High-performance liquid chromatography and coulometric electrode array detector in serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 analyses

The diode array detector in our high-performance liquid chromatography (HPLC) method for 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) did not perform satisfactorily for measuring human serum concentrations below 30 nM. Because of a need for a reliable self-managed method in ongoing and starting vitamin D studies of the laboratory, we decided to develop a chromatographic method applying coulometric electrode array detector (CEAD) and evaluate reliability of the method by participating in the Vitamin D External Quality Assessment Scheme (DEQAS). The limit of quantification for 25OHD3 and 25OHD2 of the new method was 0.36 pmol on column (3.6 nM), and linearity ranged from 5 to 2400 nM. Accuracy of the method was 90% for 25OHD3 and 69% for 25OHD2. The HPLC–CEAD results from five DEQAS rounds were in line with those of the other participating laboratories using HPLC methods. The HPLC–CEAD results for 25OHD3 also corresponded to the results obtained with the Chromsystems HPLC method in a certified laboratory. The long-term coefficients of variation for 25OHD3 were 6.2%, 7.8%, 5.2%, 6.7%, and 7.3% in concentrations of 27.5, 38.7, 48.4, 78.4, and 88.0 nM, respectively. The developed HPLC–CEAD method was shown to be applicable for determining 25OHD3 and 25OHD2 in human serum samples.