Isotope-labeling derivatization with 3-nitrophenylhydrazine for LC/multiple-reaction monitoring-mass-spectrometry-based quantitation of carnitines in dried blood spots

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dc.contributor.authorHan, Jun
dc.contributor.authorHiggins, Rehan
dc.contributor.authorLim, Mark D.
dc.contributor.authorAtkinson, Kieran
dc.contributor.authorYang, Juncong
dc.contributor.authorLin, Karen
dc.contributor.authorBorchers, Christoph H.
dc.date.accessioned2018-11-02T07:55:40Z
dc.date.available2018-11-02T07:55:40Z
dc.date.copyright2018en_US
dc.date.issued2018
dc.description.abstractCarnitines are diagnostic biomarkers of fatty acid oxidation defects and organic acidemias. Quantitative measurements of various carnitines in dried blood spot (DBS) have potential use in remote health applications for disease diagnosis and epidemiological surveillance. To provide an improved LC/multiple-reaction monitoring (MRM)-MS method for quantitation of carnitines in DBS, 3-nitrophenylhydrazine (3NPH) was tested as a high-efficiency chemical isotope-labeling reagent for pre-analytical derivatization of 24 routinely-analyzed species. Reaction conditions were optimized and carnitine structural isomers were separated by reversed-phase LC with positive-ion MRM/MS detection, giving on-column lower LOQs of sub- to low-femtomole levels. 13C6-3NPH was used to produce 13C6- or 13C12-labeled derivatives of the mono- and di-carboxylic carnitines in a “one-pot” reaction. These labeled analogues were used as stable isotope-labeled internal standards to compensate for possible ESI matrix effects. Combined with an optimized, two-step procedure for the extraction of carnitines from DBS, this isotope-labeling derivatizaiton - LC/MRM-MS method provided good linearity, high precision (intra-day CVs of ≤7.8% and inter-day CVs of ≤8.8%) and high accuracy (three levels of standard substances spiked in, with recoveries of 86.9%–109.7%) quantitation of carnitines in three sets of DBSs on cellulose or cotton filter paper. This method was then applied to determine the concentration changes of the analytes in the DBSs under two stability-testing regimes: 1) a one-time 4-h sunlight exposure and 2) a set of cycled temperature transitions (−20 °C for 2 days, 40 °C for 2 days, and back to −20 °C for 2 additional days). All of the carnitines showed good stabilities under the first testing condition. Under the second testing condition, free carnitine showed concentration increases of 9.3%–16.1%; acetyl carnitine, 3-OH butyryl carnitine, and malonyl carnitine showed concentration decreases of 12.2%–17.3%, 12.9%–17.1% and 10.7%–15.3%, respectively, and other 20 acyl carnitines showed concentration changes of <10% in three sets of DBSs on cellulose or cotton filter paper. These preliminary stability-testing results indicate a need to more systematically investigate the effects of various environmental conditions on the chemical stabilities of carnitines in DBS specimens if this sampling method is to be used in remote health applications.en_US
dc.description.reviewstatusRevieweden_US
dc.description.scholarlevelFacultyen_US
dc.description.sponsorshipCHB is grateful for support from the Leading Edge Endowment Fund (University of Victoria) and for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada). CHB is also grateful for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada). We also thank Dr. Carol Parker for careful editing and review of this manuscript.en_US
dc.identifier.citationHan, J., Higgins, R., Lim, M.D., Atkinson, K., Yang, J., Lin, K. & Borchers, C.H. (2018). Isotope-labeling derivatization with 3-nitrophenylhydrazine for LC/multiplereaction monitoring-mass-spectrometry-based quantitation of carnitines in dried blood spots. Analytics Chimica Acta, 1037, 177-187. https://doi.org/10.1016/j.aca.2018.01.045en_US
dc.identifier.urihttps://doi.org/10.1016/j.aca.2018.01.045
dc.identifier.urihttp://hdl.handle.net/1828/10222
dc.language.isoenen_US
dc.publisherAnalytics Chimica Actaen_US
dc.subjectcarnitine
dc.subjectdried blood spot
dc.subjectisotope labeling chemical derivatization
dc.subject3-nitrophenylhydrazine
dc.subjectLC/MRM-MS
dc.subjectstability testing
dc.subjectUVic Genome BC Proteomics Centre
dc.subject.departmentDivision of Medical Sciences
dc.subject.departmentSchool of Medical Sciences�
dc.subject.departmentDepartment of Biochemistry and Microbiology
dc.titleIsotope-labeling derivatization with 3-nitrophenylhydrazine for LC/multiple-reaction monitoring-mass-spectrometry-based quantitation of carnitines in dried blood spotsen_US
dc.typeArticleen_US

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