Stability of sample extracts of vitamin D₃ metabolites after chemical derivatization for LC-MS/MS analysis

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used to determine vitamin D₃ metabolites in biological samples. The ionization efficiencies of these metabolites, however, are poor under electrospray ionization conditions. Moreover, the chromatographic separation of multiple vitamin D metabolites and their epimers can be challenging. For these reasons, chemical derivatization reagents are often used to improve sensitivity and selectivity of analysis. While the derivatization schemes have been proven to be very effective, one missing aspect is the investigation of the stability of the chemical derivatization products in stored sample extracts. In this study, we investigated the long-term stability of several vitamin D₃ metabolites after 1 and 3 months of storage at - 20 °C. Five vitamin D₃ metabolites were examined after derivatization with seven different derivatization reagents. Generally, Amplifex products were the most stable in the long term in our study with 11-20% degraded after 1 month of storage and 14-35% after 3 months. The stabilities for some of the metabolites' 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP-TS), isonicotinoyl chloride (INC) and 4-phenyl-1,2,4-triazoline-3,5-dione acetylated (PTAD-Ac) products were also acceptable after 1 month of storage. Other derivatized metabolites, however, degraded extensively already after 1 month of storage, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (54-72% degradation) and 2-nitrosopyridine (PyrNO) (32-100% degradation). Importantly, for every metabolite, there was an optimum derivatization reagent that met the criteria of stability proposed by international regulatory bodies after 1 month of storage. Some derivatives were stable for even up to 3 months of storage, with degradation of less than 15%.