Sensitive and Specific Quantitation of 1alpha,25-Dihydroxy Vitamin D_(3) and Analogs Using LC-MSMS and Labeling Chemistry

摘要

Derivatization of 1alpha, 25-dihydroxy Vitamin D_(3) using QAO-Cookson reagent resulted in sensitivity enhancement of about 200 fold in Human Serum or Plasma samples. The chemistry is simple (one step) & quick (30 minutes) and imparts a more hydrophilic nature to the derivatized product (less carry over and insolubility issues). Sample preparation is completely antibody free and requires only 200 (mu)L of serum or plasma (analyte enrichment is not required). Inexpensive cartridges/ solvents are used for SPE. Derivatized 1,25 DHVD_(3) was well resolved in LC from its isobaric metabolites. LC Run time 6 min. LOD is <2 pg and LLOQ is 5 pg in Human Serum. This labeling and analysis protocol covers a wide dynamic range (up to 1000 pg/mL). The derivatization technique presented herein is generic and can be used for a broader range of Vitamin D_(3) and Vitamin D_(2) analogs.