高效液相色谱法测定牡蛎中牛磺酸含量

Development of High Performance Liquid Chromatography Method for Determination of Taurine in Oyster(Ostrea Plicatula)

目的:通过优化流动相组成,制定牛磺酸的标准工作曲线,评价牛磺酸测定的稳定性及准确性。测定不同条件下提取的牡蛎牛磺酸样品,建立并评价高效液相色谱法测定牡蛎中牛磺酸含量的方法。方法:采用水煮法提取牡蛎中的牛磺酸,分别用HCl和NaOH溶液除去其中的酸性蛋白质和碱性蛋白质,经浓缩、乙醇脱水,制得牡蛎牛磺酸粗品;用邻苯二甲醛(OPA)法柱前衍生3min;色谱柱为AgilentEclipseXDB-C18,流动相流速0.50mL/min,检测波长360nm,柱温30℃。结果:优化得到测定牡蛎牛磺酸的流动相为20%乙腈和80%水,牛磺酸的出峰时间为3.858min,走样时间为7min,此时牛磺酸和牡蛎提取物中的其它成分完全分离(R>1.5)。牛磺酸质量浓度在5～60μg/mL范围具有良好的线性(R2=0.9946),最低检出限0.02μg/mL,最低定量限0.07μg/mL,平行样品的相对标准偏差(RSD)1.31%～4.48%。相同样品4h内测定的RSD为4.93%,样品测定的回收率95.82%～98.03%。随着提取温度的升高,牡蛎牛磺酸的提取率上升,当提取温度100℃时,牡蛎中牛磺酸的提取率达219.42mg/100g。结论:所建立的方法专属性好,灵敏度高,可精确测定牡蛎提取物中的牛磺酸含量。

Objective： To establish a HPLC method for determination of taurine in oyster in concern of optimization of mobile phase, construction of the calibration curve, evaluation of the accuracy and repeability and analysis of taurine that extacted from oyster at different extraction temperature. Method： Taurine sample was obtained by the combined pro- cedure consisting of extracting the oyster with water at 100 ℃ for 1 h, removing the acid and alkaline protein by the treatment of HC1 and NaOH, concentration by the vacuum rotary evaporation, dehydration by treatment of ethanol and colorization by pre-column derivation with orthophthalaldehyde（OPA）. Taurine sample was HPLC separated on an Eclipse XDB-C18 column （4.6 mmx150 mm, 5 μm） eluted with an mobile phase in composition of 20% acetonitrile and 80% ul- trapure water at 0.50 mL/min and 30 ℃. The signal was detected by a UV detector at 360 nm. Results： The optimal mobile phase which was revealed comprised of 20% of acetonitrile and 80% of ultrapure water made taurine that had retention time at 3.858 min completely separated from other impurity in the sample and a running time of 7 min. Meanwhile, the method displayed good linearity within 5-60 μg/mL with an R2 of 0.9946, detection limit of 0.02 μg/mL, quantitative detect limit of 0.07 μg/mL, relative standard deviation （RSD） among different parallels was within 1.31%-4.48%, RSD of the same sample at different time within 4 h was 4.93%, recovery range was 95.82%-98.03%. As extraction temperature raised the yield of taurin increased and reached the climax at 100 ℃ with a yielding of 219.42 mg/100 g. Conclusion： The proposed method has good specificity and high sensitivity, and is accurate and precise to determine taurine in oyster.