建立并验证了一种 LC-MS/MS 方法，用于测定 EDTA 处理的人血浆中的羟氯喹、两种代谢物和阿奇霉素。
Development and validation of an LC-MS/MS method for determination of hydroxychloroquine, its two metabolites, and azithromycin in EDTA-treated human plasma.
机构信息
Drug Research Unit, Department of Clinical Pharmacy, School of Pharmacy, University of California at San Francisco, San Francisco, California, United States of America.
出版信息
PLoS One. 2021 Mar 5;16(3):e0247356. doi: 10.1371/journal.pone.0247356. eCollection 2021.
BACKGROUND
Hydroxychloroquine (HCQ) and azithromycin (AZM) are antimalarial drugs recently reported to be active against severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2), which is causing the global COVID-19 pandemic. In an emergency response to the pandemic, we aimed to develop a quantitation method for HCQ, its metabolites desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ), and AZM in human plasma.
METHODS
Liquid chromatography tandem mass spectrometry was used to develop the method. Samples (20 μL) are extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a PFP column (2.0 × 50 mm, 3 μm). ESI+ and MRM are used for detection. Ion pairs m/z 336.1→247.1 for HCQ, 308.1→179.1 for DHCQ, 264.1→179.1 for BDCQ, and 749.6→591.6 for AZM are selected for quantification. The ion pairs m/z 342.1→253.1, 314.1→181.1, 270.1→181.1, and 754.6→596.6 are selected for the corresponding deuterated internal standards (IS) HCQ-d4, DHCQ-d4, BDCQ-d4, and AZM-d5. The less abundant IS ions from 37Cl were used to overcome the interference from the analytes.
RESULTS
Under optimized conditions, retention times are 0.78 min for BDCQ, 0.79 min for DHCQ, 0.92 min for HCQ and 1.87 min for AZM. Total run time is 3.5 min per sample. The calibration ranges are 2-1000 ng/mL for HCQ and AZM, 1-500 ng/mL for DHCQ and 0.5-250 ng/mL for BDCQ; samples above the range are validated for up to 10-fold dilution. Recoveries of the method ranged from 88.9-94.4% for HCQ, 88.6-92.9% for DHCQ, 88.7-90.9% for BDCQ, and 98.6%-102% for AZM. The IS normalized matrix effect were within (100±10) % for all 4 analytes. Blood samples are stable for at least 6 hr at room temperature. Plasma samples are stable for at least 66 hr at room temperature, 38 days at -70°C, and 4 freeze-thaw cycles.
CONCLUSIONS
An LC-MS/MS method for simultaneous quantitation of HCQ, DHCQ, BDCQ, and AZM in human plasma was developed and validated for clinical studies requiring fast turnaround time and small samples volume.
背景
羟氯喹（HCQ）和阿奇霉素（AZM）是最近报道对导致全球 COVID-19 大流行的严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）具有活性的抗疟药物。在对大流行的紧急应对中，我们旨在开发一种用于 HCQ、其代谢物去乙基羟氯喹（DHCQ）和双去乙基氯喹（BDCQ）以及 AZM 在人血浆中的定量方法。
方法
采用液相色谱串联质谱法开发方法。样品（20 μL）通过固相萃取提取，注入配备 PFP 柱（2.0×50mm，3μm）的 LC-MS/MS 系统。ESI+和 MRM 用于检测。用于定量的离子对为 m/z 336.1→247.1 的 HCQ、m/z 308.1→179.1 的 DHCQ、m/z 264.1→179.1 的 BDCQ 和 m/z 749.6→591.6 的 AZM。选择 m/z 342.1→253.1、314.1→181.1、270.1→181.1 和 754.6→596.6 的离子对作为相应的氘代内标（IS）HCQ-d4、DHCQ-d4、BDCQ-d4 和 AZM-d5。使用丰度较低的 37Cl IS 离子克服分析物的干扰。
结果
在优化条件下，BDCQ 的保留时间为 0.78min，DHCQ 的保留时间为 0.79min，HCQ 的保留时间为 0.92min，AZM 的保留时间为 1.87min。每个样品的总运行时间为 3.5 分钟。HCQ 和 AZM 的校准范围为 2-1000ng/mL，DHCQ 的校准范围为 1-500ng/mL，BDCQ 的校准范围为 0.5-250ng/mL；超过该范围的样品可验证高达 10 倍稀释。该方法的回收率范围为 HCQ 的 88.9-94.4%、DHCQ 的 88.6-92.9%、BDCQ 的 88.7-90.9%和 AZM 的 98.6%-102%。所有 4 种分析物的 IS 归一化基质效应均在（100±10）%范围内。室温下血液样本至少稳定 6 小时。室温下血浆样本至少稳定 66 小时，-70°C 下稳定 38 天，冷冻-解冻循环 4 次。
结论
开发并验证了一种用于人血浆中 HCQ、DHCQ、BDCQ 和 AZM 同时定量的 LC-MS/MS 方法，适用于需要快速周转时间和小样本量的临床研究。
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