1 Scope
This standard specifies the determination method of nitrite and nitrate in foods.
This standard is applicable to determination of nitrite and nitrate in foods.
Method I Ion Chromatography Method
2 Principle
Settle proteins and eliminate fat in the sample, extract and purify with corresponding methods, the potash solution is used as eluate, separate with anion exchange column and detect with electrical conductivity detector. Determine the nature with retention time and determine the quantity with the external standard method.
3 Reagents and Materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 3 water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetic acid (CH3COOH).
3.1.2 Potassium hydroxide (KOH).
3.2 Reagent preparation
3.2.1 Acetic acid solution (3%): measure acetic acid 3mL form a 100mL volumetric flask, diluted to the scale with water, mix well.
3.2.2 Potassium hydroxide solution (1mol/L): weigh 6g potassium hydroxide, dissolve it with water and diluted to 100mL, mix well.
3.3 Standards
3.3.1 Sodium nitrite (NaNO2, CAS No.: 7632-00-0): reference reagent, or nitrite standard solution with reference material certificate.
3.3.2 Sodium nitrate (NaNO3, CAS No.: 7631-99-4): reference reagent, or nitrate standard solution with reference material certificate.
3.4 Preparation of standard solution
3.4.1 Standard nitrite stock solution (100mg/mL, calculated in NO2-, the same below): Dry the sodium nitrite at 110℃ to 120℃ to constant weight. Accurately weigh 0.1500g and dissolve it in water and transfer to a 1000mL volumetric flask, dilute with water to the scale, mix well.
3.4.2 Standard nitrate stock solution (1000mg/mL, calculated in NO3-, the same below): Dry the sodium nitrate at 110℃ to 120℃ to constant weight. Accurately weigh 1.3710g and dissolve it in water and transfer to a 1000mL volumetric flask, dilute with water to the scale, mix well.
3.4.3 Mixed standard intermediate solution of nitrite and nitrate: accurately transfer respectively 1.0mL standard stock solution of nitrite ion (NO2-) and nitrate ion (NO3-) to the 100mL volumetric flask, dilute with water to the scale, this solution includes 1.0mg nitrite ions and 10.0mg nitrate ions for each 1L.
3.4.4 Mixed standard working solution of nitrite and nitrate: transfer mixed standard intermediate solution of nitrite and nitrate, dilute with water at multiple rank to prepare mixed standard working solution series; the concentration of nitrite ion are 0.02mg/L, 0.04mg/L, 0.06mg/L, 0.08mg/L, 0.10mg/L, 0.15mg/L and 0.20mg/L respectively; the concentration of nitrite ion are 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L, 1.0mg/L, 1.5mg/L and 2.0mg/L respectively.
4 Apparatus and Equipment
4.1 Ion chromatograph: include electrical conductivity detector, equipped with suppressor, high volume anion exchange column and 50 μL fixed quantity ring.
4.2 Grinder.
4.3 Ultrasonic cleaner
4.4 Scale: sensibility balance is 0.1mg and 1mg.
4.5 Centrifuge: rotational speed ≥10000 rounds/minute, matched with 5mL or 10mL centrifuge tube.
4.6 0.22μm water filter membrane pinhead filter apparatus
4.7 Decontaminating column: including C18 column, Ag column and Na column or equivalent column
4.8 Injector: 1.0mL and 2.5mL
Note: All the glass wares shall be soaked with 2 mol/L potassium hydroxide and water for 4h, wash with water for 3 to 5 times and drain and set aside before use.
5 Analysis Procedure
5.1 Pretreatment for sample
5.1.1 Vegetables and fruits: wash the fresh vegetable and fruit samples with tap water, clean it with water, dry and take the edible part, mince and bed blending. Take right amount of minced sample by quartering, and make into slurry with grinder for use. If water is added, the volume of water shall be recorded.
5.1.2 Grains and other plant sample: after elimination of visible impurities, take 50g to 100g representative sample, filtrate with 0.30mm hole sizer after crushed, mix well for use.
5.1.3 Meats, eggs, aquatic products and manufactured products: take right amount or the entire part by quartering, and make into slurry with grinder for use.
5.1.4 Solid dairies (excluding cheese) such as milk powder, soy milk powder, baby formulas: put the sample into a cover-container homogenizing with a volume 2 times of the sample, sufficiency bed blending the sample by shake and bottom up the container time and again to make the sample homogenization.
