title: 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 100 μL portion of the filtrate was analyzed on a YMC-Pack ODS-AMQ C18 column, using 100 mM potassium dihydrogen phosphate-methanol-acetic acid (100 : 0.6 : 0.1) as the mobile phase, a flow rate of 1 mL/min, and photometric detection at 254 nm. PABA and APAP-cystein-S-yl (APAP-Cys) eluted at ~14.7 min and 22.7 min, respectively. Method linearity, based on on-column concentrations of APAP-Cys, was observed over the range 0.078-40 μg. 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 0.06 μg and 0.14 μg, 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, NY 11439, USA.
authors.forename: Miteshkumar
authors.initials: M
authors.lastname: Acharya
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Lau-Cam
mesh_terms: D000082:Acetaminophen / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D057230:Limit of Detection; D008099:Liver / Q000378:metabolism*; D008297:Male; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D011506:Proteins; D000082:Acetaminophen