LC-MS/MS测定人血浆中对乙酰氨基酚及其人体药动学和生物等效性研究

Determination of Paracetamol in Human Plasma Using LC-MS/MS: Application to A Pharmacokinetic and Bioequivalence Study in Healthy Human Subjects

关键词:  对乙酰氨基酚  LC-MS/MS  人血浆  药动学  生物等效性

Key words:  paracetamol  LC-MS/MS  human plasma  pharmacokinetics  bioequivalence

摘要:

目的 建立一种快速、灵敏的高效液相色谱-串联质谱（LC-MS/MS）方法以测定人血浆中对乙酰氨基酚浓度，并应用于2种对乙酰氨基酚制剂的人体药动学和生物等效性研究。方法 以替硝唑为内标，200 μL血浆样品经1 mL乙酸乙酯液液萃取，再经Waters XBridge^® C₁₈柱等度洗脱分离后导入串联质谱，以正离子多反应监测模式进行定量分析，对乙酰氨基酚和内标的选择性反应离子对分别是m/z 152.0→110.0和248.0→121.0。方法经验证后应用于19名健康受试者单剂量空腹口服2种对乙酰氨基酚制剂500 mg后药动学和生物等效性的研究。结果 血浆中对乙酰氨基酚在0.1~8.0 μg·mL^-1范围内线性良好（r²>0.99），最低检测限为0.1 μg·mL^-1，提取回收率为91.0%~98.7%，日内和日间准确度分别为98.8%~111.3%（精密度≤9.0%）和94.9%~102.6%（精密度≤10.7%）。生物等效性试验中，受试制剂与参比制剂的主要药代动力学参数C_max、AUC_{0-12 h}和AUC_0-∞几何均值比的90%置信区间分别为83.50%~105.79%，94.25%~101.54%和93.24%~101.02%，均落在生物等效可接受标准的80.00%~125.00%。结论 所建立测定人血浆中对乙酰氨基酚浓度的LC-MS/MS具有快速灵敏、回收率高、选择性好的特点，适用于对乙酰氨基酚片人体药动学和生物等效性研究。受试制剂与参比制剂在人体内吸收速度和程度相似，2种制剂生物等效。

Abstract:

OBJECTIVE To develop a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometric(LC-MS/MS) assay for the quantitative determination of paracetamol in human plasma, and apply it to a pharmacokinetic and bioequivalence study of two paracetamol formulations in human. METHODS Tinidazole was used as internal standard(IS), paracetamol were extracted from 200 μL human plasma with 1 mL ethyl acetate. Chromatographic separation was achieved on an Waters XBridge^® C₁₈ column under isocratic elution condition. Then with the mass spectrometer operated in positive electrospray ionization mode using multiple reaction monitoring, paracetamol and IS were quantitative analyzed by detecting MS/MS ion transition at m/z 152.0→110.0 and 248.0→121.0, respectively. The validated method was employed in the pharmacokinetic and bioequivalent study of two paracetamol formulations, which conducted in 19 healthy adult human subjects following single-dose oral administrations of 500 mg paracetamol under fasting condition. RESULTS The method showed linearity over the concentration range of 0.1-8.0 μg·mL^-1(r²>0.99) for paracetamol in human plasma. The lower limit of quantitation achieved was 0.1 μg·mL^-1 and the extraction recovery ranged from 91.0% to 98.7%. Intra-and inter-day accuracy were in the ranges of 98.8%-111.3% (precision ≤ 9.0%) and 94.9%-102.6% (precision ≤ 10.7%) respectively. Additionally, in the bioequivalence study, the calculated 90% confidential intervals of geometric mean (test/reference) ratios for pharmacokinetic parameters(C_max, AUC_{0-12 h} and AUC_0-∞) of the two paracetamol formulations were 83.50%-105.79%, 94.25%-101.54% and 93.24%-101.02%, respectively, which all fell within the bioequivalence acceptance range of 80.00%-125.00%. CONCLUSION A rapid and sensitive LC-MS/MS method for the determination of paracetamol in human plasma is developed, it has the characteristics of rapid sensitivity, high recovery and good selectivity and is successfully applied to the pharmacokinetic and bioequivalent study in healthy human subjects. The results show that the test formulation is similar to the reference formulations in terms of rate and extent of absorption, the two formulations are bioequivalent.