5.1.5 Rancid milk, milk, condensed milk and other liquid dairy: mix or shake and bottom up the container time and again to make the sample sufficiently bed blending.
5.1.6 Cheese: take an amount of sample, grind it into uniform muddy. To avoid moisture loss, avoid generating superabundant heat quantity in the process of lapping.
5.2 Extraction
5.2.1 Fruits and vegetables: weigh 5 g sample slurry (accurate to 0.01g, appropriately adjust the sampling quantity, the same below), put into a 100mL volumetric flask, add 80mL water and 1mL potassium hydroxide solution (1mol/L), extract with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 r/min centrifuge and take the filtrate for use.
5.2.2 Fish, meat, eggs and their products: weigh 5g sample slurry (accurate to 0.01g), put into a 100mL volumetric flask, add 80mL water and 1mL potassium hydroxide solution (1mol/L), extract with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 r/min centrifuge and take the filtrate for use.
5.2.3 Salted fish, salted meat and other salted products: weigh 2g sample slurry (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, extract 30 min with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 rounds/minute centrifuge and take the filtrate for use.
5.2.4 Milk: weigh 10g sample (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, mix well; extract for 30 min with ultrasound, add 2mL 3% acetic acid solution, keep at 4 ℃ for 20 min, take it out to get room temperature and dilute with water to the scale division. Filter the solution with filter paper and take the filtrate for use.
5.2.5 Milk power and cheese: weigh 2.5g sample (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, mix well; extract for 30 min with ultrasound, take it out to get room temperature; add 2mL 3% acetic acid solution, and dilute with water to the scale, mix well. Keep at 4 ℃ for 20 min, take it out to get room temperature. Filter the solution with filter paper and take the filtrate for use.
5.2.6 Take 15mL above standby the filtrate via 0.22μm water filter membrane pinhead filter apparatus and C18 column discard the upper 3mL (if chloride ion is more than 100mg/L, then discard the upper 7mL via pinhead filter apparatus, C18 column, Ag column and Na column) and collect the remaining eluent for use.
Solid phase extraction column shall be activated before use, for instance using C18 column (1.0mL), Ag column (1.0mL) and Na column (1.0mL). The reactivation process is: before use C18 column (1.0mL), wash it with 10mL methanol and 15mL water and keep it still and activated for 30 min. Wash Ag column (1.0mL) and Na column with 10mL water keep it still and activated for 30 min.
5.3 Reference chromatographic condition
5.3.1 Chromatographic column: hydroxide selectivity, high-capacity anion exchange column compatible gradient elution of Styrene-divinyl benzene copolymer matrix, alkoxide quaternary ammonium functional group, 4 mm× 250 mm (with guard column 4mm× 50mm), or equivalent ion chromatography columns.
5.3.2 Eluate
5.3.2.1 Common sample: potash solution, concentration is 6 mmol/L to 70 mmol/L, elution gradient is 6 mmol/L30 min, 70 mmol/L5 min, 6 mmol/L5 min ; flow speed 1.0mL/min.
5.3.2.2 Powder baby formula foods: potash solution, concentration mmol/L to 50 mmol/L; elution gradient is 5 mmol/L 33min; 50 mmol/L 5min; 5 mmol/L 5min and the flow speed is 1.3mL/min.
5.3.3 Suppressor: continuous auto-regenerated film negative ion suppressor or equivalent suppression device
5.3.4 Detector: electrical conductivity detector, the detection pool temperature is 35℃.
5.3.5 Sample injection volume: 50 μL (adjust according to ionic content in the measured sample).
5.4 Determination
5.4.1 Plotting of standard curve
Inject the standard working solution series into ion chromatograph respectively, to get the chromatogram of each concentration of standard working solution, and measure the corresponding peak height (μS) or peak area. Take the concentration of standard working solution as the lateral coordinate, peak height (μS) or peak area as the longitudinal coordinate, plot standard curve (see Figure A.1 for the standard chromatogram of nitrite and nitrate).
5.4.2 Determination of sample solution
Inject blank solution and sample solution into ion chromatograph, and measure the peak height (μS) or peak area of the blank solution and sample solution. Nitrite ion or nitrate ion concentration in the solution to be tested is obtained according to the standard curve.
1 Scope
This standard specifies the determination method of nitrite and nitrate in foods.
This standard is applicable to determination of nitrite and nitrate in foods.
