Teegarden Matthew D, Riedl Kenneth M, Schwartz Steven J
Department of Food Science and Technology, The Ohio State University, United States.
Department of Food Science and Technology, The Ohio State University, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jun 1;991:118-21. doi: 10.1016/j.jchromb.2015.04.011. Epub 2015 Apr 15.
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 250mm C18 column with 3μm 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.
通过使用4-苯基-1,2,4-三唑啉-3,5-二酮（PTAD）进行化学衍生化并结合液相色谱-串联质谱法（LC-MS/MS），有助于检测生物样品中低水平的25-羟基维生素D。25-羟基维生素D3（25OHD3）及其C-3差向异构体（C3epi）的色谱共洗脱显著阻碍了这种分析模式。这项工作的目的是通过将PTAD衍生化的25-羟基维生素D3与C3epi分离，改进目前用于分析PTAD衍生化的25-羟基维生素D3的LC-MS/MS方法。此外，还研究了该方法在人血清和小鼠皮肤中的适用性。使用PTAD衍生化的25OHD3和C3epi的混合标准评估了不同长度和粒径的C18柱的性能。血清样品采用固相萃取法处理，皮肤研磨成粉末并提取亲脂性化合物。样品用PTAD衍生化，随后采用同位素稀释LC-MS/MS，在正模式下进行大气压化学电离分析。在一根250mm、粒径为3μm的C18柱上，实现了PTAD-25OHD3与PTAD-C3epi的近基线分离。这种分离使得能够检测和定量血清和皮肤样品中的两种代谢物。PTAD-C3epi在所有样品中都是一个显著的混杂分析物，在皮肤中的状态测量中占比高达20%。该方法在PTAD衍生化的维生素D代谢物色谱分析方面有显著改进，可能会极大地影响低丰度样品中维生素D状态和C3epi生物学的评估。
