Assay of 25-hydroxyvitamin D3 in human plasma by high-performance liquid chromatography.

A method using preparative and analytical high-performance liquid chromatography (HPLC) is proposed for the assay of 25-hydroxyvitamin D3 (25-OH-D3) in human plasma. A constant volume (0.2--2.0 ml) of a plasma sample was saponified. The unsaponifiable matter was first applied to preparative HPLC using a column of the straight-phase type (Zorbax SIL) in order to separate a 25-OH-D3 fraction from lipophilic concomitants giving ultraviolet-absorbing noise. Then, the separated 25-OH-D3 fraction was applied to analytical HPLC using a column of the reversed-phase type (Zorbax ODS) in order to measure the content of 25-OH-D3 from the peak height. This is a revised method from Jones (1978): Clin. Chem., 24, 287--298). The results showed that the clean-up procedure by the first preparative HPLC was successfully performed because the peak corresponding to 25-OH-D3 on the chromatogram of the second analytical HPLC was not disturbed by any other interfering peaks. Moreover, recovery through the whole procedure was satisfacotry (about 100%) and the procedures of saponification and isolation of the unsaponifiable matter diminished the overload to the columns. These are the revised points of Jones' method. When two determinations were performed on 12 samples of plasma taken from normal adults in October, the values were 22.6 +/- 4.8 and 21.0 +/- 3.6 (mean +/- SD) ng/ml, respectively.