High-performance liquid chromatography and coulometric electrode array detector in serum 25-hydroxyvitamin D(3) and...

The diode array detector in our high-performance liquid chromatography (HPLC) method for 25-hydroxyvitamin D(3) (25OHD(3)) and 25-hydroxyvitamin D(2) (25OHD(2)) did not perform satisfactorily for measuring human serum concentrations below 30nM. 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 25OHD(3) and 25OHD(2) of the new method was 0.36pmol on column (3.6nM), and linearity ranged from 5 to 2400nM. Accuracy of the method was 90% for 25OHD(3) and 69% for 25OHD(2). 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 25OHD(3) also corresponded to the results obtained with the Chromsystems HPLC method in a certified laboratory. The long-term coefficients of variation for 25OHD(3) were 6.2%, 7.8%, 5.2%, 6.7%, and 7.3% in concentrations of 27.5, 38.7, 48.4, 78.4, and 88.0nM, respectively. The developed HPLC-CEAD method was shown to be applicable for determining 25OHD(3) and 25OHD(2) in human serum samples.