Quantitative determination of 25‐hydroxyvitamin D 3 3‐sulphate in human plasma using high performance liquid chromatography

The quantitative determination of 25‐hydroxyvitamin D 3 3‐sulphate in human plasma was completed using reversed phase high performace liquid chromatography with UV detection (265 nm) and an internal standard method. The vitamin D sulphate fraction was obtained from a plasma specimen with the combined use of a Bond Elut C18 cartridge for solid‐phase extraction and a piperidinohydroxypropyl Sephadex LH‐20 column for lipophilic ion‐exchange chromatography. Separation of the compounds was performed on a YMC‐Pack ODS‐AM column. The limit of quantitation was 5 ng/mL and the assay was linear from 5 to 50 ng/mL. The proposed method is satisfactory in its accuracy and precision.