The assay of vitamin D serum levels in patients has become a common practice in clinical laboratories due to its well-known essential role in bone mineralization and calcium/phosphate absorption. Some clinical studies indicate vitamin D deficiency as a possible cause of persistent infections and chronic inflammations, consequently different pathological may occur, including chronic tiredness, exhaustion, and depression. Clinical reports suggest a close connection between vitamin D and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Given the different biological functions performed by vitamin D in human body, tests on vitamin D in human serum have increased exponentially over the past decade. Vitamin D levels in the clinical field are usually measured using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-MS/MS). However, alternative separative methodologies are reported in literature such as gas chromatography (GC). This research aimed to develop and optimize a based-GC method as alternative HPLC strategy for the monitoring of vitamin D levels in human serum. In order to improve the sensitivity of the method, milder electron ionization conditions were applied for the detection of vitamin D metabolites. Particular emphasis was placed on optimizing sample preparation procedures. In particular, the selection of the most effective silylating agent for the conversion of vitamin D species into trimethylsilyl (TMS) derivatives and the optimization of the GC method to achieve a fast separation of the target compounds were careful. The accuracy of the optimized protocol was evaluated by analyzing the Standard Reference Material (SRM) 972a “Vitamin D Metabolites in Frozen Human Serum”, certified by National Institute Standard and Technology (NIST). [1]
Micalizzi G., Vento F., et al., Journal of Chromatography B 1227 (2023) 123813, DOI: https://doi.org/10.1016/j.jchromb.2023.123813.