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

Abstract

Carnitines 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.