advisable to

have good chromatographic separation in order to reduce matrix effects that typically
Ac

result in the suppression or, less frequently, in the enhancement of analyte signals.

Therefore, short HPLC columns are generally used, considerably speeding up the

analysis. As it is indicated in Table 4, C18 reversed phase based columns are widely used

for HPLC multi-residue analytical methods.

Because MS detection is incompatible with most mobile phases, volatile organic

modifiers should be used when HPLC is coupled to MS. Thus, formic and acetic acid or

Page 21 of 76
their ammonium salts are added to acetonitrile–water or methanol–water mixtures. The

typical concentrations of modifiers range from 2 to 20 mmol L-1. It has been observed

that the higher concentrations lead to reduced signal intensities.

The method of HPLC-MS/MS was successfully applied to the simultaneous

quantification of SA residues in porcine liver [29], pork and chicken samples [35], milk

t
and milk powder [69, 78], grass carp tissues [97], eggs [111], honey [117], feed [147] and

ip
soil [191]. Yu et al. [32] simultaneously determined 18 SA residues in poultry tissues,

cr
muscle, and the livers and kidneys of swine, cows and chickens.

us
A major drawback of a multi-residue method using HPLC–MS/MS is the high cost

of the organic solvents and the HPLC-MS/MS equipment. The development of UHPLC
an
has greatly reduced the analysis time. By reducing the particles of the stationary phase to

less than 2 µm, the resolution can be increased up to 60 %, allowing good separation in
M
less time. A highly selective and sensitive method was developed for the simultaneous
d

detection of twelve SAs in beef and milk by immunoaffinity chromatography purification

e

coupled to UHPLC-MS/MS [38]. The limit of detection (LOD) for the studied SAs
pt

ranged from 0.4 to 2.0 μg L-1 (1.6 – 8.0 μg kg-1 for beef and 1.8 – 6.4 μg kg-1for milk).
ce

She et al. [79] have developed a novel method for the rapid separation and determination

of twenty-four SAs in bovine milk by UHPLC-MS/MS. The samples were treated with
Ac

acetonitrile followed by homogenization, ultrasound treatment and centrifugation. The

analytes were detected by multiple reaction monitoring (MRM) in the positive ion scan

mode. Liu et al. [143] have presented a fast and sensitive UHPLC-MS/MS method for the

simultaneous quantitative determination of 16 SAs in animal feeds.

Recently, a hybrid triple quadrupole-linear ion trap (QqLIT) instrument has been

developed. This is a powerful technique for the large-scale screening of SAs and their

Page 22 of 76
metabolites in real samples [37, 152, 181]. The method is based on a QqQ with the third

quadrupole (Q3), which can be used as either a conventional quadrupole mass filter or a

linear ion trap combining the advantages of the classical QqQ scanning functionality and

the possibility of additional sensitive ion trap scans for structural analysis within the same

operating platform. Due to the high ion-accumulation capacity, this method has improved

t
full-spectrum sensitivity that provides very promising modes such as enhanced full mass

ip
scan, enhanced product-ion and multi-stage scans. All of these features make the

cr
technique very powerful for the identification of unknown or suspected analytes even

us
with poor fragmentation and at low concentrations. Another attractive capability of

QqLIT for semi-targeted analysis is information-dependent acquisition that automatically

an
combines a survey scan with the dependent (enhanced trap) scan during a single

experiment.
M
The progress in chromatography and mass-spectrometry resulted in development
d

of multi-class analytical methods, which are currently a significant trend in the detection
e

of several classes of veterinary drugs in food and environmental samples that can be
pt

successfully applied for both quantification and screening purposes [133]. These methods
ce

are able to detect more than 200 different compounds and are of great interest to

analytical laboratories due to their simplicity, high sample throughput and cost-
Ac

effectiveness.

A certain drawback of such procedures is the occurrence of abundant matrix

effects, which compromise the quantitative aspects and selectivity of the methods. The

extracts from solid matrices usually have high contents of organic components, such as

lipids, humic acids, etc. These interfering compounds compete with the analytes in

reaching the droplet surface positions, which affects the maximum evaporation efficiency

Page 23 of 76
and hampers ionization of the analytes. These components also increase the viscosity of

the sample and the surface tension of the droplets generated in ESI source, hindering the

evaporation of the analytes.

Different approaches have been developed to minimise the matrix effects. They

are based on the use of improved chromatography with better separation of the matrix

t
compounds from the analytes (e.g., UHPLC). The dilution of the sample extracts and the

ip
use of internal standards or matrix matched calibration are also frequently used [37, 97,

cr
181]. The main aspect in the multi-class residue analysis, however, is a sample

us
preparation step that should effectively extract a broad range of compounds from the

samples. The different physico-chemical properties of target analytes as well as the

an
presence of the high concentrations of interferences in the matrices complicate the

extraction and clean-up.

M
A variety of sample pretreatment methodologies and several multi-class HPLC-
d

MS/MS and UHPLC–MS/MS methods have been applied for the detection of SAs along
e

with veterinary drugs in animal food (see Table 2, 3), feeds, waters, soils and sediments
pt

(see Table 5). Most multi-class methods involve simple liquid extraction and
ce

QuEChERS, while clean-up is carried out by SPE. The degree of clean-up provided by

many of these methods is usually limited because the extensive purification would result
Ac

in total loss of some residues. Other simple approaches such as “dilute-and-shoot”

strategy, which utilises diminishing matrix effects in diluted samples and is usually

exploited in the mass spectrometric analysis of SAs, as well as ultra-filtration or on-line

column switching have also been developed.

For instance, a rapid and simple method for both quantification and confirmation

of nineteen antibiotics of five classes in muscle (SAs, tetracyclines, quinolones, β-lactams

Page 24 of 76
and macrolides) has been described [45]. The antibiotics were extracted by 70%

methanol, diluted with water and injected in the HPLC–MS/MS system. Cronly et al.

[138] have described a confirmatory method for the determination of fourteen prohibited

medicinal additives (metronidazole, dimetridazole, ronidazole, ipronidazole,

chloramphenicol, SMZ, SDZ, dinitolimide, ethopabate, carbadox, clopidol, tylosin,

t
virginiamycin and avilamycin) in pig and poultry feed at levels of 100 µg kg−1. The

ip
compounds were extracted using acetonitrile with the addition of sodium sulfate and

cr
cleaned with hexane before determination using HPLC–MS/MS. In another example, a

us
fast and easy HPLC-MS/MS multiclass method was developed for the detection of fifty

antimicrobials from thirteen different families in animal feeds [146]. Analytes were
an
extracted using a mixture of methanol, acetonitrile and McIlvaine buffer combined with

sonication. The feed extracts were simply diluted prior to injection. Analysis was carried
M
out by HPLC-MS/MS using an ESI source operating in the positive and negative modes.
d

A multi-class UHPLC-MS/MS method has been developed for the determination

e

of more than 160 regulated or banned compounds of various classes in eggs, honey and
pt

milk [47]. These compounds include anthelmintics such as benzimidazoles, avermectins

ce

and others; antibiotics including amphenicols, beta-lactams, macrolides, pyrimidines,

quinolones, SAs and tetracyclines; beta-agonists; corticosteroids; ionophores;

Ac

nitroimidazoles; non-steroidal anti-inflammatory agents; steroids; and tranquillisers. The

compounds were extracted with acetonitrile without any additional purification steps and

analysed by UHPLC-MS/MS. In most cases, the target value was set at 5 mg kg-1 for

unauthorised compounds. Furthermore, Zhan et al. have developed a simple and fast

extraction procedure for the determination of 226 veterinary drugs and other

contaminants in muscle [51]. The method is based on LLE, low temperature clean-up and

Page 25 of 76
dispersive SPE. The limit of quantification (LOQ) varied from 0.05 to 10 µg kg–1.

Another work [108] describes a multiclass method for the determination of forty-one

antimicrobial agents from seven families (SAs, diaminopyridine derivatives, quinolones,

tetracyclines, macrolides, penicillins and lincosamides) in eggs. The method has been

validated according to the requirements of the European Commission Decision 2002/657.

t
Compounds were extracted using PLE with a mixture of acetonitrile and succinic acid

ip
buffer (pH 6.0) (1:1, v/v) at 70◦C. No further clean-up was necessary. Analytes were

cr
determined by UHPLC–MS/MS over a chromatographic run of 13 min. The LOQs were

us
in the range of 0.5–3.8 µg kg−1 for non-authorised compounds. The proposed method

would enable to process about twenty-five samples per day.

an
A rapid and simple analytical method that was able to simultaneously determine

220 undesirable chemical residues (veterinary drugs, mycotoxins, pesticides, hormones

M
and other contaminants) in infant formula has been developed [132]. The method
d

includes extraction with acetonitrile, low temperature clean-up by water precipitation and
e

detection by UHPLC–MS/MS with ESI using the MRM mode. Most of the fat materials
pt

in the acetonitrile extract were eliminated by the low temperature clean-up. The LOQs
ce

were from 0.01 to 5 µg kg-1.

The QuEChERS multiresidue procedure simplifies extraction and clean-up and

Ac

reduces the time of the analysis (Table 3, 5). For example, the QuEChERS method has

been developed for the determination of trace amounts of thirty-one substances including

hormonal steroids, veterinary and human drugs in soil [172]. The analysis was performed

using HPLC–MS/MS. This method allowed for the detection of substances at the sub-ng

g-1 concentration level. The methodology was applied to real soil samples collected in the

several areas of France that had received different treatments with manure or sludge.

