LC-MS/MS测定人血浆中厄贝沙坦和氢氯噻嗪的含量及其应用

Determination of Irbesartan and Hydrochlorothiazide in Human Plasma by LC-MS/MS and Its Application

关键词:  厄贝沙坦|氢氯噻嗪|生物等效性|液相色谱-质谱联用

Key words:  irbesartan(IBST)|hydrochlorothiazide(HCZ)|bioequivalence|LC-MS/MS

摘要:

目的 建立可同时测定人血浆中厄贝沙坦（irbesartan，IBST）和氢氯噻嗪（hydrochlorothiazide，HCZ）浓度的LC-MS/MS测定方法，并用于厄贝沙坦氢氯噻嗪（IBST/HCZ）片人体生物等效性研究。方法 血浆样品经过蛋白沉淀法处理后上样，采用IBST-d4和甲氯噻嗪（chlorothiazide，MCTZ）作为内标。色谱柱为Waters ACQUITY UPLC^®XTERRA RP₁₈（4.6 mm×100 mm，3.5 μm），流动相A和B分别为乙腈和5 mmol·L^-1乙酸铵-水，进行梯度洗脱，流速为0.85 mL·min^-1。在电喷雾电离源负离子模式下检测离子对为m/z 427.1→193.1（IBST），m/z 431.2→193.1（IBST-d4），m/z 295.9→268.9（HCZ）和m/z 357.8→322.0（MCTZ）。对健康受试者口服相同剂量的IBST/HCZ片（150 mg/12.5 mg）受试制剂或参比制剂后的血药浓度进行定量分析。结果 血浆中内源性物质不干扰待测物定量，IBST、HCZ、IBST-d4、MCTZ之间不存在相互干扰和相互转化。人血浆中IBST和HCZ的定量范围分别为10~4 000 ng·mL^-1和1~400 ng·mL^-1，线性关系良好。IBST与内标提取回收率均在95.7%~105.4%，HCZ与内标提取回收率均在95.0%~106.4%；IBST和HCZ的内标归一化基质因子分别为101.2%~102.6%和79.8%~93.6%，变异系数（coefficient of variation，CV）均<5%；批内、批间准确度偏差均<15%，CV均<10%。用本法测定得到的IBST和HCZ的浓度水平及获取的药动学参数与文献报道一致，试验样品再分析复测值与原测值的偏差均<10%。结论 本法方便快速、特异性强、灵敏、准确、重复性好、所需样本体积少，充分满足IBST/HCZ片人体生物等效性研究的高通量定量分析需求。

Abstract:

OBJECTIVE To establish an LC-MS/MS method for simultaneous determination of irbesartan(IBST) and hydrochlorothiazide(HCZ) concentrations in human plasma and to be used for a bioequivalence study of IBST/HCZ tablets. METHODS Plasma samples were pre-treated by protein precipitation. IBST-d4 and chlorothiazide(MCTZ) were used as the internal standard. The analytes were retained and eluted by using a Waters ACQUITY UPLC^®XTERRA RP₁₈(4.6 mm×100 mm, 3.5 μm) column, with a binary mobile phase system consisting of acetonitrile(A) and 5 mmol·L^-1 ammonium acetate solution(B) at a flow rate of 0.85 mL·min^-1. The analytes were detected in an electrospray ionization source under negative ion mode, using the ion transitions of m/z 427.1→193.1(IBST), m/z 431.2→193.1(IBST-d4), m/z 295.9→268.9(HCZ) and m/z 357.8→322.0 (MCTZ). The quantitation method was applied to a bioequivalence study of IBST/HCZ tablets(150 mg/12.5 mg) in healthy subjects. RESULTS Endogenous substances in plasma did not interfere with the quantification of the substance to be measured, and no interference and transformation on and from IBST, HCZ, IBST-d4 and MCTZ. The quantitative ranges of IBST and HCZ in human plasma were 10-4 000 ng·mL^-1 and 1-400 ng·mL^-1, respectively, with good linearity. The extraction recoveries of IBST and IBST-d4 were between 95.7% and 105.4%, HCZ and MCTZ were between 95.0% and 106.4%; the internal standard normalization matrix effect factors of IBST and HCZ were recorded as 101.2%-102.6%, and 79.8%-93.6% all with coefficient of variation(CV)<5%, respectively. Intra- and inter-batch accuracy and precision were all within 15%, CV<10%. The concentrations of IBST and HCZ determined by the method and the pharmacokinetic parameters calculated thereafter were in accordance with those found in previous reports, and the difference of concentrations generated in the incurred sample reanalysis assay and the original measurement was <10%. CONCLUSION The method is rapid, specific, sensitive, accurate, and reproducible requiring a small sample volumn, and fully meets the requirements of high-throughput quantitative analysis of IBST/HCZ tablets bioequivalence studies.