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.

Key words::

• Phenacetin
• solid-phase extraction (SPE)
• nuclear magnetic resonance (NMR) spectroscopy
• mass spectrometry (MS)
• futile deacetylation
• acetaminophen

Acknowledgements

The authors would like to acknowledge the cooperation of Bruker Biospin and the Waters Corporation for their assistance with this work.