Chinese Journal of Animal Nutrition 2009, 19(5)

Simultaneous Measurement of Vitamin A, D

3
and E
in Fish Tissues by HPLC
TAN Qing-song
1
, FU Jie
2
, and HE Rui-guo
3*

(1. Fisheries College, Huazhong Agricultural University, Wuhan 430070, China; 2. Jiangxi Great Feed Industry Co., Ltd.,
Nanchang 330038, China; 3. Animal Science and Technology College, Huazhong Agricultural University, Wuhan 430070)
Abstract: A method for simultaneous determination of fat-soluble vitaminsA, D
3
and Ein fish tissues by HPLC is
presented. The sample preparation procedure, consisting of saponification and extraction by mineral ether was carried out.
The following chromatographic conditions were used: VIRIAN Res Elut 5 Cl8 90A column, mobile phases consisted of
methanol-water gradient elution with 1.0 ml /min flow velocity. All the vitamins were detected under the wave length of 292
nm. The linearity range between peak area and vitamin content was 0-15g/ml for vitamin A, 0-10g/ml for vitamin D
3
and
0-20g/ml for vitamin E. The lower limits were 0.810
- 3
ng for vitamin A, 0.0610
- 3
ng for vitamin D
3
and 9.010
-3
ng for
vitamin E, respectively. The fat-soluble vitamins A, D
3
and E in fish tissues could be effectively separated under the
described conditions and simultaneously determined by HPLC at 292 nm after mineral ether extraction.
Key words: HPLC; Vitamin A; Vitamin D
3
; Vitamin E; Fish tissue

Fat soluble vitamins, which are essential nutrients to maintain normal body metabolism and functions,
are vital for animal growth, skeleton and vision development, reproduction and immune function. In
aquatic animals, many studies have been carried out on vitamins nutrition. The vitamin requirements are
usually determined by the maximal vitamin content in fish tissue
[1]
. Thus, it is important to study the
method for vitamin assaysimplifying the sample treatments and improving the efficiency of detection.
Many methods for vitamin assay have been developed, such as chemical analysis, colorimetry, molecul
fluorometry, gas chromatographic techniques, high performance liquid chromatographic (HPLC)
techniques and microbiology method
[2-4]
. In these methods, the HPLC method has been most often used
for its superiorities, such as high sensitivity, good reproducibility, simplified operation and simultaneous
determination of various vitamins. For fat-soluble vitamins assay, the HPLC techniques are the most
convenient method. Methods for simultaneous measurement of various fat-soluble vitamins by HPLC with
fluorescence or UV-Vis detection have been published in national and international journals, mainly for
serum sample
[4-9]
, and insufficient reports in feed and food samples
[10,11]
. The method for simultaneous
determination of vitamins A, D
3
, E in animal tissue by reversed HPLC has seldom been reported.
To simplify the operation and improve the assay efficiency, present work aimed to develop a rapid,
sensitive and convenient method for simultaneous determination of fat-soluble vitamins A, D
3
and E in
animal tissues by an one-step extraction and HPLC analysis, which is of signality to fish physiology and
animal nutrition research.
1 Materials and methods
1.1 Materials
1.1.1 Instruments
High performance liquid chromatograph (VARIAN) consisted of a solvent pump (ProStar 230), a
photoelectricity diode array detector (ProStar 330) and ProStar work station. Reversed- phase
chromatographic column Res Elut 5 Cl8 90A (4.6 x150mmVarian, 12159012) was used.
