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 310.7 ng/ml and 5.2 to 311.1 ng/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.
Keywords: C3-epimer; Liquid chromatography; Mass spectrometry; Vitamin D.
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