Nonenzymatic formation of a novel hydroxylated sulfamethoxazole derivative in human liver microsomes: implications for bioanalysis of sulfamethoxazole metabolites
Abstract
Sulfamethoxazole is metabolized by microsomal CYP2C9 to a hydroxylamine that is thought to be responsible for the relatively high incidence of hypersensitivity reactions associated with the drug. Accurate quantification of the hydroxylamine requires the loss of metabolite through autoxidation to be blocked with ascorbate. In this study, a partly nonenzymatically generated arylhydroxylated derivative of sulfamethoxazole was identified by liquid chromatography/mass spectrometry in incubations of human liver microsomes, and it was found to coelute with the isomeric hydroxylamine under the conditions of three published high-performance liquid chromatography (HPLC) assays. Partial inhibition of the aryl hydroxylation by 1-aminobenzotriazole suggested some involvement of cytochrome P450. However, the formation of this compound was ascorbate-dependent, and it was enhanced by the addition of Fe2+/EDTA and inhibited by desferrioxamine but not by mannitol. These findings are consistent with the phenol being generated via an Fe2+/ascorbate/O2-oxygenating system that does not involve hydroxyl radicals. It was also produced by H2O2/ascorbate. Because the compound shares close chromatographic similarities with the hydroxylamine metabolite, it is possible that previous studies may have inaccurately characterized or quantified sulfamethoxazole metabolism.
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Previous LC-MS/MS methods would not accurately separate SMX-OH and SMX-HA, which have the same mass.
“They also showed previous LC-MS/MS methods would not accurately separate SMX-OH and SMX-HA, which have the same mass.”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [35]
Ascorbate promotes formation of SMX-OH in HLM.
“We showed that ascorbate promotes formation of SMX-OH, consistent with the findings by Sanderson et al., (2008).35”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [35]
Sanderson et al. (2008) showed the formation of a non-enzymatic arylhydroxylated derivative of SMX (SMX-OH), which is formed in the presence of ascorbate.
“A study by Sanderson et al., (2008) showed the formation of a non-enzymatic arylhydroxylated derivative of SMX (SMX-OH), which is formed in the presence of ascorbate.35”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [35]
The new LC-MS/MS method ensures accurate measurement of SMX-HA, which may have been confounded in previous analyses.
“This approach ensures accurate measurement of SMX-HA, which may have been confounded in previous analyses.35”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [35]
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- Authors
- Sanderson, J. P., Hollis, F. J., Maggs, J. L., Clarke, S. E., Naisbitt, D. J., & Park, B. K.
- Affiliations
- University of Liverpool (GB)
- Venue
- Drug Metabolism and Disposition
- Publisher
- Elsevier BV
- Published
- 2008-12
- Volume
- 36
- Issue
- 12
- Pages
- 2424-2428
- ISSN
- 0090-9556 (ISSN-L)
- OpenAlex
- W2119204170
- Cited by
- 8
- References
- 28
- Citations by year
- 2025: 1 · 2022: 1 · 2021: 3 · 2018: 2 · 2012: 1
- FWCI
- 0.00
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Drug Metabolism and Disposition (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
- Health Sciences › Medicine › Pharmacology › Drug-Induced Adverse Reactions
- Physical Sciences › Chemistry › Analytical Chemistry › Analytical Methods in Pharmaceuticals
- Keywords
- sulfamethoxazole, vitamin C, CYP2C9, hydroxylamine metabolites, human liver microsomes, 1-aminobenzotriazole, deferoxamine, drug metabolite quantification
- MeSH terms
- Ascorbic Acid, Chromatography, High Pressure Liquid, Deferoxamine, Edetic Acid, Enzyme Inhibitors, Ferrous Compounds, Humans, Hydrogen Peroxide, Hydroxylation, Kinetics, Microsomes, Liver, NADP, Sulfamethoxazole, Triazoles, Molecular Structure, Tandem Mass Spectrometry
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