FPLC-ESI-MS/MS法测定人血浆猪磺去氧胆酸浓度

FPLC-ESI-MS/MS determination of taurohyodeoxycholic acid in human plasma

目的:建立FPLC-ESI-MS/MS法测定人血浆猪磺去氧胆酸浓度。方法:人血浆样本用固相萃取法提取后,选用Shim-pack XR-ODS色谱柱(75 mm×3.0 mm,2.2μm),以甲醇-10 mmol.L-1乙酸铵(含0.5%乙酸)为流动相,梯度洗脱,流速为0.4 mL.min-1;选用API3200型三重四级杆串联质谱仪的多重反应监测(MRM)扫描方式进行监测,电喷雾离子化源,正离子方式,选择监测离子反应分别为m/z 498.3→m/z(79.9+106.9)(猪磺去氧胆酸),m/z 377.1→m/z 331.1(内标23-Nordeoxycholic)。结果:猪磺去氧胆酸和23-Nordeoxycholic的保留时间分别为3.5和5.0 min;血浆中猪磺去氧胆酸浓度线性范围为10～2 500 ng.mL-1(r>0.99),定量下限为10 ng.mL-1;日内、日间RSD均<6.22%;相对偏差(RE)均在1.19%～4.31%之间;平均提取回收率为(66.7±3.9)%;稳定性试验中,在各种储存条件下血浆中猪磺去氧胆酸均较稳定。结论:该方法快速、灵敏,专属性强,重现性好,适用于人血浆猪磺去氧胆酸浓度的测定,可应用于猪磺去氧胆酸的药代动力学研究。

Objective： To develop an FPLC-ESI-MS/MS method for determining taurohyodeoxycholic acid in human plasma.Methods： After extraction with SPE,the analyte and internal standard,23-Nordeoxycholic,were separated on a Shim-pack XR-ODS（75mm×3.0mm,2.2μm） analytical column using the mobile phase of methanol and 10mmol·L-1 ammonium acetate（contain 0.5% acetic acid）.Detection was carried out by electrospray negative ionization mass spectrometry in the multiple reaction monitoring（MRM） mode.The MRM transition of m/z 498.3→m/z（79.9＋106.9） or m/z 377.1→m/z 331.1 was used to quantify taurohyodeoxycholic acid or internal standard,respectively.Results： Taurohyodeoxycholic acid and internal standard were eluted at 3.5 and 5.0min,respectively.The calibration curve was linear over the concentration range of 10~2500ng·mL-1（r0.99）with the lower limit of quantitation（LLOQ） 10ng·mL-1.Intra-and inter-day RSD were both less than 6.22%,and the relative errors（RE） were between 1.19%~4.31%.The mean extract recoveries were（66.7±3.9）%.Conclusion： This is a rapid,sensitive,selective and reliable method for the determination of taurohyodeoxycholic acid in human plasma,and can be applied to a pharmacokinetic study of taurohyodeoxycholic acid capsules in healthy volunteers.