Streamlined Vitamin\nD Metabolite Fingerprinting Analysis\nUsing Isotope-Coded Multiplexing MS with Cost-Effective One-Pot Double\nDerivatization

In this study, we extended a previously developed one-pot\ndouble\nderivatization reaction to establish the first routine isotope-coded\nmultiplex derivatization for vitamin D and its metabolites for application\nin clinical environments, using commercial reagents, without the need\nfor specialized reagents and advanced synthesis requirements. The\noriginal derivatization process consisted of using both a Cookson-type\nreagent and derivatization of hydroxyl groups. Initially, the analytes\nare derivatized by a Diels–Alder reaction using 4-phenyl-1,2,4-triazoline-3,5-dione\n(PTAD), followed by acetylation using acetic anhydride, catalyzed\nby 4-dimethylaminopyridine at room temperature. To enable sample multiplexing,\nwe utilized acetic anhydride as well as the <i>d</i><sub>3</sub>- isotopologue of acetic anhydride, generating <i>d</i><sub>3</sub>- and <i>d</i><sub>6</sub>-products of the\ninvestigated vitamin D<sub>3</sub> metabolites. This approach not\nonly allowed for the simultaneous measurement of two samples within\na single LC-MS/MS run but also improved the LC separation of the important\n25-hydroxyvitamin D<sub>3</sub> epimers (3α-25(OH)D<sub>3</sub> and 3β-25(OH)D<sub>3</sub>) on a conventional C-18 column,\naddressing a significant challenge in vitamin D analysis. Typically,\nthe separation of these epimers after PTAD derivatization cannot be\nperformed on C-18 columns, necessitating the use of pentafluorophenylpropyl\n(PFP) stationary phases. However, PFP columns are not as stable as\nC-18 in long-term use, wherein the acetylation of the C-3 hydroxyl\ngroup provided a solution by enhancing chromatographic selectivity\nand achieving the baseline separation of the metabolites 24,25(OH)<sub>2</sub>D<sub>3</sub>, 3α-25(OH)D<sub>3</sub>, 3β-25(OH)D<sub>3</sub>, and vitamin D<sub>3</sub> using a C-18 column with methanol/water\ngradient elution. The described duplex derivatization was tested on\n40 serum samples of patients with chronic liver diseases (CLD). Additionally,\nthe method was evaluated in terms of linearity, accuracy, precision,\nand interferences between heavy and light tag samples using both commercial\nquality control samples and in-house quality control and calibration\nsamples.