GB/T 15399-1994 Determination of sulfur-containing amino acids in feeds--Ion exchange chromatography
Some standard content:
National Standard of the People's Republic of China
Determination of sulfur amino acids in feedstuffs-Ion exchange chromatography
Determination of sulfur amino acids in feedstuffs-Ion exchange chromatography1 Subject content and scope of application
This standard specifies the determination method of sulfur amino acids in feedstuffs. This standard is applicable to single feed, compound (mixed) feed and concentrated feed. 2 Reference standards
GB/T6432 Determination method of crude protein in feed GB/T6439 Determination method of water-soluble chloride in feed 3 Principle
GB/T 15399-94
Sulfur-containing amino acids (cystine, cysteine and methionine) in feed are oxidized with performic acid and hydrolyzed with hydrochloric acid to generate sulfalanine and methionine sulfone. These two products can be separated and determined by ion exchange chromatography. 4 Reagents
Except where otherwise specified, all reagents are analytically pure, and water is deionized water with a conductivity of less than 1MS. 4.1 Performic acid solution
4.1.1 Conventional performic acid solution: Mix 30% hydrogen peroxide (GB6684) and 88% formic acid (HG3--1296) at a ratio of 1:9 (V/V), place at room temperature for 1 hour, cool in an ice water bath for 30 minutes, and prepare before use. 4.1.2 Performic acid solution for concentrates: Add silver nitrate (GB670) at 3 mg/mL to conventional performic acid solution. This solution is suitable for concentrates with a sodium chloride content of less than 3%. When the sodium chloride content in the concentrate is greater than 3%, the concentration of silver nitrate in the oxidant can be calculated using the following formula: Ck ≥ 1. 454 X m × Cn
Where: Ck—the concentration of silver nitrate in performic acid, mg/mL; Cn—the sodium chloride content in the sample, mg/mL; m—the mass of the sample, mg.
4.2 Oxidation terminator
4.2.1 48% hydrobromic acid (GB621)
4.2.2 Sodium metabisulfite solution: add 33.6g sodium metabisulfite (HG3--909) to 100mL with water. 4.3 Acid hydrolysis agent
Approved by the State Administration of Technical Supervision on December 30, 19941)
Implemented on July 1, 1995
GB/T 15399--94
4.3.1 6.0mol/L hydrochloric acid solution: mix high-grade pure hydrochloric acid (GB622) with water at a ratio of 1:1 (V/V). 4.3.2 6.8mol/L hydrochloric acid solution: dilute 1133mL of high-grade pure hydrochloric acid (GB622) with water to 2000ml. 4.4 7.5 mol/L sodium hydroxide solution: Take 30g of high-grade pure sodium hydroxide (GB629), dissolve it in water and make up to 100ml. 4.5 Dilute sodium citrate buffer for use on the machine, pH 2.2, 0.2 mol/L Na: Weigh 19.6g of trisodium citrate (HG3-1298), dissolve it in water, add 16.5mL of high-grade pure hydrochloric acid, 5.0mL of thiodiglycol, 1g of phenol (HG3-1165), and finally add water to make up to 1000ml, and filter it with a G4 vertical fused glass sand core funnel. 4.6 Sodium citrate buffer for elution with different pH and ionic strength: Prepare according to the instrument manual. 4.7 Triketone solution: Take an appropriate amount of triketone and prepare according to the instrument manual. 4.8 Standard stock solution of cysteic acid-methionine sulfone, 2.50 μmol/mL: Accurately weigh 105.7 mg of cysteic acid and 113.3 mg of methionine sulfone, dissolve in water and dilute to 250 mL. 4.9 Amino acid mixed standard stock solution: Contains 17 kinds of L-amino acids for conventional protein hydrolysis analysis such as L-aspartic acid and L-threonine, and the concentration of each component is 2.50 μmol/mL.
4.10 Mixed amino acid standard working solution: Pipette 1.00 mL of cysteic acid-methionine sulfone standard stock solution (4.7) and amino acid mixed standard stock solution (4.8), place in a 50 mL volumetric flask, add sodium citrate buffer (4.4) for dilution to dilute to volume, and mix well. The concentration of each component is 50 nmol/mL.
5 Instruments and equipment
5.1 Laboratory sample crusher;
5.2 Sample sieve: pore size 0.45mm (40 mesh); 5.3 Analytical balance: sensitivity 0.0001g;
5.4 Blowtorch;
5.5 Rotary evaporator or concentrator: can adjust the temperature between room temperature and 65℃, the temperature control accuracy is ±1℃, and the vacuum degree can be as low as 3.3×10°Pa (25 mmHg column).
5.6 Constant temperature box or hydrolysis furnace;
5.7 Amino acid automatic analyzer requires the resolution of methionine sulfone to be greater than 90%. 6 Samplesbzxz.net
Take a representative feed sample, reduce it to about 25g by quartering, crush it and pass it through a 0.45mm pore size (40 mesh) sieve, mix it thoroughly and put it into a ground-mouth bottle for use.
