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This standard replaces GB/T 5009.179-2003 Determination of Trimethylamine Nitrogen in Ham.
The following main changes have been made with respect to GB/T 5009.179-2003:
——This standard is renamed as "National Food Safety Standard - Determination of Trimethylamine in Foods";
——The original Spectrophotometric Method is modified as Method I Headspace Gas Chromatography - Mass Spectrometry and Method II Headspace Gas Chromatography;
——Application scope of this standard is extended to aquatic animals and their products as well as meat and meat products;
——Trimethylamine nitrogen is changed as trimethylamine.
National Food Safety Standard
Determination of Trimethylamine in Foods
1 Scope
This standard specifies determination methods for trimethylamine in aquatic animals and their products as well as meat and meat products.
This standard is applicable to the determination of trimethylamine in the aquatic animals and their products as well as meat and meat products.
Method I Headspace Gas Chromatography - Mass Spectrometry
2 Principle
Extract the specimen with 5% trichloroacetic acid solution, put the extract into a sealed headspace bottle, transform the trimethylamine hydrochloride to trimethylamine under the action of alkali liquor, balance at 40℃ for 40 min, so that the trimethylamine realizes dynamic balance in gas-liquid phases. Pipet the gas in the headspace bottle and inject into the gas chromatography - mass spectrometer for detection, carry out the qualitative determination by retention time (RT), assisted qualitative ion (m/z 59 and m/z 42) and quantitative ion (m/z 58) and the quantitative determination by external standard method.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Class-I water (defined in GB/T 6682) are adopted for the purpose of this method.
3.1 Reagents
3.1.1 Sodium hydroxide (NaOH)
3.1.2 Trichloroacetic acid (C2HCl3O2).
3.2 Preparation of reagents
3.2.1 50% sodium hydroxide solution: weigh 100 g sodium hydroxide and dissolve in 100mL water (20℃~30℃).
3.2.2 5% trichloroacetic acid solution: weigh 25 g trichloroacetic acid, dissolve in water and scale the volume to 500 mL.
3.3 Standard product
Trimethylamine hydrochloride (CAS: 593-81-7), molecular formula: (CH3)3NHCl, purity ≥98%, put it into a dryer and preserve at 4℃.
3.4 Preparation of standard solutions
3.4.1 Trimethylamine standard stock solution: weigh 0.0162 g trimethylamine hydrochloride standard product, dissolve with 5% trichloroacetic acid solution and scale the volume to 100 mL, equivalent to the trimethylamine standard stock solution with the concentration of 100 μg/mL, and preserve at 4℃.
3.4.2 Trimethylamine standard working solution: pipet certain volume of trimethylamine standard stock solution, dilute with 5% trichloroacetic acid solution into the trimethylamine standard working solution with the concentrations of 1.0 μg/mL, 2.0 μg/mL, 5.0 μg/mL, 10.0 μg/mL, 20.0 μg/mL and 40.0 μg/mL respectively step by step.
4 Instruments and Apparatus
4.1 Gas chromatography - mass spectrometer, equipped with split/splitless injection port and electron impact ionization source (EI source).
4.2 Balance: with the sensibility of 0.1 mg and 1 mg.
4.3 Thermostat water bath: with temperature control accuracy of ±2℃.
4.4 Headspace bottle: 40mL volume, equipped with PTFE silicone rubber gasket and sealing cap, bake for 2 h at 120℃ prior to use.
4.5 Microinjector: 1 mL.
4.6 Medical plastic syringe: 5 mL.
4.7 Homogenizer.
4.8 Mincer.
4.9 Low-speed centrifuge.
5 Analysis Steps
5.1 Preparation of specimens
5.1.1 Pretreatment and preservation of specimens
Remove the fat and skin for livestock and poultry meat and their products, while remove the scales or skin for aquatic animals such as fish and shrimp and their products; all samples shall be about 100g taken from the muscle part, minced with a mincer or cut up with a knife and mixed uniformly. The well-prepared specimen, if not determined immediately, shall be sealed in a polyethylene plastic bag and preserved by freezing at -18℃, and may be unfrozen by placing under room temperature prior to use.
5.1.2 Extraction of specimens
Weigh about 10 g (accurate to 0.001 g) well-prepared sample, put into a 50 mL plastic centrifuge tube, add 20 mL trichloroacetic acid solution (5%), homogenize for 1 min with homogenizer, centrifuge for 5 min at the rotation speed of 4000 r/min, add a small amount of cotton wool on the glass funnel, filter the supernatant into a 50 mL volumetric flask, repeat the above extraction process twice for the residues with 15 mL and 10 mL trichloroacetic acid solution (5%) respectively, combine the filtrate and scale the volume with 5% trichloroacetic acid solution to 50 mL.
5.1.3 Headspace treatment for extract
Accurately pipet 2.0 mL extract into a 20 mL headspace bottle, place the cover for sealing, and accurately inject 5.0 mL sodium hydroxide solution (50%) with medical plastic syringe for standby use.
5.1.4 Headspace treatment for standard solution
Put 2.0 mL all standard working solutions given in 3.4.2 into 20 mL headspace bottles respectively, place the covers for sealing, and accurately inject 5.0 mL sodium hydroxide solution (50%) with medical plastic syringe for standby use.
5.2 Reference conditions of instruments
5.2.1 Chromatographic conditions
Chromatographic conditions are as follows:
a) Quartz capillary chromatographic column: 30 m (length) × 0.25 mm (inside diameter) × 0.25 μm (film thickness) with polyethylene glycol as stationary phase, or other equivalent chromatographic column;
b) Carrier gas: high-purity helium;
c) Flow rate: 1.0 mL/min; temperature of injection port: 220℃;
d) Splitting ratio: 10: 1;
e) Temperature rise procedures: maintain for 3 min at 40℃, rise to 220℃ at a rate of 30℃/min and maintain for 1 min.
5.2.2 Mass spectrometer conditions
Mass spectrometry conditions are as follows:
a) Ion source: electron impact ionization source (EI source); temperature: 220℃;
b) Ionization energy: 70 eV;
c) Temperature of transmission line: 230℃;
d) Solvent delay: 1.5 min;
e) Scanning mode: selective ion monitoring (SIM)
5.3 Determination
5.3.1 Headspace sample injection: balance the well-prepared specimen at 40℃ for 40 min. Under the chromatographic and mass spectrometry conditions given in 5.2, take 100 μL head space gas from the headspace bottle with a sample injection needle and inject into the GC-MS for determination.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Accuracy
8 Other
9 Principle
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Accuracy
15 Other
Annex A Chromatogram and Mass Spectrum
