GB 5009.294-2023National food safety standard - Determination of tryptophan in foods (English PDF)

3.1 Reagents

3.1.1 Methanol (CH3OH): chromatographically pure.

3.1.2 Acetic acid (C2H4O2): chromatographically pure.

3.1.3 Ammonium acetate (C2H7NO2): chromatographically pure.

3.1.4 Hydrochloric acid (HCl): 12 mol/L.

3.1.5 2-Amino-2-hydroxymethyl-1,3-propanediol (C4H11NO3, Tris base).

3.1.6 Streptomyces griseus protease (type XIV; derived from Streptomyces griseus):

The activity is >=3.5 U/mg.

3.2 Reagent preparation

3.2.1 Hydrochloric acid solution (1.0 mol/L): Measure 83 mL of hydrochloric acid, pour

it into 300 mL of water, mix well, and then add water to dilute to 1 L.

3.2.2 Tris buffer (0.1 mol/ L, pH=8.5): Weigh 6.06 g of Tris base into a 1000 mL beaker, add 500 mL of water to dissolve, and adjust the pH of the solution to 8.5+/-0.05 with 1.0 mol/L hydrochloric acid solution. Store it at room temperature, and the shelf life is 6 months.

3.2.3 Protease solution (17.5 U/mL): Weigh 0.05 g of Streptomyces griseus protease, dissolve it in Tris buffer, and make the volume up to 10 mL, so that the protease mass concentration is 17.5 U/mL. Prepare it fresh just before use.

3.2.4 Ammonium acetate solution (10 mmol/ L, pH=4.0): Weigh 0.77 g of ammonium

acetate, add 900 mL of water to dissolve, adjust the pH of the solution to 4.0+/-0.1 with acetic acid, add water to 1000 mL, and filter through a 0.22 um aqueous phase microporous filter membrane for later use.

3.3 Standards

L-Tryptophan standard (C11H12N2O2, CAS number: 73-22-3): purity of >=99%, or a reference material with national authentication and a Reference Material Certificate.

3.4 Preparation of standard solutions

3.4.1 L-tryptophan standard stock solution (1.00 mg/mL): Accurately weigh 25 mg

(accurate to 0.0001 g) of L-tryptophan standard in a beaker, add 10mL of water to dissolve and transfer to a 25 mL volumetric flask, dilute to volume with water, and mix well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf life is 6 months.

3.4.2 L-Tryptophan standard intermediate solution (100 ug/mL): Take 1.00 mL of L-

tryptophan standard stock solution into a 10 mL volumetric flask, add water to make the volume up to the mark, and mix well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf life is 6 months.

3.4.3 L-tryptophan standard series working solution: Pipette 20.0 uL, 100 uL, 500 uL,

1.00 mL, 2.00 mL, and 5.00 mL of L-tryptophan standard intermediate solution into 10 mL volumetric flasks respectively, add 2.40 mL of methanol to each, add water to make the volume up to the mark, and mix well. The mass concentrations of L-tryptophan standard series working solutions are 0.200 ug/mL, 1.00 ug/mL, 5.00 ug/mL, 10.0 ug/mL, 20.0 ug/mL, and 50.0 ug/mL, respectively. Prepare the solutions fresh just before use.

3.5 Materials

3.5.1 Threaded centrifuge tubes with caps.

3.5.2 Microporous filter membrane: 0.22 um, organic phase.

3.5.3 Microporous filter membrane: 0.22 um, aqueous phase.

4.1 High-performance liquid chromatograph: equipped with a fluorescence detector or

ultraviolet detector.

4.2 Analytical balance: The sensitivities are 0.01 g, 0.001 g, and 0.0001g, respectively.

4.3 Constant temperature incubator: 50 degrees C+/-0.5 degrees C.

4.4 Ultrasonic generator.

4.5 pH meter: The accuracy is +/-0.01.

4.6 Vortex mixer.

5.1 Sample processing

5.1.1 Sample preparation

Formula foods for infants and young children and formula foods for special medical purposes shall be mixed evenly. Cereal supplementary foods for infants and young children shall be crushed in advance and mixed.

5.1.2 Sample extraction

Accurately weigh 0.2 g of the sample (accurate to 0.001 g) into a 50 mL threaded centrifuge tube with a cap, add 0.5 mL of protease solution, 3.0 mL of Tris buffer, and 200 uL of methanol in sequence, vortex to mix, then dissolve with ultrasound, and keep it in an incubator at a constant temperature of 50 °C to enzymatic hydrolysis for 16 hours.