1.1.2 Chemicals and Reagents
Vitamin A (all-trans-Retinol, R2500), vitamin E (-Tocopherol, T3251) and vitamin D
3
(Cholecalciferol,
47763) were purchased for Sigma Chemical Co. (USA). Ethanol and methanol were
Author information: Tan Qing-song, male, doctor, E-mail: tanqs2000@163.com
*
Corresponding author: He Rui-guo, professor, E-mail: qstan@hotmail.comTAN Qing-song et al.: Simultaneous
Measurement of Vitamin A, D3 and E in Fish Tissues by HPLC
HPLC-grade. NaOH solution was prepared by dissolving 500g of NaOH in 1L of double-distilled
water. The 10 g/L ascorbic acid solution was prepared as follows: Dissolving 1.0 g of ascorbic acid in 4 ml
of hot distilled water, then diluted to 100 ml with ethanol (made up just before using). The phenolphthalein
indicator was prepared by dissolving 10 g of phenolphthalein in ethanol then diluted to 1 L. Mineral ether
was sulfonated by concentrated sulphuric acid as follows: 100 ml mineral ether was added to a separating
funnel with volume of 150 ml, and washed twice with 10 ml of concentrated sulphuric acid, then washed
with saturated solutions composed of 10% sulphuric acid and KMnO
4
untill the purple color in water layer
keep constantly, then washed twice with distilled water. The washed mineral ether was dried with
anhydrous calcium chloride for 1h and then distilled. The ultrapure water and nitrogen gas (99.9%) was
used. The other reagents used were analytical reagent.
1.1.3 Tested samples
Hepatopancreas and muscle tissue of rice field eel were used as tested samples.
1.2 Working standard solutions
1.2.1 Stock standard solutions
1Vitamin A stock standard solution was prepared by dissolving 11.0 mg of vitamin A (all-trans-Retinol)
in ethanol and diluting to 50 ml to provide a concentration of 220 g/ml, then was stored at 4.
2Vitamin D
3
stock standard solution was prepared by dissolving 10 mg of vitamin D
3
(Cholecalciferol)
in ethanol and diluting to 50 ml to provide a concentration of 200 g/ml, then was stored at 4.
3Vitamin E stock standard solution was prepared by dissolving 11.0 mg of -Tocopherol in ethanol and
diluting to 50 ml to provide a concentration of 200 g/ml, then was stored at 4.
1.2.2 Mixed stock standard solution of vitamins A, D
3
and E
The mixed stock standard solution was prepared to provide concentrations of 22 g/ml vitamin A,
10g/ml vitamin D
3
and 50 g/ml vitamin E: 5 ml of vitamin A stock standard solution, 12.5 ml of vitamin
E stock standard solution and 2.5 ml of vitamin D
3
stock standard solution were accurately measured and
mixed, en diluted with ethanol to the final volume of 50 ml.
1.2.3 Calibration curve
The working standard solutions with vitamin concentrations ranged from 0.10 to 20 g/ml were prepared
by appropriate dilution of the mixed stock solution with ethanol, and filtered through a 0.45m membrane
before being injected into the system. Then, each of the working standard solution with the volume of 20
m was injected to the system and determined. The calibration curve was accessed by setting the vitamin
mass in X-axis and the peak area in Y-axis.
1.3 Sample treatments
1.3.1 Saponification
The hepatopancreas and muscle tissue were fully homogenized, then sampled and weighed accurately to
0.0001 g. Each of the weighed samples was put into a stoppled erlenmeyer flask, then 30 ml of anhydrous
alcohol and 10 ml of 10 g/L ascorbic acid solution, 10 ml of 50 g/L sodium hydroxide solution were added.
Then the solutions in the stoppled erlenmeyer flasks were mixed and saponified in 70 for 30 min.
During the saponification, the flasks were vibrated constantly, avoiding the sample sticking to the flask
wall. After saponification, the flasks were immediately cooled to 40 by water flow.
