title: Identification of phenacetin metabolites in human urine after administration of phenacetin-C2H3: measurement of futile metabolic deacetylation via HPLC/MS-SPE-NMR and HPLC-ToF MS.
abstract: The metabolism of acetyl-labelled phenacetin-C2H3 was investigated in man following a single (150 mg) oral dose. Urine samples were collected at predose, 0-2 h and >2-4 h post-dose, and samples from each time-point were then analysed directly using 1H-nuclear magnetic resonance (NMR) spectroscopy. The phenacetin metabolites acetaminophen (paracetamol) glucuronide, sulphate and the N-acetyl-L-cysteinyl conjugate were identified by this method, and all showed clear evidence of the loss of the original 2H3-acetyl label and its replacement with 1H3 (futile deacetylation). The observed percentage futile deacetylation by 1H-NMR spectroscopy was measured as approximately 20% in each metabolite (about 2% of the recovered dose). After sample preparation by solid-phase extraction on a C18 solid-phase extraction (SPE) cartridge, further profiling was performed using high-performance liquid chromatography/mass spectrometry-solid-phase extraction-nuclear magnetic resonance (HPLC/MS-SPE-NMR) confirming futile deacetylation had taken place as indicated by NMR spectroscopy on both the isolated acetaminophen glucuronide and L-cysteinyl-metabolites. Additional analysis by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-ToF MS) identified further phenacetin metabolites, and from these data the mean percentage of futile deacetylation was measured as 31% +/- 2% for the acetylated phenacetin metabolites. A number of non-acetylated metabolites were also detected in the sample via HPLC-ToF MS. The results showed that phenacetin underwent a transient formation via a number of toxic intermediates to a much greater extent than had been observed in similar studies on acetaminophen. These results may contribute to the understanding of the analgesic nephropathy reported following chronic phenacetin consumption.
authors.affiliation: Metabometrix Ltd, RSM, London, UK. andrew.w.nicholls@gsk.com
authors.forename: A W
authors.initials: AW
authors.lastname: Nicholls
authors.forename: I D
authors.initials: ID
authors.lastname: Wilson
authors.forename: M
authors.initials: M
authors.lastname: Godejohann
authors.forename: J K
authors.initials: JK
authors.lastname: Nicholson
authors.forename: J P
authors.initials: JP
authors.lastname: Shockcor
mesh_terms: D000107:Acetylation; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010615:Phenacetin / Q000008:administration & dosage / Q000378:metabolism*; D051381:Rats; D014556:Urine / Q000737:chemistry*
chemical_list: D010615:Phenacetin