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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） fish tissues by HPLC is presented. The in sample preparation procedure, consisting of saponification and extraction by mineral ether was carried out. The following chromatographic conditions were used: VIRIAN Res Elut 5µ 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 0-15µg/ml for vitamin A, 0-10µg/ml for vitamin D3 and 0-20µg/ml for vitamin E. The lower limits were 0.8×10- 3 ng for vitamin A, 0.06×10- 3 ng for vitamin D3 and 9.0×10-3 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 assay，simplifying 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
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 5µ Cl8 90A (4.6 x150mm；Varian, 12159012) was used. 1.1.2 Chemicals and Reagents Vitamin A (all-trans-Retinol, R2500), vitamin E (α-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
Author information: Tan Qing-song, male, doctor, E-mail: firstname.lastname@example.org * Corresponding author: He Rui-guo, professor, E-mail: email@example.com
Chemical Co. (USA). Ethanol and methanol were
NaOH solution was prepared by dissolving 500g of NaOH in 1L of double-distilled water.TAN Qing-song et al. the flasks were immediately cooled to 40℃ by water flow. and washed twice with 10 ml of concentrated sulphuric acid. 10 ml of 50 g/L sodium hydroxide solution were added.2. D3 and E in Fish Tissues by HPLC HPLC-grade.3 Calibration curve The working standard solutions with vitamin concentrations ranged from 0. 1. then was stored at 4℃.2.3.2 Extraction The saponified mixture was transferred to a separating funnel (Volume: 250ml).0 mg of vitamin A (all-trans-Retinol) in ethanol and diluting to 50 ml to provide a concentration of 220 µg/ml. 1.1 Saponification The hepatopancreas and muscle tissue were fully homogenized. avoiding the sample sticking to the flask wall. During the saponification.5 ml of vitamin D3 stock standard solution were accurately measured and mixed. D3 and E The mixed stock standard solution was prepared to provide concentrations of 22 µg/ml vitamin A.3 Tested samples Hepatopancreas and muscle tissue of rice field eel were used as tested samples.3.2 Mixed stock standard solution of vitamins A.3 Sample treatments 1. 1. then was stored at 4℃. The mixture was fully mixed and 50 ml of mineral ether was added. The washed mineral ether was dried with anhydrous calcium chloride for 1h and then distilled. Then. Again.0 g of ascorbic acid in 4 ml of hot distilled water.10 to 20 µg/ml were prepared by appropriate dilution of the mixed stock solution with ethanol.9%) was used. then washed with saturated solutions composed of 10% sulphuric acid and KMnO4 untill the purple color in water layer keep constantly. The flask was washed three times with 60 ml of ultrapure water and the washings were transferred to the same funnel. The calibration curve was accessed by setting the vitamin mass in X-axis and the peak area in Y-axis.1 Stock standard solutions 1）Vitamin A stock standard solution was prepared by dissolving 11. After saponification. 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. 1. Then the solutions in the stoppled erlenmeyer flasks were mixed and saponified in 70℃ for 30 min. the mixture in the separating funnel was fully vortexed for 2min and placed to demix.2 Working standard solutions 1. The ultrapure water and nitrogen gas (99.0001 g. 3）Vitamin E stock standard solution was prepared by dissolving 11.: Simultaneous Measurement of Vitamin A. then 30 ml of anhydrous alcohol and 10 ml of 10 g/L ascorbic acid solution. each of the working standard solution with the volume of 20 µm was injected to the system and determined. the flasks were vibrated constantly. 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. The other reagents used were analytical reagent. Each of the weighed samples was put into a stoppled erlenmeyer flask.1.5 ml of vitamin E stock standard solution and 2. then washed twice with distilled water.2. too (Note: The volume ration of ethanol to water in the funnel should be larger than 1). then sampled and weighed accurately to 0. en diluted with ethanol to the final volume of 50 ml. 10µg/ml vitamin D3 and 50 µg/ml vitamin E: 5 ml of vitamin A stock standard solution. 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 . 1. 12. then was stored at 4℃. 2）Vitamin 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 µg/ml. 1. The 10 g/L ascorbic acid solution was prepared as follows: Dissolving 1. The water phase was transferred to another separating funnel and extracted with 50 ml of mineral ether.45µm membrane before being injected into the system.0 mg of α-Tocopherol in ethanol and diluting to 50 ml to provide a concentration of 200 µg/ml. and filtered through a 0.
