Central Laboratory Division, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.
Laboratório de Investigação Médica (LIM 03), Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.
J Appl Lab Med. 2021 Jul 7;6(4):868-880. doi: 10.1093/jalm/jfab031.
Hydroxychloroquine (HCQ) blood levels are used to monitor efficacy, safety, and patient adherence during treatment. Oral fluid has emerged as an alternative noninvasive, easily accessible, and low-complexity matrix for drug monitoring. However, there is no analytical method to measure HCQ in oral fluid. Therefore, we developed and validated an ultra-high-performance liquid chromatography-tandem mass (UHPLC-MS/MS) method for the measurement of HCQ and its main metabolites in oral fluid and compared to whole blood.
Ten microliters of matrices were used for sample preparation by protein precipitation with acetonitrile followed by online solid phase extraction. The validation process included assessment of lower limit of quantification, linearity, precision, recovery, matrix effect, interferences assessment, carryover, and sample dilution validation.
The lower limit of quantification was 50 ng/mL for HCQ and metabolites in both oral fluid and whole blood. The calibration curve was linear from 50 to 2000 ng/mL (r2 = 0.999). The coefficient of variation for precision assay was 1.2% to 9.7% for intraday and 1.1% to 14.2% for interday for both HCQ and metabolites in oral fluid and whole blood samples at 150, 750, and 1250 ng/mL. The recovery was 85.3% to 118.5% for 150, 750, and 1250 ng/mL of HCQ and metabolites in both oral fluid and whole blood. Dilution factor up to 5-fold was validated for concentrations higher than the upper limit of quantification.
The validated method is specific, precise, and accurate to determine the analytical range for therapeutic monitoring of HCQ and its main metabolites in oral fluid and blood.
羟氯喹 (HCQ) 的血药浓度用于监测治疗期间的疗效、安全性和患者依从性。口服液已成为替代非侵入性、易于获取和低复杂性的药物监测基质。然而，目前尚无分析方法可用于测量口服液中的 HCQ。因此，我们开发并验证了一种超高效液相色谱-串联质谱 (UHPLC-MS/MS) 方法，用于测量口服液和全血中的 HCQ 及其主要代谢物，并与全血进行比较。
使用 10 微升基质通过乙腈沉淀蛋白进行样品制备，随后进行在线固相萃取。验证过程包括评估定量下限、线性、精密度、回收率、基质效应、干扰评估、交叉污染和样品稀释验证。
HCQ 和代谢物在口服液和全血中的定量下限均为 50ng/mL。校准曲线在 50 至 2000ng/mL 范围内呈线性（r2=0.999）。在 150、750 和 1250ng/mL 时，口服液和全血中 HCQ 和代谢物的日内精密度测定变异系数为 1.2%至 9.7%，日间精密度测定变异系数为 1.1%至 14.2%。150、750 和 1250ng/mL 时，HCQ 和代谢物在口服液和全血中的回收率为 85.3%至 118.5%。验证了稀释倍数高达 5 倍的稀释因子，用于浓度高于定量下限的情况。
该验证方法专属性强、精密度高、准确度高，可用于测定口服液和全血中 HCQ 及其主要代谢物的治疗监测分析范围。
