This standard supersedes Determination of Taurine in Foods (GB/T 5009.169-2003) and National Food Safety Standard - Determination of Taurine in Foods for Infants and Young Children, Milk and Milk Products (GB 5413.26-2010).
Compared with GB/T 5009.169-2003, the main changes in this standard are as follows:
——the name of this standard is revised as "National Food Safety Standard - Determination of Taurine in Foods";
——the OPA post-column derivatization high performance liquid chromatography is added as Method I; the dansyl chloride pre-column derivatization high performance liquid chromatography is added as Method II;
——the thin layer chromatography is canceled.
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
中华人民共和国国家标准
GB 5009.169-2016
National Standard of Food Safety
Determination of Taurine in Foods
食品安全国家标准
食品中牛磺酸的测定
1 Scope
This standard specifies the method for determining taurine in foods.
This standard is applicable to determination of taurine in formula foods for infant and young children, milk powder, soya flour, soybean milk, milk beverage, beverages for special uses, flavored drinks, solid beverage and jelly.
Method I O-Phthalaldehyde (OPA) Post-column Derivatization High Performance Liquid Chromatography
2 Principle
Dissolve the specimen with water, precipitate protein with metaphosphoric acid, extract through ultrasonic vibration, centrifuge and filter with microporous membrane, separate with sodium ion chromatographic column, carry out derivation reaction with o-Phthalaldehyde (OPA), detect with fluorescence detector and quantify by using external standard method.
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 Metaphosphoric acid.
3.1.2 Trisodium citrate.
3.1.3 Phenol.
3.1.4 Nitric acid.
3.1.5 Methanol: chromatographically pure.
3.1.6 Boric acid.
3.1.7 Potassium hydroxide.
3.1.8 O-Phthalaldehyde (OPA).
3.1.9 2-mercaptoethanol.
3.1.10 Polyoxyethylene ether lauric acid (Brij-35).
3.1.11 Potassium ferricyanide.
3.1.12 Zinc acetate.
3.1.13 Amylase: activity ≥1.5U/mg.
3.2 Reagent preparation
3.2.1 Metaphosphoric acid solution (30g/L)
Weigh 30.0g of metaphosphoric acid (3.1.1), dissolve it and scale the volume to 1,000mL with water.
3.2.2 Trisodium citrate solution
Weigh 19.6g of trisodium citrate (3.1.2), add 950 mL of water to dissolve it, add 1mL of phenol (3.1.3), adjust the pH value to 3.10 ~3.25 with nitric acid (3.1.4), and filter it with 0.45 μm microfiltration membrane.
3.2.3 Post-column fluorescence derivatization solution (o-Phthalaldehyde solution)
3.2.3.1 Potassium borate solution (0.5mol/L): weigh 30.9g of boric acid (3.1.6) and 26.3g of potassium hydroxide (3.1.7), dissolve them and scale the volume to 1,000mL with water.
3.2.3.2 O-Phthalaldehyde derivatization solution: weigh 0.60g of o-Phthalaldehyde (3.1.8), dissolve it with 10mL of methanol (3.1.5), add 0.5 mL of 2-mercaptoethanol (3.1.9) and 0.35g of Brij-35 (3.1.10), scale the volume to 1,000mL with 0.5 mol/L potassium borate solution (3.2.3.1), and filter the solution with 0.45 μm microfiltration membrane; prepare the solution immediately before use.
3.2.4 Precipitator
3.2.4.1 Precipitator I: weigh 15.0g of potassium ferrocyanide (3.1.11), dissolve it and scale the volume to 100mL with water. Such precipitator maintains stable for 3 months at ambient temperature.
3.2.4.2 Precipitator II: weigh 30.0g of zinc acetate (3.1.12), dissolve it and scale the volume to 100mL with water. Such precipitator maintains stable for 3 months at ambient temperature.
3.3 Standard products
Purity ≥99%, CAS: 107-35-7.
3.4 Preparation of standard solution
3.4.1 Taurine standard stock solution (1 mg/mL)
Accurately weigh 0.1000g of standard product of taurine (3.3), dissolve it and scale the volume to 100mL with water.
3.4.2 Taurine standard working solution
Dilute the taurine standard stock solution (3.4.1) with water to prepare a series of standard solutions with the concentrations of 0 μg/mL, 5.0μg/mL, 10.0μg/mL, 15.0μg/mL, 20.0μg/mL and 25.0μg/mL; prepare the solutions immediately before use.
4 Instruments and Apparatus
4.1 High-performance liquid chromatograph: with fluorescence detector.
4.2 Post-column reactor.
4.3 Infusion pump of fluorescence derivatization solvent.
4.4 Ultrasonic vibrator.
4.5 pH meter: with a precision of 0.01.
