HPLC测定人血浆中霉酚酸及其代谢物浓度

Determination of Mycophenolic Acid and its Metabolins in Human Plasma by RP-HPLC

关键词:  霉酚酸  酚化葡萄糖醛麦考酚酸  酰基化MPAG  反相高效液相色谱法  血浆浓度

Key words:  mycophenolic acid  phenolic glucuronide of mycophenolic acid  acyl-MPAG  RP-HPLC  plasma concentration

摘要:

目的 建立反相高效液相色谱法，同时测定人血浆中霉酚酸(mycophenolic acid, MPA)、酚化葡萄糖醛麦考酚酸(phenol glucuronide metabolite, MPAG)、酰基化MPAG(acyl-MPAG, AcMPAG)的浓度。方法 用蛋白沉淀法对样品进行处理。固定相为Zorbax Eclipse XDB C₁₈柱(4.6 mm×250 mm，5 mm)，流动相：甲醇: 20 mmol×L^-1 NaH₂PO₄(用20%磷酸调至pH 3.0)为55∶45，流速：1.2 mL×min^-1，检测波长：304 nm，柱温：45 ℃。结果 MPA、MPAG、AcMPAG浓度在0.2~50 mg×mL^-1(r=0.999 7)、2.8~531 mg×mL^-1(r=0.999 9)、0.3~24 mg×mL^-1( r=0.999 4)内呈良好的线性关系。MPA及其代谢物的绝对回收率均大于80%，MPA、MPAG、AcMPAG的相对回收率分别为94.0%~101.4%，98.4%~101.9%和96.1%~104.2%。日内及日间RSD远低于15%。结论 采用反相高效液相色谱法测定人血浆中MPA、MPAG、AcMPAG的浓度和进行药物代谢动力学研究，方法灵敏度高、重复性强

Abstract:

OBJECTIVE RP-HPLC method detection for simultaneous determination of Mycophenolic acid (MPA), its phenol glucuronide metabolite (MPAG) and acyl-MPAG (AcMPAG) in human plasma was established. METHODS The plasma samples were prepared with protein-precipitating reagent, and the supernatant was eluted on Zorbax column (250 mm×4.6 mm, 5 mm) with 20 mmol×L^-1 NaH₂PO₄ buffer (pH 3.0, adjusted with 20% phosphoric acid) and methanol (45∶55) at 304 nm. The column temperature was 45 ℃, and the flow rate was 1.2 mL×min^-1. RESULTS The assay was linear within the range of 0.2-50 mg×mL^-1 for MPA (r=0.999 7), 2.8-531 mg×mL^-1 for MPAG(r=0.999 9), and 0.3-24 mg×mL^-1 for AcMPAG(r=0.999 4). Mean absolute recovery of MPA and its metabolites was more than 80%. The average recoveries of MPA, MPAG and AcMPAG were 94%.0–101.4%, 98.4%–101.9% and 96.1%–104.2 %, respectively. The RSD of within-day and between-day were all lower than 15%. CONCLUSION The method described is sensitive, reproducible, and will be useful in TDM or pharmacokinetic studies of MPA.