A novel high-performance liquid chromatographic assay for vitamin D metabolites using a coulometric electrochemical detector

Abstract A new, highly sensitive HPLC assay method using an electrochemical detector (ECD) for multiple assay of vitamin D metabolites is reported. The assay involves extracting lipids from plasma with methylene chloride and methanol, purification on Zorbax SIL column with 5.5% (v/v) iso-propanol in hexane and quantification by HPLC-ECD. A coulometric system, composed of the dual electrode analytical cell and a guard cell, was used for ECD of the eluting compounds. The potentials applied to detectors 1 and 2 in a dual electrode analytical cell were adjusted to +0.20 V and +0.60 V, respectively. This method is sensitive to 20 pg of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and of 24 R ,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ]. Calibration curves gave linearity from 20–1000 pg for 25(OH)D 3 and 24,25(OH) 2 D 3 . The detection limit was approximately 50 pg ml −1 for 25(OH)D 3 and 24,25(OH) 2 D 3 in plasma. This sensitivity combined with an overall recovery of 25(OH)D 3 (81.5 ± 2.6%, mean ± S.E.) allows the measurement of trace amount of 25(OH)D 3 with only 20 μl of plasma. Intra- and interassay RSD values were 5.3 and 9.7% for 25(OH)D 3 and 6.3 and 9.7% for 24,25(OH) 2 D 3 , respectively. Plasma levels of 25(OH)D 3 and 24,25(OH) 2 D 3 in normal adults were 15.9 ± 2.8 ng ml −1 ( n = 10) and 1.4 ± 0.5 ng ml −1 ( n = 10), respectively. This method allows the determination of 25(OH)D 2 and 25(OH)D 3 for evaluating their nutritional and clinical status. From these results, it is concluded that the proposed HPLC-ECD assay system is useful for the determination of vitamin D metabolites in biological fluids as a highly sensitive physicochemical method.