Method I Ion Chromatography Method
2 Principle
Settle proteins and eliminate fat in the sample, extract and purify with corresponding methods, the potash solution is used as eluate, separate with anion exchange column and detect with electrical conductivity detector. Determine the nature with retention time and determine the quantity with the external standard method.
3 Reagents and Materials
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 3 water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Acetic acid (CH3COOH).
3.1.2 Potassium hydroxide (KOH).
3.2 Reagent preparation
3.2.1 Acetic acid solution (3%): measure acetic acid 3mL form a 100mL volumetric flask, diluted to the scale with water, mix well.
3.2.2 Potassium hydroxide solution (1mol/L): weigh 6g potassium hydroxide, dissolve it with water and diluted to 100mL, mix well.
3.3 Standards
3.3.1 Sodium nitrite (NaNO2, CAS No.: 7632-00-0): reference reagent, or nitrite standard solution with reference material certificate.
3.3.2 Sodium nitrate (NaNO3, CAS No.: 7631-99-4): reference reagent, or nitrate standard solution with reference material certificate.
3.4 Preparation of standard solution
3.4.1 Standard nitrite stock solution (100mg/mL, calculated in NO2-, the same below): Dry the sodium nitrite at 110℃ to 120℃ to constant weight. Accurately weigh 0.1500g and dissolve it in water and transfer to a 1000mL volumetric flask, dilute with water to the scale, mix well.
3.4.2 Standard nitrate stock solution (1000mg/mL, calculated in NO3-, the same below): Dry the sodium nitrate at 110℃ to 120℃ to constant weight. Accurately weigh 1.3710g and dissolve it in water and transfer to a 1000mL volumetric flask, dilute with water to the scale, mix well.
3.4.3 Mixed standard intermediate solution of nitrite and nitrate: accurately transfer respectively 1.0mL standard stock solution of nitrite ion (NO2-) and nitrate ion (NO3-) to the 100mL volumetric flask, dilute with water to the scale, this solution includes 1.0mg nitrite ions and 10.0mg nitrate ions for each 1L.
3.4.4 Mixed standard working solution of nitrite and nitrate: transfer mixed standard intermediate solution of nitrite and nitrate, dilute with water at multiple rank to prepare mixed standard working solution series; the concentration of nitrite ion are 0.02mg/L, 0.04mg/L, 0.06mg/L, 0.08mg/L, 0.10mg/L, 0.15mg/L and 0.20mg/L respectively; the concentration of nitrite ion are 0.2mg/L, 0.4mg/L, 0.6mg/L, 0.8mg/L, 1.0mg/L, 1.5mg/L and 2.0mg/L respectively.
4 Apparatus and Equipment
4.1 Ion chromatograph: include electrical conductivity detector, equipped with suppressor, high volume anion exchange column and 50 μL fixed quantity ring.
4.2 Grinder.
4.3 Ultrasonic cleaner
4.4 Scale: sensibility balance is 0.1mg and 1mg.
4.5 Centrifuge: rotational speed ≥10000 rounds/minute, matched with 5mL or 10mL centrifuge tube.
4.6 0.22μm water filter membrane pinhead filter apparatus
4.7 Decontaminating column: including C18 column, Ag column and Na column or equivalent column
4.8 Injector: 1.0mL and 2.5mL
Note: All the glass wares shall be soaked with 2 mol/L potassium hydroxide and water for 4h, wash with water for 3 to 5 times and drain and set aside before use.
5 Analysis Procedure
5.1 Pretreatment for sample
5.1.1 Vegetables and fruits: wash the fresh vegetable and fruit samples with tap water, clean it with water, dry and take the edible part, mince and bed blending. Take right amount of minced sample by quartering, and make into slurry with grinder for use. If water is added, the volume of water shall be recorded.
5.1.2 Grains and other plant sample: after elimination of visible impurities, take 50g to 100g representative sample, filtrate with 0.30mm hole sizer after crushed, mix well for use.
5.1.3 Meats, eggs, aquatic products and manufactured products: take right amount or the entire part by quartering, and make into slurry with grinder for use.
5.1.4 Solid dairies (excluding cheese) such as milk powder, soy milk powder, baby formulas: put the sample into a cover-container homogenizing with a volume 2 times of the sample, sufficiency bed blending the sample by shake and bottom up the container time and again to make the sample homogenization.
5.1.5 Rancid milk, milk, condensed milk and other liquid dairy: mix or shake and bottom up the container time and again to make the sample sufficiently bed blending.
