Quantitative analysis of primaquine and its metabolites in human urine using liquid chromatography coupled with tandem mass spectrometry

•UPLC-MS/MS method for simultaneous quantification of primaquine and its metabolites in human urine.•Simple sample preparation with no apparent matrix effect.•The validated method was accurate, reliable, and complies with FDA guidelines.•Implementation of developed method on clinical pharmacokinetic analysis.•Primaquine orthoquinone, and glucuronide conjugation of PQ and carboxyprimaquine were the major metabolites in urine. Primaquine (PQ), a prototype 8-aminoquinoline (8-AQ) drug used to treat malaria, is rapidly metabolized into different inactive and active metabolites. Due to the hemolytic toxicity, the uses of PQ have been confined. To understand its overall metabolism and its relation to drug efficacy and toxicity, profiling of urine for the parent drug and its metabolites is important. The current study presents a convenient and rapid method for simultaneously quantifying primaquine (PQ) and its metabolites in human urine. A simple liquid–liquid extraction followed by chromatographic separation and quantification through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to quantify PQ and its eleven metabolites in the urine of healthy human volunteers who received a single oral dose of PQ. The developed method separated fourteen analytes, including internal standards, within nine minutes of run time. The linearity of all analytes was suitable in the range of 1–500 ng/mL. The extraction recovery for all concentrations of analytes from urine was ranged from 90.1 to 112.9 %. The relative standard deviation for intra- and inter-day precision were