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al Sciences
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ICESB 2011: 25-26 November 2011, Maldives

Determination of Fat-soluble Vitamins in Food and

Pharmaceutical Supplements Using Packed-fiber Solid Phase
Extraction (PFSPE) for Sample Preconcentration/ Clean-up
Liqin Chena, Zhiyong Liub, Xuejun Kanga,, Xiaoling zhoua, shenglan zhenga ,
Zhongze Guc
a

Key Laboratory of Child Development and Learning ScienceMinistry of Education, Research Center For Learning Science,
Southeast University, Nanjing (210096)
b
Beijing Administration for Industry and Commerce, Beijing (100080)
c
State Key Laboratory of Bioelectronics, Southeast University, Nanjing (210096)

Abstract
A novel and rapid method has been developed for separation and preconcentration of fat-soluble vitamins using
packed-fiber solid phase extraction from complex matrices including food and pharmaceutical supplements. The
extraction time is reduced to 5 minutes which considerably shorter than in conventional methods. Other advantages
are the reduction of manual manipulations leading to lower labour costs and reduced consumption of organic solvents
in the sample preparation step.The analytical parameters including linearity, r > 0.9990; limit of detection,
1.0g/mL,0.1g/mL,1.0g/mL and 0.1g/mL for retinyl esters, cholecalciferol, tocopherol acetate and phylloquinone
respectively; precision of the method, R.S.D. < 5% and recovery more than 90%, show that the method is appropriate
for measuring fat-soluble vitamins in food and pharmaceutical supplements sample preparations.

Asia-Pacific
2011
2011 Published
Published by
by Elsevier
Elsevier Ltd.
Ltd. Selection
Selection and/or
and/or peer-review
peer-reviewunder
underresponsibility
responsibility of
of the
ICESB
2011
Chemical, Biological & Environmental Engineering Society (APCBEES)
Keywords: Fat-soluble vitamins; packed-fiber solid phase extraction;

1. Introduction
Fat-soluble vitamins have important roles in several functions of the human body, such as vision

Corresponding author. Tel.: +86-25-8379-5664; fax: +86-25-8379-5929.

E-mail address: xjkang64@163.com.

1878-0296 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the Asia-Pacific Chemical, Biological &
Environmental Engineering Society (APCBEES)
doi:10.1016/j.proenv.2011.10.091

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(Vitamin A), calcium absorption (Vitamin D), antioxidative protection in cell membranes (Vitamin E),
and blood coagulation (vitamin K) [1-4]. These vitamins are substances often found associated in food
and pharmaceutical [5-6]. The widespread use of this kind of food and pharmaceutical supplements
demands simple, fast and reliable method for determination of active compounds in commercial forms [68].
The most commonly used methods of sample preparation for the analysis of fat-soluble vitamins in
foods and pharmaceuticals include sample saponification and later extraction of the vitamins from the
unsaponifiable matter [9-11] or direct solvent extraction of the vitamins from the sample [12-14].
Saponification or alkaline digestion readily causes oxidation of fat-soluble vitamins although some
investigators have tried to overcome this obstacle by adding antioxidants such as butylated
hydroxytoluene [15] and ascorbic acid [16]. Saponification procedures also have the potential to
introduce large variation and have low recovery and reproducibility, in addition to being time-consuming
[4, 8]. Some studies suggest that organic solvents alone do not extract vitamins quantitatively from
vitamin-fortified foods and pharmaceutical supplements. This is due to gelatin beadlets that are often
added in vitamin supplements to enhance their stability [17].
Electrospun nanofibers showed higher adsorption capability due to specific adsorbateadsorbent
interactions for their very high specific surface area, unsaturated atoms and smaller average micropores
[18]. Packed-fiber solid phase extraction (PFSPE) with electrospun nanofibers as packing sorbents for
solid-phase extraction was developed and has been applied in the purification and concentration of
several analytes from complex matrices, such as biological and environmental samples in our group [19].
The objective of this study was to develop a simple, rapid and highly efficient procedure for the
simultaneous assay of fat-soluble vitamins in food and pharmaceutical supplements using one step
extraction without saponification, which is suitable for application to fortified products containing
encapsulated fat-soluble vitamins. Validation parameters for the overall method were determined to
ensure the method validity.
2. Material and methods
All reagents used in this work were of analytical grade, methanol and ethanol were HPLC-grade from
YuWang Group (Shangdong, China) and double distilled water was used throughout the experiments. The
standards used were retinyl esters from Sigma-Aldrich (Shanghai, China), cholecalciferol, tocopherol
acetate and phylloquinone from the National Instituite for the Control of Pharmaceutical and Biological
Products (Beijing, China). Polystyrene (PS, Mw=185,000), Polyacrylonitrile (PANMw=30,000)
dimethylformamide (DMF), tetrahydrofuran (THF), sodium sulfate, sodium chloride and sodium acetate
were obtained from Shanghai chemical agents Institute (Shanghai, China). The pipette tip (200L) was
from Yonghua glasswork (Haimen, China). The procedures for preparing polystyrene (PS), nanofibers
were described detailed in our previous paper [19]and 10% Polyacrylonitrile (PAN) and 10Poly
(styrene-co-methacrylic acid) (PS-COOH) were dissolved in a mixture of DMF and THF (4:6, v/v) for
preparing nanofibers, other procedures were similar to that for preparing PS nanofibers.
The chromatographic system consists of a Shimadzu LC-20A pump and SPD-M20A photodiode-array
detector (Kyoto, Japan). A HPLC software package (Shimadzu, Japan) was used for the data analysis. An
Agilent ZORBAX SB-C18 column was used for HPLC separation (particle size 5m; column dimensions
250mm4.6 mm). The injection volume was 20 L. The mobile phase, 100% methanol, was used at the
flow rate of 1.0ml/min. All the measurement operations were performed at room temperature. Detection
was at 327 nm for retinyl esters, 264 nm for cholecalciferol, 284 nm for tocopherol acetate, and 247 nm
for phylloquinone. The total run time was 20 minutes between each routine injection.
Stock solutions of fat-soluble vitamins were prepared by dissolving appropriate quantity of the

