高效毛细管电泳法同时测定人血浆D,L-乳酸对映体

Simultaneous determination of D,L-lactic acid in plasma by high performance capillary electrophoresis

目的:用高效毛细管电泳法测定血浆中D,L-乳酸对映体。方法:以2-羟丙基-β-环糊精(2-HP-β-CD)作为手性选择剂,采用阳离子表面活性剂十四烷基三甲基溴化铵(TTAB)改变电渗流的方向,应用未涂层毛细管分离测定D,L-乳酸对映体。生物样品的处理为乙腈沉淀蛋白后干燥浓缩,超纯水25μL溶解进样。结果:乳酸对映体约42min分离结束,其分辨率≥1.25。不经过浓缩处理直接进样的D,L-乳酸标准溶液的检测限分别为80μmol/L和50μmol/L(S/N=3),在空白血浆中加入D,L-乳酸,经样品浓缩步骤处理,其检测限分别为20μmol/L和15μmol/L(S/N=3)。D-乳酸标准工作液浓度范围为0.025～2.50mmol/L,相关系数0.9969;L-乳酸标准工作液浓度范围为0.025～5.0mmol/L,相关系数0.9952。D,L-乳酸峰面积及迁移时间的相对标准偏差(RSD)小于6.27%,样品回收率大于68.2%。结论:该法适合临床样品检测,结果令人满意。

Aim：To establish a high performance capillary electrophoresis （HPCE） method for the simultaneous determination of D, L-lactic acids in plasma. Methods：The separation was performed in an uncoated fused-silica electrophoresis capillary. 150 mmol/L phosphate-Tris buffer （pH 7.0） consisting of 220 mmol/L 2-hydroxypropyl-β-cyclodextrin and 0.2 mmol/L tetradecyhrimethylammonium bromide was utilized as the running buffer. Results：The chiral separation of D, L-lactic acids was achieved about 42 rain at the voltage of - 25 kV. The resolution of lactic acid enantiomers was above 1.25. The limits of detection of D, L-lactic acids were 80 μmaol/L and 50 μmaol/L （S/N = 3） respectively. With a pre-concentration procedure as the plasma sample, the limits of detection of D, L-lactic acids were 20 μmol/L and 15 μmol/L （S/N = 3）, respectively. Linear calibration curves for the assay of D, L-lactic acid were validated in the ranges of 0.025 - 2.50 mmol/L （r^2 = 0.996 9） and 0.025 -5.0 mmol/L （r^2 = 0.995 2）, respectively. The relative standard deviation of the peak area and the migration time of D, L-lactic acids was less than 6.27%. The recovery of spiked blood plasma samples was more than 68.2%. Conclusion：This proposed method could be satisfactorily applied to the clinical chiral separation of D, L-lactic acid in plasma.