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 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%.
Ortiz Boyer
F
F
Analytical Chemistry Division, Faculty of Sciences, University of Córdoba, Córdoba E-4004, Spain.
Fernández Romero
J M
JM
Luque de Castro
M D
MD
Quesada
J M
JM