HPLC-MS/MS测定人血浆中氟比洛芬的浓度及其生物等效性研究

Determination of Flurbiprofen in Human Plasma by HPLC-MS/MS and Its Application to Bioequivalence Study

关键词:  氟比洛芬  液相色谱串联质谱法  药动学  生物等效性

Key words:  flurbiprofen  HPLC-MS/MS  pharmacokinetics  bioequivalence

摘要:

目的 建立并验证一种简单、快速、灵敏的测定人血浆中氟比洛芬的液相色谱-串联质谱分析方法,研究氟比洛芬凝胶贴膏在中国健康受试者体内的药动学特征并评价生物等效性。方法 液相条件:使用InertSustain C₁₈色谱柱(3.0 mm×100 mm,3 μm);柱温设定为40℃;流动相A为甲醇-乙腈(40:60),B为0.1%甲酸水溶液;流速为0.6 mL·min^-1;梯度洗脱,血浆样品采用蛋白沉淀的前处理方法。质谱条件:离子源为电喷雾离子化源(ESI);负离子模式;氟比洛芬和氟比洛芬-d5的检测离子对分别为m/z 243.1→199.2和m/z 248.0→204.3。结果 本分析方法总运行时间为4.5 min,氟比洛芬和氟比洛芬-d5的保留时间分别为2.43,2.44 min。该方法在0.5~100 ng·mL^-1内线性关系良好。批内精密度在1.3%~12.5%,准确度在97.2%~115.2%。批间精密度在1.9%~11.6%,准确度在99.5%~107.8%。提取回收率范围为103.5%~104.9%,基质效应不显著。在不同的测试条件下,氟比洛芬在溶液和血浆中都是稳定的。验证方法成功应用于一项在中国健康志愿者开展的随机、开放、两周期、四序列、交叉给药的生物等效性研究。结果表明,2种每40 mg氟比洛芬凝胶贴膏在外用给药下,生物等效性符合接受标准,安全和耐受性良好。参比制剂和受试制剂的药动学参数为C_max(19.695±13.159)和(18.135±11.772)ng·mL^-1、T_max(19.375±3.807)和(19.575±4.138)h、AUC_0-∞(534.402±272.792)和(506.529±256.746)h·ng·mL^-1。结论 该方法可应用于氟比洛芬凝胶贴膏的生物等效性研究。

Abstract:

OBJECTIVE To establish and validate a simple, fast and sensitive method for the determination of flurbiprofen in human plasma by HPLC-MS/MS, to study pharmacokinetic characteristics and bioequivalence of flurbiprofen gel paste in Chinese healthy subjects. METHODS Chromatographic separation was accomplished on a InertSustain C₁₈ column(3.0 mm×100 mm, 3 μm) column, temperature was set at 40℃, using a binary gradient with mobile phase methanol- acetonitrile(40:60)(A) and water containing 0.1% formic acid(B) at a flow rate of 0.6 mL·min^-1 and sample preparation was by one- step protein precipitation via methanol. The mass instrument was operated in the negative ion mode, and the monitored transition was set at m/z 243.1→199.2 and m/z 248.0→204.3 for flurbiprofen and IS(flurbiprofen-d5), respectively. RESULTS The total run time in this study was 4.5 min and the retention time of flurbiprofen and flurbiprofen-d5 were 2.43, 2.44 min, respectively. The method was developed and validated over the concentration range of 0.5-100 ng·mL^-1 for flurbiprofen. The inter-batch precision was 1.3%-12.5% and accuracy 97.2%-115.2%. The intra-batch precision was 1.9%-11.6% and accuracy 99.5%-107.8%. The extraction recovery ranged from 103.5%-104.9% and the matrix effect was not significant. Flurbiprofen was proved to be stable in solution and human plasma under the different tested conditions. The validated method was successfully applied to a randomized, open-label, 2-period, 4 sequences, crossover bioequivalence study in healthy Chinese subjects, and the results indicated that bioequivalence was achieved for 2 formulations of per 40 mg paste flurbiprofen gel plaster under external conditions, and both treatments were safe and well tolerated by all study subjects. The pharmacokinetic parameters of flurbiprofen for the reference and test formulations under external conditions were as follows:C_max(19.695±13.159) and (18.135±11.772)ng·mL^-1, T_max(19.375±3.807) and (19.575±4.138)h, AUC_0-∞(534.402±272.792) and (506.529±256.746)h·ng·mL^-1. CONCLUSION The validated method can applied to a bioequivalence study of flurbiprofen in human plasma.