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Use of high-resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples.
Rapid Commun Mass Spectrom. 2010 Jul 15; 24(13):1902-10.RC

Abstract

During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose-dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high-resolution mass spectrometry led to unequivocal identification of the interfering peak as an N-desmethyl metabolite of the parent analyte. High-resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the (13)C-isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples.

Authors+Show Affiliations

Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

20533320

Citation

Furlong, Michael, et al. "Use of High-resolution Mass Spectrometry to Investigate a Metabolite Interference During Liquid Chromatography/tandem Mass Spectrometric Quantification of a Small Molecule in Toxicokinetic Study Samples." Rapid Communications in Mass Spectrometry : RCM, vol. 24, no. 13, 2010, pp. 1902-10.
Furlong M, Bessire A, Song W, et al. Use of high-resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples. Rapid Commun Mass Spectrom. 2010;24(13):1902-10.
Furlong, M., Bessire, A., Song, W., Huntington, C., & Groeber, E. (2010). Use of high-resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples. Rapid Communications in Mass Spectrometry : RCM, 24(13), 1902-10. https://doi.org/10.1002/rcm.4587
Furlong M, et al. Use of High-resolution Mass Spectrometry to Investigate a Metabolite Interference During Liquid Chromatography/tandem Mass Spectrometric Quantification of a Small Molecule in Toxicokinetic Study Samples. Rapid Commun Mass Spectrom. 2010 Jul 15;24(13):1902-10. PubMed PMID: 20533320.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Use of high-resolution mass spectrometry to investigate a metabolite interference during liquid chromatography/tandem mass spectrometric quantification of a small molecule in toxicokinetic study samples. AU - Furlong,Michael, AU - Bessire,Andrew, AU - Song,Wei, AU - Huntington,Christopher, AU - Groeber,Elizabeth, PY - 2010/6/10/entrez PY - 2010/6/10/pubmed PY - 2010/9/3/medline SP - 1902 EP - 10 JF - Rapid communications in mass spectrometry : RCM JO - Rapid Commun Mass Spectrom VL - 24 IS - 13 N2 - During routine liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis of a small molecule analyte in rat serum samples from a toxicokinetic study, an unexpected interfering peak was observed in the extracted ion chromatogram of the internal standard. No interfering peaks were observed in the extracted ion chromatogram of the analyte. The dose-dependent peak area response and peak area response versus time profiles of the interfering peak suggested that it might have been related to a metabolite of the dosed compound. Further investigation using high-resolution mass spectrometry led to unequivocal identification of the interfering peak as an N-desmethyl metabolite of the parent analyte. High-resolution mass spectrometry (HRMS) was also used to demonstrate that the interfering response of the metabolite in the multiple reaction monitoring (MRM) channel of the internal standard was due to an isobaric relationship between the (13)C-isotope of the metabolite and the internal standard (i.e., common precursor ion mass), coupled with a metabolite product ion with identical mass to the product ion used in the MRM transition of the internal standard. These results emphasize (1) the need to carefully evaluate internal standard candidates with regard to potential interferences from metabolites during LC/MS/MS method development, validation and bioanalysis of small molecule analytes in biological matrices; (2) the value of HRMS as a tool to investigate unexpected interferences encountered during LC/MS/MS analysis of small molecules in biological matrices; and (3) the potential for interference regardless of choice of IS and therefore the importance of conducting assay robustness on incurred in vitro or in vivo study samples. SN - 1097-0231 UR - https://www.unboundmedicine.com/medline/citation/20533320/Use_of_high_resolution_mass_spectrometry_to_investigate_a_metabolite_interference_during_liquid_chromatography/tandem_mass_spectrometric_quantification_of_a_small_molecule_in_toxicokinetic_study_samples_ L2 - https://doi.org/10.1002/rcm.4587 DB - PRIME DP - Unbound Medicine ER -