Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 Dihydroxyvitamin D2&3 measurement using a novel derivatization agent

• 1,25 dihydroxyvitamin D2, D3 is the active form of vitamin D and requires sensitive methods for measuring in human serum.
• A new ionizing agent, Amplifex, was tested for its ability to enhance liquid chromatography/mass spectrometry measurement.
• The Amplifex method significantly correlated with values measured by standard RIA and mass spectrometry.
• Sensitivity of 10 fold resulted over other derivatizing methods.
• This new method resulted in increased sensitivity and required half the sample volume of other methods.

Active vitamin D metabolites 1,25-dihydroxyvitamin D2 [1,25-(OH)2-D2; derived from ergocalciferol] and D3 [1,25-(OH)2-D3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)2-D2 and -D3, whereas liquid chromatography–tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)2-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)2-D2 and -D3 ranges (Amplifex and RIA, pc = 0.97; Amplifex and PTAD, pc = 0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc = 0.81; Amplifex and PTAD, pc = 0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)2-D2 and -D3 while using the smallest sample volume.