1. Nitrosamine Detection in Meat By Beryl Ombaso, Chemistry 4101, Fall 2008 1

2. BACKGROUND Nitrite is added to meat both for preservation and color enhancement. Amines in the meat react with the nitrite to form nitrosamines which are known carcinogens. 1 NO 2 + H + HNO 2 2HNO 2 N 2 0 3 + H 2 0 N 2 0 3 + R 2 NH R 2 N.NO + HNO 2 (a nitrosamine) 2

3. Tumorgenic levels of Nitrosamines USDA recommended exposure level-10 ppb. 2 NitrosamineTD50, mg/kg body weight/day Expected levels in typical sample, µg/kg Nitrosodimethylamine0.011.90 – 2.37 Nitrosodiethylamine0.110.33 – 0.54 Nitrosopyrrolidine2.102.32 – 8.94 Nitrosopiperidine1.300.5 – 1.31 Nitrosodibutylamine0.69 0.16 – 0.24 3

4. Methods for Nitrosamine detection Method Limit of Detection Linear range Special notes Spectrophotometry 3 0.78ppb25 to 2000 ppb Derivatization with Griess reagent. Electroanalysis 4 6.0 x 10 -8 mol L -1 2 x 10 -6 to 1.35 x 10 -5 mol L -1 Determine total nitrosamines HPLC/Fluorescence 5 0.04 ppb0.04 to 1.05 ppb. Derivatization with Dansyl chloride. 4

5. Proposed Method for Nitrosamine Detection Gas Chromatography with tandem MS would be most ideal. 6  Nitrosamines are highly volatile and thermally stable.  Limit of detection – 0.01 ppb.  Linear range – 0.01 to 10 ppb.  Percent recovery – 70-80 %.  Efficient separation and ease of use. 5

6. Instrumentation Carrier gas and flow meter Sample injection into column GC oven and Column Ionization and uptake into MS Mass analyzer 1 Interaction cell Mass analyzer 2 DetectorData analysis 6

7. SAMPLE PREPARATION Obtain meat sample to be tested Grind sample and soak in NaOH to extract nitrosamines. Use solid phase extraction to obtain nitrosamines from other species in the matrix. Evaporate sample to concentrate analytes. Add an internal standard Interfering species: amines, amino acids. SPE limits amount of interfering species. Blank sample consists of 0.1N NaOH. 7

8. Experimental conditions 6 Combination of Extrelut-Florisil cartridge for SPE. Column – 30 m, 0.25 mm i.d, 0.25 μm film thickness containing 1:6 cyanopropylphenyl:methyl polysiloxane. Carrier gas – Helium gas at 1.0 mL/min. Oven – initial temp. 50 0 C, final temp. 260 0 C Injection volume- 2.0 μl Outlet pressure – 54.8 kPa Electrospray ionization at 70eV. Reagent for chemical ionization- ammonia gas. Quadrupole mass selective detectors for analysis. 8

9. Sample Chromatogram 9

10. Sample Mass Spectrum Quantification accomplished by using calibration curve obtained from standards. Standards obtained easily from manufacturer. 10

11. Equipment model HP 5890A/5970 GC/MS Cost: $ 9,950 Oven temp: up to 450 0 C Compact size Power supply: 120V Resolution: 0.1amu S/N: 20:1 Scan speed: 1500 amu/sec Mass range:10 – 800 amu Gentechscientific.com 11

12. References 1. Mirvish, S.; Cancer letters, 1995, 93, 17-48. 2. Jay, J.; Modern Food Microbiology, 6 th Ed, 2000, pg 261. 3. Perez, E. L.; Rios, A.; Valcarcel, M.; J Anal. Chem, 2001, 371, 891-895. 4. Oliveira, R. T.; Salazar-Banda, G. R.; Machado, S. A.; Avaca, L. A.; Electroanalyis, 2008, 20, 396-401. 5.Cardenes, L.; Ayala, J. H.; Venerando, G.; Afonso, A. M.; J. Liq. Chrom. & Rel. Technol., 2002, 25, 977-984. 6.Jurtchenko, S.; Tenno, T.; Molder, U.; Reinik, M.; Proc. Estonian Acad. Sci. Chem., 2002, 51, 169-184. 12