7 Analysis steps
7.1 Oxidation and hydrolysis: Weigh two samples containing 7.5~~25mg of protein (accurate to 0.0001g, sample size not exceeding 75mg), place in a 20mL concentrator bottle or concentrator tube of a rotary evaporator, cool in an ice-water bath for 30min, then add 2mL of cooled performic acid solution (4.2). When adding liquid, the sample should be completely wetted, but do not shake. Cover the bottle stopper and place it in a 0℃ refrigerator together with the ice bath for 16h. The following steps vary depending on the oxidation terminator used: If hydrobromic acid is used as the terminator, add 0.3mL of hydrobromic acid (4.1) to each tube, shake, return to the ice bath, let stand for 30min, then move to a rotary evaporator or concentrator (5.5), and concentrate to dryness at 60℃ and below 3.3×10°Pa (25mmHg column). Use about 15mL of hydrochloric acid solution (4.3.1) to quantitatively transfer the residue into 20mL of anhydrous ammonium sulfate, seal it, and place it in a thermostat at 110±1℃ for hydrolysis for 22~24h. Alternatively, use about 25ml of 6.0mol/L hydrochloric acid solution to transfer the residue into a 50mL digestion tube, and reflux and hydrolyze it in a hydrolysis furnace at 110±3℃ for 22~24h.
Take out the anhydrous ammonium sulfate or hydrolysis tube, cool it, and quantitatively transfer the contents into a 50mL volumetric flask with water to make up the volume. Mix thoroughly, filter, take 1~2mL of the filtrate, place it in a rotary evaporator or concentrator, and evaporate it to dryness under reduced pressure at a temperature below 50℃. Add a little water and repeat the evaporation 2~3 times. Accurately add a certain volume (2~5mL) of sodium citrate buffer (4.5) for dilution and shake, centrifuge after sufficient dissolution, and take the supernatant 211
for instrument measurement.
GB/T15399—94
If sodium metabisulfite is used as the terminator, add 0.5 ml of sodium metabisulfite solution (4.2.2) to the sample oxidation solution, shake well, then directly add 17.5 ml of hydrochloric acid solution (4.3.2), and place at 110±3℃ for hydrolysis for 22~24 hours. Take out the hydrolysis tube, cool it, transfer the contents to a 50 ml volumetric flask with water, neutralize it with sodium hydroxide solution (4.4) to pH about 2.2, and dilute it with dilution buffer (4.5), centrifuge it, and take the supernatant for instrument measurement. If the amino acid analysis is affected by the Na+ concentration in the sample solution, and the chromatographic peak time drifts too much, the hydrolyzate needs to be filtered and then 2-5 mL of the filtrate is taken and evaporated to about 0.5 mL under reduced pressure at 50°C (do not evaporate to dryness). It is transferred to a 10 mL volumetric flask with diluted buffer for the machine (4.5), and adjusted to pH 2.2 with sodium hydroxide solution (4.4), and the volume is fixed with diluted buffer for the machine (4.5). Mix well, centrifuge, and take the supernatant for instrument determination. The concentrated material is first determined according to GB6439 for its NaCl content. The sample processing steps are the same as above, except that the oxidant is 4.1.2. 7.2 Determination: Use the mixed amino acid standard working solution (4.9) and adjust the instrument operating parameters and (or) the pH of the elution citric acid buffer (4.5) according to the instrument manual to make the resolution of methionine sulfone reach the best state (greater than or equal to 90%), inject the prepared sample and amino acid standard working solution (4.9) for analysis and determination. Each group consists of 5 samples (i.e. 10 single samples), and the mixed amino acid standard working solution (4.9) is inserted between the groups for calibration.
8 Expression of analysis results
The analysis results are expressed as the mass percentage of cystine and methionine in the sample, and the calculation formula is as follows: Cystine (methionine) (%)
X10-6×D×100
Where: A per ml.The content of cystine in the sample solution, ng; m—sample mass, mg;
D-sample dilution multiple.
Report the result as the arithmetic mean of the results of two parallel samples, retaining two decimal places. 9 Repeatability
The relative deviation of the values of the two parallel samples shall not exceed 5% when the content of cystine is less than 0.50%; and shall not exceed 4% when it is greater than 0.50%.
Additional remarks:
This standard is proposed by the Ministry of Agriculture of the People's Republic of China. This standard was jointly drafted by the Analysis and Testing Center of the Chinese Academy of Agricultural Sciences [also the National Feed Quality Supervision and Inspection Center (Beijing), the Animal Husbandry Research Institute of the Chinese Academy of Agricultural Sciences and the Soybean Research Institute of the Jilin Academy of Agricultural Sciences. The main drafters of this standard are Chang Biying, Li Jianfan, Zhang Ming, Yan Huiwen and Zuo Jiangwan. 215
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