5.1.6 Cheese: take an amount of sample, grind it into uniform muddy. To avoid moisture loss, avoid generating superabundant heat quantity in the process of lapping.
5.2 Extraction
5.2.1 Fruits and vegetables: weigh 5 g sample slurry (accurate to 0.01g, appropriately adjust the sampling quantity, the same below), put into a 100mL volumetric flask, add 80mL water and 1mL potassium hydroxide solution (1mol/L), extract with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 r/min centrifuge and take the filtrate for use.
5.2.2 Fish, meat, eggs and their products: weigh 5g sample slurry (accurate to 0.01g), put into a 100mL volumetric flask, add 80mL water and 1mL potassium hydroxide solution (1mol/L), extract with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 r/min centrifuge and take the filtrate for use.
5.2.3 Salted fish, salted meat and other salted products: weigh 2g sample slurry (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, extract 30 min with ultrasound, shake every other 5 min and keep the solid phase spreading around utterly. Put the sample into the 75℃ water-bath for 5 min, then take it out to get the room temperature and dilute with water to the scale division. Filter the solution with the filter paper, take some solution to centrifuge for 15 min with 10000 rounds/minute centrifuge and take the filtrate for use.
5.2.4 Milk: weigh 10g sample (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, mix well; extract for 30 min with ultrasound, add 2mL 3% acetic acid solution, keep at 4 ℃ for 20 min, take it out to get room temperature and dilute with water to the scale division. Filter the solution with filter paper and take the filtrate for use.
5.2.5 Milk power and cheese: weigh 2.5g sample (accurate to 0.01 g), put into a 100mL volumetric flask, add 80mL water, mix well; extract for 30 min with ultrasound, take it out to get room temperature; add 2mL 3% acetic acid solution, and dilute with water to the scale, mix well. Keep at 4 ℃ for 20 min, take it out to get room temperature. Filter the solution with filter paper and take the filtrate for use.
5.2.6 Take 15mL above standby the filtrate via 0.22μm water filter membrane pinhead filter apparatus and C18 column discard the upper 3mL (if chloride ion is more than 100mg/L, then discard the upper 7mL via pinhead filter apparatus, C18 column, Ag column and Na column) and collect the remaining eluent for use.
Solid phase extraction column shall be activated before use, for instance using C18 column (1.0mL), Ag column (1.0mL) and Na column (1.0mL). The reactivation process is: before use C18 column (1.0mL), wash it with 10mL methanol and 15mL water and keep it still and activated for 30 min. Wash Ag column (1.0mL) and Na column with 10mL water keep it still and activated for 30 min.
5.3 Reference chromatographic condition
5.3.1 Chromatographic column: hydroxide selectivity, high-capacity anion exchange column compatible gradient elution of Styrene-divinyl benzene copolymer matrix, alkoxide quaternary ammonium functional group, 4 mm× 250 mm (with guard column 4mm× 50mm), or equivalent ion chromatography columns.
5.3.2 Eluate
5.3.2.1 Common sample: potash solution, concentration is 6 mmol/L to 70 mmol/L, elution gradient is 6 mmol/L30 min, 70 mmol/L5 min, 6 mmol/L5 min ; flow speed 1.0mL/min.
5.3.2.2 Powder baby formula foods: potash solution, concentration mmol/L to 50 mmol/L; elution gradient is 5 mmol/L 33min; 50 mmol/L 5min; 5 mmol/L 5min and the flow speed is 1.3mL/min.
5.3.3 Suppressor: continuous auto-regenerated film negative ion suppressor or equivalent suppression device
5.3.4 Detector: electrical conductivity detector, the detection pool temperature is 35℃.
5.3.5 Sample injection volume: 50 μL (adjust according to ionic content in the measured sample).
5.4 Determination
5.4.1 Plotting of standard curve
Inject the standard working solution series into ion chromatograph respectively, to get the chromatogram of each concentration of standard working solution, and measure the corresponding peak height (μS) or peak area. Take the concentration of standard working solution as the lateral coordinate, peak height (μS) or peak area as the longitudinal coordinate, plot standard curve (see Figure A.1 for the standard chromatogram of nitrite and nitrate).
5.4.2 Determination of sample solution
Inject blank solution and sample solution into ion chromatograph, and measure the peak height (μS) or peak area of the blank solution and sample solution. Nitrite ion or nitrate ion concentration in the solution to be tested is obtained according to the standard curve.
