Nitrosamines-excess detection in smoked fish and sausages

Abstract

Nitrosamines possess general formula R1R2N-N=O, they are toxic cancerogenic compounds damaging the human liver and all metabolism. The nitrosamines content is need to control after es of fuming (also called “smoking”) of fish or meat, or were nitrites as a food additive the process ∼ are used. An acceptable limit content of nitrosamines in food is 4 micrograms per kg (4 μg/kg). UV absorption at 220…255 nm with molar extinction ε 5000 L∙mol-1∙cm-1, allows HPLC/PDA use for detection or for exclusion of its dangerous excess in smoked foods. Reversed-phase gradient elution HPLC with C18-column (150mm ∙ 4,6mm ∙ 5μm ∙ 80nm, “Phenomenex”), assisted with Photo Diode Array detector (“Shimadzu”), was used. Phase A: Water with 0.01% Trichloroacetic Acid. Phase B: Acetonitrile with 0,1% Acetic Acid. Oven temperature: 25°C. Flow: 0,8 mL/min., 5% Phase B. Gradient points: 7 min. – flow, 24 min. – 65% Phase B, 25 min.

– 95% Phase B, 26min. – 95% Phase B, 27 min. – flow, 35 min. – flow, stop data acquisition. PDA detection: 200…800 nm, spectrum resolution 512, cell temperature 31°C, slit width: 8nm. Sampling: 12.5Hz. Basic wavelength: 230 nm (corresponding with maximum for majority of

N-nitrosamines). Standards from “Merck” of dimethyl-nitrosamine (CH3)2N-N=O (or Me2NNO), and of dipropyl-nitrosamine (C3H7)2N-N=O (or Pr2NNO), 0.2mg/mL were used, injected volume being 10μL. Experimental absorption maximums in conditions of elution: 227 and 231 nm. Smoked fish and sausages samples, 5,00 g, were homogenized, extracted with 5,00 mL of

Acetonitrile and centrifugated 10 minutes at 10000 rpm. Obtained liquid phase was filtered through Polytetrafluoroethylene (PTFL) filter with 0.22 μm pores. To increase targeted signal

expression, volumes of 100μL of these filtrates were injected. Solutions of Me2NNO and Pr2NNO, give tR = 4.00-4.10 min. and tR = 19.15-19.35 min., baseline-related pick heights, h, of 1350 mAU ∼ and 890 mAU, pick areas, A, of 15.1∙106 and 8.5∙106, respectively. In this condition, calculated nitrosamines, and this characterize used method as good. Very high resolution, RS ∼ 15, is good for separation and analysis of these two nitrosamines. According to these data, acceptable molar areas, Amolar, are 5.6∙109 and 5.5∙109, that is, they practically coincide for the studied concentrations∼ of 4 ng/kg in food correspond with Me2NNO and Pr2NNO pick areas of ∼ 1500 and 850, respectively – low values for this type of detection. Thus, HPLC/PDA allows with a

very low probability to reliably detect concentrations of nitrosamines that are less than the maximum permissible level. But 3-10-fold excess of the maximum permissible level of nitrosamines can be reliably determined by this method. It is quite enough to detect a danger. Thus, in the smoked capelin and herring samples, the maximum permissible level of nitrosamines was

exceeded by 20-30 times. At the same time, were not find chromatographic signals confirming the great exceed of studied nitrosamines in common types of smoked sausages.