In order to examine the effect of each individual factor, possible interactions between them and to work with at realistic number of experimental setups, factorial designs were employed. Acetonitrile (extraction solvent) was of HPLC grade (Rathburn Chemicals Ltd., Y27632 Walkerburn, Scotland). Formic acid (purity 98–100% for

analysis), pipecolic acid (purity 98%), and the methanol for LC eluent (purity ⩾ 99.9%, Fluka-Analytical) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Erythorbic acid, ascorbyl palmitate, tripolyphosphate, iron(III)sulphate hydrate and myoglobin from equine heart were also purchased from Sigma–Aldrich Co. The pure standards of N-nitrososarcosine (NSAR), N-nitrosohydroxyproline (NHPRO), N-nitrosodibenzylamine (NDBzA), N-nitrosoproline (NPRO), N-nitrosomethylaniline (NMA), N-nitroso-2-methyl-thiazolidine 4-carboxylic acid (NMTCA)

and N-nitroso-thiazolidine-4-carboxylic acid (NTCA) were purchased from Toronto Research Chemicals (Toronto, Canada), whereas the standards N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosomorpholine (NMOR) and N-nitrosodimethylamine (NDMA) were purchased from Sigma–Aldrich Co. N-nitrosomethylethylamine (NMEA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP) were purchased from Dr. Ehrenstorfer (Ausburg, Germany). The internal standards N-nitrosodimethylamine-d6 (NDMA-d6) and N-nitrosopyrrolidine-d8 (NPYR-d8) were purchased from Sigma–Aldrich Co. and CDN Isotopes (Quebec, Canada), respectively. The purity of all of the NA standards was ⩾98% except for NMA which was of DZNeP purchase 95% purity. Sodium nitrite (Fluka-Analytical) was purchased from Sigma–Aldrich Co. Cooked pork sausages were

chosen as representative meat products, because sausages account for a major part of the total consumption of processed meat products by the Danish, buy Baf-A1 as well as other European populations (Linseisen et al., 2002). By choosing a minced meat product the ingredients are also more evenly distributed and any relevant equilibria are reached faster. Trimmed fresh pork loin with a fat content of approximately 12% (www.foodcomp.dk) was minced (Kenwood, MG470, Elgiganten, Glostrup Denmark) and all ingredients common for all samples in the setup were added during mixing (Bear Varimixer, AR5A, A/S Wodschow & Co., Broendby, Denmark). The sausage meat was prepared from tap water (26%), minced meat (67%), potato flour (4%), ground black pepper (Piper nigrum, Santa Maria A/S, Broendby, Denmark) (0.125 or 0.5%), sodium chloride (2%), paprika (0.5%), nitrite (0–350 mg kg−1 depending on the setup). Aliquots of the thoroughly mixed sausage meat were transferred to a mini chopper (Phillips hand blender with chopper, HR1372, Punkt1, Roedovre, Denmark) and further mixed with the ingredients/factors to be tested and chopped until they were evenly distributed. The sausage meat that needed no further additions was chopped in the same way.