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standards in methanol. These solutions were stored in dark flask at 20 C. The working solutions for
calibration were prepared provisionally by diluting suitable volumes of stock solutions of fat-soluble into
0.5 mol/L sulfate sodium salt. In addition, samples used for the recovery studies of PFSPE were prepared
by adding appropriate aliquots of fat-soluble vitamins into food and pharmaceutical supplements. In all
cases, the methanol concentration in the solution before PFSPE was restricted to 10% (v/v).
Samples used in this study were commercially fortified milk power and multivitamin tablets. All
samples were prepared with protection in subdued light. The PFSPE columns were prepared following
our previous works [19]and it was preconditioned with 100 L of ethanol and 200 L of water. Figure
1 shows the system for the extraction of the analytes from samples. Aliquots of 1.0 g samples (milk
power or multivitamin tablets ) was added with 5.0 mL of ethanol and was diluted with 0.5 mol/L sodium
sulfate to 50 mL, After vortex mixing for two minutes, the mixture was centrifuged at 10,000g for two
minutes. Then the supernatant was loaded and pushed through the sorbent by the pressure of air forced by
a gas tight plastic syringe (2 mL). The flow was carefully controlled in a slow dropwise manner. After 0.5
mL mixture was eluted through the sorbent, the mini-column was washed with 100 L of water, and the
analytes were eluted with 100 L of ethanol. Finally, 20 L aliquot of ethanol extract was analyzed by
high performance liquid chromatography.

Figure 1. Schematic representation of PFSPE device: (1) packed nanofibers; (2) pipette tip; (3) gastight syringe; (4) eppendorf
centrifuge tube

3. Results and discussion

The PFSPE strategy comprises the isolation and concentration of the analytes from complex matrices
by adsorption onto an appropriate sorbent, the removal of interfering impurities simultaneously and then
the selective recovery of the retained analytes with solvent system of suitable elution strength. This
process is necessary to be performed by selection of sorbent and solvent systems, so that the analytes are
retained mostly by a sorbent and the analytes are then recovered in the eluate. Three kinds of nanofibers
including PS-COOHPS and PAN were tested in order to found which nanofiber has the best retention
to fat-soluble vitamins. As presented in Figure 2, PS-COOH nanofiber showed better extraction efficiency
than others. It is suspected that these carboxyl groups improve nanofibers wettability with more affinity
for target compounds. Again from Table 1, 100 L of ethanol were sufficient to elute analytes absorbed
on the nanofibers.

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Figure 2. Influence of different kinds of nanofibers on the extraction. Extraction conditions as follows: a mixed spiked water
solution (0.5 g/mL, 500 L); each kind of nanofiber had an average diameter of 450 nm; no salt addition; eluting solvent, 100 L
of ethanol. retinyl esterscholecalciferoltocopherol acetatephylloquinone.
Table 1 Influence of the eluting solvent and eluting volume on the extraction.

retinyl esters

Relative Recovery
methanol
50
100
200
0.57 0.91 0.92

cholecalciferol

0.60

0.94

0.93

0.74

0.98

0.51

0.82

0.89

Tocopherol acetate

0.55

0.83

0.84

0.65

0.43

0.75

0.80

phylloquinone

0.61

0.92

0.95

0.77

0.54

0.81

0.91

ethanol
50
100
0.69 1

200
1

acetonitrile
50
100
0.46 0.80

200
0.87

Extraction efficiency from samples was influenced by two aspects mostly; loading certain amount of
nanofibers would be needed for absorbing analytes, because trace amounts of fat-soluble vitamins could
not be retained onto the sorbents completely if smaller mass of nanofibers were used. We estimate that
loading 2.0 mg of nanofibers on the PFSPE could retain sub-milligramme of the analytes completely from
Figure 3. In addition, it was investigated that dissolving agents containing salt like sodium sulfate played
an important role on the complete retention of fat-soluble vitamins onto sorbent. The effect of salt added
into dissolving agents has been examined in many other papers [20]. In general, it is a common practice
to add salt to the aqueous samples in order to decrease the solubility of organic compounds in the water
phase, and to increase the partition coefficients of the targets. It was found that sodium sulfate was the
suitable salt for the present and the optimum concentration was 0.5mol/L (as shown in Figure 4).

