High-throughput measurement of 25-hydroxyvitamin D by LC-MS/MS with separation of the C3-epimer interference for pediatric populations

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for the measurement of 25-hydroxyvitamin D₃ (25OHD₃) and 25-hydroxyvitamin D₂ (25OHD₂) in blood. However, the presence of 25OHD₃ C3-epimer (3-epi-25OHD₃) may cause interference and overestimation of the 25OHD₃ level. We developed a rapid and simple assay for measurement of 25OHD that separates this interference and to investigate its impact on 25OHD₃ measurement in adult and pediatric populations. Methods: Sample preparation consisted of protein precipitation followed by solid-phase extraction with an LC run time of 4.8. min. Method comparison with another LC-MS/MS method of a major reference laboratory that does not separate the C3-epimer interference was performed using adult (n. =. 52) and pediatric (n. =. 40) samples. Results: This method is free from significant ion suppression, carryover and interference. The assay can separate 25OHD₃ from the 3-epi-25OHD₃, and can measure 25OHD₂ and 25OHD₃ from 4.2 to 310.7ng/ml and 5.2 to 311.1ng/ml, respectively. Method comparison with a LC-MS method that does not separate the interference revealed biases of -0.15 and 4.54 for 25OHD₃ measurement in adult and pediatric samples, respectively. Conclusion: A fast and simple LC-MS/MS method for quantification of 25OHD₃ and 25OHD₂ without 3-epi-25OHD₃ interference was developed. This assay is required for accurate quantitation of 25OHD₃ in pediatric samples and suitable for routine use in a high volume clinical laboratory.