1.3.2 Extraction
The saponified mixture was transferred to a separating funnel (Volume: 250ml). The flask was washed
three times with 60 ml of ultrapure water and the washings were transferred to the same funnel, too (Note:
The volume ration of ethanol to water in the funnel should be larger than 1). The mixture was fully mixed
and 50 ml of mineral ether was added. Again, the mixture in the separating funnel was fully vortexed for
2min and placed to demix. The water phase was transferred to another separating funnel and extracted with
50 ml of mineral ether. The water phase was transferred to the third separating funnel and extracted with
50ml of mineral ether once more. Then the water phase in the third funnel was discarded and the mineral
Chinese Journal of Animal Nutrition www.chinajan.com
ether phase in the three separating funnels were put into the first separating funnel together. The combined
ether phases were washed three times, each with 50 ml of water saturated with mineral ether. When first
washing, the mixture should be vortexed slightly to avoid emulsification. And after the last washing, the
water phase in the funnel should be neutral, tested by the phenolphthalein indicator. Then, the mineral
ether extractive was dehydrated by anhydrous sodium sulfate. At last, the dehydrated ether extractive was
transferred to a brown volumetric flask and 100mg of BHT was added to the extractive to avoid oxidation
of vitamins in the following operations, then diluted with mineral ether to a final volume of 250 ml (V
1
).
All the operations should be conducted in a photophobic fume cupboard.
1.3.3 Condensation
A given amount of samples (V
2
) was measured from the prepared mineral ether extractive, then put into a
rotatory evaporator to evaporate the mineral ether in a water bath at 50 . Then the vitamins were dried
with nitrogen gas and dissolved with 1 ml of ethanol. The solution was then filtered through a 0.45m
membrane and injected into the system for analysis.
1.4 Analysis procedure
1.4.1 Appropriately graded dilution of the stock standard solutions was conducted and the dilutions were
injected into the system to determine the remain time and the optimal concentrations of the vitamins.
1.4.2 Following the procedure of 1.2.3, appropriate concentrations of various mixed working standard
solutions were prepared to determine the degree of separation of the three vitamins and the calibration
cureve between the peak area and the vitamin mass for each vitamin.
1.4.3 Test for recoveries of retinol, -tocopherol, and cholecalciferol before applying the proposed
methods: A given amount of vitamin standards were treated as the procedure same to the tested samples,
and the recoveries of the three vitamins were tested.
1.5 Calculations
The contents of various vitamins in the tissue samples were calculated as the following equation
(Equation 1):
Of which, C is the amount (IU or mg) of vitamin A, D or E in 1 kg of sample; m is the sample mass (g); V
1
(ml) is the total volume of each vitamin extractive; V
2
(ml) is the volume of the sampled extractive for
analysis; V
3
(m1) is the final volume of the tested sample solution after dried and redissolved; (g/m1) is
the concentration of the working standard solution of the tested vitamin; V
st
(l) is the volume of the
injected working standard solution; V
4
is the final volume of the injected tested sample solution; S
st
is the
peak area corresponding to the volume of the injected working standard solution V
st
; S
1
is the peak area
corresponding to the volume of the injected tested sample solution (V
4
); f is the conversion coefficient,
1IU vitamin A=0.3g all-trans-retinol, 1IU vitamin D
3
=0.025g cholecalciferol.
2 Results and discussion
2.1 Sample extraction
Methods for the extraction of vitamins ADE from animal tissues have been reported in several
papers
[7,12]
.Because of lower concentrations of vitamin D in the fish tissue, greater weight (1g) of fish
tissue and larger volume of KOH solution to meet the requirement of sample saponification were used in
the present study, in contrast with the method by Lpez-Cervantes et al.
[12]
.
2.2 Chromatographic conditions for sample separation
For better separation of vitamins A, D
3
and E, the chromatographic conditions were studied and selected as
follows: The injection volume was 20l; Mobile phases were the mixture of methanol and water with a
gradient elution, as showed in Table 1. Res Elut 5 Cl8 90A (4.6 x150mmVarian, 12159012) was used;
Detection was at 292 nm; The flow rate was 1.0 ml/min. Sensitivity was 0.05AUFS. As shown in Fig.1,
the three vitamins were well separated under the conditions. fVVmSVVVC42stst311S TAN
Qing-song et al.: Simultaneous Measurement of Vitamin A, D3 and E in Fish Tissues by HPLC
Table 1 The mobile phases at
different time in the analysis
(Volume ratios) Timemin
MethanolWater
0 955
7 955
15 1000
20 955