Simultaneous measurement of 1-hydroxylated and 24-hydroxylated vitamin D metabolites by liquid-chromatography tandem mass-spectrometry: an innovative tool in CKD management

Chronic kidney disease (CKD) reduces renal 1alpha- and 24-hydroxylation of vitamin D metabolites, which hampers the assessment of vitamin D status with traditional methods. Therefore, we developed and validated a liquid-chromatography tandem mass-spectrometry (LC-MS/MS) method capable of simultaneously tracking 25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxy vitamin D (1,25[OH]2D), 24,25-dihydroxyvitamin D (24,25[OH]2D), and 1,24,25-trihydroxyvitamin D (1,24,25[OH]3D). Sample preparation combined protein precipitation, liquid-liquid-extraction and derivatisation. A Nexera UHPLC (SHIMADZU, Kyoto, Japan) with a Kinetex® 5 μm F5 100 Å LC column (150×4.6 mm, Phenomenex, Torrance, CA, USA) coupled to a QTRAP 6500 (SCIEX, Framingham, MA, USA) was used for analysis. Clinical utility was demonstrated analysing serum from individuals with 24-hydroxlase deficiency, and kidney donors and recipients. For all metabolites, linear ranges and limits of quantitation cover the concentrations range physiologically observed in humans. Recovery ranged between 90.8 to 105.5% for all four metabolites. Imprecision was ≤11.5% across the entire analytical range for all metabolites. Analysis of patient samples showed that kidney transplantation and 24-hydroxylase deficiency are associated with profound alterations of 24-hydroxylated, but not 1alpha-hydroxylated, vitamin D metabolites. Hence, the simultaneous measurement 25(OH)D and its 1-alpha and 24-hydroxlated metabolites by LC-MS/MS is a promising tool to identify abnormalities in vitamin D metabolism.