LC-MS/MS法测定多粘菌素E血药浓度的方法建立及在MDRGN感染患儿中的临床应用

Development and Validation of an LC-MS/MS Assay for the Quantification of Colistin in Human Serum and its Clinical Application in Children with MDRGN Infections

关键词:  多粘菌素E  多粘菌素E甲磺酸钠  LC-MS/MS  MDRGN  儿童治疗药物监测

Key words:  colistin  CMS  LC-MS/MS  MDRGN  therapeutic drug monitoring

摘要:

目的 建立一种液相色谱/串联质谱（LC-MS/MS）方法，用多黏菌素B1（Polymyxin B1）作为内标测定耐药革兰氏阴性菌（multidrug-resistant Gram-negative，MDRGN）感染患儿血清中的多黏菌素E1（Colistin A）和多黏菌素E2（Colistin B）的含量以及对MDRGN患儿多黏菌素E药代动力学进行研究。方法 采用Waters Acquity UPLC? BEH C18 (2.1 mm*50 mm，1.7 μm)色谱柱对多黏菌素E的主要成分进行色谱分离，建立时间程序为5.0 min的LC-MS/MS法，以含2%甲酸的乙腈（ACN）为沉淀剂进行蛋白沉淀的样品前处理，用正离子扫描以及多反应监测模式进行分析物测定。结果 血清中多黏菌素E1在0.11～4.14 μg·mL-1内线性关系良好（r2>0.997），多黏菌素E2在0.15～5.60 μg·mL-1内线性关系良好（r2>0.990）。批内和批间精密度和准确度的RSD均≤15%。测定1例使用多黏菌素EMDRGN患儿的体内暴露量， MDRGN患儿的多黏菌素E血药浓度药代动力学表明，首次负荷剂量给药（4 mg·kg-1）时，静脉滴注开始后0.5h，多黏菌素甲磺酸钠（colistimethate sodium, CMS）的浓度达到峰值；静脉滴注开始后4h，CMS在体内水解吸收，多黏菌素E达到峰浓度。第二剂开始改为维持剂量（2.5 mg·kg-1 CMS，q12h），连续治疗10 d，患儿在第3d开始，多黏菌素E的谷浓度趋于稳态。根据两点法估算AUC0-24h为41.40±1.25 mg·h·L-1。结论 本研究建立了一种稳健的测定多黏菌素E总含量的方法。该方法快速、简单、经济，适合应用于临床MDRGN患儿多黏菌素E的药物浓度监测研究。该方法是临床医生精确监测多药耐药（MDR）革兰氏阴性菌引起的感染MDRGN患儿中多黏菌素E治疗的有价值的工具。

Abstract:

OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying serum concentrations of colistin A and colistin B in pediatric patients with multidrug-resistant Gram-negative (MDRGN) infections, using polymyxin B1 as an internal standard, and to characterize the pharmacokinetics of colistin in this population. METHODS The Waters Acquity UPLC? BEH C18 (2.1 mm*50 mm，1.7 μm) column was used to separate the main components of colistin, and an LC-MS/MS with a time program of 5.0 min was established. Acetonitrile (containing 2% formic acid) was used as precipitator for sample pretreatment of protein precipitation, positive ion scanning and multi-reaction detection mode were used for analyte determination. RESULTS A linear response was observed for colistin E1 in serum concentrations ranging from 0.11 to 4.14 μg·mL-1 and for colistin E2 from 0.15 to 5.60 μg·mL-1, with all calibration curves yielding a correlation coefficient (r2) of >0.99. The LLOQs were 0.11 μg·mL-1 (colistin A) and 0.15 μg·mL-1 (colistin B) with accuracy and precision (RSD) within ±15%. Application of this validated method to characterize the pharmacokinetics of colistin in pediatric patients with MDRGN infections revealed that following an initial loading dose (4 mg·kg-1), the prodrug CMS rapidly attained its maximum plasma concentration (Cmax) at 0.5 h after the initiation of intravenous infusion. The active metabolite, colistin reached its Cmax at 4 hours post-dose, consistent with the known hydrolysis and conversion process of CMS in vivo. The maintenance dose (2.5 mg·kg-1 CMS, q12h) was initiated starting from the second dose and continued for 10 days. The trough concentration of polymyxin E in the pediatric patient began to approach steady state from day 3 onward. The area under the concentration-time curve from 0 to 24 hours (AUC0–24h), estimated using a limited sampling strategy, was 41.40 ± 1.25 mg·h·L-1. CONCLUSION In this study, serum samples were pretreated using a simple protein precipitation method. A quantitative assay for colistin was established employing a Waters ACQUITY UPLC I-Class/Xevo TQD IVD LC-MS/MS system. The developed method is rapid, straightforward, and cost-effective, making it well-suited for therapeutic drug monitoring (TDM) of colistin in pediatric patients with MDRGN infections. This approach serves as a valuable tool for clinicians to precisely monitor polymyxin E therapy in children suffering from MDRGN infections caused by multidrug-resistant Gram-negative bacteria.