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

作者：Wan Debin; Yang Jun; Barnych Bogdan; Hwang Sung Hee; Lee Kin Sing Stephen; Cui Yongliang; Niu Jun; Watsky Mitchell A; Hammock Bruce D^*

来源：The Journal of Lipid Research, 2017, 58(4): 798-808.

DOI：10.1194/jlr.D073536

• 1.25-dihydroxyvitamin D
• major vitamin D metabolites
• matrix effect
• smaller sample volume
• quantification

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1,25-dihydroxyvitamin D [1,25(OH)(2)VitD], 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 1,25(OH)(2)VitD, 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 mu L 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.

• 出版日期2017-4