Continuous cleanup/preconcentration procedure of hydroxyvitamin D3 metabolites in plasma as an alternative to batch...

A continuous automatable cleanup procedure coupled on-line with a liquid chromatograph and UV detector for hydroxyvitamin D3 metabolites [24,25-(OH)2, 1,25-(OH)2 and 25-(OH)] as an alternative to batch solid-phase extraction is reported. The method, based on continuous solid-phase cleanup/preconcentration of the analytes, requires only the passage of the sample through a single minicolumn, which also results in a preconcentration effect which increases the sensitivity. The proposed method is also compared with a conventional batch, two-step solid-phase extraction method previously improved by the authors. The method has been checked by applying it to plasma samples spiked with the target analytes (linear range between 0.05 and 100 ng/ml with coefficient of variation values lower than 6.5%) and acceptable recoveries ranging between 94.6 and 101% have been obtained. The sampling frequency was 4 h-1.