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Figure 3. Influence of loading amount on the extraction basing on PS-COOH nanofiber. A value of 1 was assigned to the peak area
of PS-COOH nanofibers with 2.0mg, and the rest of the areas were correlated to this value. Extraction conditions as follows: a
mixed spiked water solution (5005.05005.0g/mL for retinyl esters cholecalciferoltocopherol acetate and phylloquinone
respectively500L); PS-COOH nanofiber with an average diameter of 450 nm; no salt addition; eluting solvent, 100 L of
ethanol.

Figure 4. Influence of sulfate sodium salt concentration on the extraction recoveries. Extraction conditions as follows: a mixed
spiked water solution (0.5g/mL, 500L); PS-COOH nanofiber with an average diameter of 450 nm; eluting solvent, 100L of
ethanol.

The new method has been validated under optimized conditions. Linearity was achieved in the
concentration range 0.5500g/mL (r2= 0.9979) for retinyl esters, 0.05-5 g/mL (r2=0.9965) for
cholecalciferol, 0.5-500 g/mL for tocopherol acetate (r2=0.9983) and 0.05-5.0g/mL for phylloquinone
(r2=0.9972) respectively. The LOD calculated as signal-to-noise ratio equal to 3 were: 0.1g/mL for
retinyl esters, 0.01g/mL for cholecalciferol, 0.2g/mL for tocopherol acetate and 0.01g/mL for
phylloquinone respectively.
The reproducibility of the presented method was assessed by analyzing six replicates of the plasma
samples spiked with analytes at three different concentrations (1.0, 50, 100 g/mL) for retinyl esters, (0.1,
1.0, 3.0g/mL) for cholecalciferol,1.0, 50, 100g/mLfor tocopherol acetate and (0.1, 1.0, 3.0g/mL)
for phylloquinone. The average coefficients of variation of within-day and between-day assays were 1.5%
and 4.6% respectively, which were assessed by analyzing duplicate injections for each amount over six

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consecutive days.
Recovery was calculated at (1.0, 50, 100g/mL) for retinyl esters, (0.1, 1.0, 3.0g/mL) for
cholecalciferol,1.0, 50, 100g/mLfor tocopherol acetate and (0.1, 1.0, 3.0 g/mL) for phylloquinone
(n=3) in spiked samples by comparing the peak areas of analytes from extracted samples with those of
external standard in water, which was subjected to PFSPE procedure. The relative recoveries of 91.7% to
104.5% were obtained respectively. Figure 5 shows a typical chromatogram of extraction and separations
of fat-soluble vitamins in different medium by PFSPE. It clearly shows that the fat-soluble vitamins in
food and pharmaceutical supplements could be extracted and separated well.
In order to evaluate the applicability of the proposed method, multivitamin products were analyzed and
results were compared with those reported by manufacturer. As can be seen in Table 2, there is a good
agreement between the results obtained by the proposed method and reported values.
Table 2 Quantification of fat-vitamins in fortified milk power and multivitamin tablets

retinyl esters
cholecalciferol
tocopherol acetate
phylloquinone

Fortified milk powder(IU/100g)

Found
Label
meanS.D. (n = 3)
1353.7
1284.24.0
309.0
286.71.5
a
a
a
a

Multivitamin tablets(mg/g)
Found
Label
meanS.D. (n = 3)
33
31.70.1
0.003
0.0030.0002
9.8
9.30.05
0.025
0.0210.001

a no addition in samples

Figure 5. Typical HPLC extraction and separations of fat-soluble vitamins (1 retinyl esters, 2cholecalciferol, 3 tocopherol acetate, 4
phylloquinone) by PS-COOH nanofiber. (A) spiked water sample; (B) fortified milk powder sample; (C) multivitamin tablets

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sample.

4. Conclusions
This work proposes a new method for the separation and concentration of four fat-soluble vitamins (A,
D3, E, and K1) in sample pretreatment. Packed-fiber solid phase extraction proved to be an effective tool
for performing adequate separation of the four fat-soluble vitamins from complex matrices with satisfied
recoveries ranging from 91.7 to 104.5%. The simplicity of the procedure should make it highly desirable
for quality control of multi-vitamin products in the pharmaceutical and health food industries.
Acknowledgements
This work was jointly supported by Science and Technology Pillar Program of Jiangsu Province
(Grant No.BE2010088), the Scientific Research Foundation of Graduate School of Southeast University,
and Suzhou Science and Technology Department Foundation (Grant No. SYJG0912SYN201006).
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