Abstract
A U(H)PLC-MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5min/sample, requires only 5μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44μg/ml (APAP-C), 0.58μg/ml (APAP-SG), 0.84μg/ml (APAP-NAC), 2.75μg/ml (APAP-S), 3.00μg/ml (APAP-G) and 16μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.
Highlights
The analgesic and antipyretic drug acetaminophen (paracetamol, N-(4-hydroxyphenyl) acetamide, APAP) was launched over 60 years ago
Two further methods have been described offering validated HPLC [16] or UPLC [17]-ESI-MS/MS methods that enable the quantification of APAP, its glucuronide (APAP-G), sulfate (APAP-S), GSH (APAP-GS), cysteinyl (APAP-C), N-acetylcysteinyl (APAP-NAC) [16,17] and methoxy-(APAP-OMe) [16] metabolites in human plasma
Materials & methods Chemicals & reagents APAP, APAP-G and the deuterated internal standard APAP-d3 were purchased from Sigma Aldrich (Gillingham, UK), its sulfate (APAP-S, potassium salt), cysteinyl (APAP-C, trifluoroacetic acid salt), N-acetylcysteinyl (APAP-NAC, disodium salt), GSH (APAP-SG, and 3-methoxy (APAP-OMe) conjugates, and deuterated internal standards, APAP-S-d3, APAP-G-d3, APAP-C-d5 (TFA salt) APAP-NAC-d5 and APAP-SG-d3, were purchased from Toronto Research Chemicals (Toronto, Canada) and were used as supplied
Summary
The analgesic and antipyretic drug acetaminophen (paracetamol, N-(4-hydroxyphenyl) acetamide, APAP) was launched over 60 years ago It remains one of the most widely used drugs for the treatment of pain in the general human population. Several LC–MS-based methods have been developed that offer the opportunity of reduced sample size and improved specificity compared with LC-UV [11,12,13,14]. For some of these LC–MS methods metabolite coverage was limited to the drug and its glucuronide and/or sulfate conjugates [11,12,13].
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