UPLC-MS/MS测定患者血浆多黏菌素B的浓度及用药方案优化研究

Determination of polymyxin B in plasma by UPLC-MS/MS and dosage optimization

目的:建立血浆中多黏菌素B浓度测定的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法。方法:采用ACQUITY UPLC HSS T3(2.1 mm×100 mm,1.7μm)色谱柱,流动相组成为0.2%甲酸的水-甲醇和乙腈(1∶1,V/V)溶液,梯度洗脱3.5 min。多反应监测的离子对为多黏菌素B1 m/z 602.4→241.1,多黏菌素B2 m/z 595.5→100.9,多黏菌素E1 m/z 585.5→202.0。按照一房室模型公式,根据多黏菌素B峰浓度和谷浓度进行AUCss,24 h的推算。结果:血浆中多黏菌素B1在0.05~5μg·mL^(-1)、B2在0.022~0.553μg·mL^(-1)内线性关系良好。多黏菌素B1、B2批内和批间的精密度均小于10.9%,准确度为88.8%~105.7%。平均回收率在88.0%~103.2%之间,基质效应为100.0%~113.7%。血浆样本在室温放置6 h、4℃放置24 h、3次冻融循环和-80℃冻存30 d均稳定。按照经验用药,纳入观察的6例患者中1例AUC_(ss,24 h)明显低于50 mg·h·L^(-1),及时和临床医生反馈,调整后达标。结论:建立了一种快速、准确、灵敏度高、特异性强的UPLC-MS/MS法用于人血浆中多黏菌素B的分析。方法学验证符合生物样本定量分析方法指导原则的要求。本方法适于在临床上开展多黏菌素B的治疗药物监测。

OBJECTIVE To develop an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for determination of polymyxin B in plasma.METHODS The analytical column was ACQUITY UPLC HSS T3 column(2.1 mm×100 mm,1.7μm).The mobile phase consisted of aqueous phase A as water and organic phase B composed of methanol and acetonitrile(1∶1,V/V).Formic acid in all mobile phase was adjusted to 0.2%.The gradient elution lasted 3.5 mins.Electron spray ionization was set at positive mode.Multiple reaction monitoring was performed at m/z 602.4→241.1 for PB1,595.5→100.9 for PB2 and 585.5→202.0 for internal standard PE1.According to one compartment model,trough and peak plasma concentration were obtained to calculate AUCss,24h.RESULTS The linear range of PB1 in plasma was 0.05-5μg·mL^(-1)and PB2 was 0.015-0.372μg·mL^(-1).The intra-day and inter-day precision were less than 10.9%,and the accuracy was between 88.8%and 105.7%.For the average recoveries was 88.0%to 103.2%,and the matrix effect was 100.0%to 113.7%.The analyte was stable when the plasma samples were stored at room temperature for 6 h,at 4℃for 24 h,after 3 freeze-thaw cycles and-80℃for 30 days.By empirical medication one of six patients included in the observation had an AUCss,24h lower than 50 mg·h·L^(-1),which was reported to doctors immediately and after dosage optimization AUC_(ss,24h)met requirements.CONCLUSION The established UPLC-MS/MS method was rapid,accurate,sensitive and specific and was successfully used to monitor major polymyxin B.The method validation is in according with guidelines for biological samples quantitative analysis.Our method is suitable for polymyxin B therapeutic drug monitoring in clinical practice.