Various calibration procedures result in optimal standardization of routinely used 25(OH)D ID-LC-MS/MS methods

The variety of LC-MS/MS methods measuring total 25(OH)D used today is vast and the comparability among these methods is still not well assessed.

Here, we performed a comparison in samples of healthy donors between the currently routinely used 25(OH)D LC-MS/MS methods in the Netherlands and the Ghent University reference measurement procedure to address this issue (n=40). Additionally, an interlaboratory comparison in patient serum samples assessed agreement between the Dutch diagnostic methods (n=37).

The overall correlation of the routine methods for 25(OH)D₃ with the reference measurement procedures and with the mean of all diagnostic methods was excellent (r>0.993 and r>0.989, respectively). Three out of five methods aligned perfectly with both the reference measurement procedure and the median of all methods. One of the routine methods showed a small positive bias, while another showed a small negative bias consistently in both comparisons.

The biases most probably originated from differences in calibration procedure and may be obviated by reassessing calibration of stock standards and/or calibrator matrices. In conclusion, five diagnostic centers have performed a comparison with the 25(OH)D Ghent University reference measurement procedure in healthy donor serum samples and a comparison among themselves in patient serum samples. Both analyses showed a high correlation and specificity of the routine LC-MS/MS methods, yet did reveal some small standardization issues that could not be traced back to the technical details of the different methods. Hence, this study indicates various calibration procedures can result in perfect alignment.