0 ml/min.2 Following the procedure of 1.3 Test for recoveries of retinol. Then the vitamins were dried with nitrogen gas and dissolved with 1 ml of ethanol. 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. D or E in 1 kg of sample. α-tocopherol. 1IU vitamin A=0. in contrast with the method by López-Cervantes et al. The flow rate was 1.4. 1.1 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.5 Calculations The contents of various vitamins in the tissue samples were calculated as the following equation (Equation 1): C＝ S 1 × V 1 × V 3 × ρ × V st S st × m × V 2 × V 4 × f Of which. then diluted with mineral ether to a final volume of 250 ml (V1). The solution was then filtered through a 0. 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.12].chinajan.Chinese Journal of Animal Nutrition www. 2 Results and discussion 2. V3 (m1) is the final volume of the tested sample solution after dried and redissolved. And after the last washing. V2 (ml) is the volume of the sampled extractive for analysis. the three vitamins were well separated under the conditions. and the recoveries of the three vitamins were tested. tested by the phenolphthalein indicator. 1. 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. Res Elut 5µ Cl8 90A (4.Because of lower concentrations of vitamin D in the fish tissue. Detection was at 292 nm.com ether phase in the three separating funnels were put into the first separating funnel together. 1. the mineral ether extractive was dehydrated by anhydrous sodium sulfate. 1IU vitamin D3=0. the mixture should be vortexed slightly to avoid emulsification. m is the sample mass (g).6 x150mm；Varian.3. 12159012) was used.4 Analysis procedure 1. as showed in Table 1. The combined ether phases were washed three times.1 Sample extraction Methods for the extraction of vitamins A、D、E from animal tissues have been reported in several papers[7. All the operations should be conducted in a photophobic fume cupboard. Mobile phases were the mixture of methanol and water with a gradient elution. As shown in Fig. Vst (µl) is the volume of the injected working standard solution. then put into a rotatory evaporator to evaporate the mineral ether in a water bath at 50 ℃. D3 and E.4. the chromatographic conditions were studied and selected as follows: The injection volume was 20µl. 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.. At last. the water phase in the funnel should be neutral. Sensitivity was 0. Then.4. and cholecalciferol before applying the proposed methods: A given amount of vitamin standards were treated as the procedure same to the tested samples. 1.025µg cholecalciferol.3µg all-trans-retinol.3 Condensation A given amount of samples (V2) was measured from the prepared mineral ether extractive. each with 50 ml of water saturated with mineral ether. 1. V1 (ml) is the total volume of each vitamin extractive. When first washing. f is the conversion coefficient.05AUFS. C is the amount (IU or mg) of vitamin A. S1 is the peak area corresponding to the volume of the injected tested sample solution (V4).126.96.36.199 Chromatographic conditions for sample For better separation of vitamins A. 2. ρ (µg/m1) is the concentration of the working standard solution of the tested vitamin. separation .45µm membrane and injected into the system for analysis.
which required a multichannel detecte.TAN Qing-song et al. 2 The chromatography of the three vitamins in fish samples.2380×10-4x-4.3 Linearity and limits of detection Under the selected chromatographic condition.9999. D3 and E in fish tissue can be simultaneously determined by HPLC at 292 nm with UV-Vis detecter after mineral extraction. -5 D3 and E were generated as y=3.69 3. The detection limits under the described conditions were 0. and y=3.4091 (r＝0. In the present study. n=3) in the tested range of 0-20 µg/ml. 2. which decreased the requirement of detector and simplified the method. the three vitamins were detected at 292nm.9998.13.8788×10 x-0. n=3) in the tested range of 0-10µg/ml.3 ng for vitamin D3. Similar results were also reported by Qian and Shen  . . the vitamins were detected at their respective wavelength with maximum absorption peak in most studies. for better separation of three fat-soluble vitamins.0339 (r=0. enabling the processing of a large number of samples.9999. 0. vitamins D3 and E could not be well separated if the mobile phase was 95% methanol with 5% water. However. 2.0 ×10-3 ng for vitamin E with a sensitivity of 0.8×10-3 ng for vitamin A. The present study showed that vitamins A. Table 2 Analytical results of the samples (n=3) Vitamins samples Vitamin A（µg /g） Vitamin D3 (µg /g) Vitamin E (µg/g) in the Concentration 21. single mobile phase can be used to separate vitamins A and E .46 Relative deviation (%) 2.1225×10-3 (r=0. 1 The chromatography of the three vitamins in standard solution. For simultaneous determination of several fat-soluble vitamins.4 Quantification and recovery The vitamins A.06×10. n=3) in the tested range of 0-15µg/ml.33 0.05 AUFS and a VA VE VD signal-to-noise ratio of 3. y=1. µg) of vitamin A.70 Commonly. the peak area of the vitamins showed linear relations Fig. which is effective and fast. 2. the results Fig. As shown in Table 2 and Table 3. The separation of the three vitamins in fish tissue was shown in Fig. the mobile phases should be gradient detergents [6. However. and 9.19 16. respectively.19 4. In the present study.1874×10-4x － 0. 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) Time（min） 0 7 15 20 Methanol﹕Water 95﹕5 95﹕5 100﹕0 95﹕5 relating peak area (y) to injected amounts (x. 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. assayed in fish samples and the recoveries of vitamins before applying the proposed methods showed reasonable. D3 and E in the tissue samples of rice field eel were separated after saponification and extraction with mineral ether.: Simultaneous Measurement of Vitamin A.14]  .
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