4-Diazomethylpyridine as a Derivatization Reagent and Its Application to the Determination of Prostaglandin E2 by LC–MS/MS

A high ionization efficiency derivatization reagent with a diazonium moiety, 4-diazomethylpyridine, has been designed for prostaglandins labeling. The labeled prostaglandins are then determined by LC–MS/MS. Prostaglandin E 2 (PGE 2 ) in rat brain homogenate was used for evaluation of the analytical potential of this reagent. 4-Diazomethylpyridine was synthesized in three steps. Analytes were purified in one step via C 18 solid-phase extraction, derivatized with 4-diazomethylpyridine, separated by reverse-phase HPLC and quantified on a triple-quadrupole mass spectrometer using selected reaction monitoring in positive ionization. The derivatization reaction was completed at 40 °C within 10 min without catalyst. Chromatographic separation of PGE 2 derivative was achieved on a Waters Symmetry C 18 column (100 × 2.1 mm, 3.5 μm) using a mobile phase consisting of 10-mM ammonium acetate in deionized water and methanol (25:75, v/v ). The flow rate was 0.2 mL min −1 and the total run time was 5 min. The mass transition was m/z 444 → 110. The calibration curve was linear in the range from 0.65 to 7.10 pmol per injection volume of 5 μL and the detection limit was about 0.21 pmol on column. Recoveries of spiked PGE 2 were above 70 % with intra-day relative standard deviation