Merck
  • Home
  • Search Results
  • LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2013-12-25)
Arne Trettin, Jens Jordan, Dimitrios Tsikas
ABSTRACT

Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Potassium phosphate dibasic solution, 1.0 M
Sigma-Aldrich
N-Acetyl-L-cysteine, Sigma Grade, ≥99% (TLC), powder
Sigma-Aldrich
Acetaminophen, BioXtra, ≥99.0%
Sigma-Aldrich
Acetaminophen, meets USP testing specifications, 98.0-102.0%, powder
Sigma-Aldrich
Potassium phosphate dibasic, reagent grade, ≥98.0%
Sigma-Aldrich
Acetaminophen, analytical standard
Sigma-Aldrich
Potassium phosphate dibasic, meets USP testing specifications
Sigma-Aldrich
N-Acetyl-L-cysteine, BioXtra, ≥99% (TLC)
Sigma-Aldrich
N-Acetyl-L-cysteine, BioReagent, suitable for cell culture
Supelco
Acetonitrile, analytical standard
Supelco
Ethyl acetate, analytical standard
Sigma-Aldrich
L-Glutathione reduced, suitable for cell culture, BioReagent, ≥98.0%, powder
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
L-Glutathione reduced, BioXtra, ≥98.0%
Supelco
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% (GC)
Sigma-Aldrich
Potassium phosphate dibasic, anhydrous, for luminescence, for molecular biology, BioUltra, ≥99.0% (T)
Sigma-Aldrich
Sodium nitrite, 99.999% trace metals basis
Sigma-Aldrich
Ethyl acetate, natural, ≥99%, FCC, FG
Sigma-Aldrich
Ethyl acetate, ≥99%, FCC, FG
Sigma-Aldrich
N,N-Diisopropylethylamine, ReagentPlus®, ≥99%
Sigma-Aldrich
2,3,4,5,6-Pentafluorobenzyl bromide, 99%
Sigma-Aldrich
Ethyl acetate, anhydrous, 99.8%
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
Acetonitrile, ≥99.5%, ACS reagent
Supelco
Nitrite ion standard solution, 0.1 M NO2-, for ion-selective electrodes
Sigma-Aldrich
Ethyl acetate, ACS reagent, ≥99.5%
Supelco
Acetaminophen, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Potassium phosphate dibasic, 99.95% trace metals basis
Sigma-Aldrich
Ethyl acetate, ReagentPlus®, ≥99.8%