Page 26 of 76
Veterinary antibiotics, which were mainly from the sulfonamide family, were found in

the manure-treated soils (0.02 – 0.12 ng g-1).

Another good possibility for the multi-component analysis is a sensitive full mass

scan MS technique such as time-of-flight (ToF-MS) or quadrupole time-of-flight mass

spectrometry (QTOF-MS). These analysers provide high specificity due to both the high

t
mass accuracy and the high mass resolution. The advantage of a ToF-MS analyser is its

ip
ability to analyse a sample for a large number of compounds. The HPLC–ToF-MS

cr
approach is capable of high-sensitivity screening of several hundreds of compounds

us
within a single run. Furthermore, the data can be acquired and processed without any

prior knowledge about the presence of certain compounds. In other words, no analyte-
an
specific information is required before injecting a sample, and the presence of newly

identified compounds can be confirmed in the previously analysed samples simply by

M
reprocessing the data. The advantage of ToF-MS can be further improved by combining
d

it with UHPLC [46, 81, 94, 103]. A fast liquid chromatography ToF-MS method has
e

been developed for the simultaneous multiclass determination of selected antibiotics and
pt

other veterinary drugs (benzalkonium chloride, ethoxyquin, leucomalachite green,

ce

malachite green, mebendazole, SDZ, SDM, SMZ, SMT, SAM, SPY, STZ and

trimethoprim) in shrimps [103]. Different sample pretreatment methodologies based on

Ac

either liquid partitioning with different solvents and SPE or MSPD were tested for the

extraction of the targeted species. The extraction method that was finally selected was

liquid extraction with acetonitrile followed by a clean-up step with PSA (QuEChERS).

The LOD ranged from 0.06 to 7 µg kg−1.

Deng et al. [46] have developed an UHPLC-QTOF-MS method for a

comprehensive screening of 105 veterinary drugs and metabolites including beta-

Page 27 of 76
agonists, benzimidazoles, corticoids, triphenylmethane, nitromidazoles, quinolones, SAs,

tetracyclines, and benzodiazepams in meat, milk, and eggs. Acetonitrile containing 0.1%

formic acid was used to extract the drug residues from these matrices and to precipitate

the proteins. Sample clean-up was then conducted on an Oasis HLB column. The

separation was achieved within 30 min at the optimised chromatographic conditions. A

t
HPLC-QTOF-MS method has been developed to analyse veterinary drug residues in milk

ip
[81]. Drugs were extracted with acetonitrile and a molecular weight cut-off filter was the

cr
only clean-up step used in the procedure. A set of target compounds (including

us
representative SAs, tetracyclines, β-lactams, and macrolides) was used for validation.

Although the method was intended to be qualitative, the evaluation of the MS data
an
indicated a linear response and the acceptable recoveries for a majority of target

compounds. Finally, milk samples from cows dosed with veterinary drugs including
M
SMZ, flunixin, cephapirin or enrofloxacin were analysed. In addition to the parent
d

residues, several metabolites were detected in these samples. The proposed identification
e

of these residues could be made by evaluating the MS and MS/MS data. For example,
pt

several plausible metabolites of enrofloxacin, which has not been previously observed in
ce

milk, were reported in this study.

3.1.2. Detection with other techniques

Ac

Classical reversed-phase HPLC with ultraviolet (UV) [22, 25, 26, 33, 34, 41, 57,

58, 63, 64, 66, 72, 76, 77, 82, 101, 104, 112, 115,116, 135, 142, 159, 165, 166, 168, 169,

179], photodiode array (DAD) [23, 48, 59, 60, 62, 65, 67, 70, 95, 107, 128, 130, 144,

161,163, 178, 186] and fluorescence (FL) [24, 27, 28, 30, 61, 71, 74, 83, 95, 114,

117,121, 129, 145, 167, 175, 178, 190] detectors is still widely used for the routine

quantification of SAs in different types of food, feed and environmental samples. All of

Page 28 of 76
these approaches are quantitative but not confirmatory, as they cannot provide direct

evidence of the structure or composition of a substance. Electrochemical detection is used

less frequently [105, 114, 163, 181]. UV detection is often carried out at 270–280 nm or

in some cases at 255 nm. DAD detection is often performed at 267, 268 or 263 nm. FL

detection is carried out at the excitation wavelength of 405–420 nm and at the emission

t
wavelength of 485–495 nm after pre-column derivatization with fluorescamine. UV

ip
detection is the most affordable and versatile but the least selective and sensitive. FL

cr
detection is much more sensitive and selective, but its use is connected to the additional

us
derivatization procedure for transformation of SAs into their fluorescent derivatives. The

less frequent use of electrochemical detection seems to be due to the complexity of

selection of the detection conditions.

an
The vast majority of the chromatographic separations of SAs have been performed
M
with conventional silica-based reversed phased columns (mainly C18) with spherical
d

sorbent particles of 3 – 5 μm in diameter. Some types of commercially available

e

stationary phases used for the determination of SAs are represented in Table 6.
pt

The speed of the analysis can be increased through the use of a monolithic column
ce

[27, 105], high temperature [159] or an ultra-high-pressure system [164, 171]. A fast

analytical method based on HPLC-UV that provides pressure up to 600 bar, and a 150
Ac

mm column operated at high temperature (600C) was used for the separation and

determination of nine SAs in surface and wastewater samples in the shortest possible

time (3 min) [159]. Herrera et al. [164] used magnetic multi-walled carbon nanotubes

combined with UHPLC-DAD to extract eleven SAs (i.e., SAM, SAA, SDZ, STZ, SMR,

SDD, SMP, SDO, SMX, SSA and SDM) from different water samples [164]. A DLLME

Page 29 of 76
procedure combined with UHPLC-DAD has been developed for the determination of

eleven SAs and fourteen quinolones in mineral and run-off water [171].

The mobile phases mainly consist of acetonitrile–water or methanol–water

mixtures (Table 6). Furthermore, there are papers reporting the application of three-

component mixtures such as water:methanol:acetonitrile [58, 72, 144]. In most cases, the

t
mobile phase was modified with acetic [41, 30, 79, 104, 119, 25, 165] or formic acid

ip
[119, 152, 168, 120], and acetate [27, 61], phosphate [72, 169] or formate buffer [146].

cr
The application an elution gradient allows the separation and simultaneous detection of

us
SAs in complex mixtures with other antibiotics.

3.2. Electrophoretic methods

an
Capillary electrophoresis (CE) is another good quantitative analytical approach

that is mainly used when only small amounts of a sample are available. Some advantages
M
of CE are its high separation efficiency, ability to analyse several samples simultaneously
d

in multicapillary systems, and low consumption of reagents and accessories (packaged

e

columns are not required). Several CE methods for the analysis of SAs were published
pt

and reviewed in 2009 [17]. Recent advances in the analysis of antibiotics, including SAs,
ce

by CE from 2009 to 2011 are summarised in another review [194].

For the past five years, CE has been used for the determination of SAs in chicken
Ac

and edible pig tissues [25, 40, 43], milk [80], shrimp [99] and water samples [170].

Phosphate [40, 43] and borate [80, 99] buffers, which sometimes contain additional

organic modifiers such as sodium polystyrene sulfonate [170], were used as a running

buffer.

The simultaneous determination of six SAs (SMZ, SDM, SMR, STZ, SDZ and

SMX) in chicken and edible pig tissues was accomplished by CE with electrochemical

Page 30 of 76
detection [25]. The complete separation of SAs was achieved within 17 min, using 40

mmol L−1 Na2 B4O7/25 mmol L−1 KH2PO4 (pH 6.2) at the applied voltage of 18 kV. Chu

et al. [40] used CE for the separation and detection of SMX, SMZ, SMR and SDM in

chicken and beef tissue samples. The analysis was performed under the following

conditions: the stationary phase was a quartz capillary; the mobile phase was 45 mmol L-1

t
phosphate buffer (pH 6.3); detection was performed using a photodiode array; and the

ip
voltage was 20 kV. The LODs of the SAs were 4 – 6 μg kg-1. Four SAs were detected in

cr
meat samples by CE following clean-up of the acetonitrile extracts on graphene-filled

us
cartridges [43]. A hollow-fibre liquid-phase microextraction method was developed for

the preconcentration of six SAs, including SMZ, SMR, SDZ, SDM, SMX and STZ,
an
which were detected by CE with electrochemical detection. Under the optimum

conditions, these compounds could achieve the baseline separation within 35 min. The
M
LODs were in the range of 0.033–0.44 ng mL-1 [170].
d

Microfluidic chip electrophoresis is an alternative to the conventional capillary

e

electrophoresis that usually has better performance [56, 185]. Wang et al. [56] have
pt

proposed a sensitive microchip electrophoresis method for the efficient separation and
ce

detection of four SAs (SMZ, SMX, SQX and SAM) in milk and chicken drumstick

muscle using the laser-induced fluorescence detection. Separation of fluorescamine-

Ac

labelled SAs was accomplished using a buffer containing 5 mmol L-1 boric acid and 1%

(w/v) polyvinyl alcohol. Under optimised conditions, the separation of four SAs was

achieved within 1 min with the LODs of 0.2−2.3 μg L-1. A compact and low-cost light-

emitting diode-induced fluorescence detection coupled to a microchip electrophoresis

system was applied to the detection of fluorescamine-labelled SAs in commercial

Page 31 of 76
preparations and in a rabbit plasma sample [185]. The LODs for SDZ, SMZ and SGD

were 0.36–0.50 μg mL-1.

