Chinese Journal of Animal Nutrition

2007, 19(5)

Simultaneous Measurement of Vitamin A, D3 and E in Fish Tissues by HPLC

TAN Qing-song1, FU Jie2, and HE Rui-guo3*
(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 vitamins A, D3 and E in 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 D3 and 0-20g/ml for vitamin E. The lower limits were 0.810- 3 ng for vitamin A, 0.0610- 3 ng for vitamin D3 and 9.010-3 ng for vitamin E, respectively. The fat-soluble vitamins A, D3 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 D3; 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 . 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] [1]

and insufficient reports in feed and food samples [10,11]. The method for simultaneous determination of vitamins A, D3, 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, D3 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. Reversedphase 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 D3 (Cholecalciferol, 47763) were purchased for Sigma

. 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]

Author information: Tan Qing-song, male, doctor, E-mail: tanqs2000@163.com * Corresponding author: He Rui-guo, professor, E-mail: qstan@hotmail.com

Chemical Co. (USA). Ethanol and methanol were

TAN 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 KMnO4 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 D3 stock standard solution was prepared by dissolving 10 mg of vitamin D3 (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, D3 and E The mixed stock standard solution was prepared to provide concentrations of 22 g/ml vitamin A, 10g/ml vitamin D3 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 D3 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 (V1). All the operations should be conducted in a photophobic fume cupboard. 1.3.3 Condensation A given amount of samples (V2) 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):

S 1 V 1 V 3 V st S st m V 2 V 4 f

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); V1 (ml) is the total volume of each vitamin extractive; V2 (ml) is the volume of the sampled extractive for analysis; V3 (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; Vst (l) is the volume of the injected working standard solution; V4 is the final volume of the injected tested sample solution; Sst is the peak area corresponding to the volume of the injected working standard solution Vst; S1 is the peak area corresponding to the volume of the injected tested sample solution (V4); f is the conversion coefficient, 1IU vitamin A=0.3g all-trans-retinol, 1IU vitamin D3=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 For better separation of vitamins A, D3 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. separation

TAN Qing-song et al.:

Simultaneous Measurement of Vitamin A, D3 and E in Fish Tissues by HPLC

with the vitamins mass: the regression equation

Table 1 The mobile phases at different time in the analysis (Volume ratios) Timemin 0 7 15 20 MethanolWater 955 955 1000 955

relating peak area (y) to injected amounts (x, g) of vitamin A,

-5

D3

and

were

generated

as

y=3.878810 x-0.0339 (r=0.9999; n=3) in the tested range of 0-15g/ml; y=1.238010-4x-4.122510-3 (r=0.9999; n=3) in the tested range of 0-10g/ml, and y=3.187410-4x 0.4091 (r0.9998; n=3) in the tested range of 0-20 g/ml, respectively. The detection limits under the described conditions were 0.810-3 ng for vitamin A, 0.0610- 3 ng for vitamin D3, and 9.0 10-3 ng for vitamin E with a sensitivity of 0.05 AUFS and a

VA VE

VD

signal-to-noise ratio of 3. 2.4 Quantification and recovery The vitamins A, D3 and E in the tissue samples of rice field eel were separated after saponification and extraction with mineral ether. The separation of the three vitamins in fish tissue was shown in Fig. 2. As shown in Table 2 and Table 3, the results

Fig. 1 The chromatography of the three vitamins in standard solution.

assayed in fish samples and the recoveries of vitamins before applying the proposed methods showed reasonable. The present study showed that vitamins A, D3 and E in fish tissue can be simultaneously determined by HPLC at 292 nm with UV-Vis detecter after mineral extraction, which is effective and fast, enabling the processing of a large number of samples.
Table 2 Analytical results of the samples (n=3) Vitamins samples Vitamin Ag /g Vitamin D3 (g /g) Vitamin E (g/g) in the Concentration 21.33 0.19 16.46 Relative deviation (%) 2.19 4.69 3.70

Commonly, single mobile phase can be used to separate vitamins A and E . However, for better separation of three fat-soluble vitamins, the mobile phases should be gradient detergents
[6,13,14] [8]

. In the

present study, vitamins D3 and E could not be well separated if the mobile phase was 95% methanol with 5% water. Similar results were also reported by Qian and Shen
[10]

. For simultaneous determination of

several fat-soluble vitamins, the vitamins were detected at their respective wavelength with maximum absorption peak in most studies, which required a multichannel detecte[7]. However, some studies also showed that no significant difference was observed when a single wavelength was used in the simultaneous determination of three fat-soluble vitamins[10]. In the present study, the three vitamins were detected at 292nm, which decreased the requirement of detector and simplified the method. 2.3 Linearity and limits of detection Under the selected chromatographic condition, the peak area of the vitamins showed linear relations

Fig. 2 The chromatography of the three vitamins in fish samples.

