N-nitroso-triazolopyrazine(NTTP/7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine), an NDSRI associated with sitagliptin, poses a significant nitrosamine risk due to its very low acceptable intake (37 ng/day). A robust UHPLC–MS/MS method was developed and validated for the simultaneous quantification of N-nitroso-triazolopyrazine and its precursor, triazolopyrazine(3-(Trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazine), in both drug substance and drug product within a single run. The method uses an Agilent Poroshell C18 column with 10 mM ammonium formate as mobile phase A and 0.1% formic acid in methanol as mobile phase B, detected in ESI-positive MRM mode. The method demonstrated excellent linearity (R2 ≈0.9992–0.9994), high sensitivity (LOD/LOQ: 0.037/0.11 ppm), good accuracy (recoveries: ∼90.9–115.3%), and precision (%RSD ≤ 4.2%). Mechanistic studies showed N-nitroso-triazolopyrazine formation is strongly pH-dependent, favoring acidic, nitrite-rich conditions. Degradation studies confirmed the formation of both impurities under all stress conditions, predominantly acidic. Scavenger studies identified ascorbic acid and 2,5-dihydroxybenzoic acid as effective in suppressing N-nitroso-triazolopyrazine formation.#https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/sscp.70250
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