Background

This study aimed to establish a robust, simple method to detect 25-hydroxyvitamin D3 (25(OH)D3), 25-hydroxyvitamin D2 (25(OH)D2), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1,25-dihydroxyvitamin D2 (1,25(OH)2D2), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), and 24,25-dihydroxyvitamin D2 (24,25(OH)2D2) simultaneously with efficient separation of 3-epi 25(OH)D3, 3-epi 24,25(OH)2D3, 23R,25(OH)2D3, and 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3).

Method

This method was validated according to procedures established by CLSI and then applied in a healthy population to determine the distribution of the vitamin D metabolites by liquid chromatography-tandem mass Spectrometry (LC-MS/MS).

Results

The total-run CV% of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, 24,25(OH)2D2, 1,25(OH)2D3, and 1,25(OH)2D2 were 6.30–8.40%, 5.00–8.40%, 5.90–9.00%, 5.60–9.00%, 5.60–8.00%, and 7.00–9.70%, respectively. The linearity correlation coefficients r of these six vitamin D metabolites were > 0.99. The matrix effects of 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, 24,25(OH)2D2, 1,25(OH)2D3, and 1,25(OH)2D2 were 90.6%~103.3%, 99.4%~106.3%, 90.7%~106.3%, 100.7%~114.5%, 97.9%~104.6%, and 97.0%~111.0%. The trueness values of 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were 93.8–103.0%, 101.0%, and 96.3–100%, respectively.

Conclusion

This study successfully established an efficient, accurate, robust method for simultaneous measurement of serum 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D by LC-MS/MS with efficient separation of 3-epi analogs, 23R,25(OH)2D3, and 4β,25(OH)2D3.