基于超滤离心前处理的液相色谱-串联质谱法手性拆分人血浆中的亚叶酸和5-甲基四氢叶酸非对映异构体及其药代动力学应用  

Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to

作　　者：徐陈凤 惠文凯 孙莉莉 高倩倩 邹巧根 XU Chenfeng;HUI Wenkai;SUN Lili;GAO Qianqian;ZOU Qiaogen(Nanjing Tech University, Nanjing 211800, China;Chia Tai Tianqing Pharmaceutical Group Co., Ltd, Lianyungang 222000, China;Nanjing Healthnice Medical Technology Co., Ltd, Nanjing 210009, China)

机构地区：[1]南京工业大学,江苏南京211800 [2]正大天晴药业集团股份有限公司,江苏连云港222000 [3]海纳医药科技股份有限公司,江苏南京210009

出　　处：《色谱》2019年第6期581-588,共8页Chinese Journal of Chromatography

摘　　要：建立了液相色谱-串联质谱(HPLC-MS/MS)同时测定人血浆中的亚叶酸和5-甲基四氢叶酸两对非对映异构体的方法。血浆经蛋白质沉淀-超滤离心处理后,以甲氨蝶呤为内标,乙腈-10 mmol/L pH 8.0醋酸铵为流动相,通过手性HSA色谱柱(150 mm×4 mm, 5 μm)进行梯度洗脱。亚叶酸非对映异构体在25~ 5 000 μg/L 范围内、5-甲基四氢叶酸非对映异构体在12.5~ 2 500 μg/L 范围内,线性关系均良好。本方法在灵敏度、精密度、准确度、基质效应、提取回收率、稳定性等方面均得到充分验证,并成功应用于125 mg/m^2 亚叶酸和62.5 mg/m^2 左旋亚叶酸的药代动力学研究。结果显示:在125 mg/m^2 亚叶酸剂量组,左亚叶酸和左旋-5-甲基四氢叶酸的血浆峰浓度( Cmax)为( 3 137.917 ±408.837)和( 1 679.633 ±244.132)μg/L ,从时间点0到最后可定量时间点的药物代谢动力学时间曲线下面积(AUC0- t)为( 7 504.883 ± 1 185.101 )和( 14 001.214 ± 2 868.949 )μg/L;在62.5 mg/m^2左亚叶酸剂量组,左亚叶酸和左旋-5-甲基四氢叶酸的Cmax为( 3 187.917 ±387.298)和( 1 739.204 ±224.755)μg/L , AUC0- t 为( 7 426.664 ±854.825)和( 14 884.331 ± 1 843.353 )μg/L 。两剂量组主要药物代谢动力学参数均无显著差异,特征一致,吸收的速度和程度一致,能够为后期进行左亚叶酸钠生物等效性研究提供技术支持。A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 μm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25- 5 000 μg/L and 12.5- 3 000 μg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers , respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m 2 6 R,S -leucovorin and 62.5 mg/m^2 6S -leucovorin. The results showed that the maximum observed concentrations ( Cmax ) of 6 S -leucovorin and L-5-methyltetrahydrofolate were ( 3 137.917 ±408.837) and ( 1 679.633 ±244.132)μg/L , respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t ) were ( 7 504.883 ± 1 185.101 ) and ( 14 001.214 ± 2 868.949 )μg/L in the 125 mg/m^2 6R,S -leucovorin dose group. The C max values of 6S -leucovorin and L-5-methyltetrahydrofolate were ( 3 187.917 ±387.298) and ( 1 739.204 ±224.755)μg/L , respectively, and AUC0- t values were ( 7 426.664 ±854.825) and ( 14 884.331 ± 1 843.353 )μg/L in the 62.5 mg/m^2 6S -leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leuco

关 键 词：液相色谱串联质谱 超滤离心 亚叶酸 药代动力学 手性拆分 

分 类 号：O658[理学—分析化学]