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J Lipid Res. 2017 Apr;58(4):798-808. doi: 10.1194/jlr.D073536. Epub 2017 Feb 1.

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

Author information

1
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
2
Department of Cell Biology and Anatomy, Georgia Regents University, Augusta, GA 30912.
3
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616 bdhammock@ucdavis.edu.

Abstract

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)2VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

KEYWORDS:

1,25-dihydroxyvitamin D; major vitamin D metabolites; matrix effect; quantification; smaller sample volume

PMID:
28148721
PMCID:
PMC5392736
DOI:
10.1194/jlr.D073536
[Indexed for MEDLINE]
Free PMC Article

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