Simple reversed-phase HPLC method with Spectrophotometric detection for measuring acetaminophen-protein adducts in rat liver samples

Abstract

A simple reversed-phase HPLC method for measuring hepatic levels of acetaminophen- (APAP-) protein adduct following an overdose of APAP was developed. An aliquot of liver homogenate in phosphate-buffered saline pH 7.4 (PBS) was placed on a Nanosep centrifugal device, which was centrifuged to obtain a protein residue. This residue was incubated with a solution of p-aminobenzoic acid (PABA), the internal standard, and bacterial protease in PBS, transferred to a Nanosep centrifugal device, and centrifuged. A 100L portion of the filtrate was analyzed on a YMC-Pack ODS-AMQ C18 column, using 100mM potassium dihydrogen phosphate-methanol-acetic acid (100:0.6:0.1) as the mobile phase, a flow rate of 1mL/min, and photometric detection at 254nm. PABA and APAP-cystein-S-yl (APAP-Cys) eluted at 14.7min and 22.7min, respectively. Method linearity, based on on-column concentrations of APAP-Cys, was observed over the range 0.07840g. Recoveries of APAP-Cys from spiked blank liver homogenates ranged from 83 to 91. Limits of detection and of quantification of APAP-Cys, based on column concentrations, were 0.06g and 0.14μg, respectively. RSD values for interday and intraday analyses of a blank liver homogenate spiked with APAP-Cyst at three levels were, in all cases, ≤1.0 and <1.5%, respectively. The proposed method was found appropriate for comparing the antidotal properties of N-acetylcysteine and taurine in a rat model of APAP poisoning. © Copyright 2012 Miteshkumar Acharya and Cesar A. Lau-Cam.