A robust method for simultaneous measurement of serum 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D by liquid chromatography‐tandem mass spectrometry with efficient separation of 3‐epi analogs, 23R,25(OH)2D3, and 4β,25(OH)2D3

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.

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