Capillary electrochromatography is a hybrid separation technique that combines

the stationary phase of HPLC and the electroosmotic flow of CE. Presently, capillary

electrochromatography is carried out on particle-packed or polymeric monolithic

t
columns. For the separation and detection of nine SAs in meat samples, the authors [39]

ip
suggested using capillary electrochromatography with MS-detection. The separation was

cr
carried out on several monolithic stationary phases, which were synthesised using one-

us
step co-polymerization of divinylbenzene with different alkyl methacrylates (butyl-,

octyl-, lauryl- or stearyl methacrylate). Among these, the poly(divinylbenzene-octyl

an
methacrylate) monolithic column gave the best results. Optimization of the mobile phase

composition and the gradient elution strategy allowed for the successful detection of
M
sulfonamide antibiotics in meat samples at levels of 10 μg L-1.
d

A micellar electrokinetic capillary chromatographic method with UV detection

e

was used for the simultaneous detection of SAs and amphenicols in poultry tissue [42].
pt

The analytes were isolated from tissue samples by SPE with C18 cartridges followed by
ce

protein precipitation with acetonitrile. The compounds were separated on the unmodified

quartz capillary (57 cm). A solution of 25 mmol L-1 sodium dodecylsulfate and 10 mmol
Ac

L-1 sodium borate was used as the mobile phase. The total time of analysis was less than

8 min.

3.3. Microbiological assays

The microbiological inhibition assays are based on the use of SA-sensitive bacteria

as indicators. These assays assess the ability of microbes to reproduce in milk. When

growth of these bacteria is suppressed, which is determined directly or indirectly by the

Page 32 of 76
metabolic activity of the bacteria, a conclusion is made about the presence of drugs. The

presence of the residual substances is determined from inhibition plots of the bacterial

growth obtained from an agar-diffusion method that uses cylinders, wells on the agar

surface or discs of filter paper. The test microbes are streptococci, micrococci and aerobic

spore-forming bacteria.

t
These types of techniques usually meet the requirements of a screening approach.

ip
The microbiological method was used for screening samples of milk for their SA and

cr
antibiotic contents before these compounds could be detected by enzyme immunoassay

us
[82] or HPLC [83]. Additional information on the application of microbial screening

methods for antibiotic residues, including SAs, can be found in a previous review [195].
an
That review presents an overview of the developments in the field of microbial screening

methods for antibiotic residues and the efforts expended to bring antibiotic screening
M
methods into compliance with EU legislation.
d

3.4. Immunoassays
e

Immunoassays are semi-quantitative methods characterised by high specificity,

pt

high sensitivity, simplicity and cost effectiveness, which make them particularly useful
ce

for routine uses. These assays are based on a specific reaction between an antibody and
Ac

an antigen, and they are capable of detecting the low concentration of residues in short

time and often do not require laborious extraction or clean-up steps. A large number of

papers have been devoted to the detection and identification of SAs using different

immuno-methods. Many of them are summarised in the reviews [14 – 16].

Enzyme-linked immunosorbent assays (ELISA) are the most widely used

immunoassays due to their high sample throughput. These methods can drastically reduce

the number of analyses required to detect sulfonamide contamination in food samples.

Page 33 of 76
Therefore, ELISA methods have been developed for sulfonamide screening in honey [53,

122], chicken muscle [54], fish [106], feed [53, 141], milk [82, 196] and farm animal hair

samples [197].

For the high-throughput monitoring of SA residues in edible animal tissues, a

novel hapten and monoclonal-based indirect competitive ELISA has been developed. The

t
novel hapten was synthesised and conjugated to a carrier protein as the immunogen. The

ip
spleen cells of the inoculated mice, which expressed group-specificity against SAs, were

cr
used. The obtained monoclonal antibody showed cross-reactivity to 16 structurally

us
different SAs. Based on this antibody, an optimised ELISA protocol with only

phosphate-buffered saline was carried out for the fast extraction of SAs from tissues. The
an
LODs of SAs in chicken ranged from 1.5 to 22.3 µg kg-1. The developed ELISA method

would be a useful tool in screening for SAs residues in edible animal tissues [54].
M
A method of detecting the most commonly used SAs (SMR, SDM and SDZ) in
d

commercial fish samples has been developed [106]. The important advantage of this
e

method is that only one pair of immunoreagents is used for the detection of three
pt

analytes. Therefore, it is possible to use the same ELISA to determine the total
ce

concentration of SAs or quantify any of these analytes by using the calibration curves

that correspond to each sulfonamide.

Ac

Currently, there are many commercially available immunoassay kits used for the

fast and highly specific detection of SAs and other veterinary drug residues. For example,

ELISA test kits (RIDASCREEN, R-Biopharm AG, Darmstadt, Germany) were used to

assess the contamination of milk with SAs, tetracyclines and quinolones residues [196].

The declared LODs were 1.5 μg L-1 for tetracyclines, 3.5 μg L-1 for sulfonamides, 10 μg

L-1 for sulfamethazine and 0.25 μg L-1 for quinolones.

Page 34 of 76
 One of the most useful immunoassay formats is the lateral flow immunoassay

(LFIA). With respect to SAs, this method has been developed [123] in the competitive

reaction format and applied to test for STZ residues in honey samples. To prepare the

assay test, a hapten conjugate was used as the capture and goat antirabbit antiserum was

the control reagent. These reagents were dispensed on nitrocellulose membrane. A

t
polyclonal antiserum against STZ was conjugated to colloidal gold nanoparticles and

ip
used as the detection reagent. The visual LOD (cut-off value) of STZ was 15 ng g-1. The

cr
qualitative results were reached in 10 min, and the test was highly specific and showed

us
no cross-reactivity to other chemically similar antibiotics.

Examples of immunoassays for the determination of SAs are given in Table 7.

3.5. Biosensors
an
Immunochemical methods, which use antibodies as the specific recognition
M
elements, are excellent complementary analytical alternatives for detecting SA residues
d

in small sample volumes without complex sample preparation and purification steps. In
e

fact, over the past five years, it has been demonstrated that the electrochemical and
pt

optical biosensors are excellent tools for detecting contaminants in food and
ce

environmental matrices [203, 204]. Their main advantages are their technical simplicity,

low cost, and the possibility of being used in field analyses. Electrochemical sensors
Ac

based on the use of receptors fabricated through different imprinting approaches have

been developed for the detection of SAs [89, 91, 125 – 127, 205].

One of the prospective trends in this field is the development of miniaturised

disposable electrodes which could potentially avoid a labour regeneration step simply by

changing the electrode. The main role in this type of methods is played by screen-printed

carbon electrodes. Thus, the authors [89, 91] have suggested an amperometric

Page 35 of 76
immunosensor based on an antibody covalently immobilised onto a 4-aminobenzoic acid

film grafted onto disposable screen-printed carbon electrodes and a direct competitive

immunoassay using horseradish peroxidase-labelled tracers. The developed methodology

showed very low LOD (in the low ppb level) for SA antibiotics in milk samples and good

selectivity to other antibiotic residues frequently detected in milk and dairy products.

t
Another interesting approach is the application of nanoparticles, quantum dots and

ip
nanocomposites. For example, an electrochemical immunosensor based on a

cr
nanocomposite-modified glass carbon electrode has been developed [205]. The

us
biospecific surface was a CeO2-chitosan-modified nanocomposite with an attached anti-

SMX polyclonal antibody. The presence of the CeO2-chitosan nanocomposite

an
significantly enhanced the conductivity of the electrode. The large electroactive surface

area of the electrode resulted in high loading of the antibody. The LOD of SMX was
M
shown to be 3.25×10−7 mg mL−1. No cross-reactivity with other antibiotics of the
d

sulfonamide family was found. The immunosensor was successfully applied to the
e

analysis of milk, honey and egg samples.

pt

The immunosensor [125] was based on biofunctionalised magnetic particles and

ce

electrochemical nanoprobes prepared by labelling the specific antibodies with CdS

nanoparticles (CdSNPs). After the immunochemical reaction, the CdSNPs were dissolved
Ac

and the released metal ions were reduced at the electrode and read in the form of the

current or charge signal by using the well-known anodic stripping technique. Due to the

amplification effect on the amperometric/coulombimetric signal produced by CdSNPs,

high detectability could be reached. For example, SPY could be detected at 0.20 µg L-1.

The immunosensor has been applied to the detection of residues of this antibiotic in

Page 36 of 76
honey samples. The use of magnetic particles minimised the matrix effect and allowed

LOD values of up to 0.11 µg kg-1.

This effect has conditioned the application of magnetic particles in

immunosensing methods. The use of magnetic particles offers unique advantages such as

permitting interferences in the sample matrix, minimizing the matrix effect, improving

t
immunoreaction kinetics, and ease of magnetic manipulation. For example, a new

ip
electrochemical magnetoimmunosensor (EMIS) has been developed [127] for the

cr
detection of sulfonamide antimicrobials in honey samples. The improved EIMS was

us
based on well-characterised immunoreagents, biofunctionalised magnetic beads and

homemade graphite–epoxy composite electrodes containing an internal magnet. The

an
EMIS was able to detect SAs in honey at levels below 25 μg kg−1.

