Abstract
Abstract Background 25-hydroxyvitamin D is the recommended target to evaluate vitamin D status. The accurate measurement of 25-hydroxyvitamin D by liquid chromatography/mass spectrometry (LC/MS/MS) is impacted by the presence of the biologically inactive epimer of 25-hydroxyvitamin D2 and D3, 3-EPI-25-hydroxyvitamin D2 and D3. The epimers are isomers and share the same mass and fragment transitions as 25-hydroxyvitamin D2 and D3. For an accurate measurement the epimer should be chromatographically separated from 25-hydroxyvitamin D. Herein we assess the significance of 3-EPI-25-hydroxyvitamin D in our patient population. Methods A LC/MS/MS method was developed to chromatographically separate 25-hydroxyvitamin D2 and D3 from 3-EPI-25-hydroxyvitamin D2 and D3 on a Waters Xevo TQ-S Micro with an Acquity UPLC System using a 2.6 μm Pentafluorophenyl (PFP) column (Phenomenex). There were 231 residual serum samples analyzed. Results The cohort tested consisted of 16 infant samples (<1 years), 62 pediatric samples (1–17 years), adult samples (≥18 years), and 20 pregnant samples. 3-EPI-25-hydroxyvitamin D2 was not detected in any sample analyzed (<1 ng/mL). The prevalence and concentration of 3-EPI-25-hydroxyvitamin D3 was variable (Fig. 1). Conclusion The specificity of 25-hydroxyvitamin D measurement by LC/MS/MS is not affected by 3-EPI-25-hydroxyvitamin D2 as it is not present in our patient population. However, 3-EPI-25-hydroxyvitamin D3 is a significant interference. The presence of 3-EPI-25-hydroxyvitamin D3 is variable with the highest prevalence and concentration in infants. The prevalence and concentration decrease with age and is minimal in pregnancy. For infant and pediatric samples, an assay that chromatographically separates 3-EPI-25-hydroxyvitamin D3 from 25-hydroxyvitamin D3 is needed. For adult samples, it may be appropriate not to separate 25-hydroxyvitamin D3 from the epimer. For this patient population a balance between accuracy and throughput must be established.
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