Assessment of a two-step high-performance liquid chromatographic assay using dual-wavelength ultraviolet monitoring for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol in human serum or plasma

Abstract

The technique of dual-wavelength monitoring was used to verify the purity of high-performance liquid chromatographic (HPLC) peaks quantified as 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. The data obtained show the need for a second HPLC step prior to quantitation. Potential inaccuracy arising from inadvertent collection of radio-labelled decomposition products was assessed. Between-day coefficients of variation were 7.3, 5.0 and 3.6%, respectively for 11.3 (n = 12), 17.1 (n = 14), and 32.9 (n = 8) ng/ml of 25-hydroxycholecalciferol. For 25-hydroxyergocalciferol, these values were 6.4 and 3.8% for 11.1 (n = 12) and 20.1 (n = 8) ng/ml concentrations, respectively. Comparison of total 25-hydroxycalciferol with a competitive protein binding assay was made. The comparison produced a correlation coefficient (r) of 0.94 and a relationship of y = 1.03x + 3.3. Four of the samples contained more than 10 ng/ml of 25-hydroxyergocalciferol and the results are consistent with the reported 100% cross-reactivity of the competitive binding protein method for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. A simple regeneration procedure is also described which enables Sep-Pak C18 cartridges to be reused up to eighteen times. Samples may be stored at -18 degrees C for upto several months before assay and either serum or plasma may be used.