柱前衍生化的人血浆中(+),(-)棉酚的HPLC测定法

本文报道分别定量检测人血浆中(+),(-)棉酚含量的高效液相色谱测定法。棉酚人血浆样品经乙腈沉淀蛋白后,上清液加手性试剂——(+)-2-氨基-1-丁醇,75℃加热45min。反应液加NaCl进行溶剂反混合处理后,取乙腈层50～100μl进样。选用ODS固定相,紫外254nm检测,以甲醇—异丙醇—水—磷酸(80:20:20:0.1 V/V)为流动相,流速1.2ml/min时,(+),(-) 棉酚衍生物的保留时间分别为4.2,7.7 min。检测人血浆中(+),(-)棉酚在0.125～2.0μg/ml范围内呈线性关系。棉酚回收率达94～98%,此法已用于棉酚的临床血药浓度监测和动物实验研究。

DETERMINATION OF (+)- AND (-)-GOSSYPOL IN HUMAN PLASMA USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH PRE-COLUMN CHEMICAL DERIVATIZATION

A new assay method for the determination of (+)- and (-)- gossypol using HPLC with pre-column chemical derivatization has been developed. 2, 6-dimethyl-naphthalene was used as internal standard and (+)-2-amino-l-butanol as the chiral derivatizing agent. (+)- and (-)- gossypol derivatives were separated completely on an ODS column. Methanol-isopropanol-water-phosphorie acid (80:20:20:0.1) was used as the mobile phase. The wave length of UV detector was settled at 254 nm. The retention times of (-)- and (+)-gossypol derivatives were 4.2 and 7.7 min, respectively.Acetonitrile-treated protein-free plasma samples were prepared, and (+)-2-amino-l-butanol was added to a final concentration of 2%. The system was kept at 75℃ for 45 min. After solvent demixing by adding sodium chloride, 50～100μl of acetonitrile phase was injected into the column.The linearities with (+)- and (-)-gossypol plasma sample ranged from 0.125 to 2.0μg/ml. The absolute recoveries of (+)-, (-)-gossypol and internal standard were found to be 98.2%, 94.1% and 94.6%, respectively, with within-day and dayto-day variations less than 6% for both (+)- and (-)-gossypol. The sensitivity for detection of (+)- or (-)- gossypol was found to be 0.125μg/ml (S/N 3:1). This method is considered to be suitable for some clinical and experimental studies of gossypol.