Iran. J. Chem. Chem. Eng. Research Article Vol. 39, No.

4, 2020

Determination of Fat-Soluble Vitamins A, D3, and E

in Infant Formula and Milk Powder
Using High-Performance Liquid Chromatography
with Photodiode Array Detection: Jordan Market as a Case Study

Alali, Feras*+
Pharmaceutical Sciences College of Pharmacy, QU Health, Qatar University, QATAR

Amayreh, Mohammad●
Department of Chemistry, Mut’ah University, Al-Karak 61710, JORDAN

Massadeh, Adnan
Department of Medicinal Chemistry and Pharmacognosy, Jordan University of Science and Technology,
Irbid 22110, JORDAN

El-Alali, Abdullah
Department of Chemistry, Mut’ah University, Al-Karak 61710, JORDAN

ABSTRACT: A new, simple, rapid, and sensitive reversed-phase High-Performance Liquid

Chromatography-Photodiode Array (HPLC-PDA) method was developed and validated for the
simultaneous analysis of fat-soluble vitamins A, D3, and E. The method required a simple sample
preparation step of saponification with aqueous KOH and extraction with n-hexane. The method
was validated in terms of linearity, accuracy, precision, stability, detection limits, and recovery.
The method has the advantage of simultaneous determination of vitamins A, D 3, and E in a short run
time of 10 min. The method was applied successfully for the determination of vitamins A, D3, and E
in some infant formula and milk powder from the Jordanian market. Based on this study,
vitamin Content in all brands was within 90.0-410% of the labeled value. Vitamin D3 content
in the studied brands was within 100.0-850%, while vitamin E content in all brands was less than 48%.
The results showed large variation and discrepancy of most vitamins’ contents, which were not
in good agreement with the manufacturer label value, though they were below toxic levels.

KEYWORDS: Fat-soluble vitamins; Infant formula; Milk Powder; HPLC-PDA.

* To whom correspondence should be addressed.

+ E-mail: feras.alali@qu.edu.qa
● Other Address: Department of Chemistry, Faculty of Science, Al-Balqa Applied University,P.O.Box 19117, Al-Salt, JORDAN
1021-9986/2020/3/173-181 11/$/6.01

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INTRODUCTION of origin. The Jordan Food and Drug Administration

Human milk is commonly believed to be complete finds practical difficulty in analyzing vitamins in every
and perfect food for human infant, with breast feeding batch and from all sources. Some brands are also
is the most important mode of delivery [1,2]. Moreover, smuggled into the country, which because of their
breast milk may also protect infants against certain cheaper prices, attract poor and middle class section of
diseases; while it provides complete nutrition when the society. Thus, this study was aimed to develop an
feeding is successfully established [1-4]. accurate, sensitive, and simple analytical method utilizing
Infant formulas are designed as nutritionally complete a simple extraction procedure and reverse phase HPLC
feeds to replace breast milk. This is may be for reasons coupled with diode array detector (PDA) for
of convenience, illness of the mother or inadequate simultaneous determination of the fat-soluble vitamins A,
supply of breast milk. These formulas are soy and cow's D3 and E in infant formula, milk powder, and cereal food.
milk- based formulas to mimic the composition of mature The measured values were compared with the specified
breast milk. However, no formula can provide fully amounts on the manufacturer label. We believe this study
the whole benefits of breast milk [1,2,5]. will increase the public and government awareness
According to the global standard for the composition of this particular vital issue.
of infant formula; infant formula must contains energy,
proteins, lipids, carbohydrates, vitamins, and minerals [6]. EXPERIMENTAL SECTION
Fortified food such as milk powder and infant formula General
are usually supplemented with vitamin A Analysis was performed using a Merck–Hitachi
in the form of synthetic retinyl acetate or retinyl HPLC, (Merck–Hitachi, Tokyo, Japan) equipped with
palmitate[7].The acetate ester of d-α-tocopheryl acetate L-7150 isocratic pump, solvent degasser L-7612,
(RRR-α-tocopheryl acetate) form is usually used autosampler L-7200, diode array detector L-7455,
in the fortification of foods[8,9]. However, vitamin D is added interface D-7000 and D-7000 HSM. The analytical
to food in the form of cholecalciferol. Milk continues chromatographic column was a reversed-phase C18, 250
to be the food of choice for vitamin D fortification, mm (length) × 4.6 mm (internal diameter) packed with 10 μm
due to the presence of calcium [10]. particles (Waters, Dublin, Ireland). Samples were dried
The Recommended Dietary Allowance (RDA) of using RE 200 rotary evaporator (Bibby, Liverpool, UK).
fat-soluble vitamins[11,12] are listed in Table 1. Samples were sonicated using Eurosonic 22 Sonicator
The addition of vitamins to certain foods, (Eurosonic Neumann GmbH, Kelten Germany). Digital
fortification, is a common practice in most micro pipettes (1000-100 μL and 100-10 μL)
countries[13,14]. In other cases, vitamins are added (witegLabortechnik GmbH, Wertheim, Germany).
to restore the vitamin content to that originally present Glass syringes (100 and 10 μL) were obtained from
before processing. Many studies were conducted Hamilton (Hamilton, Bonaduz, Switzerland). A filtration
to calculate the amount of fat-soluble vitamins added unit for mobile phase degassing was obtained from Schott
to milk powder and infant formula in comparison with Duran,
the labeled values. Several studies in the USA showed Samples were weighed using an AT Delta Range,
that milk and infant formulas rarely contain the same Mettler Toledo analytical balance (Mettler Toledo,
amount of vitamin D stated on the manufacturer label; Switzerland).
documented fortification errors in fortified milk products L-(+)-ascorbic acid and potassium hydroxide pellets
across the US milk industry were reported [15,16]. were obtained from Lonver House, England and
In other studies, hypervitaminosis D was shown Scharlau, Spain. Water was purified using Seradest S600,
to be resulted from drinking milk which is incorrectly water purification systems D-5412, Ransbach,
and excessively fortified with vitamin D [17-19]. Thus, Baumbach. Methanol (HPLC grade), n-hexane (analytical
the fortification process must be carefully monitored. grade), ethanol absolute (analytical grade), all
Jordan market hosts varieties of powdered milk and were obtained from Scharlau Chemie S.A, Spain. Standard
infant formulas of different manufacturers and countries vitamin A (all-trans-retinol) 99.0% purity for HPLC

