Optimised liquid chromatography tandem mass spectrometry method for the simultaneous quantification of serum vitamin D analogues while also accounting for epimers and isobars.

The accurate quantification of multiple vitamin D analogues simultaneously is challenging. This study set out to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method capable of measuring a comprehensive vitamin D profile, encompassing twelve vitamin D analogues (vitamin D2, D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi-25(OH)D3, 7αC4 and1α(OH)D3) in a single run. Serum samples were prepared using double liquid-liquid extraction and analysed on an Agilent 6460 QQQ LC-MS/MS equipped with a Pursuit 3 Pentafluorophenyl (4.6 x 100 mm, 3 μm) column. Recovery rates for all analytes were above 95 % with a coefficient of variation (CV) below 10 %. The method exhibited good linearity (r > 0.995) and had a range of detection limits between 0.01 and 0.35 ng/mL and quantification limits between 0.15 and 0.96 ng/mL. Repeatability and within-lab precision were acceptable, with CV values below 10 % and 15 %, respectively. Method accuracy was excellent, with a systematic error below 6.60 %. additionally, all analytes-maintained stability for 48 h following sample preparation, and no interferences were observed among co-eluting analytes. Lastly, this method achieved "world-class" status according to the Sigma metric scale specifications, requiring minimal quality control to ensure data quality. This successfully validated method has the potential not only for improving vitamin D profiling procedures but also for aiding in the diagnosis of other genetic disorders where measuring beyond 25(OH)D is crucial.