Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood

Abstract

ObjectivesHydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described.MethodsBlood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 μL/min. The analysis time was 8 min.ResultsFor each analyte linear calibration curves were obtained over the concentration range 50-2000 μg/L, the lower limit of quantification (LLOQ) was 13 μg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 μg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively.ConclusionThis method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.

Review of the Use of Liquid Chromatography-Tandem Mass Spectrometry in Clinical Laboratories: Part II–Operations

“…Approaches differ in the predilution concentrations used for assessment. In one approach, samples are fortified to a concentration above the analytical measurement range (AMR) and measured only after dilution to within the AMR [ 10 , 11 ]. Alternatively, samples measured to lie within the AMR are diluted and re-assayed [ 12 , 13 ].…”