Nitrosamine Detection in Meat

By Beryl Ombaso,

Chemistry 4101,

Fall 2008

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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

NO2 + H+ HNO2

2HNO2 N203 + H20

N203 + R2NH R2N.NO + HNO2

(a nitrosamine)

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Tumorgenic levels of Nitrosamines

• USDA recommended exposure level-10 ppb.2

Nitrosamine TD50, mg/kg body weight/day

Expected levels in typical sample, µg/kg

Nitrosodimethylamine 0.01 1.90 – 2.37

Nitrosodiethylamine 0.11 0.33 – 0.54

Nitrosopyrrolidine 2.10 2.32 – 8.94

Nitrosopiperidine 1.30 0.5 – 1.31

Nitrosodibutylamine 0.69 0.16 – 0.24

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Methods for Nitrosamine detectionMethod Limit of

DetectionLinear range

Special notes

Spectrophotometry3 0.78ppb 25 to 2000 ppb

Derivatization with Griess reagent.

Electroanalysis4 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/Fluorescence5 0.04 ppb 0.04 to 1.05 ppb.

Derivatization with Dansyl chloride.

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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.

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Instrumentation

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SAMPLE PREPARATION

• Interfering species: amines, amino acids.

• SPE limits amount of interfering species.

• Blank sample consists of 0.1N NaOH.

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Experimental conditions6

• 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. 500C, final temp. 2600C• 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.

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Sample Chromatogram

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Sample Mass Spectrum

• Quantification accomplished by using calibration curve obtained from standards.

• Standards obtained easily from manufacturer.

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Equipment model

• HP 5890A/5970 GC/MS• Cost: $ 9,950• Oven temp: up to 4500C• Compact size• Power supply: 120V• Resolution: 0.1amu• S/N: 20:1 • Scan speed: 1500 amu/sec• Mass range:10 – 800 amu• Gentechscientific.com

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References1. Mirvish, S.; Cancer letters, 1995, 93, 17-48.

2. Jay, J.; Modern Food Microbiology, 6th 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.

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