Development and validation of the simultaneous measurement of four vitamin D metabolites in serum by LC MS/MS for clinical laboratory applications
The quantification of serum 25 hydroxyvitamin D [25(OH)D] as an indicator of vitamin D status is currently primarily conducted by immunoassays, yet LC MS/MS would allow more accurate determination. Furthermore, LC MS/MS would allow simultaneous measurement of multiple analytes. The aim of this study was to develop and validate an LC MS/MS method to simultaneously measure four vitamin D metabolites (25(OH)D3, 3 epi 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3) in serum for clinical laboratory applications. Serum samples were first prepared in a 96 well supported liquid extraction plate and the eluate was derivatized using the Cookson type reagent 4 (4' dimethylaminophenyl) 1,2,4 triazoline 3,5 dione (DAPTAD) which rapidly and quantitatively reacts with the s cis diene structure of vitamin D metabolites. The derivatized samples were subjected to LC MS/MS, ionized by electrospray ionization (positive ion mode) and detected by selected reaction monitoring. The lower limits of quantification for 25(OH)D3, 3 epi 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 0.091, 0.020, 0.013, and 0.024 ng/mL, respectively. The accuracy values and the extraction recoveries for these four metabolites were satisfactory. Serum 25(OH)D levels determined by our LC MS/MS were compared with those obtained by conventional radioimmunoassay (RIA) that cannot distinguish 25(OH)D3 and 25(OH)D2. The values obtained by the RIA method exhibited a mean bias of about 8.35 ng/mL, most likely as a result of cross reaction of the antibody with low abundance metabolites, including 24,25(OH)2D3. Various preanalytical factors, such as long sample sitting prior to serum separation, repeated freeze thaw cycles, and the presence of anticoagulants, had no significant effects on these determinations. This high throughput LC MS/MS simultaneous assay of the four vitamin D metabolites 25(OH)D3, 3 epi 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 required as little as 20 mL serum. This method will aid further understanding of low abundance vitamin D metabolites, as well as the accurate determination of 25(OH)D3 and 25(OH)D2.
Analytical and Bioanalytical Chemistry