LC-MS/MS测定人血浆中甲磺司特含量的方法开发及验证

Bioanalytical Method Development and Validation for Determination of Suplatast Tosilate in Human Plasma by LC-MS/MS

关键词:  甲磺司特  LC-MS/MS  稳定性  生物等效性

Key words:  suplatast tosilate  LC-MS/MS  stability  bioequivalence

摘要:

目的 建立可靠的全血采集及生物分析方法，用于甲磺司特(suplatast tosilate，ST)颗粒的生物等效性研究。方法 冰浴条件下，将全血采集到含有NaF/EDTA抗凝剂的采血管中，分离的血浆分成2份至采血管中，分别加入体积比为0.5%的甲酸。20 μL血浆样品经蛋白沉淀处理后上样，以ST-D5为内标。采用正相硅胶分离体系，色谱柱为Agilent Polaris 3 Si-A (3.0 mm×100 mm，3 μm)，流动相为10 mmol·L^–1乙酸铵水溶液和甲醇-乙腈溶液(1︰1)，流速0.80 mL·min^–1，以50%有机相进行等度洗脱。在电喷雾电离源正离子模式下检测，ST和ST-D5离子通道(m/z)分别为328.1→266.3，333.1→271.3。结果 定量范围为1~200 ng·mL^–1，线性关系良好(r=0.999 8)；ST与TS-D5提取回收率均在105.0%~117.1%；批内和批间准确度分别为95.67%~108.00%(精密度≤9.10%)和99.10%~105.00%(精密度≤7.66%)。ST在全血、血浆中的稳定性符合要求。结论 所建方法稳定、准确、重复性好，且所需样本体积少，满足ST颗粒生物等效性研究的需求。

Abstract:

OBJECTIVE To establish a reliable method for whole blood collection and bioanalysis method for the bioequivalence study of suplatast tosilate(ST) granules. METHODS Blood was collected into heparin tubes containing NaF/EDTA under ice bath conditions. After processing, the separated plasma was split into 2 aliquots into each heparin tube containing 0.5% formic acid. Plasma sample(20 μL) was pre-treated by protein precipitation, and ST-D5 was used as the internal standard. Chromatographic separation was achieved through a normal phase silica column[Agilent Polaris 3 Si-A column(3.0 mm× 100 mm, 3 μm)], the mobile phases was 10 mmol·L^–1 ammonium acetate solution and methanol-acetonitrile(1︰1). The flow rate was 0.80 mL·min^–1, the organic proportion was 50% with isocratic elution. Then with the mass spectrometer operated in positive electrospray ionization mode using multiple reaction monitoring, ST and ST-D5 were quantitative analyzed by detecting the ion transitions of m/z 328.1→266.3, 333.1→271.3. RESULTS The method showed linearity over the concentration range of 1 -200 ng·mL^–1was good(r=0.999 8). The extraction recoveries of ST and ST-D5 were between 105.0% -117.1%. Intra- and inter-day accuracy were in the ranges of 95.67% -108.00%(precision≤9.10%) and 99.10% -105.00%(precision≤7.66%), respectively. Stability of ST in whole blood and plasma were acceptable. CONCLUSION The established method is stable, accurate, and reproducible for ST with small sample volume, and meets the requirements of bioequivalence studies of ST granules.