4.6 Centrifuger: not less than 5,000 r/min.
4.7 Microfiltration membrane: 0.45μm.
4.8 Balance: with a sensibility of 0.0001g.
5 Analysis Steps
5.1 Specimen preparation
Accurately weigh 1g~5g of solid specimen (to the nearest of 0.01g) and put it in conical flask, add 20mL of warm water with a temperature of about 40℃, shake it well so that the specimen is dissolved, and carry out ultrasonic extraction by putting the solution in ultrasonic vibrator for 10min. Add 50mL of metaphosphoric acid solution (3.2.1) and shake it well. Carry out 10min~15min of ultrasonic extraction by putting it into ultrasonic vibrator, take it out and cool to ambient temperature, transfer it into a 100mL volumetric flask, dilute it with water to the scale and shake it well; centrifuge the specimen solution at 5,000 r/min for 10 min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
As for cereal products, weigh 5g of specimen (to the nearest of 0.01g) and put it in conical flask, add 40 mL of warm water with a temperature of about 40℃, add 0.5g of amylase (enzyme activity ≥1.5U/mg) and mix them uniformly; fill the conical flask with nitrogen, cover the stopper, place the conical flask in a 50℃~60℃ incubator for 30min, take it out and cool to ambient temperature, add 50mL of metaphosphoric acid solution (3.2.1) and shake it well. Carry out 10min~15min of ultrasonic extraction by putting it into ultrasonic vibrator, take it out and cool to ambient temperature, transfer it into a 100mL volumetric flask, dilute it with water to the scale and shake it well; centrifuge the specimen solution at 5,000 r/min for 10 min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
Accurately weigh 5g~30g of liquid specimen (except milk beverage) (to the nearest of 0.01 g) and put it in a conical flask, add 50mL of metaphosphoric acid solution (3.2.1) and shake it well. Carry out 10min~15min of ultrasonic extraction by putting it into ultrasonic vibrator, take it out and cool to ambient temperature, transfer it into a 100mL volumetric flask, dilute it with water to the scale and shake it well; centrifuge the specimen solution for 10 min at 5,000 r/min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
As for beverages with high taurine content, dilute them until the concentration is proper; during the last dilution, add 50mL of metaphosphoric acid solution (3.2.1) and shake it well. Carry out 10min~15min of ultrasonic extraction by putting it into ultrasonic vibrator, take it out and cool to ambient temperature, transfer it into a 100mL volumetric flask, dilute it with water to the scale and shake it well; centrifuge the specimen solution for 10 min at 5,000 r/min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
As for jelly, weigh 5g of specimen (to the nearest of 0.01 g) and put it in a conical flask, add 20mL of water, carry out water bath at 50℃~60℃ for 20min to dissolve it, cool it down, add 50mL of metaphosphoric acid solution (3.2.1) and shake it well. Carry out 10min~15min of ultrasonic extraction by putting it into ultrasonic vibrator, take it out and cool to ambient temperature, transfer it into a 100mL volumetric flask, dilute it with water to the scale and shake it well; centrifuge the specimen solution for 10 min at 5,000 r/min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
As for milk beverage, weigh 5g~30g of specimen (to the nearest of 0.01g) and put it in conical flask, add 30mL of warm water with a temperature of about 40℃, mix them uniformly, put the conical flask on ultrasonic vibrator so as to carry out ultrasonic extraction for 10min, and cool it down to ambient temperature. Add 1.0 mL of precipitator I (3.2.4.1), eddy to mix them, add 1.0mL of precipitator II (3.2.4.2) and eddy to mix them, transfer the solution in a 100mL volumetric flask, dilute with water to the scale and mix it uniformly; centrifuge the specimen solution for 10 min at 5,000 r/min, take the supernatant and filter it with 0.45 μm microporous membrane (4.7), and collect the intermediate filtrate for specimen injection.
5.2 Reference condition of instruments
5.2.1 Chromatographic column: special column (25cm×4.6mm) for analysis of sodium ion and amino acid or equivalent.
5.2.2 Mobile phase: trisodium citrate solution (3.2.2).
5.2.3 Mobile phase flow velocity:0.4 mL/min.
5.2.4 Fluorescence derivatization solvent flow velocity: 0.3 mL/min.
5.2.5 Column temperature: 55℃.
5.2.6 Detection wavelength: excitation wavelength is 338nm, and emission wavelength is 425nm.
5.2.7 Injection volume: 20μL.
5.3 Standard curve drawing
Inject the standard series working solution into high-performance liquid chromatograph respectively, determine the corresponding chromatogram peak height or peak area, draw the standard curve by taking the concentration of standard working solution as horizontal coordinate and the response value (peak area or peak height) as longitudinal coordinate.
5.4 Determination of specimen solution
Inject the specimen solution into high-performance liquid chromatograph so as to obtain the chromatogram peak height or peak area, and obtain the concentration of taurine in to-be-determined solution according to standard curve.
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Apparatus
5 Analysis Steps
6 Expression of Analysis Results
7 Precision
8 Others
9 Principle
10 Reagents and Materials
11 Instruments and Apparatus
12 Analysis Steps
13 Expression of Analysis Results
14 Precision
15 Others
Appendix A Chromatogram
