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Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Ayala DC, Morin D, Buckpitt AR - PLoS ONE (2015)

Bottom Line: Subsequent biotransformation results in urinary elimination of several conjugated metabolites.The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide.The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, United States of America; University of California Davis, Forensic Science, Graduate Program, 1909 Galileo Ct., Suite B, Davis, CA 95618, United States of America.

ABSTRACT
Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

No MeSH data available.


Related in: MedlinePlus

Extracted ion chromatograms of urinary naphthalene metabolites and deuterated internal standards.(A) QC Sample #2. Naphthalene mercapturate, N-acetyl glutathione conjugate, naphthol glucuronide, and naphthol sulfate at 40, 5, 70, 20 ng on column, respectively. (B) Pooled urine sample from day 6–7 of exposure treatments (n = 3 animals). Calculated amounts of each metabolite on column were: 124.8, 7.9, 45.3, 25.9 ng for mercapturic acid, N-acetyl GSH, naphthol glucuronide and naphthol sulfate, respectively.
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pone.0121937.g002: Extracted ion chromatograms of urinary naphthalene metabolites and deuterated internal standards.(A) QC Sample #2. Naphthalene mercapturate, N-acetyl glutathione conjugate, naphthol glucuronide, and naphthol sulfate at 40, 5, 70, 20 ng on column, respectively. (B) Pooled urine sample from day 6–7 of exposure treatments (n = 3 animals). Calculated amounts of each metabolite on column were: 124.8, 7.9, 45.3, 25.9 ng for mercapturic acid, N-acetyl GSH, naphthol glucuronide and naphthol sulfate, respectively.

Mentions: We achieved optimal separation of all four major naphthalene metabolites and their corresponding deuterium-labeled internal standards with a 0.1% acetic acid water/acetonitrile gradient. All targeted metabolites eluted between 38.9 and 74.4 min (Table 1). Strong signals were observed for all metabolite-specific MS/MS fragments under all conditions tested. Fig 2 shows representative extracted ion chromatograms (EIC) of both a QC standard and typical composite urine sample from mice exposed to naphthalene. Two other metabolites were successfully separated, and were tentatively identified as the mercapturic acid conjugate derived from the dihydrodiol epoxide and dihydroxynaphthalene diglucuronide based on molecular ion (m/z) values 340 and 511, respectively (data not shown). Both were verified by high resolution mass spectrometry.


Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Ayala DC, Morin D, Buckpitt AR - PLoS ONE (2015)

Extracted ion chromatograms of urinary naphthalene metabolites and deuterated internal standards.(A) QC Sample #2. Naphthalene mercapturate, N-acetyl glutathione conjugate, naphthol glucuronide, and naphthol sulfate at 40, 5, 70, 20 ng on column, respectively. (B) Pooled urine sample from day 6–7 of exposure treatments (n = 3 animals). Calculated amounts of each metabolite on column were: 124.8, 7.9, 45.3, 25.9 ng for mercapturic acid, N-acetyl GSH, naphthol glucuronide and naphthol sulfate, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390350&req=5

pone.0121937.g002: Extracted ion chromatograms of urinary naphthalene metabolites and deuterated internal standards.(A) QC Sample #2. Naphthalene mercapturate, N-acetyl glutathione conjugate, naphthol glucuronide, and naphthol sulfate at 40, 5, 70, 20 ng on column, respectively. (B) Pooled urine sample from day 6–7 of exposure treatments (n = 3 animals). Calculated amounts of each metabolite on column were: 124.8, 7.9, 45.3, 25.9 ng for mercapturic acid, N-acetyl GSH, naphthol glucuronide and naphthol sulfate, respectively.
Mentions: We achieved optimal separation of all four major naphthalene metabolites and their corresponding deuterium-labeled internal standards with a 0.1% acetic acid water/acetonitrile gradient. All targeted metabolites eluted between 38.9 and 74.4 min (Table 1). Strong signals were observed for all metabolite-specific MS/MS fragments under all conditions tested. Fig 2 shows representative extracted ion chromatograms (EIC) of both a QC standard and typical composite urine sample from mice exposed to naphthalene. Two other metabolites were successfully separated, and were tentatively identified as the mercapturic acid conjugate derived from the dihydrodiol epoxide and dihydroxynaphthalene diglucuronide based on molecular ion (m/z) values 340 and 511, respectively (data not shown). Both were verified by high resolution mass spectrometry.

Bottom Line: Subsequent biotransformation results in urinary elimination of several conjugated metabolites.The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide.The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616, United States of America; University of California Davis, Forensic Science, Graduate Program, 1909 Galileo Ct., Suite B, Davis, CA 95618, United States of America.

ABSTRACT
Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

No MeSH data available.


Related in: MedlinePlus