National Food Safety Standard
Determination of Amino Acid in Foods
食品安全国家标准
食品中氨基酸的测定
1 Scope
This standard specifies a method of determining amino acid in foods with amino acid analyzer (ninhydrin post-column derivatization ion exchange chromatograph).
This standard is applicable to the determination of 16 kinds of amino acids like aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine.
2 Principle
Via hydrochloric acid hydrolyzing, protein in foods becomes free amino acid and then has color reaction with ninhydrin solution after it is separated by ion exchange column, then the content of amino acid is determined with visible light spectrophotometer.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade I water (defined in GB/T 6682) are adopted for the purposes of this method.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl): concentration≥36%, and guaranteed reagent.
3.1.2 Phenol (C6H5OH).
3.1.3 Nitrogen: with the purity of 99.9%.
3.1.4 Sodium citrate (Na3C6H5O7·2H2O): guaranteed reagent.
3.1.5 Sodium hydroxide (NaOH): guaranteed reagent.
3.2 Reagents preparation
3.2.1 Hydrochloric acid solution (6mol/L): take 500mL of hydrochloric acid, dilute it to 1 000mL with water, and mix well.
3.2.2 Refrigerant: mix the commercially available common salt with ice at the mass ratio of 1:3.
3.2.3 Sodium hydroxide solution (500g/L): weigh 50g of sodium hydroxide, dissolve it into 50mL water, cool to room temperature, then dilute to 100mL with water and mix well.
3.2.4 Sodium citrate buffer solution [c(Na+)=0.2mol/L]: weigh 19.6g of sodium citrate, add into 500mL of water to dissolve it and then add 16.5mL of hydrochloric acid; dilute it to 1 000mL with water, mix well and adjust the pH value to 2.2 with 6mol/L hydrochloric acid solution or 500g/L sodium hydroxide solution.
3.2.5 Buffer solution for elution with different pH and ion strength: prepare or purchase by reference to the apparatus instruction.
3.2.6 Ninhydrin solution: prepare or purchase by reference to the apparatus instruction.
3.3 Standards
3.3.1 Standard solution of mixed amino acid: the standard solution that is approved and awarded with reference material certificate by the nation.
3.3.2 16 kinds of single amino acid standards: solid and purity≥98%.
3.4 Preparation of standard solution
3.4.1 Standard stock solution of mixed amino acid (1μmol/mL): respectively and accurately weigh single amino acid standards (accurate to 0.000 01g) into one 50mL beaker, dissolve with 8.3mL of 6mol/L hydrochloric acid solution, accurately transfer it to a 250mL volumetric flask, dilute and bring the volume to the scale with water and then mix well (see Table 1 for the reference value of the weighing mass of each amino acid standard).
3.4.2 Standard working solution of mixed amino acid (100nmol/mL): accurately pipet 1.0mL of the standard stock solution of mixed amino acid to 10mL volumetric flask, bring the volume to the scale with the sodium citrate buffer solution in pH of 2.2, mix well to obtain the standard upper liquid.
4 Apparatuses
4.1 Tissue pulverizer or grinder used in laboratory.
4.2 Refiner.
4.3 Analytical balance: with sensitivity of 0.000 1g and 0.000 01g respectively.
4.4 Hydrolysis tube: glass tube with pressure-resistant screw cap or ampoule bottle, with the volume of 20mL~30mL.
4.5 Vacuum pump: exhaust volume≥40L/min.
4.6 Alcohol blast burner.
4.7 Electric heating air-blowing thermostat or hydrolysis furnace.
4.8 Test tube concentrator or parallel evaporimeter (equipped with auxiliary test tube of 15mL~25mL).
4.9 Amino acid analyzer: ninhydrin post-column derivatization ion exchange chromatograph.
5 Analysis Procedures
5.1 Specimen preparation
Solid or semi-solid specimen is pulverized with tissue pulverizer or grinder; liquid specimen is made into homogenate with refiner and preserved in sealing and freezing state and used after defrosting during analysis.
5.2 Specimen weighing
For the sample with good uniformity, like milk powder, accurately weigh a certain amount of specimen (accurate to 0.000 1g) to make the protein content in the specimen within the range of 10mg~20mg. For the sample with unknown protein content, determine the protein content in sample first and place the weighed sample into the hydrolysis tube.
For the specimen with low uniformity, like fresh meat, appropriately increase the sampling weight to reduce error and dilute the specimen before determination.
For the sample with low protein content, like vegetable, fruit, beverage and starchy food, the sampling weight of solid or semi-solid specimen is not greater than 2g and that of liquid specimen is not greater than 5g.
5.3 Specimen hydrolysis
Add 10mL~15mL of 6mol/L hydrochloric acid solution into the hydrolysis tube according to the protein content of specimen. For the specimen with high water content and low protein content, like beverage, fruit and vegetable, first add into the hydrochloric acid with about same volume and mix well, then supplement with 6mol/L hydrochloric acid solution to about 10mL. Continuously add 3~4 drops of phenol into the hydrolysis tube.
Put the hydrolysis tube into coolant to freeze for 3min~5min, connect it to the extraction pipe of vacuum pump for vacuum-pumping (get close to 0Pa), then seal or tighten the screw cover in the nitrogen-filling state after repeating the vacuum-pumping - filling nitrogen for 3 times.
Put the sealed hydrolysis tube into the electric heating air-blowing thermostat of 110℃±1℃ or hydrolysis furnace to hydrolyze for 22h, then take it out and cool to room temperature.
Open the hydrolyze tube, filter the hydrolysate to a 50mL volumetric flask and flush the hydrolyze tube for many times with a small amount of water; transfer the washing liquid into that volumetric flask, dilute with water to the scale and then shake to mix well.
Accurately pipet 1.0mL of filtrate into 15mL or 25mL test tube and dry under reduced pressure with test tube concentrator or parallel evaporimeter at the heating environment of 40℃~50℃; after drying, dissolve the residues with 1mL~2mL water, dry under reduced pressure again and finally evaporate to dryness.
Add 1.0mL~2.0mL of sodium citrate buffer solution in pH of 2.2 into dried test tube to dissolve, after shaking and mixing well, pipet solution to make it pass through 0.22μm filter membrane and transfer it to sample-injecting bottle to serve as sample determination solution for apparatus determination.
5.4 Determination
5.4.1 Apparatus conditions
Inject the adopted standard working solution of mixed amino acid into automatic amino acid analyzer, properly adjust the apparatus operation procedure and parameter as well as the reagent mix ratio of the buffer solution for elution and confirm the apparatus operation condition, by reference to "Verification Regulation of Automatic Amino Acid Analyzer" (JJG 1064 - 2011) and apparatus instruction.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
Appendix A Chromatogram