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Table 1: Recommended Dietary Allowance of vitamin A, D3 and E(Units/day).

Age Vitamin A(µg RE)11 Vitamin D (µg)11 VitaminE(mg/g) PUFA12

0-3 and 4-6 months 350 8.5-10 0.4

7-9 and 10-12 months 350 8.5-10 0.4

1-3 years 350 10 0.4

*RE, retinol equivalents; PUFA, polyunsaturated faty acids.

analysis was purchased from Fluka Chemie GmbH, USA, for 30 min at 50 °C with frequent shaking, then it was left
vitamin E (dl-α-tocopherol) 98.0% purity for HPLC for 5 min in cold water, afterwards, the content
analysis from Applichem Biochemica Synthesis Services, was transferred to 250 mL separatory funnel. A volume of
Germany, and vitamin D3 (cholecalciferol) 99.0% purity 25 mL of n-hexane were added, followed by vigorous
for HPLC analysis from ACROS, Europe. shaking for one min and phase left to separate for 5 min;
this step was repeated three times, ratio of sample-
Samples' collection extraction solvent (w/v, g/mL)1:10. Organic phases
Two different batches of eight brands of infant were combined into 100 mL round-bottomed flask.
formula, and three brands of full cream milk powder The collected solvent was evaporated in a rotary evaporator
were collected randomly from Jordanian local markets. at 50 °C under vacuum. The residues were then dissolved
Brands of infant formula and full cream milk powder in 2 mL methanol, filtrated by 0.45 μm Teflon filter and
were cow's milk-based formula. transferred into 2 mL amber HPLC vials. An aliquot of
50 μL was injected into HPLC within 1 h. Three
Chromatographic conditions extraction replicates were prepared for each batch of
The optimized mobile phase was an isocratic blend samples.
of methanol and water (95:5, v/v) with a 2 mL/min flow Standard stock solutions of the vitamins: retinol
rate, monitoring at 280 nm (optimum absorbance (vitamin A) (500 μg/mL), cholecalciferol (vitamin D3)
for the detection of vitamins A, E, and D3, simultaneously), (100 μg/mL), and α–tocopherol (vitamin E ) (500 μg/mL)
and a total analysis time of 10 min. were prepared by accurately weighing 5, 10 and 50 mg of
vitamins A, D3 and E reference standards into 10, 100
Standard solutions and samples preparation and 100 mL volumetric flasks, respectively. In sequence,
Milk powder and infant formula samples were kept a volume of 2, 5 and 5 mL of absolute ethanol were
in a refrigerator at 4 °C to avoid temperature and light added to each flask to aid solvation. Afterwards, each
degradation. Portion, 2.50 g (±0.01) of each sample flask was filled to the mark using methanol. The standard
was accurately weighed and placed into 100 mL Erlenmeyer stock solutions were stored in a refrigerator at -20 °C
flask, a volume of 25 mL of hot water (50 °C) for a period of not more than one week for vitamins A and
were added, followed by the addition of 0.8 g of ascorbic acid D3 and two weeks for vitamin E. All standards
as antioxidant to avoid vitamins oxidation. The solution were protected from light during the storage to minimize
was swirled for 5 min to assure good mixing. A volume degradation.
of 5 mL aqueous KOH (60% w/v) and 15 mL of absolute
ethanol were added to the mixture. The Erlenmeyer flask Calculations
was tightly closed to prevent air oxidation of vitamins Vitamins A and D3 content in milk powder and infant
during the saponification process, which depends formula were calculated per 100 g milk powder using
on the saturation degree of ethanol vapor. The Erlenmeyer the following equation: Content (µg/100 g) = C × FV ×
flask was covered by dark sheet to protect from light. Wl/Ws. While vitamin E content in milk powder and
All steps were carried out in dark place. The saponification infant formula were calculated by the following equation:
step converts all the ester forms of vitamin A and vitamin E Content (mg/100 g) = C × FV × Wl/Ws ×1/1000; Where,
to parent alcohol (Fig. 1). The mixture was then sonicated C: is the sample's vitamin content (μg/mL) extrapolated

