A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.
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.
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Debin
D
Wan
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Jun
J
Yang
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Bogdan
B
Barnych
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Sung Hee
SH
Hwang
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Kin Sing Stephen
KSS
Lee
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Yongliang
Y
Cui
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616.
Jun
J
Niu
Department of Cell Biology and Anatomy, Georgia Regents University, Augusta, GA 30912.
Mitchell A
MA
Watsky
Department of Entomology and Nematology and University of California Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616. Electronic address: bdhammock@ucdavis.edu.
Bruce D
BD
Hammock
D002853:Chromatography, Liquid; D057930:Click Chemistry; D006801:Humans; D011725:Pyridines / Q000737:chemistry*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D014230:Triazoles / Q000737:chemistry; D014807:Vitamin D / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D011725:Pyridines; D014230:Triazoles; D014807:Vitamin D; C053540:4-phenyl-1,2,4-triazoline-3,5-dione; C023666:pyridine
1,25-dihydroxyvitamin D; major vitamin D metabolites; matrix effect; quantification; smaller sample volume