In a few articles [86 – 88], the application of aptamer-based sensors and assays for
M
the detection of SAs has been mentioned. Aptamers are single-stranded DNA or RNA
d

oligonucleotides able to bind to their target with high affinity and specificity. The
e

application of aptamers in biosensors offers the possibility of fast and easy detection of
pt

SAs. A polymer-based aptasensor using fluoresceinamidite-modified aptamers and

ce

coordination polymer nanobelts has been developed for detecting SDM residues in milk

[86]. Using the fluorescence quenching effect, the proposed aptasensor showed high
Ac

sensitivity for SDM (LOD = 10 ng mL-1). In another study [86], SDM-binding DNA

aptamers and unmodified AuNPs have been proposed for the sensitive detection of SDM.

The assay used aptamer-conjugated gold nanoparticles to combine the selectivity and

affinity of aptamers and the spectroscopic advantages of gold nanoparticles. The red-to-

blue colour change of AuNPs in the presence of SDM was easily observed by the naked

eye or measured using a UV–vis spectrometer. The linear dynamic range and the

Page 37 of 76
detection sensitivity were found to be 50 to 1000 ng mL-1 and 50 ng mL-1, respectively.

Adrian et al. [88] have developed a portable wavelength-interrogated optical system

(WIOS) exploiting class-selective bioreceptors for the fast screening of antibiotics (e.g.,

SAs, fluoroquinolones, b-lactams and tetracyclines) in milk. The label-free sensor used

the evanescent-wave principle. The changes in the refractive index close to the modified

t
chip surface were detected by scanning the resonance conditions when a light wave was

ip
coupled in the waveguide through a conveniently designed grating. The bioreagents used

cr
in this study were developed to detect a wide range of congeners of the each selected

us
family of antibiotics below the MRL values established for milk samples.

3.6. Other methods

an
In addition to the methods described above, different physical-chemical methods
M
have been used for the detection of SAs. These methods are all quantitative and include

fluorescence [73, 84, 85, 124, 184], spectrophotometry [90, 128, 188, 189, 206], surface
d

enhanced Raman spectroscopy [207, 208] and electrochemical methods [55, 187, 209 –
e

215]. The use of these methods is justified when it is necessary to carry out the routine
pt

quality control of relatively simple sample compositions. The advantages of these

ce

methods include their simplicity, compactness, and relatively low cost of the analysis.
Ac

Molecular fluorescence spectrometry can be used as a screening method to detect

SAs in milk [73, 84, 85], honey [124] and pharmaceuticals [184]. For the derivatization

of SAs, fluorescamine [73, 84, 85] and β-cyclodextrins [124] have been employed. In a

previous paper [184], a new, simple and accurate method has been proposed to detect

SAs based on the direct quenching effect produced by several SAs (SSZ, SAM and

SMX) on the luminescence of terbium (III). The proposed method allowed for the

detection of up to 50 samples per hour.

Page 38 of 76
 A net analyte signal standard addition method has been used for the simultaneous

determination of SDZ and trimethoprim by spectrophotometry in some bovine milk and

veterinary medicines. The method combines the advantages of a standard addition

method with the net analyte signal concept that allows for the acquisition of information

about certain analytes from the spectra of multi-component mixtures. This method has

t
some advantages, such as the use of a full spectrum realization. Therefore it does not

ip
require calibration and prediction steps and only a few measurements are required for

cr
antibiotic detection [90].

us
A simple, sensitive and accurate spectrophotometric method for the detection of

SMX, SGD, SQX, SML and SDD in different pharmaceutical preparations has been
an
developed [189]. The charge-transfer reactions between SAs as π-electron donors and

7,7,8,8-tetracyanoquinodimethane, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and 2,5-

M
dichloro-3,6-dihydroxy-1,4-benzoquinone as π-acceptors result in highly coloured
d

complexes. p-Dimethylaminocinnamaldehyde (p-DAC) has been suggested [206] as a

e

spectrophotometric reagent for the detection of SAs. It was shown that in acetonitrile
pt

medium, this reagent participated in a condensation reaction with SAM, SMP, SCP,
ce

SMX and SMZ, forming coloured products. The method was developed for the

spectrophotometric detection of SAs with LODs of n·10-2 g mL-1. For the detection of
Ac

SAs in honey samples, a screening method based on flow injection analysis coupled to a

liquid waveguide capillary cell has been developed [128]. The proposed method was

based on the reaction between SAs and p-DAC in the presence of sodium dodecylsulfate

in dilute acid medium (hydrochloric acid), with the reaction product being measured

spectrophotometrically at 565 nm. The non-compliant and false non-compliant samples

Page 39 of 76
were also analysed by a confirmatory HPLC method. The proposed system enables the

screening of SAs in honey samples with a low number of the false non-compliant results.

Electroanalytical techniques pose viable alternatives for the detection of SAs,

which have advantages such as simplicity, portability and sensitivity that make them very

attractive for the monitoring of pharmaceutical compounds. Owing to the electroactive

t
properties of SAs, the anodic oxidation of these substances occurs at the aromatic amino

ip
group with the formal oxidation–reduction potentials range of 0.75– 1.05 V. In the last

cr
five years, there have been several reports concerning the electrochemical properties of

us
SAs on different types of electrodes, such as a boron-doped diamond [187], a glassy

carbon [209] and a multi-walled carbon nanotube paste electrode [210, 211], carbon

nanotubes-poly(1,5-diaminonapthalene)
an
[212] or phthalocyanine [213] modified

electrodes, and a MIPs-modified carbon paste electrode [214]. These electrodes were
M
used to develop a variety of voltammetric methods for the detection of SDZ [209, 213],
d

SMX [210], SAA [212] and SSZ [214] in commercial pharmaceutical formulations as
e

well as SMT in human blood plasma [211] and SSZ in human serum [214].
pt

Trace quantities of SMZ, SMT, SMM, SQX and SDM were successfully
ce

preconcentrated, separated, and detected in a microfluidic device by employing

electrokinetic separation and electrochemical detection using an Al2O3–AuNPs modified

Ac

carbon paste electrode [55]. The method was used for the direct analysis of SAs in real

meat samples.

A method based on a multicommutation stopped-flow system has been developed

and applied to the simultaneous detection of SMX and trimethoprim in pharmaceutical

formulations by using a differential pulse voltammetry with a boron-doped diamond

electrode [187]. Almeida et al. [215] described the construction of an SDZ-selective

Page 40 of 76
electrode made from stainless steel tubular syringes of different lengths. The selective

electrode was used as a potentiometric detector in a flow-injection analysis system for

on-site detection of SDZ in aquaculture waters.

4. Conclusions and outlook

t
Over the past five years, different methods for monitoring the residues of SAs in

ip
various types of samples (e.g., food, feed, environmental, clinical and pharmaceutical

cr
samples) have been proposed. The key roles in these methods are played by sample

us
preparation techniques, and the main efforts in this field have been focused on the

optimization of the preparation, extraction and clean-up steps and on the enhancement of
an
the environmental safety of these procedures. The methods with the most promise in

achieving these goals are QuEChERS and SPE. The main advantages of these approaches
M
are good compatibility with high throughput multi-residue analytical procedures and their

relatively low cost. Therefore, these techniques are expected to have the most
d

pronounced development in the future.

e
pt

The currently proposed analytical approaches for the detection of SAs are mainly

based on HPLC–MS or HPLC–MS/MS. Great advances in HPLC-MS/MS have made it a

ce

key technique for the determination of not only SAs but also other antibiotic residues.

The main trend in this field is the combination of MS detectors with modern
Ac

chromatographic approaches such as UHPLC and the application of the powerful QqTOF

and Orbitrap instruments. These hybrid approaches have made a great contribution to the

analysis of trace organic contaminants, including SAs, and have contributed to the

development of multi-analyte techniques for the detection of a wide range of substances

in a single analytical run. These methods seem poised to be the most frequently used

techniques for the purposes of analysis in the future. The main disadvantages of these

Page 41 of 76
methods are their complex equipment and high costs. This fact currently stimulates a

great interest in the development of screening methods based on microbiological,

immunoassays and biosensors, which have the main advantages of low cost, short

analysis times and the possibility of their on site use. The clear trend in this field is the

miniaturization of the screening systems (chips, microarrays, microtiter plates) as well as

their automation. We think that these features will maintain the sustainable progress of

t
ip
these methods in the near future.

cr
Acknowledgments

us
The work was financially supported by the Russian Foundation for Basic Research (grant N 13-

03-00100)

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[204] N. Sanvicens, I. Mannelli, J.P. Salvador, E. Valera, M.P. Marco, Trend. Anal.

Chem. 30 (2011) 541 –553.

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2623–2627.

[206] E. V. Klokova, S. G. Dmitrienko, Mosc. Univ. Chem. Bull. 63 (2008) 284–287.

[207] M. Lamshoft, B.B. Ivanova, M.Spiteller, Talanta. 85 (2011) 2562 − 2575.

