The analytical column was a Poroshell PhenylHexyl column 150 × 2.1 mm, 3 μm column (Agilent Technologies). A mobile phase gradient programme was applied, using 0.1% formic acid in water and methanol, respectively. The injection volume was 3.5 μl. Quantitative and qualitative

analysis were performed by external calibration (0.334 to 1000 ng ml−1) and compared with the retention times and quantifier ion/qualifier ion ratios obtained by analysing NA standard solution and/or spiked QC samples (Herrmann et al., 2014). By increasing the ingoing amount of nitrite (0, 60, 100, 150, 250, 350 mg kg−1) the levels of NHPRO, NPRO, NTCA, NMTCA (Fig. 1A), NSAR and NPIP Everolimus mw (Fig. 1C) increased in the sausages. A steep increase in the level of NMTCA was observed by adding 60 mg kg−1. Higher levels of nitrite only increased the NMTCA levels slightly, indicating that other factors than nitrite is the limiting factor for the formation of NMTCA. In sausages prepared with 150 mg kg−1 nitrite, which

is the amount of nitrite allowed to be added to sausages for the common European market (https://webgate.ec.europa.eu/sanco_foods), NPIP (Fig. 1C), NHPRO, NPRO, NTCA and NMTCA (Fig. 1A) were found in levels of approximately 2, 10, PCI32765 40, 70 and 25 μg kg−1, respectively. NSAR was at LOD if more than 150 mg kg−1 nitrite was added, and by further increasing the nitrite level a clear increase in the NSAR level was found (Fig. 1C). The levels of NDMA and NPYR were relatively unaffected by the increase in added nitrite. The levels of NDMA and NPYR remained at or below 2 μg kg−1, which is at the limit of quantification (LOQ) for the method applied (Herrmann et al., 2014). Increasing the level of nitrite was also found by others to have a limited effect on the level of NDMA (Drabik-Markiewicz et al., 2011). If the sausages were further

prepared by pan frying (Fig. 1B and C) the levels of NSAR, NPIP (Fig. 1D), NTCA and NMTCA (Fig. 1B) increased by up to about 2, 2, 1.5 and 4 times, respectively. For NTCA the difference in the content between the not fried (Fig. 1A) and the fried sausages (Fig. 1B) increased with increasing amount of ingoing nitrite. This resulted in a more PtdIns(3,4)P2 linear correlation between added nitrite and NTCA level and with a steeper slope than found for the not fried sausages. For these fried sausages a slightly higher level of NDMA and NPYR were indicated for the sausages prepared with 60 or 100 mg kg−1 nitrite than in those prepared without nitrite (Fig. 1D). In the sausages prepared with 150 mg kg−1 nitrite the levels of NPIP (Fig. 1D), NHPRO, NPRO, NTCA and NMTCA (Fig. 1B) amounted to 2.6, 10, 40, 70 and 80 μg kg−1, thus frying induced an increase in the NPIP (2.6 μg kg−1) and the NMTCA (80 μg kg−1) levels.