Determination of vitamin D3 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 D3 (24,25-(OH)2-D3, 1,25-(OH)2-D3 and 25-OH-D3) in plasma is reported. The method is based on the integration of three subsystems: continuous cleanup/preconcentration, HPL separation and post-column fluorimetric derivatisation. The derivatising subsystem is based on the dehydration reaction undergone 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 with excellent regression coefficients (≥0.9933) in all cases. The precision at two concentration levels was established with acceptable RSDs (%) in all instances (values between 2.1 and 5.2%). The method was also checked by applying it to human plasma samples spiked with the target analytes and the recoveries ranged between 86 and 106%.