Chromatographic separation of PTAD-derivatized 25-hydroxyvitamin D3 and its C-3 epimer from human serum and murine skin

• We demonstrate HPLC separation of PTAD-derivatized 25OHD3 and its C-3 epimer.
• Both analytes were detected in serum and skin at nM and pmol/g levels, respectively.
• C-3 epimer measurement may be important for studies of potential biological impact.

The detection of 25-hydroxyvitamin D at low levels in biological samples is facilitated by the use of chemical derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in concert with liquid chromatography–tandem mass spectrometry (LC–MS/MS). This mode of analysis is notably hampered by chromatographic co-elution of 25-hydroxyvitamin D3 (25OHD3) and its C-3 epimer (C3epi). The objective of this work was to improve upon current LC–MS/MS methods used for the analysis of PTAD-derivatized 25-hydroxyvitamin D3 by resolving it from C3epi. Additionally, the applicability of this method in human serum and murine skin was investigated. C18 columns of increasing length and varying particle sizes were assessed for performance using a mixed standard of PTAD-derivatized 25OHD3 and C3epi. Serum samples were processed using solid phase extraction, and skin was powdered and extracted for lipophilic compounds. The samples were derivatized with PTAD and subsequently analyzed using isotope dilution LC–MS/MS with atmospheric pressure chemical ionization operated in positive mode. Near baseline resolution of PTAD-25OHD3 from PTAD-C3epi was achieved on a 250 mm C18 column with 3 μm sized particles. This separation allowed for detection and quantification of both metabolites in serum and skin samples. PTAD-C3epi represented a significant confounding analyte in all samples, and comprised up to 20% of the status measurement in skin. This method is a significant improvement on the chromatography of PTAD-derivatized vitamin D metabolites that could greatly influence the assessment of vitamin D status and C3epi biology in low abundance samples.