Enhanced sensitivity by laser-induced fluorescence for the determination of calcitriol and other vitamin D3 metabolites in plasma

Abstract

SummaryA sensitive method for the determination of hydroxymetabolites of vitamin D3, , particularly calcitriol (1,25-(OH)2-D3), in human plasma is reported. The method is based on the use of laser-induced fluorescence detection as an alternative to conventional fluorimetry in an integrated cleanup/preconcentration, HPLC separation and post-column derivatization system. The derivatization step is based on a dehydration reaction which takes place in secosteroid structures at high temperature in a strong-acid medium. A LOD of 0.01 pg mL -1 (SNR=3) was obtained for each analyte with a linear dynamic range over 4 order of magnitude with excellent regression coefficients (-> 0.9922) in all cases. The precision was studied at two concentration levels and the RSDs values (for n= 5) were acceptable (between 2.6 and 4.7 %). The method was also checked by applying it to human plasma spiked with the target analytes and excellent recoveries were obtained. This is the first time that these species have been determined at the sub-pg mU 1 level.

Automated method for the determination of vitamin�D3 hydroxymetabolites in serum

“…The method provides precision similar to methods in which a post-column reaction with photometric [8] (where the precision was 2.1%-5.2%) or laser-induced fluorescence [9] (precision between 2.6% and 4.7%) was developed.…”

“…The determination of these metabolites in serum or plasma has been carried out by HPLC using several detectors (conventional fluorimetric [8], laser-induced fluorimetric [9], photometric [10], coulometric [11], mass-spectrometry [12]). ELISA has been used for the single determination of 25-hidroxyvitamin D 3 .…”