[208] K. Lai, F. Zhai, Y. Zhang, X. Wang, B.A. Rasco, Y. Huang, Sens. Instrumen. Food

t
Qual. 5 (2011) 91 – 96.

ip
[209] O. C. Braga, I. Campestrini, I. C. Vieira, A. Spinelli, J. Braz. Chem .Soc. 21(2010)

cr
813-820.

us
[210] M. Arvand, R. Ansari, L. Heydari, Mater. Sci. Eng. C. 31 (2011) 1819–1825.

[211] S. M. Ghoreishi, M. Behpour, A. Khoobi, Z. Moghadam, Anal. Lett. 46 (2013)

323–339.
an
M
[212] S.K. Yadav, P.K. Choubey, B. Agrawal, R.N. Goyal, Talanta 118 (2014) 96–103.

[213] X.P. Hong, J.Y. Ma. Chin. Chem. Lett. 24 (2013) 329–331.
d

[214] S. Sadeghi, A. Motaharian, A. Z. Moghaddam. Sensor. Actuat. B 168 (2012) 336–

e

344.
pt

[215] S. A. A. Almeida, L. R. Amorim, A. H. Heitor, M. C. B. S. M. Montenegro, J.

ce

Barbosa,L. C. Sá, M. G. F. Sales, Anal. Bioanal. Chem. 401 (2011) 3355–3365.

Ac

Page 57 of 76
 Table 1 .Non-classical extraction techniques for the sample preparation prior to the
detection of SAs

Extraction technique Advantages Disadvantages References

Liquid-liquid extraction with Relatively low toxic Extremely low 29, 44
fast partition at very low solvent temperature
temperature (LLE-FPVLT) Low solvent Requires liquid
consumption nitrogen
Free of additional Expansive

t
ip
purification
Pressurized liquid extraction High performance Requires high 32, 99,

cr
(PLE) Ease of automation pressure 108
Possibility to use water Relatively
as an extractant complex

us
equipment
Expansive
Matrix solid-phase dispersion Easy preparation of Additional 30, 31, 48,
(MSPD)
samples
an
solid and viscous purification is
usually required
97

QuEChERS Simplicity Partial lack of 49, 50, 70,

M
Quickness the extraction 71, 102,
Low cost selectivity 103, 109,
110, 131
d

Dispersive liquid-liquid Quickness Additional 33, 60, 61,

microextraction (DLLME) High efficiency solvent is 71, 78,
e

required 101, 128,

pt

130
ce

Table 2 Examples of SAs extraction along with other veterinary drugs and their multi-
class detection.
Compounds Matrix Method Detection Ref.
Ac

37 veterinary drugs, Porcine muscle Extraction with ACN at HPLC–MS/MS 44

including 16 SAs very low temperature

19 veterinary drugs, Porcine and Extraction with 70% HPLC–MS/MS 45

including 4 SAs bovine muscle methanol

105 veterinary Meat, eggs Extraction with ACN UHPLC–MS 46

drugs, including 18 containing 0.1% formic
SAs acid

160 veterinary Meat, eggs, Extraction with ACN UHPLC–MS/MS 47

drugs, including 16 honey
SAs

Page 58 of 76
226 veterinary Meat Extraction with UHPLC–MS/MS 51
drugs, including 18 ACN/EtOH (5:1, v/v) and
SAs Na2EDTA

120 veterinary Bovine kidney Extraction with ACN/Н2О HPLC–MS/MS 52

drugs, including 21 (4:1, v/v)
SAs

25 veterinary drugs, Milk Extraction with ACN HPLC–MS 81

including 8 SAs

t
ip
17 SAs, 5 tetracycli- Fish Extraction with acidic HPLC–MS/MS 92
nes MeOH/ACN (50:50, v/v)

cr
70 veterinary drugs, Fish fillets Extraction with acidic UHPLC–MS 94
including 5 SAs ACN/Н2О (80:20, v/v)

us
9 SAs, Catfish Extraction with acidic HPLC–MS/MS 96
5 tetracyclines MeOH

32 veterinary drugs,
including 13 SAs
Fish
ACN
an
Extraction with acidic HPLC–MS/MS 98
M
100 veterinary Fish, eggs Extraction with ACN/Н2О UHRLC–MS 100
drugs, including 15 (6:4, v/v)
SAs
d

41 veterinary drugs, Eggs Extraction with acidic HPLC–MS/MS 108

e

including 14 SAs ACN

pt

25 veterinary drugs, Eggs Extraction with acidic UHPLC–MS/MS 109

including 4 SAs ACN and Na2EDTA
ce

220 veterinary Baby food Extraction with ACN and UHPLC–MS/MS 132
drugs, including 18 Na2EDTA
SAs
Ac

Page 59 of 76
 Table 3 Examples of the QuEChERS methods used in the extraction and clean-up of SAs
along with other veterinary drugs and their multi-class detection.
Compounds Matrix Method Detection Ref.

48 veterinary drugs, Chicken Extraction with ACN containing HPLC–MS/MS 49

including 16 SAs 0.1% (v/v) acetic acid. Shaking
with anhydrous Na2SO4. DSPE
with Bondesil NH2 and Na2SO4.
Filtration and evaporation of
solvent. Dissolving in ACN – H2O

t
(90:10, v/v)

ip
21 veterinary drugs, Chicken Extraction with acidic ACN/Н2О UHPLC–MS/MS 50

cr
including 6 SAs (80:20, v/v). Shaking with sodium
citrate dibasic sesquihydrate,
sodium citrate dihydrate and

us
anhydrous MgSO4. DSPE with
PSA. Filtration and dilution with
acidic ACN/Н2О (1:1, v/v)

32 veterinary drugs,
including 13 SAs
Fish
an
Extraction with ACN/MeOH
(75:25, v/v). Shaking with
UHPLC–MS/MS 102

anhydrous MgSO4 and NaAc.

M
Filtration and evaporation of
solvent. Dissolving in acidic
ACN/Н2О (1:1, v/v).
d

14 veterinary drugs, Shrimps Extraction with acidic ACN. HPLC–MS 103

e

including 7 SAs Shaking with anhydrous MgSO4

and NaCl. DSPE with PSA and
pt

anhydrous MgSO4. Filtration and

evaporation of solvent. Dissolving
ce

in MeOH/Н2О (20:80, v/v)

21 veterinary drugs, Eggs Extraction with acidic MeOH/Н2О HPLC–MS/MS 110

Ac

including 3 SAs (80:20, v/v). Shaking with

anhydrous Na2SO4 and NaAc.
Filtration

29 veterinary drugs, Baby Extraction with acidic ACN. UHPLC–MS/MS 131

including 5 SAs food Shaking with anhydrous MgSO4
and NaAc. Filtration

Page 60 of 76
 Table 4 Examples of HPLC-MS/MS methods for the detection of SAs.
Matrix Sample Analytical Mobile phase LODs Ref.
preparation column

Porcine Extraction with Zorbax SB C18 A: ACN/Н2О (5:95, 5.58 − 16.75 29

liver fast partition at (250×4.6 mm, 5 v/v) / 0.1% formic acid μg kg-1
very low μm) B: ACN/Н2О (95:5,
temperature with v/v)/ 0.1% formic acid
ACN

t
Pork Extraction with Acquity BEH C18 A: 0.2% formic acid in 112 − 129 μg kg-1 31

ip
phosphate buffer (100×2.1 mm, 1.7 Н2 О
(pH 6) μm) B: МеОН, gradient

cr
SPE on multi-
walled carbon

us
nanotubes

Porcine PLE with ACN Zorbax SB C18 A: ACN 10 μg kg-1 32

and (250 mm×4.6 mm B: 0.1% formic acid,
swine
liver,
SPE on HLB
cartridge. , 5 μm) an
gradient

bovine
M
muscle

Pork, Extraction with Kinetex C18 – 0.0012 − 0.0146 35

chicken ACN (100×3 mm, 2.6 μg kg-1
d

Monolith-based μm)
e

stir bar sorptive

extraction
pt

Pork, Extraction with Agilent-Plus C18 А: 10 mmol L-1 0.1 − 0.3 μg kg-1 36
mutton ACN (100×2.1 mm, 1.8 ammonium formate /
ce

SPE on μm) 0.1% formic acid

AccuBONDII B: ACN, gradient
Ac

SCX cartridge

Pork, STEMIT or Agilent Zorbax A: 0.02% formic acid 1.0 – 7.5 μg kg-1 37
chicken, SEP/MAC Eclipse AAA in Н2О
fish, QuEChERS-like (150×4.6 mm, 3.5 B: 0.02% formic acid
honey, method μm) in ACN, gradient
milk

Beef, Extraction with Acquity BEH C18 A: 0.1% formic acid in 1.6 − 8.0 μg kg-1 38
milk Н2О/EtОН (100×2.1 mm, 1.7 Н2 О
(20:80, v/v). μm) B: MeOH, gradient
Immunoaffinity
chromatographic

Page 61 of 76
 purification

Milk Magnetic SPE Shim-pack VP- A: 0.2% formic acid in 0.0005 − 0.0495 68
on ODS Н2 О μg L-1
magnetite/silica/ (250×2 mm, 5 B: 0.2% formic acid in
poly(methacryli μm) MeOH, gradient
c acid–co–
ethylene glycol
dimethacrylate)
composite

t
microspheres

ip
Milk C18-Stir bar Zorbax ODS C18 МеОН/Н2О, gradient 0.9 − 10.5 μg L-1 69

cr
sorptive (150×2.1 mm, 3.5
extraction μm)

us
Milk Acidic Kinetex C18 A: 0.1% formic acid in 8 − 96 μg kg-1 75
deproteinization core–shell Н2 О
SPE on Oasis B: 0.1% formic acid in

