title: Determination of free and glucuronated hexane metabolites without prior hydrolysis by liquid- and gas-chromatography coupled with mass spectrometry.
abstract: Since n-hexane metabolites are excreted as glucuronide conjugates, most conventional analytical procedures require preliminary hydrolysis, yielding to the 'total' 2,5-hexanedione (2,5-HD), but also giving rise to a number of artifacts. The whole pattern of n-hexane metabolites, both conjugated and unconjugated, as well as different methods of sample pretreatment have been evaluated by hyphenated techniques (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS)). Aliquots of urine from rats exposed to n-hexane underwent enzymatic or acid hydrolysis or both; whereas one aliquot was applied to LC-MS, dichloromethane extracts were analyzed by GC-MS. In untreated urine, four glucuronides (-G) were identified and characterized by LC-MS: 2-hexanol-G, 5-hydroxy-2-hexanone-G, 4,5-dyhydroxy-2-hexanone-G, and 2,5-hexanediol-G. 'Free' 2,5-HD was detectable in non-hydrolyzed samples by both GC- and LC-MS. Whereas enzymatic hydrolysis did not increase the amount of 2,5-HD, acid hydrolysis led to increase 2,5-HD in variable amount and produced gamma-valerolactone as a result of a complete transformation of 4,5-dihydroxy-2-hexanone-G and the partial conversion from 5-hydroxy-2-hexanone-G. Further experiments showed that both 5-hydroxy-2-hexanone-G and 4,5-dihydroxy-2-hexanone-G, isolated by solid-phase extraction and hydrolyzed, yield comparable amount of 2,5-HD and gamma-valerolactone. In samples treated by acid hydrolysis, GC-MS only does not allow to understand the true source of 'total' 2,5-HD, which may be produced not only from 4,5-dihydroxy-2-hexanone-G but also from the more abundant 5-hydroxy-2-hexanone-G, which thus represents the main source of analytical artifacts. 'Free' 2,5-HD seems to be both suitable from an analytical point of view and meaningful for biological monitoring purposes, provided that conjugate metabolites are rapidly removed from the body leading to a negligible neurotoxic risk.
authors.affiliation: Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma Medical School, Italy. scot@unipr.it
authors.forename: P
authors.initials: P
authors.lastname: Manini
authors.forename: R
authors.initials: R
authors.lastname: Andreoli
authors.forename: A
authors.initials: A
authors.lastname: Mutti
authors.forename: E
authors.initials: E
authors.lastname: Bergamaschi
authors.forename: I
authors.initials: I
authors.lastname: Franchini
mesh_terms: D000143:Acids; D000818:Animals; D002849:Chromatography, Gas / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004798:Enzymes; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005965:Glucuronates / Q000652:urine*; D006586:Hexanes / Q000378:metabolism* / Q000633:toxicity / Q000652:urine; D000441:Hexanols / Q000652:urine; D006588:Hexanones / Q000652:urine; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000143:Acids; D004798:Enzymes; D005965:Glucuronates; D006586:Hexanes; D000441:Hexanols; D006588:Hexanones; C054726:4,5-dihydroxy-2-hexanone; C026385:n-hexane; C036260:1-hexanol; C037076:2-hexanol; C011269:2,5-hexanedione