Development of the Liquid Chromatography Tandem Mass Spectrometry Method for Determination of Chloroquine and Desethylchloroquine in Human Plasma

Abstract

Objective: Several studies reported a relationship between adverse effect and elevated blood concentration of chloroquine (CQ) and its active metabolite, desethylchloroquine (DCQ). Therefore, therapeutic drug monitoring of these drugs might be useful. This study aims to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of CQ and DCQ in human plasma.

Methods: Chromatographic separation was carried out on the Synergi® 2.5μm polar RP (150 x 4.6 mm.I.D.) and used 0.3% formic acid/acetonitrile at 70/30,v/v as a mobile phase. The mass spectrometry was operated with positive electro spray ionization and multiple reactions monitoring (MRM) mode. The mixture of methyl t-butyl ether and isooctane at 90/10,v/v with adjusted pH to 12 by ammonium hydroxide were used for extraction. The method was developed and fully validated according to USFDA guidelines.

Results: The ion transitions were 320.01 to 247.01 and 142.12 m/z for CQ and 292.0 to 179.01 and 114.10 m/z for DCQ. The lower limit of quantification (LLOQ) were 0.2 and 0.4 ng/mL for CQ and DCQ, respectively. This method had accept- able accuracy and precision with good linearity (r 2 >0.997) and high recovery of extraction (89.34-108.42%). The ranges of quantification were 0.2-1,000 ng/mL for CQ and 0.4-1,000 ng/mL for DCQ. Neither anticoagulant nor matrix had any effect which interfered with this analysis.

Conclusion: A highly selective, sensitive and reproducible LC-MS/MS method to detect CQ and DCQ level in human plasma was developed and validated. This method was successfully applied to determine the steady state concentration of CQ and DCQ in plasma of rheumatoid arthritis patients.