Milk
HLB cartridge

Acidic Polar-RP 80A

an ACN, gradient

A: 0.1% formic acid in 12.5−25.5 μg kg-1 78

deproteinization (50×2 mm, 4 μm) Н2 О
M
B: 0.1% formic acid
in ACN, gradient
d

Milk Extraction with Acquity BEH C18 A: 0.2% acetic acid in 0.04 – 1.35 μg kg- 79
1
ACN (100×2.1 mm, 1.7 Н2 О
e

Clean-up with μm) B: ACN, gradient

pt

hexane

Fish Extraction with Acquity BEH C18 A: 5 mМ formic acid 3 − 6 μg kg-1 93

ce

ACN (100×2.1 mm, 1.7 in Н2О

μm) B: 5 mМ formic acid
in ACN, gradient
Ac

Grass Extraction with Halo fused-core A: 0.1% formic acid in 0.75 − 3.0 μg kg-1 97
carp ACN/water C18 silica (50×2.1 Н2 О
(50/50, v/v). mm, 2.7 μm) B: ACN, gradient
On-line MSPD

Eggs SPE on Zorbax SB C18 A: 0.5% acetic acid in 1.4 − 2.8 μg kg-1 111
magnetic (250×4.6 mm, 5 Н2 О
MWCNTs μm) B: МеОН, gradient

Honey Extraction with MGIII C18 A: 50 mmol L-1 1.0 μg kg-1 113
ACN column (150 ×2.1 ammonium acetate
mm, 1.8 μm) (including 0.3%

Page 62 of 76
 formic acid)
B: МеОН, gradient

Honey SPE on Oasis Kinetex XB C18 0.1% formic acid in 0.01 − 0.5 μg kg-1 117
HLB (100×3 mm, 2.6 Н2О : ACN (80:20,
μm) v/v, pH 2.6)

Honey SPE on α- Agilent SB-C18 A: 0.15% formic acid 0.25 − 0.5 μg kg-1 118
zirconium (250×4.6 mm, 5 in Н2О
phosphate μm) B: МеОН, gradient
intercalated by

t
hexadecyl

ip
trimethyl
ammonium

cr
bromide
Honey SPE on XTerra C18 А: 0.5% acetic acid in 1.5 − 4.3 μg kg-1 119

us
magnetic Н2 О
molecular B: ACN, gradient
imprinted
polymer an
Honey Hollow fiber NovaPak C18 (150 А: МеОН 5.1 − 27.4 μg kg-1 120
liquid membrane ×3.9 mm, 4μm) B:0.1% formic acid
M
extraction and 10 mmol L−1
ammonium acetate,
gradient
d

Feeds Extraction with Eclipse Plus C18 A: 0.1% FAc in Н2О 0.5 − 20 μg kg-1 143
e

ACN. (100×2.1 mm, 1.8 B: ACN, gradient

µm)
pt

SPE on basic
alumina column
ce

Feeds Modified Zorbax Eclipse А: 0.1% formic acid in 0.5 − 4.2 μg kg-1 147
QuEChERS XDB C18 Н2О/ACN (95:5, v/v)
procedure (150×4.6 mm, 5 B: 0.1% formic acid −
Ac

including DSPE µm) Н2О/ACN (5:95, v/v),

on PSA gradient

Ground Online SPE on Atlantis C18 A: 10 mМ formic acid 0.00009 − 0.011 151
water Oasis HLB (150×2.1 mm, 3 in Н2О μg L-1
cartridge µm) B: 10 mМ formic acid
in ACN, gradient

Environ Online SPE on Atlantis C18 A: 0.1% formic acid in 0.00005 − 152
mental Oasis HLB, (150×2.1 mm, 3 Н2 О 0.00784 μg L-1
water PLRP-s or µm) B: 0.1% formic acid in
Hysphere C18 ACN, gradient
EC cartridges

Page 63 of 76
Soil Microwave Pinnacle II C18 А: 0.2% acetic acid in 1.4 – 4.8 μg kg-1 173
assisted (250×4.6 mm, 5 Н2 О
extraction with µm) B: ACN, gradient
ACN
SPE on alumina

Soil Ultrasonic Zorbax SB C18 А: 0.5% acetic acid in 0.37–6.74 μg kg-1 174
assisted (250×4.6 mm,5 Н2 О
extraction with μm) B: ACN, gradient

t
ACN

ip
Magnetic SPE
on Fe3O4/Al2O3

cr
nanoparticles

Soil Extraction with Gemini C18 and А: 1 mmol L-1 0.31 – 2.74 μg L-1 176

us
MeOH Gemini C6 NH4Ac/acetic acid in
Clean-up on (150×4.6 mm, 5 Н2О/ACN (90:10, v/v,
Strata-X µm) pH 3.5)
Polymeric
Reversed Phase
an
B: ACN, gradient

SPE-column
M
Soils PLE with Atlantis C18 A: 10 mmol L-1 formic 0.01 − 4.2 μg kg-1 181
and ACN/water (150×2.1 mm, 3 acid in Н2О
sewage (25:75, v/v) or µm) B: 10 mmol L-1 formic
d

sludge MeOH/water acid in ACN, gradient

(90:10, v/v)
e

SPE on Oasis
HLB.
pt

Plasma Acidic Hypersil Gold 2 mmol L-1 NH4Ac LOQs: 30; 880 191
deproteinization (50×4.6 mm, 5 (pH 3, formic acid) in μg L-1
ce

SPE on µm) Н2О/ACN (40:60, v/v)

Orpheous DVB-
HL cartridge
Ac

Page 64 of 76
 Table 5 Examples of HPLC-MS/MS methods for the detection of SAs along with other
veterinary drugs in feed and environmental samples.
Compounds Matrix Method Analytical Mobile phase Ref.
column

18 veterinary Animal Extraction with C12 Hydro-RP A: 0.1% formic acid 136
drugs, feed Н2О/MeOH (95:5, v/v). (50×2 mm, 4 in Н2О
including 7 SPE (Hysphere C18 µm) B: 0.1% formic acid
SAs HD) in МеОН, gradient

t
ip
33 veterinary Feeding Extraction with Zorbax XDB A: 0.1% formic acid 137
drugs, stuffs MeOH/ACN/McIlvaine plus (150×2.1 in Н2О
including 1 buffer, pH 4.6 (37.5: mm, 3.5 µm) B: 0.1% formic acid

cr
SA 37.5:25, v/v/v). SPE in ACN/МеОН
(C18 or HLB) (70:30, v/v),

us
gradient

15veterinary Pig and Extraction with ACN. Luna C18 А: 0.2% acetic acid 138
drugs, poultry Shaking with (100×2 mm, 3 in Н2О
including 2
SAs
compound
feed
anhydrous Na2SO4.
Filtration. Extraction
an
µm) B: 0.2% acetic acid
in ACN, gradient
with hexane,
M
evaporation of solvent.
Dissolving in
Н2О:ACN (85:15, v/v)
d

48 veterinary Pig feed Extraction with acidic Altima HP A: 0.5% formic acid 139
drugs, ACN/MeOH/1% C18 (150×3.2 in Н2О
e

including 12 formic acid in Н2О (65 mm, 5 µm) B: 0.5% formic acid
pt

SAs :25:10, v/v/v) in МеОН, gradient

96 veterinary Corn Extraction with Altima HP C18 A: 0.5% formic acid 140
ce

drugs, ACN/MeOH/0.1% (150×3.2 mm, in Н2О

including 13 formic acid in Н2О 5 µm) B: 0.5% formic acid
SAs (80:10:10, v/v/v) in МеОН, gradient
Ac

50 veterinary Animal Extraction with Kinetex XB- A: 5 mmol L-1 146

drugs, feed MeOH/ACN/McIlvaine C18 (100×2.1 formic acid in Н2О
including 11 buffer (37.5:37.5:25, mm, 1.7 μm) B: 50 mmol L-1
SAs v/v/v) formic acid in
Н2О/ACN (10:90,
v/v), gradient

22 veterinary Animal PLE with MeOH. SPE C12 Hydro-RP A: 0.1% formic acid 148
drugs, feed (Oasis HLB) (50×2 mm, 4 in Н2О
including 8 μm) B: 0.1% formic acid
SAs in МеОН, gradient

13 veterinary Swine SPE (Oasis HLB) Dionex A: ACN 154

Page 65 of 76
drugs, wastewater Acclaim C18 B: 0.1% formic acid
including 4 and (150×2.1 mm, in Н2О, gradient
SAs environme 4.6 μm)
ntal water

