Determination of vitamin D-3 hydroxymetabolites in plasma at the sub-part per trillion levels using on-line cleanup/preconcentration and HPLC-fluorimetric post-column derivatisation

A new method for the determination of the hydroxymetabolites of vitamin D-3 (24,25-(OH)(2)-D-3 1,25-(OH)(2)-D-3 and 25-OH-D-3) in plasma is reported. The method is based on the integration of three subsystems: continuous clea nup/preconcentration, HPL separation and post-column Fluorimetric derivatis ation. The derivatising subsystem is based on the dehydration reaction unde rgone by the secosteriod molecules in a strong-acid medium. The calibration graphs were run between 0.1 pg ml(-1) and 100 ng ml(-1) for each analyte w ith excellent regression coefficients (greater than or equal to 0.9933) in all cases. The precision at two concentration levels was established with a cceptable RSDs (%) in all instances (values between 2.1 and 5.2%). The meth od was also checked by applying it to human plasma samples spiked with the target analytes and the recoveries ranged between 86 and 106%. (C) 1999 Els evier Science B.V. All rights reserved.