Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples

Objectives: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)(2)D-3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods. Methods: 24,25(OH)(2)D-3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liege (LC-MS/MS 2). Results: In DEQAS samples, 24,25(OH)(2)D-3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively. Conclusions: This study is the first to assess the comparability of 24,25(OH)(2)D-3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)(2)D-3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)(2)D-3 in serum samples and in the DEQAS program.

Automated summary

This study assessed the agreement of 24,25-dihydroxyvitamin D3 (24,25(OH)(2)D-3) measurements by two in-house LC-MS/MS methods. The results showed high precision measurements of 24,25(OH)(2)D-3 with both methods, despite minor differences between them.