反相高效液相色谱法同时测定血清中5种水溶性维生素

Simultaneous Determination of Five Water-soluble Vitamins in Human Serum by High Performance Liquid Chromatography

目的建立血清中水溶性维生素B1(VB1)、B6(VB6)、烟酸、烟酰胺和叶酸的高效液相色谱同时测定方法。方法取全血40μL,用200μL甲醇沉淀蛋白,冰箱冷冻(-18℃)30 min后离心(8000 r/min,8 min),上清液通过固相萃取柱进一步净化,收集洗脱液,50℃水浴中氮气吹至0.20 mL,取50μL进样色谱柱分析。对样品的固相萃取条件和色谱条件进行优化后,用于测定人血清样品中5种水溶性维生素的含量。结果优化后的固相萃取条件:固相萃取柱AGTcleanertTMODS C18柱;洗脱液:甲醇(2.4 mL)-水(0.6 mL)混合液;洗脱流速:0.5 mL/min。本法的色谱分离条件为:分离柱Phenomenex C18 Luna色谱柱(250×4.6 mm,5μm);柱温:室温(20℃);流动相:0.05 mol/L KH2PO4溶液(pH6.0)-甲醇梯度洗脱;流速:0.9 mL/min;紫外检测波长:266 nm。5种水溶性维生素标准曲线的线性范围为0～100 ng,相关系数均大于0.999,相对标准偏差(RSD)小于5%,方法检出限(信噪比S/N=2)为0.076～0.170μg/mL,加标回收率为80.2%～115.0%。人血清样品中除烟酰胺未检出外,其余4种均检出。结论所建立的方法快速、简便、灵敏、准确,可用于人血清中水溶性维生素VB1、VB6、烟酸和叶酸的同时测定,但烟酰胺的检测灵敏度有待提高。

Objective To establish a method for the simultaneous determination of 5 water-soluble vitamins including VBa, VB6, Nicotinic acid, Nicotinamide and Folio acid by high performance liquid chromatography. Methods Methanol （200μL） was added to 50 μL human blood sample to precipitate proteins. The sample was frozen （-18 ℃） for 30 min, and then centrifuged at 8000 r/rain for 8 rain. The supernatant was subsequently applied to the solid-phase cartridge for further purification. The elute was collected and evaporated to 0. 20 mL under nitrogen in a water bath at the temperature of 50 ℃ and finally 50μL of the sample was injected onto HPLC column for analysis. The five water-soluble vitamins were determined under optimized condition of solid phase extraction and chromatograph. Results The optimized condition for solid phase extraction （SPE） was identified as： AGT cleanertTM ODS C18 column as SPE column and CHaOH（2.4 mL）-H2O（0.6 mL） mixed liquor as eluent, with a flow rate of elution of 0.5 mL/min. The optimized chromatographic condition was identified as： Phenomenex C18 Luna column （250×4.6 mm, 5 μm） as analytical column under a temperature of 20 ℃ and 0.05 mol/L potassium dehydrogenates phosphate buffer （pH6.0） and methanol as gradient elution with mobile phase with a flow rate of 0.9 mL/min. The detection wavelength was 266 nm. The correlation coefficients for the standard curves were greater than 0. 999 in the range from 0 to 100 ng （RSD〈5%）. The detection limits （S/N=2） ranged from 0. 076 to 0. 170 μg/mL. The recovery rate ranged from 80. 2% to 115. 0%. Except for Nicotinamide, the other four vitamins were detected successfully in the blood sample. Conclusion This quick and convenient method can be applied to the simultaneous determination of 4 water-soluble vitamins in human serum samples. But the sensitivity for detecting Nicotinamide needs to be improved.