Chinese Journal of Animal Nutrition

www.chinajan.com

A and E in human serum by HPLC. Journal of Table 3 Recoveries of the samples (n=4) Vitami Samples VA (g) VD3 (g) VE (g) n added 50 5 40 Vitamin determined 48.89 5.04 38.75 Recovery (%) 97.4-102.6 96.3-103.7 93.5-97.8 Relative deviation (%) 2.57 3.97 3.44 Pharmaceutical and Biomedical Analysis. 35: 575-582. [7] Lpez-Cervantes J, Snchez-Machado D I, Ros-Vzquez N J. 2006. High-performance liquid chromatography method for the simultaneous quantification of retinol, -tocopherol, and cholesterol in shrimp waste hydrolysate. Journal of Chromatography A. 1105: 135-139. [8] Urbnek L, Solichov D, Melichar B, Dvrk J, Svobodov I, Solich P. 2006. Optimization and validation of a high performance liquid chromatography method for the simultaneous determination of vitamins A and E in human serum using monolithic column and diode-array detection. Analytica Chimica Acta. 573574, 267-272. [9] Alvarez J C, De Mazancourt P. 2001. Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, a-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection. Journal of Chromatography B. 755: 129-135. [10] Qian H, Sheng M. 1998. Simultaneous determination of fat-soluble vitamins A, D and E and pro-vitamin D2 in animal feeds by one-step extraction and high-performance liquid chromatography analysis. Journal of Chromatography A. 825: 127-133. [11] Salo-Vnnen P, Ollilainen V, Mattila P, Lehikoinen K, Salmela-Mls E, Piironen V. 2000. Simultaneous HPLC analysis of fat-soluble vitamins in selected animal products after small-scale extraction. Food Chemistry. 71: 535-543 [12] Botsoglou N, Fletouris D, Psomas I, Mantis A P. 1998. Rapid gas chromatographic method for simultaneous determination of cholestrol and a tocopherol in eggs. Journal of AOAC International. 81: 1177. [13] Panfili G, Manzi P, Pizzoferrato L. High-performance liquid chromatographic method for the simultaneous determination of tocopherols, carotenes, and retinol and its geometric isomers in Italian cheeses. Analyst, 1994, 119: 1161-1165. [14] Hewavitharana A K, van Brakel A S, Harnett M. 1996. Simultaneous liquid chromatographic determination of vitamins A, E and -carotene in common dairy foods. International Dairy Journal. 6: 613-624.

2.5 Precautions 2.5.1 The preparations of the vitamin samples should be carried out at low temperature and photophobic condition. 2.5.2 The air in the stoppled Erlenmeyer flask should be discharged before saponification; the time for saponification should be strictly commanded and the cooling after saponification should be quickly finished; All of the separating funnel used should be with good capacity of liquid seal.

3 Conclusion
The fat-soluble vitamins A, D3 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. References:
[1] Li A J, 1996. Aquatic animal nutrition and feed science. Beijing: China Agricultural Press, 44-46. (In Chinese). [2] He Z F, Zhang D Q. 1998. Functional food chemistry and determination techniques. Beijing: China Light Industry Press, 89. (In Chinese). [3] Yu J G, Wang W Z. 1994. Practical analysis techniques for modern instrument. Beijing: China Forestry Publishing House, 388. (In Chinese). [4] Yakushina L, Aranova A. 1995. Simultaneous determination of fat soluble vitamins in human serum by HPLC. Journal of Pharmaceutical and Biomedical Analysis. Anal., 13: 715-718 [5] Xu J, Zhang H F, Shao Y K, Wang Y. 2006. Simultaneous Determination of Fat-Soluble Vitamins A, D3, 25-OH-D3, E and -Carotene in Human Serum by HPLC. Chinese Pharmaceutical Journal. 41: 147-149. (In Chinese). [6] Mata-Granados J M, Luque de Castro M D, Quesada J M. 2004. Fully automated method for the determination of 24,25(OH)2 and 25(OH) D3 hydroxyvitamins, and Vitamins

(Editor: JI Peng)