液相色谱-串联质谱法检测人血浆中阿兹夫定的浓度

Determination of Azvudine Concentration in Human Plasma by Liquid Chromatography-Mass Spectrometry

目的建立液相色谱-串联质谱法(LC-MS/MS)检测人血浆中阿兹夫定(azvudine,AZV)的浓度,并应用于新型冠状病毒感染患者的临床样本测定。方法血浆经OstroTM板除蛋白及磷脂后,以15N4-次黄嘌呤为内标,采用LC-MS/MS测定。色谱柱采用HILIC Silica柱,以乙腈(0.1%甲酸)-10 mmol·L^(-1)甲酸铵(0.1%甲酸及5%乙腈)为流动相进行梯度洗脱,流速0.30 mL·min^(-1),柱温40℃,进样量0.50μL。采用电喷雾离子源,以多反应监测模式进行正离子扫描,用于定量分析的离子对为m/z 287.3→112.0(AZV)、m/z 141.0→113.0(内标)。结果血浆中AZV在0.10~20.00 ng·mL^(-1)内线性良好,定量下限日内、日间相对标准偏差(RSD)均不大于4.46%,相对误差为12.00%~15.13%;低、中、高质控样本日内、日间RSD均不高于2.88%,相对误差为3.10%~12.11%。平均提取回收率为75.69%,基质效应及残留均不影响待测物的准确定量。该方法经验证后,成功应用于慢性透析合并新型冠状病毒感染患者血浆中AZV浓度的测定。结论本试验所建立的LC-MS/MS简单、灵敏、准确,可用于AZV的治疗药物监测。

OBJECTIVE To establish an LC-MS/MS method for the determination of azvudine(AZV)concentration in plasma samples from patients with corona virus disease 2019(COVID-19).METHODS After protein precipitation and phospholipid removal with 96-well OstroTM plate,AZV in human plasma was determined by LC-MS/MS method.15N4-hypoxanthine was used as the internal standard(the IS).Gradient elution was performed on a HILIC silica column.The gradient mobile phase consisted of acetonitrile(0.1%formic acid)and 10 mmol·L^(-1)ammonium formate(0.1%formic acid and 5%acetonitrile)at a flow rate of 0.3 mL·min^(-1).The column temperature was maintained at 40℃and the injection volume was 0.50μL.An ESI source was used on positive mode.MRM transitions were m/z 287.3→112.0(AZV)and m/z 141.0→113.0(the IS).RESULTS The assay was linear within the range of 0.10-20.00 ng·mL^(-1).Intra-and inter-day precision for LLOQ was less than 4.46%and the relative error was 12.00%-15.13%.For quality control samples,the intra-and inter-day precisions were less than 2.88%and the relative error was 3.10%-12.11%.The average extraction recovery was 75.69%.The matrix effect and carryover were negligible.After validation,the method was applied to the determination of AZV in plasma of chronic dialysis patients with COVID-19.CONCLUSIONS The established LC-MS/MS method is simple,sensitive and accurate,and it can be applied to the therapeutic drug monitoring of AZV in clinical practice.