HPLC法同时测定霉酚酸及其代谢产物的方法学研究

目的 建立测定器官移植患者血浆中霉酚酸（Mycophenolic acid,MPA）与其代谢物（霉酚酸葡萄糖苷,MPAG和霉酚酸酰基葡萄糖苷,AcMPAG）的一种新颖简单的高效液相色谱（high performance liquid chromatography,HPLC）方法.方法 以10%的偏磷酸为沉淀剂采用蛋白沉淀的方法提取血浆中的待测物,MPA的类似物（the carboxybutoxy ether of MPA,MPAC）被用作内标物质.色谱分析条件是以乙腈和磷酸缓冲液（pH2.7）作为流动相的梯度洗脱,流速1.2 mL·min-1,紫外检测波长为254 nm.结果 所有待测物基线分离好,在待测范围内与峰面积线性关系良好（r2≥0.999）,方法精密（日间和日内变异度均≤6.02%）且准确（日间和日内准确度均在92.17% ~104.78%之间）.MPA、MPAG和AcMPAG在血浆中的回收率分别超过83.11%、98.62% 和93.94%,最低检测限则分别为0.1、0.2、0.1 mg·L-1.三种物质的在酸化过的血浆中,室温放置6 h、4℃条件下放置12 h、-40℃条件下放置4周及反复冻融三次均稳定无明显降解.提取后的三种物质在进样盘中放置至12 h同样稳定无降解.结论 该方法简便、灵敏且准确,可应用于MPA与其代谢物的临床监测.

肾移植病人血浆霉酚酸酯的浓度与UGT2B7 211G>T基因多态性相关性研究

AIM: To clarify whether Mycophenolic acid(MPA) or its metabolite AcMPAG can cause gastrointestinal disturbances, and to explore the effect of UGT2B7 SNP 211G>T on the pharmacokinetic of AcMPAG. METHODS: Twenty-four renal transplant patients were enrolled in this study. Pharmacokinetic study was performed on day 14 after transplantation and symptoms were recorded on the same day. Multiple blood samples were collected before dosing and 0.5,1,1.5,2,4,6,8,10 and 12 hours after morning dosing. Plasma concentrations of MPA, MPAG and AcMPAG were detected by HPLC. Genotype of UGT2B7 211G>T was determined using PCR-RFLP method. RESULTS: No significant difference was observed between patients with and without side effects for AUC(0-12) of MPA and MPAG. The values of AUC(0-12)/dose of MPA, MPAG and AcMPAG were (39.7±12.3), (1063.7±646.5) and (10.2±3.0) ng·h·mL-1·mg-1 in TT genotype group and (42.0±20.0),(1466.5±683.3) and (20.7±5.6) ng·h·mL-1·mg-1 in GG genotype group, respectively. The ratio of AUCAcMPAG(0-12)/AUCCMPA(0-12), which was used to describe the change of UGT2B7 enzyme activity, was 0.49±0.20,0.34±0.26 and 0.28±0.14 in recipients with 211GG, GT and TT genotype, respectively(P<0.05). CONCLUSION: There was no significant differences in pharmacokinetics of MPA and its metabolites between side effect group and non-side effect group. It seems that the polymorphism 211G>T in the UGT2B7 gene results in the reduction of enzyme acitivity.

液-质联用法同时测定人血浆中霉酚酸及其代谢物浓度及其在肝移植患者药动学研究中的应用

目的：建立LC-MS/MS法同时测定血浆中霉酚酸（MPA）及其代谢物MPAG与AcMPAG浓度,用于治疗药物监测与药动学研究。方法：100μl血浆标本加入300μl含内标吲哚美辛的乙腈沉淀,离心后取上清液进样。色谱柱为Agilent E-clipse XDB-C18柱（3.5μm,2.1 mm×100 mm）,流动相为2 mmol·L-1甲酸铵水溶液-甲醇,采用梯度洗脱的方法,流速0.3 ml·min-1。用多反应监测进行定量,ESI负离子方式进行检测,用于定量分析的检测离子分别为m/z319.1→274.9（MPA）、m/z495.3→174.4（MPAG与AcMPAG）与m/z 356.1→312.0（内标吲哚美辛）。24例肝移植患者,采用包括霉酚酸钠肠溶片（EC-MPS）的三联免疫抑制方案,测定服药后MPA及其代谢物浓度并计算药动学参数。结果：MPA在0.10～50.5μg·ml-1,MPAG在1.13～4501μg·ml-,AcMPAG在0.11～22.4μg·ml-1范围内线性良好（r2〉0.99）。MPA、MPAG、AcMPAG提取回收率为77.3%～92.6%;基质效应为76.0%～86.7%;回收率为94.2%～116.4%;日内及日间变异均小于15%。24例肝移植患者用药1周后MPA、MPAG、AcMPAG的主要药动学参数分别如下：峰浓度Cmax为（18.8±10.9）,（154±118）,（3.07±2.85）μg·ml-1;达峰时间Tmax为（3.82±2.66）,（4.74±2.51）,（4.51±2.72）h;曲线下面积AUC0-12为（45.2±20.3）,（1 456±1 195）,（18.3±16.2）μg·h·ml-1;消除半衰期t1/2为（3.21±2.56）,（9.26±4.33）,（5.57±5.76）h。结论：本研究所建立的方法快速准确、灵敏、专属性强,适用于MPA及其代谢物的血药浓度监测和人体药动学研究。