Rapid liquid chromatography-tandem mass spectrometry method for determination of 24,25(OH)₂D and 25OHD with efficient separation of 3-epi analogs

This study establishes and validates a rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization steps to simultaneously measure of 24,25(OH)₂D₂, 24,25(OH)₂D₃, 25OHD₂, and 25OHD₃, while efficiently separating the 3-epi analogs. Samples were prepared by precipitation and liquid-liquid extraction. The linearity, precision, accuracy, recovery and matrix effect of the method were thoroughly evaluated according to the Clinical & Laboratory Standards Institute guidelines. Additionally, the four vitamin D metabolites in the serum of 38 apparently healthy Chinese volunteers were evaluated. The total analysis time was 8.0 min, with efficient separation of 3-epi 24,25(OH)₂D₃ and 3-epi 25OHD₃, without interference from isomers such as 23,25(OH)₂D₃ or 1,25(OH)₂D₂, 1,25(OH)₂D₃. Good reproducibility was obtained for all four metabolites with within-run coefficient variations (CVs) of 4.07%-6.55%, 4.26%-7.84%, 2.46%-7.21%, and 4.90%-6.87% for 25OHD₃, 25OHD₂, 24,25(OH)₂D₃, and 24,25(OH)₂D₂, respectively, and the total CVs were 4.29%-6.64%, 6.14%-7.84%, 4.33%-7.21%, 5.82%-9.90%, respectively. The limit of quantification was 0.625 ng/mL for 25OHD₃ and 25OHD₂, and 0.5 ng/mL for 24,25(OH)₂D₃ and 24,25(OH)₂D₂. The relative bias of the LC-MS/MS method compared to the certified results of SRM 972a for 25OHD₃, 25OHD₂ and 24,25(OH)₂D₃ was -2.21% to 1.01%, 3.38% to 6.73%, and -7.72% to -3.9%, respectively. The mean±SD values for 25OHD, 24,25(OH)₂D and 25OHD/24,25(OH)₂D in the volunteers were 13.5±4.4 ng/mL(range:7.6-27.5 ng/mL), 0.84±0.42 ng/mL (range:0.26-2.1 ng/mL), and 18±7(range:8-37), respectively. Thus, a simple, precise LC-MS/MS method for appropriate retention and separation of vitamin D metabolites and their epi analogs was developed.