49 veterinary Wastewate Extraction with Zorbax A: 0.2% formic acid 155

drugs, r, sludge ACN/citric acid buffer eclipse plus and 2 mmol L-1
including 15 (pH 3) (1:1, v/v). SPE C18 (100×2.1 NH4Ac in Н2О
SAs (Oasis HLB) mm, 1.8 μm) B: ACN, gradient

t
5 veterinary Livestock SPE (Oasis HLB and Shim-pack A: 0.3% formic acid 156

ip
drugs, wastewater MCX) FC-ODS and 0.1% NH4Ac
including 3 (75×3 mm, 3 in Н2О
SAs μm) B: МеОН/ACN

cr
(50:50, v/v),
gradient

us
11 veterinary Surface SPE (Oasis HLB) Nucleodur C18 A: 0.1% formic acid 157
drugs, water, ISIS (125×2 in Н2О
including 4 wastewater mm, 3 μm) B: 0.1% formic acid
SAs an in ACN, gradient

9 veterinary Suspended Extraction with Waters A: 0.2% formic acid 158

МеОН/0.2 mol L-1
M
drugs, solids of Symmetry C18 in Н2О
including 5 swine citric acid buffer (pH (150×2.1 mm, B: МеОН
SAs wastewater 4.7) (1:1, v/v). SPE 5 μm) C: ACN, gradient
(Oasis HLB and SAX)
d

28 veterinary Surface SPE (Strata-X) Synergi Polar- A: 0.1% formic acid 160
e

drugs, water RP (50×2 mm, in ACN

pt

including 9 2.5 μm) B: 0.1% formic acid

SAs in Н2О, gradient
ce

14 veterinary Soil Extraction with Zorbax Eclipse A: 0.01% FAc in 172

drugs, Н2О/ACN (1:1.5, v/v). plus C18 Н2О (pH 3.3)
including 7 SPE (SAX and Strata- (50×2.1 mm, B: МеОН, gradient
Ac

SAs X) 1.8 μm)

10 veterinary Broiler Extraction with Xterra MS C18 A: 0.3% formic acid 177
drugs, manure, MeOH/ACN/0.1 mol (100×2.1 mm, and 0.1% NH4F in
including 1 soil, L-1 EDTA/Mc 3.5 μm) Н2 О
SA manure Ilvaine buffer (pH 4), B: ACN/МеОН
compost 30:20:25:25, v/v/v/v. (1:1, v/v), gradient
SPE (Oasis HLB)

17 veterinary Soils, PLE with water. SPE Sunfire C18 A: 0.1% formic acid 180
drugs, sediments (SAX and Oasis HLB) (150×4.6 mm, in МеОН
including 1 3.5 μm) B: 0.1% formic acid
SA in Н2О, gradient

Page 66 of 76
8 veterinary Biosolids PLE with МеОН/0.2 Luna C18 A: 1% acetic acid in 182
drugs, mol L-1 citric acid (pH (150×4.6 mm, Н2 О
including 4 3) (50:50, v/v). SPE 5 μm) B: МеОН, gradient
SAs (Oasis HLB)

12 veterinary Sediments MSPD (C18 and Synergy A: 0.1% formic acid 183
drugs, Florisil). Extraction Fusion C18 in Н2О
including 4 with ACN/ 5% oxalic (150×2 mm, 4 B: 0.1% formic acid
SAs acid (6:4, v/v). μm) in ACN, gradient

t
ip
Table 6 Examples of HPLC–UV, HPLC–FL and HPLC–AD methods for the detection of
SAs.

cr
Matrix Sample preparation Analytical Mobile phase Detection Ref.
column (LODs)

us
Swine muscle Salting-out assisted Mightysil RP-18 A: ACN:1% DAD, 23
extraction with ACN (250×4.6 mm, 5 acetic acid (10:90, 280 nm
coupled with back- μm) v/v)
extraction with
water/ACN/dichlorom
ethane
an B: ACN, gradient
(0.2 − 1.0
μg kg-1)

SPE on magnetic
M
Pork, liver, C18 column ACN:1% acetic UV, 26
chicken molecular imprinted (250×4.6 mm, 5 acid (20:80, v/v) 270 nm
polymer 34
μm) (0.1 − 0.5
d

μg L-1)
e

Poultry Extraction with acidic Chromolith A: 0.05 mol L-1 FL, 27

muscle, eggs ACN Performance acetate buffer (pH 406 nm, 496
pt

SPE on Nexus Abselut RP-18 (100×4.6 3.4) nm

mm) B: MeOH, (2 − 17
ce

gradient μg kg-1)

Livers MSPD with Zorbax Bonus- A: 2% acetic acid FL, 30

diatomaceous earth,
Ac

RP (150×4.6 in Н2О 401 nm,

silica gel or neutral mm, 3.5 µm) B: ACN, gradient 495 nm
alumina
(1.33 − 2.47
μg kg-1)

Milk Deproteinization with Belta ODS MeOH : 1% acetic UV, 57

acidic ACN/water (150×3.9 mm, 5 acid (14:86, v:v) 260 nm
Polypropylene μm)
membrane protected (0.38 − 0.62
micro-SPE on μg L-1)
poly(methacrylic acid-
ethylene glycol
dimethacrylate)

Page 67 of 76
Milk Acidic deproteinization NovaPak C18 А: NaAc (5mМ) UV, 58
Extraction with ethyl (150×3.9 mm, 5 B: ACN 280 nm
acetate μm) C: МеОН, (–)
gradient

Milk Centrifugation Luna octyl silica 0.05 mol L-1 AD, 62

Restricted access (150×4.6 mm, KH2PO4 (pH 5) : 1.25B
media (RAM) octyl- 10 μm) ACN (82:18, v/v) (15.0; 25.0
bovine serum albumin μg L-1)

t
column

ip
Milk Monolith-based stir bar Kromasil C18 ACN:Н2О (35:75, UV, 64
sorptive extraction (250×4.6 mm, 5 v/v) 268 nm

cr
μm) (1.3 − 7.9
μg L-1)

us
Milk DLLME and C18 Ascentis™ A: 2% acetic acid FL 71
QuEChERS (10cm×4.6cm, 3 in Н2О(pH 2.5) 405 nm,
μm) B: ACN, gradient 495 nm
an (0.6 − 2.7
μg L-1)
M
Milk Centrifugation Luna C18 А: 0.1 mol L-1 UV, 72
Restricted access- (250×4.6 mm, 5 phosphate buffer 268 nm
molecular imprinted μm) (pH 6.0)/МеОН (0.8 μg L-1)
(95:5, v/v)
d

material
B: 0.1 mol L-1
e

phosphate buffer
(pH 7.0)/ МеОН
pt

(83:17, v/v)
C: 0.1 mol L-1
ce

phosphate buffer
(pH 8.0)/ МеОН
(94:6, v/v)
Ac

Fish, shrimp Extraction with 1% Venusil XBP А: acetic acid : UV, 104
acetic acid in water. C18 (250×4.6 Н2О (1:99, v/v) 270 nm
SPE on group- mm, 5 μm) B: ACN, gradient (8.4 − 10.9
selective molecular μg kg-1)
imprinted polymer

Shrimp Extraction with Chromolith® 0.1M KH2PO4 AD, 105

McIlvaine’s buffer. Performance (pH 3) : 1.2B
SPE on Oasis HLB RP-18e ACN:EtOH (1.2 − 3.4
cartridge (100×4.6 mm) (80 : 15 : 5, v/v/v) μg L-1)

Honey Sugaring-out assisted Cosmosil 5C18- A: 2% acetic acid FL, 114

AR-II (250×4.6 405 nm,

Page 68 of 76
 LLE mm, 5 μm) B: ACN, gradient 495 nm
(0.6 − 0.9
μg kg-1)

Honey Salting-out LLE Belta ODS МеОН:Н2О:NH4 UV, 115

(150×3.9 mm, 5 Ac (15:75:1, 260 nm
μm) v/v/v) (0.11 − 0.27
μg L-1)

Honey SPE on Oasis HLB, Varian A: 25 mmol L-1 FL, 117

t
ip
Strata-XL OmniSpher C18 KH2PO4 in Н2О 420 nm,
(250×4.6 mm, 5 (pH 5) 480 nm
μm) B: МеОН

cr
(1.0 − 15.0
C: ACN, gradient μg kg-1)

us
Honey Extraction with Ascentis RP- A: 0.02 mol L-1 FL, 129
MeOH. Amide (250×4.6 NaAc (pH4.5) 403 nm,
mm, 5 μm) B: ACN, gradient 492 nm
an (1.3 − 5.0
μg kg-1)

Animal feeds Extraction with ACN. C8 Inertsil A: 0.01 mol L-1 UV, 135
M
SPE on Oasis HLB, (250×4.6 mm, 5 acetic acid in Н2О 268nm
Bond Elut C18, Bond μm) – NaAc buffer
Elut Plexa, and Bond (5.0 − 18.0
(pH 4.7) μg L-1)
Elut Plexa PCX
d

B: ACN, gradient
e

Poultry feed SPE on core-shell Shimadzu VP- МеОН : Н2О : UV, 142
magnetic molecular ODS C18 acetic acid 270nm
pt

imprinted polymers (150×4.6 mm, 5 (30:70:0.1, v/v/v)

(Fe3O4@MIPs) (14.6 μg L-1
μm)
ce

Feeds Extraction with Luna C18 А: 0.02 mol L-1 DAD, 144
chloroform/acetone (250×4.6 mm, 5 NH4Ac (pH 4.5) 270 nm
(50:50; v/v) μm) B: MeOH : ACN
Ac

