Comprehensive Assessment of Nitrosamine Formation in Meat Products Using UHPLC-HRMS: Analytical Challenges and Potential Dietary Implications

Nitrosamines (NAs) pose a risk due to their carcinogenic properties, especially in processed and cured meats where nitrites and nitrates are widely used. The objective of this study was to develop an integrated Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS) workflow for detecting both volatile (VNAs) and non-volatile (NVNAs) nitrosamines in meat matrices. Comparison of two ionization techniques showed that heated electrospray ionization (HESI) and atmospheric pressure chemical ionization (APCI) provided complementary coverage and sensitivity. Extraction and cleanup were optimized for meat, although recovery rates remained variable, underscoring the analytical complexity. The method was applied to raw, cooked, cured, and grilled meats, as well as to in vitro gastric digestion and co-digestion with spinach. Results revealed that some NAs were present even in untreated raw meat (≈3.0 µg/kg, N-nitrosodi-n-butylamine), while the addition of nitrites and nitrates significantly increased their levels (more than 10 µg/kg, N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosodi-n-butylamine). Gastric digestion was the most critical condition, further promoting nitrosamine formation, particularly for N-nitrosodiethylamine, N-nitrosodi-n-butylamine, and N-nitrosopiperidine. Ascorbate exhibited a dual role, acting as an inhibitor at low nitrite concentrations but becoming pro-oxidant at high levels (300 mg/kg). Cooking alone had limited impact, whereas cooking combined with digestion yielded the highest and most consistent nitrosamine concentrations. The inclusion of spinach during digestion modestly altered nitrosamine levels, reflecting both its nitrate content and polyphenolic profile. Nonparametric ANOVA (aligned rank transform) confirmed that preservative treatment, rather than processing or interaction effects, was the main driver of variability (total nitrosamines: H = 24.15, <i>p</i> = 2.33 × 10^-5), with the combination of preservative ascorbate plus nitrite producing significantly higher levels than other treatments (q = 0.000656). N-nitrosodimethylamine consistently emerged as the most relevant marker for dietary exposure, in agreement with EFSA guidance. Overall, this study underscores both the analytical and biochemical complexity of nitrosamine detection and formation in meat products, while highlighting the importance of preservative formulation and the potential role of dietary antioxidants in mitigating exposure.