HPLC-MS/MS法同时测定人血浆中替诺福韦艾拉酚胺及其代谢物替诺福韦的浓度和临床应用

目的：建立快速、灵敏的高效液相色谱-串联质谱（HPLC-MS/MS）方法同时测定人血浆中替诺福韦艾拉酚胺（TAF）及其活性代谢产物替诺福韦（TFV）的浓度，并研究该药物在人体内的药动学特征。方法：采用Capcell pake ADME色谱柱（75 mm×2.1 mm，5 μm），以0.1%甲酸水溶液-甲醇为流动相，梯度洗脱，流速0.4 mL·min^-1，进样量5 μL；采用多反应监测（MRM），选择质荷比（m/z）477.3/346.1（TAF）、288.2/176.0（TFV）、484.3/346.1（内标1：TAF-d₇）及294.2/182.0（内标2：TFV-d₆）进行检测。结果：TAF与TFV质量浓度分别在0.1~400 ng·mL^-1及0.4~20 ng·mL^-1范围内线性关系良好，定量下限分别为0.100 0 ng·mL^-1及0.400 0 ng·mL^-1，日内、日间精密度均小于9.0%，准确度分别为94.1%~99.2%和95.5%~112.8%。健康中国人服用富马酸替诺福韦艾拉酚胺片25 mg后，TAF主要药动学参数：C_max=（207.97±61.08）ng·mL^-1，T_1/2=（0.54±0.15）h，AUC_0-∞=（126.07±42.67）h·ng·mL^-1；TFV主要药动学参数：C_max=（8.76±1.46）ng·mL^-1，T_1/2=（29.47±4.16）h，AUC_0-∞=（193.19±45.75）h·ng·mL^-1。结论：该方法适用于TAF及其代谢产物TFV的血药浓度检测，并提供药动学参数。

Objective:To develop a selective and rapid HPLC-MS/MS method for simultaneously determination of tenofovir alafenamide(TAF)and its active metabolite,tenofovir(TFV),in human plasma,in order to study the pharmacokinetics in healthy volunteers.Methods:The fractionation of the plasma sample and determination of TAF/TFV was achieved using HPLC-MS/MS equipped with Capcell pake ADME column(75 mm×2.1 mm,5 μm),using 0.1% formic acid water solution and methanol as mobile phase,respectively,by a gradient elution at 0.4 mL·min^-1 flow rate.The mass was captured by multiple reactions monitoring(MRM),with m/z of 477.3/346.1(TAF),288.2/176.0(TFV),484.3/346.1(internal standard 1:TAF-d₇)and 294.2/182.0(internal standard 2:TFV-d₆)as the characteristic ion masses.Results:Under the concentration range 0.1-400 ng·mL^-1 for TAF and 0.4-20 ng·mL^-1 for TFV,good linearity was achieved with 0.100 0 ng·mL^-1 and 0.400 0 ng·mL^-1 as lower limit of quantification,respectively.Both inter-and intra-day precisions were bellow 9.0% and the accuracy was between 94.1%-99.2% for TAF and 95.5%-112.8% for TFV.The main pharmacokinetic parameters of TAF and TFV measured in healthy Chinese volunteers after oral administration TAF 25 mg were as follows:C_max=(207.97±61.08)ng·mL^-1,(8.76±1.46)ng·mL^-1;T_1/2=(0.54±0.15)h,(29.47±4.16)h;AUC_0-∞=(126.07±42.67)h·ng·mL^-1,(193.19±45.75)h·ng·mL^-1.Conclusion:The HPLC-MS/MS method is validated for application in quantification of TAF and its metabolite TFV,and proved adaptable for following pharmacokinetic studies.

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