Quantification of 3,4-Dimethyl-1H-Pyrazole Using Ion-Pair LC–MS/MS on a Reversed-Phase Column

BACKGROUND: Few methods exist for the analysis of the soil nitrification inhibitor 3,4-dimethyl-1H-pyrazole (3,4-DMP), which is a pesticide with the ability to reduce the production of nitrogenous greenhouse gases in soils as a result of fertilizer application. Due to its small size and polar nature, 3,4-DMP can be difficult to retain on an LC column, which makes diversion of a co-extracted soil matrix away from the MS/MS impossible. OBJECTIVE: The current study aims to better control the retention time (RT) of 3,4-DMP. Additionally, 3,4-DMP-15N2 was synthesized and used as an internal standard for the soil extraction of 3,4-DMP. METHODS: Perfluoroalkanoic acids were used as ion-pair reagents and were compared for their abilities to improve peak shape and RT, to better separate 3,4-DMP from the soil matrix without the need for cleanup during soil extraction. RESULTS: RTs increased with both the carbon number and the concentration of the perfluoroalkanoic acid, and this improved peak shape and height. Perfluorooctanoic acid performed best, and improved peak height (PH) and shape were obtained by increasing the flow rate, resulting in a better S/N than from formic acid. The method provided a 10-fold improvement limit of quantitation on the most sensitive existing method and the use of 3,4-DMP-15N2 as an internal standard resulted in recoveries of 101–107%. CONCLUSION: Ion-pair reagents drastically increased the retention of 3,4-DMP and allowed the re-use of old LC columns that may otherwise be discarded. Improved separation of 3,4-DMP from the soil matrix allowed much of the matrix to be diverted from the MS/MS spray chamber. HIGHLIGHTS: Greater control of 3,4-DMP retention by the LC column resulting in the ability to separate 3,4-DMP from the soil matrix. The inclusion of ion-pair reagents only in the aqueous phase reduced ionization suppression of the analytes in the MS source.