UHPLC-MS/MS测定人血浆中的阿普斯特浓度及生物等效性研究

Determination of apremilast concentration in human plasma UHPLC-MS/MS and its bioequivalence study

目的采用超高效液相色谱-串联质谱(UHPLC-MS/MS)法,建立快速、简单、灵敏的测定人血浆中阿普斯特浓度的方法并用于阿普斯特人体生物等效性试验研究。方法采用蛋白沉淀法处理,以阿普斯特-d5作为内标,色谱柱为Phenomenex Kinetex C_(18)柱(2.1 mm×50 mm,2.6μm),流动相为0.1%甲酸水-0.1%甲酸乙腈,流速0.45 mL·min^(-1)梯度洗脱,采用电喷雾(ESI)离子源以正离子方式进行MRM检测,用时3 min。结果阿普斯特在2~600 ng·mL^(-1)(r^(2)=0.9977)范围内线性关系良好,准确度和精密度均小于15%。空腹和餐后条件下阿普斯特片受试制剂AUC_(0~t)、AUC_(0~∞)和C_(max)的90%CI为参比制剂相应参数的80.00%~125.00%范围内。结论受试制剂与参比制剂具有生物等效性。

Objective To develop an UHPLC-MS/MS method for determination of apremilast in human plasma and the bioequivalence study.Methods Plasma sample was treated with methanol for protein precipitation while apremilast-d 5 was used as internal standard(IS).Separation was achieved on a Phenomenex Kinetex C_(18) column(2.1 mm×50 mm,2.6μm)by gradient elution at a flow rate of 0.45 mL·min^(-1),with acetonitrile(containing 0.1% of formic acid)and water(containing 0.1%formic acid)as mobile phase.Detection was performed using electrospray ionization in positive ion multiple reaction monitoring(MRM)mode for three minutes.Results Calibration curves of apremilast were linear in the concentration range of 2~600 ng·mL^(-1)(r^(2)=0.9977),with precisions and accuracies were less than 15%.The 90% confidence intervals of AUC_(0~t),AUC_(0~∞)and C_(max) of the test preparations of Apremilast Tablets under fasting and postprandial conditions were within the ranges of 80.0%~125.0% of the corresponding parameters of the reference preparation.Conclusion The test preparation and reference preparation are bioequivalent.