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

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)2 D3 ), 1,25-dihydroxyvitamin D2 (1,25(OH)2 D2 ), 24,25-dihydroxyvitamin D3 (24,25(OH)2 D3 ), and 24,25-dihydroxyvitamin D2 (24,25(OH)2 D2 ) simultaneously with efficient separation of 3-epi 25(OH)D3 , 3-epi 24,25(OH)2 D3 , 23R,25(OH)2 D3 , and 4β,25-dihydroxyvitamin D3 (4β,25(OH)2 D3 ) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).This method was validated according to procedures established by Clinical and Laboratory Standards Institute (CLSI) and then applied in healthy population to determine the distribution of the vitamin D metabolites by LC-MS/MS.The total-run CV% of 25(OH)D3 , 25(OH)D2 , 24,25(OH)2 D3 , 24,25(OH)2 D2 , 1,25(OH)2 D3 , and 1,25(OH)2 D2 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)2 D3 , 24,25(OH)2 D2 , 1,25(OH)2 D3 , and 1,25(OH)2 D2 were 90.6%-103.3%, 97.3%-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)2 D3 were 93.8%-103.0%, 101.0%, and 96.3%-100%, respectively.This study successfully established an efficient, accurate, robust method for simultaneous measurement of serum 25(OH)D, 1,25(OH)2 D, and 24,25(OH)2 D by LC-MS/MS with efficient separation of 3-epi analogs, 23R,25(OH)2 D3 , and 4β,25(OH)2 D3 .