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Fig. 1: Hydrolysis of vitamin A palmitate (1, A) and vitamin A acetate (2, A) and vitamin E acetate (B).

from calibration curve's linear regression; FV: is the final using reversed phase liquid chromatography. A reverse
volume of the sample extracted (mL); Wl: is the weight phase and isocratic system of methanol-water (95:5 v/v)
of milk powder (which is equal to 100 g); Ws: is using HPLC-PDA was adapted. Flow rate of 2 mL/min
the weight of portion sample (g) (which is equal to 2.5 g). and a run time of 10 min were set as a result of
The values obtained from the equations were normalized optimization process. In order to improve baseline level,
by the % recovery measured for each vitamin. an injection volume of maximum 50 μL was used.
The recovery for vitamins A, D3 and E were 95.33, 73.77 The three vitamins were investigated for maximum absorption
and 83.47%, respectively. Vitamin E was calculated using PDA. A compromise wavelength of maximum
as α-tocopherol, so the value in mg was multiplied by 1.2 absorption at 280 nm for the three vitamins was selected.
to account for other vitamins that are present which gives Identification of vitamins in the samples depended
an approximation value for total vitamin E activity as mg on the retention times of standard vitamins. Standard vitamins
of α-tocopherol equivalents [9]. and milk powder samples were first injected into HPLC
to check the resolution and retention time variability
RESULTS AND DISCUSSION (Fig. 2 and Fig. 3).
Development and validation of HPLC-PDA method
for vitamins A, D3 and E analysis Method validation
Method development The method was validated in terms of linearity,
The main aim of the chromatographic system precision, recovery, detection limits, and stability
developed was to obtain better resolution, faster and according to the Food and Drug Administration (FDA)
simultaneous analysis of fat-soluble vitamins A, D3 and E guidelines for analytical method validations [20].

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0.08 0.028

0.07 0.020
Absorbance (a.u.)

0.06 0.018
0.05
0.010
0.04
0.008
0.03
0.02 0.000

0.01 -0.008
0.00 -0.001
0 1 2 3 4 5 6 7 8 9 10 11 12 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Retention time (min)

Fig. 2: A chromatogram for the standard solutions of Fig. 3: A chromatogram of fat-soluble vitamins in milk
fat-soluble vitamins: vitamin A (1) (2.79 min):5.0µg/mL, powder: vitamin A (2.72 min), Vitamin D3 (5.55 min),
vitamin D3 (2) (6.09 min):4.0µg/mL and vitamin E (3) (7.36 min): and vitamin E (7.36 min).
3.0µg/mL; retention time varied within 0.5 min.

