2 nitrosopyridine

The investigated reagents for the chemical derivatization reactions were PTAD, FMP-TS (< 5% 2-hydroxy-1-methylpyridinium p-toluenesulfonate, technical grade, ≥ 90%), isonicotinoyl chloride hydrochloride (95%), acetic anhydride (≥ 99%), pyridine (anhydrous, 99.8%), acetonitrile (ACN) (anhydrous, 99.8%); 4-dimethylaminopyridine (DMAP) (99%) obtained from Sigma-Aldrich (Steinheim, Germany); acetic acid and triethylamine (≥ 99,5%) from Carl Roth GmbH & Co. KG (Karlsruhe, Germany); DMEQ-TAD from Enzo Life Sciences (City, NY, USA); 2-nitrosopyridine from MedChemExpress (City, NJ, USA); and Amplifex Diene Reagent Kit from Sciex (Darmstadt, Germany).
Standards were purchased as follows: 3β-25(OH)D₃ and 1,25(OH)₂D₃ from Cayman Chemical (Ann Arbor, MI, USA); vitamin D₃ and 24,25-dihydroxyvitamin D₃ ((24R)-24,25(OH)₂D₃) from Toronto Research Chemicals (Toronto, ON, Canada); and 3α-25(OH)D₃ from Sigma-Aldrich (Steinheim, Germany).
Acetonitrile and methanol (MeOH) (UHPLC-MS grade) were obtained from Chemsolute (Th. Geyer GmbH & Co. KG, Renningen, Germany), while formic acid (97%) was from Alfa Aesar (Karlsruhe, Germany). A Millipore (Bedford, MA, USA) Direct-Q8 purification system was used to produce organic free water.
The human vitamin D₃ free serum was purchased from Sigma-Aldrich (VD-DDC Mass Spect Gold serum).

Standards of 3β-25(OH)D₃ and 1,25(OH)₂D₃ were purchased from Cayman Chemical (Ann Arbor, MI, USA); vitamin D₃ and (24R)-24,25(OH)₂D₃ from Toronto Research Chemicals (Toronto, ON, Canada); 3α-25(OH)D₃, 4-phenyl-1,2,4-triazoline-3,5-dione, 2-fluoro-1-methylpyridinium-p-toluenesulfonate, isonicotinoyl chloride hydrochloride (95%), nicotinoyl chloride hydrochloride (97%), acetic anhydride (≥ 99%), pyridine (anhydrous, 99.8%), acetonitrile (anhydrous, 99.8%), and 4-dimethylaminopyridine (99%) from Sigma-Aldrich (Steinheim, Germany); acetic acid and triethylamine (≥ 99.5%) from Carl Roth (Karlsruhe, Germany); 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione from Enzo Life Sciences (NY, USA); 2-nitrosopyridine from MedChemExpress (Monmouth Junction, NJ, USA); Amplifex Diene Reagent Kit from Sciex (Darmstadt, Germany); and 3β-25(ΟΗ)D₃-[26,26,26,27,27,27-d6] monohydrate from IsoSciences (Ambler, PA, USA). UHPLC-MS-grade acetonitrile and methanol were obtained from Chemsolute (Th. Geyer, Renningen, Germany) and formic acid (97%) from Alfa Aesar (Karlsruhe, Germany). Organic-free water was generated by a Millipore (Bedford, MA, USA) Direct-Q8 purification system. Human vitamin D₃-free serum (VD-DDC Mass Spect Gold® serum) was purchased from Sigma-Aldrich.