(390 − 640
SPE on Strata SCX (50:50, v/v), μg kg-1)
cartridge gradient

Animal feeds Extraction with LiChrospher A: 0.01 mol L-1 FL, 145
AcEt/H2O (99:1, v/v) 100 RP-18 formic acid − 405 nm,
(250×4 mm, 5 HCOONa (pH 485 nm
μm); Kinetex 3.4) (0.1 − 5.7
C18 (150×4.6 B: ACN, gradient μg L-1)
mm, 2.6 μm)

Environmental SPE on Bond Elut- C18 column МеОН : Н2О DAD, 161
water ENV, polystyrene- (70×4.6 mm, 5 (55:45, v/v) (pH 250 or 400

Page 69 of 76
 divinylbenzene μm) 3.2) nm
(0.02 − 0.03
μg L-1)

Environmental SPE on Luna C18 ACN : 2% acetic AD, 163

water hypercrosslinked (150×3.0 mm, 5 acid in Н2О 1.2B
polystyrene, Strata-X, μm) (20:80, pH 3.2). (3 μg L-1)
Strata SDB-L, carbon
nanomaterial Taunit or
Diasorb-100C16T

t
cartriges

ip
Environmental DSPE using magnetic Hypersil Gold A: 0.3% formic DAD, 164

cr
water or non magnetic C18 (100×2.1 acid in Н2О 260 or 280
MWCNTs mm and 1.9 μm) B: ACN, gradient nm

us
(0.008 −
0.032 μg L-1)

Environmental Magnetic SPE on Inertsil ODS-4 ACN : phosphate UV, 166

water Fe3O4@SiO2/graphene
μm)
an
(150×4.6 mm, 5 buffer solution
(20 mmol L-1, pH
269 nm
(0.09 − 0.16
4.9) (25:75, v/v) μg L-1)
M
Environmental Ionic liquid-based TC-C18 А: 0.1% formic UV, 168
water single-drop liquid- (150×4.6 mm, 5 acid in Н2О 265 nm
phase microextraction μm) B: ACN, gradient
d

(0.5 − 1.0
μg L-1)
e

Environmental SPE on Oasis HLB Zorbax 0.2% acetic DAD, 159

pt

water StableBond C18 acid:ACN (80:20, 272 nm

(150×4.6 mm, v/v) (1 − 10
ce

1.8 μm) μg L-1)

Soil samples Microwave-assisted Zorbax SB C18 A: 1.0 % acetic UV, 179

extraction with non-
Ac

column acid in Н2О 270 nm

ionic surfactant Triton (250×4.6 mm, 5 B: МеОН,
X-114 (3.2 − 5.7
μm) gradient μg kg-1)

Biological Extraction with acidic Inertsil ODS-3 A: 0.05 mol L-1 FL, 190
fluids ACN. (250×4 mm, 5 acetate buffer (pH 406 nm,
SPE on Nexus Abselut μm) 3.4) 496 nm
B: МеОН, (0.1 − 0.3
gradient μg L-1)

Page 70 of 76
 Table 7 Examples of immunoassays for the detection of SAs.
Matrix Assay format Solid Immunogen and antibody LODs Ref.
support
Edible Indirect 96-well 2-[[(4-aminophenyl)sulfonyl]- 1.5 – 22.3 54
animal competitive Maxisorp amino]-4-methyl)-5-pyrimidine- μg kg-1
tissues enzyme-linked microtitre carboxylic acid, 6-(4-aminoben-
(chicken) immunosorbent plates zensulfanylamino)pyridine-3-
assay (ELISA) carboxylic acid and (4-(4-amino-
benzensulfonylamino)benzoic

t
acid linked to bovine serum

ip
albumin (BSA); monoclonal
antibodies (4E5)

cr
Fish Indirect Polystyrene Synthesized haptens against 0.6 – 1 106
competitive 96-well sulfonamides and the μg L-1
ELISA microtiter commercial hapten N4-

us
plates phthalylsulfathiazole linked to
BSA and OVA; peroxidase-
labeled goat anti-rabbit

Honey Enzyme
immunoassay
Costar
9018
an
immunoglobulins
N-sulfonyl-4-aminobutyric acid
linked to ovalbumin (OVA);
0.05 μg L-1 122

(EIA) microplate rabbit polyclonal antibodies

M
Feed ELISA Nunc 5-[4-aminophenylsulfonamide]- 40 –200 141
Maxisorp 5-oxopentanoic acid linked to μg kg-1
Microtiter OVA; female white New
d

polystyrene Zealand rabbits antibodies

plates
e

Milk ELISA According to the RIDASCREEN ELISA test 3.5 – 10 196

μg L-1
pt

kits for sulfonamide (R3004) and

sulfamethazine (R3001)
Chicken Generic ELISA Polystyrene 6-(4-aminophenylsulfonamido)- ~ 0.1 μg L- 198
ce

1
muscle microplate hexanoic acid, 4-(4-(4-amino-
phenylsulfonamido)phenylsul-
fonamido)benzoic acid and 4-(4-
Ac

(4-aminophenylsulfonamido)-
phenyl)butanoic acid linked to
BSA; New Zealand white rabbits
polyclonal antibodies
Milk Hybrid The opaque Hapten-OVA conjugates; anti- 0.17 μg L-1 199
immunosorbent white sulfonamide and anti-quinolone
assay polystyrene broad-specificity monoclonal
(fluorescence- microtiter antibodies
linked plates and
immunosorbent ELISA
assay (FLISA) microtiter
and ELISA) plates
Milk Dual- Microtiter 4-(4-(4-aminophenylsulfon- 5.8 μg L-1 200

Page 71 of 76
 colorimetric plates amido)phenyl)butanoic acid
ELISA linked to BSA; monoclonal and
polyclonal antibodies
Milk and Lateral flow Nitrocellul Hapten conjugate N1-[4- 0.10 – 2.13 201
Swine immunoassay ose (carboxymethyl)-2-thiazolyl] μg L-1
Urine (LFA) membrane sulfanilamide linked to OVA;
(vivid 170) goat anti-rabbit antibody
conjugated to colloidal gold
particles
Milk Integrated 4-amino- Horseshoe crab hemocyanin 0.12 – 8.41 202

t
amperometric benzoic linked to 5-(6-(4-aminophenyl- μg L-1

ip
immunosensor acid film sulfonyl)pyridine-3-yl)-2-
based on direct grafted on methylpentanoic acid; female

cr
competitive a dispo- white New Zealand rabbits
immunoassay sable polyclonal antibody
electrode

us
an
M
e d
pt
ce
Ac

Page 72 of 76
 O
H2N
O
NH S NH2
O S
H2N S NH2 N O
S
O N NH N
O

Sulfanilamide (SAM) Sulfadiazine (SDZ) Sulfathiazole (STZ)

O NH2 N
NH H3C
N
S

t
N O O
S
O

ip
N
S NH S NH2
H3C N NH
NH2 O O
Sulfapyridine (SPY) Sulfamerazine (SMR) Sulfamethizole (SMT)

cr
CH3
CH3 H3C
NH2 O
NH2

us
O O
N O O NH
O H3C S
S
N S N N O
H3C N NH
O N NH

Sulfamethazine (SMZ) Sulfamethoxypyridazine (SMP)

an O

Sulfadoxine (SDO)
NH2

H3C O
M
O N NH
S NH2
NH2
O Cl
O
N N O N O
N OH
H3C S N S
O NH
d

OH N NH
O O
e

Sulfadimethoxine (SDM) Sulfasalazine (SSZ) Sulfachlorpyridazine (SCP)

pt

N
H3C
H3C O H2N
NH2
O CH3
O
ce

O
O H3C NH S NH2
S S
N NH NH O
O O
O
Ac

Sulfamethoxazole (SMX) Sulfisoxazole (SSA) Sulfacetamide (SAA)

CH3
O CH3
N NH O
NH2
S O
NH
O N O S
N N
NH2 S N O
H3C N NH S
O
NH2
Sulfaquinoxaline (SQX) Sulfadimidine (SDD) Sulfametrole (SML)

Fig. 1. Structures of the most commonly used sulfonamides.

Page 73 of 76
 N
%
3000 14

12
2500

10
2000

8
1500
6

t
ip
1000
4

500

cr
2

0 0

us
93

95

97

99

01

03

05

07

09

11
Year
19

19

19

19

20

20

20

20

20

an 20

Fig. 2. The annual production of publications with titles, abstracts or keywords

M
containing the term “sulfonamide” (■ histogram) and the percentage of these articles
containing the term “sulfonamide” in the title (-♦- line).The data were derived from
Scopus (on December 2013).
e d
pt
ce
Ac

Page 74 of 76
 Other Milk
19% 23%

Eggs

t
8%

ip
cr
Honey Meat
10% 16%

us
Fish Chicken
11% 13%

an
M
d

Fig. 3. Types of food samples that have usually been analysed for sulfonamides over the
e

past 5 years.The data were derived from Scopus (on December 2013).
pt
ce
Ac

Page 75 of 76
 Biosensors Fluorimetry
3% 3%
Spectro-
photometry,

t
Colorimetry

ip
9% HPLC-MS(/MS)
38%
Immunoassays

cr
10%

us
Electrophoresis
15%
an
HPLC-other
22%
M
e d
pt
ce

Fig. 4. Analytical methods that have usually been applied to the detection of
Ac

sulfonamides over the past 5 years.The data were derived from Scopus (on December
2013).

Page 76 of 76