Quantitative LC/MS/MS analysis of acetaminophen–cysteine adducts (APAP–CYS) and its application in acetaminophen overdose patients

摘要

A more sensitive approach by liquid chromatographya€“tandem mass spectrometry (LC/MS/MS) method using the AB Sciex QTRAP 5500 was developed and validated for the detection and quantification of acetaminophena€“cysteine adducts (APAPa€“CYS) in human plasma. Chromatographic separation was achieved using a Protecol P C18 column (2.1 mm i.d. ?— 100 mm, 3 microns). The mobile phase consists of A: 2 mM ammonium formate in water (0.2% formic acid) and B: 2 mM ammonium formate in acetonitrile (0.2% formic acid). The analysis was performed using positive and negative ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM) with a total run time of 7 min per assay. The MS/MS ion transitions monitored were m/z 271 a?’ 140 for APAPa€“CYS (positive mode) and m/z 154 a?’ 111 for acetaminophen-D4 (negative mode). The newly developed method showed good linearity in the range of 1.0 to 100 ng mLa?’1. Inter and intra batch precision and accuracy of the method ranged from 0.28 to 5.30% and 87.0 to 113% respectively. LOD was 0.5 ng mLa?’1 and LLOQ was 1.0 ng mLa?’1. Analytes were extracted from plasma samples by simple protein precipitation using acetonitrile. The method was successfully applied in a preliminary clinical study to determine APAPa€“CYS concentration from 46 acetaminophen overdose patients treated at outpatient clinic in the University Malaya Medical Centre.