Linearity saponification, extraction with n-hexane, evaporation

Reference standard solutions for each vitamin and analysis by HPLC. Water as a blank was used since
were prepared. Triplicate injections of each preparation milk powder matrix without fat-soluble vitamins were not
from the reference standard were made. Measuring peak available. The average recoveries for vitamins A, D3 and E
height, a linear calibration curve was constructed for each were 95.33±1.25, 73.77±1.91 for and 83.47±4.86,
with regression coefficient (r2) values of 0.9983, 0.9998 respectively.
and 0.9984 in the ranges of 0.5-50 μg/mL, 0.05-10 μg/mL
and 10-200 μg/mL for vitamins A, D3 and E, respectively Detection limits
, as shown in Table 2. The detection limits (DL) for vitamins A, D3 and E
In order to check the accuracy, three quality control were found to be 0.1, 0.01, and 0.1 μg/mL, respectively.
points at 4, 20, and 40 µg/mL for vitamin A, 2, 4 and 8 µg/mL The Quantitation Limits (QL) were determined by
for vitamin D3, and 40, 70 and 120 µg/mL for vitamin E choosing the lowest quantifiable concentration on the
were injected each month for a period of five months. calibration curve. Thus, the QL for vitamin A, D 3 and E
The accuracy for each vitamin was found to be within were found to be 0.3, 0.03, and 0.3 μg/mL, respectively.
15% during this period.
Stability
Precision Stability data of standard vitamins A, D3 and E showed
The inter- and intra-day precision was tested that the standard solution of vitamin A has a noticeable
by successive injections with 5 replicate determinations degradation by 10% after 6 h, 15.50% after 16 h and 42.60%
for two different concentrations of each vitamin. after 24 h at room temperature. Vitamin D3 showed
The repeatability is expressed as relative standard a noticeable degradation of 12.80% after 6 h, 16.8% after 16 h
deviation (RSD) as shown in Table 3. and 22.09% after 24 h at room temperature. Vitamin E
showed no significant degradation after 6 h, 8.28% after 16 h
Recovery and 29.70% after 24 h (Table 4).
Recovery was tested using a blank of an ultra-pure The slight increase in RSD values observed
water (deionized water) which was spiked with a known for vitamins A, D3 and E can largely be attributed to the
concentration at 30.0, 20.0 and 15.0 μg/mL for vitamin A, delicacy of vitamins analysis. RSD values were 2.4-19.70,
10.0, 5.0 and 2.0 μg/mL for vitamin D3, and 40.0, 30.0 6.14- 39.76 and 0.34-41.36 for vitamins A, D3 and E,
and 20.0 μg/mL for vitamin E, respectively. These known respectively. Relative standard deviations data
concentrations were subjected to the following procedure: were found to be comparable to the RSD values reported

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Table 2: Linearity parameters for vitamin (A, D3 and E).

Vitamin Concentration range (μg/mL) b* R² §

A 0.5-50 1623 0.9983

D3 0.05-10 1352.2 0.9998

E 10–200 149.26 0.9984

* §
slope, regression value.

Table 3: Precision of the HPLC method for determination of vitamins A, D3 and E in infant milk and milk powder.
Vitamin Con. (μg/mL) RSDs %

30 0.86-4.21
A
20 0.64-6.84

8 0.56-3.97
D3
4 0.79-3.19

E 160 0.17-3.16

Table 4: Stability data of standard solutions of vitamins A, D3 and E kept at room temperature.
Time intervals (hour) Vitamin A (30 μg/mL)* Vitamin D3 (2 μg/mL)* Vitamin E (40 μg/mL)*

0 30.45 1.72 41.66

3 27.57 1.66 41.24

6 27.40 1.50 40.96

16 25.73 1.44 38.21

24 17.49 1.34 29.29

*
Initial concentration

by other workers in the world. RSD values of 3.88-38.46 one of the three brands (33.33%) of milk powder contained
and 3.45-52.83 for vitamins D3 and E, respectively, less than 80% of the amount stated on the label. Two of
were reported [21]; RSD values of 2.88-39.13 and 3.33-37.78 the 8 brands (25%) of infant formula and none of the 3
for vitamins D3 and E, respectively[22]; RSD values of brands of milk powder contained more than 300%
3.27-7.16 and 5.50-10.20 by applying the AOAC method, of the amount stated on the label (Tables 5 and 6).
6.69-9.23 and 2.81-3.65 by applying the proposed For vitamin D3, none of the 8 brands of infant formula,
method for vitamins A and E, respectively [23]. and 2 of the 3 brands (66.67%) of milk powder contained
80-120 percent of the labeled value. None of the 8 brands
Samples analysis of infant formula, and none of the 3 brands of milk powder
The collected samples of infant formula, milk powder, contained less than 80% of the value stated on the label. Six of
and cereal food from the local market in Jordan the 8 brands (75%) of infant formula, food and none
were analyzed using the developed and validated method. of the 3 brands of milk powder contained more than 300%
Each run was carried out in triplicate and the results of the value stated on the label (Tables 5 and 6).
obtained are given in Tables 5 and 6. For vitamin E, none of the 8 brands of infant formula,
In our study, only 1 of 8 brands (12.5%) of infant and none of the 3 brands of milk powder contained
formula, and 1 of the 3 brands of milk powder (33.33%) 80-120% of the labeled value. All the brands infant formula
contained 80-120% of the labeled amount of vitamin A. and milk powder contained less than 60% of the labeled
Two of the 8 brands (25%) of infant formula and value (Tables 2 and 3).

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Table 5: A comparison between vitamin A, D3 and E measured content (mean ± SD) with label values for the samples tested.

Vitamin A Vitamin D3 Vitamin E

Sample Name Label
Measured value Label value Measured value Label value Measured value
value
(μg/100g)¥ (μg/100g) (μg/100g) ¥ (μg/100g) (mg/100g) ¥
(mg/100g)
Infant formula

A 2500±100 480 40±4 8.2 1.72±0.16 4.6

B 430±100 473.10 67±31 7.88 1.68±0.11 10.74

C 2400±100 520 47±11 7.5 1.07±0.18 4.03

D 370±100 600 28±3 10 0.63±0.33 4.03

E 300±100 470 25±7 12 0.69±0.64 4.56

F  540 38±7 7.75 4.12±0.09 13.42

G  570 45±5 8.75 0.55±0.18 3.36

H 250±100 510 38±5 9.75 0.33±0.11 4.56

Full Cream Milk Powder

A 700±100 630 13±6 12.5 —§ —¤

B 1800±100 630 14±1 12.5 < 0.96 —¤

C 320±100 540 16±15 5.75 < 0.96 3.36

¥
Measured values are the average values derived from triple experiments, each run in triplicate for each batch of samples.
Can't be observed due to fatty acid overlapping; §Not added; ¤No labeled value.

Table 6: The percentage of vitamins in infant formula, cereal food, and milk powder in comparison with the labeling values.
Sample Name Vitamin A Vitamin D3 Vitamin E

Infant Formula

A 520.0 485.0 37.0

B 90.0 848.0 16.0

C 470.0 630.0 27.0

D 160.0 280.0 16.0

E 64.0 200.0 15.0

F —* 490.0 31.0

G —* 510.0 16.0

H 49.0 390.0 7.2

Full Cream Milk Powder

A 110.0 100.0 —§

B 290.0 110.0 —§

C 60.0 280.0 17.0

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There were a significant variation in the vitamins samples and among different batches of the same
content of the studied brands, infant formula and milk samples. Morover, there was disagreement between
powder. For vitamin A, the highest and lowest measured the vitamins’ content found and those typed in the
values compared to the manufacturer labeled values fall manfugacturerer label, though they were still beyond
in the range 34.52-544.20% (Tables 5 and 6). This is still the toxic levels. Errors in the fortification process, incorrect
far away from the toxic level which is 25 times of the label value and fatty acid content may contribute to some
Recommended Daily Allowance (RDA). For vitamin D3, significant differences in vitamins’ content in infant
the vitamin content was significantly higher than the label formula and milk powder. In light of the findings of the
amount in all brands of infant formula. The range of current study, it is highly recommended that heath
vitamin D3 was 103.60-1177.60% of the label values. authorities must analyze fortified infant products for their
Vitamin D3 content measured in our study is still far content of vitamins A, D3, and E.
below the toxic level which amount to 50 times the RDA.
A factor to worry about regarding higher values of Acknowledgments
vitamin D3 is the amount of infant formula consumed We are grateful to Dr. Tamam El-Elimat (Jordan
by the infants. University of Science and Technology, Department of
Vitamin E content in all brands was significantly Medicinal Chemistry and Pharmacognosy) for reviewing
below the labeling value with a range of 7.24-58.48% and proofreading the manuscript. The authors would like
(Tables 2 and 3). to thank Mut’ah University and Jordan University of
The significant differences in vitamin content Science and Technology for financial support.
observed for the infant formula with respect to the stated
values might be due to an error in the fortification process Received : Oct. 13, 2018 ; Accepted :Mar. 11, 2019
or incorrect labeled values. Companies report usually the
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Research Article 181