Take out the centrifuge tube, cool to room temperature, add 12 mL of methanol, transfer and dilute the sample to the mark with water in a 50 mL volumetric flask, and mix well;

filter through a 0.22 um organic phase microporous filter membrane, and use the filtrate for liquid chromatography measurement.

5.2 Liquid chromatography reference conditions

rho -- the tryptophan content in the sample solution obtained from the standard curve, in micrograms per milliliter (ug/mL);

rho0 -- the tryptophan content in the blank solution obtained from the standard curve, in micrograms per milliliter (ug/mL);

V -- the metered volume of the sample, in milliliters (mL);

100 -- conversion factor;

m -- the mass of the sample, in grams (g);

1000 -- conversion factor.

The calculation result is rounded to 3 significant figures.

7 Precision The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

8 Others When the sample amount is 0.2 g, the detection limit of the method is 1.5 mg/100 g, and the quantitation limit is 5.0 mg/100 g.

Method B - Alkaline hydrolysis-liquid chromatography

9 Principle The sample is hydrolyzed under alkaline conditions, and the hydrolysate is adjusted to neutrality with hydrochloric acid, separated with a reversed-phase liquid chromatography column, detected with a fluorescence detector, and quantified with the internal standard method.

10 Reagents and materials Unless otherwise specified, all reagents are analytical grade and the water is first-grade water specified in GB/T 6682.

10.1 Reagents

10.1.1 Methanol (CH3OH): chromatographically pure.

10.1.2 Acetic acid (C2H4O2): chromatographically pure.

10.1.3 Ammonium acetate (C2H7NO2): chromatographically pure.

10.1.4 Lithium hydroxide (LiOH - H2O).

10.1.5 Hydrochloric acid (HCl): 12 mol/L.

10.2 Reagent preparation

10.2.1 Hydrochloric acid solution (3 mol/L): Measure 100 mL of hydrochloric acid, pour into 300 mL of water, and mix well.

10.2.2 Lithium hydroxide solution (4 mol/L): Weigh 167.8 g of lithium hydroxide, add an appropriate amount of water to dissolve, and make the volume up to 1000 mL with water.

10.2.3 Ammonium acetate solution (10 mmol/L, pH=4.0): Weigh 0.77 g of ammonium acetate, add 900 mL of water to dissolve, and adjust the pH of the solution to 4.0+/-0.1 with acetic acid; dilute to 1000 mL with water, shake well, and filter with a 0.22 um aqueous phase microporous filter membrane for later use.

10.3 Standards

10.3.1 L-Tryptophan standard (C11H12N2O2, CAS number: 73-22-3): a purity of >=99%, or a reference material with national authentication and a Reference Material Certificate.

10.3.2 alpha-Methyl-D-tryptophan standard (C12H14N2O2, CAS number: 110117-83-4): a purity of >=98%.

10.4 Preparation of standard solutions

10.4.1 L-tryptophan standard stock solution (1.00 mg/mL): Accurately weigh 25 mg (accurate to 0.0001 g) of L-tryptophan standard into a beaker, add 10 mL of water to dissolve and transfer to a 25 mL volumetric flask, dilute to volume with water, and mix well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf life is 6 months.

10.4.2 L-tryptophan standard intermediate solution (100 ug/mL): Take 1.00 mL of L-tryptophan standard stock solution into a 10 mL volumetric flask, dilute to volume with water, and mix well. Transfer it to a brown glass container and store it in a 4 °C refrigerator. The shelf life is 6 months.

10.4.3 Internal standard working solution (1 mg/mL): Weigh 10 mg (accurate to 0.001 g) of alpha-methyl-D-tryptophan standard into a beaker, add 5 mL of water to dissolve, and transfer to a 10 mL volumetric flask; dilute to volume with water and mix well. Transfer it to a brown glass container and store it in a refrigerator at 4 °C. The shelf life is 6 months.

10.4.4 L-tryptophan standard series working solution: Pipette 20 uL, 100 uL, 500 uL,

1.00 mL, 2.00 mL, and 5.00 mL of L-tryptophan standard intermediate solution into 10 mL volumetric flasks, and add 100 uL internal standard working solution to each; add water to make it up to the mark and mix well. The mass concentrations of L-tryptophan standard series working solutions are 0.20 ug/mL, 1.00 ug/mL, 5.00 ug/mL, 10.0 ug/mL, 20.0 ug/mL, and 50.0 ug/mL, respectively. Prepare the solutions fresh just before use.

10.5 Materials

10.5.1 Hydrolysis tube: It is made of polytetrafluoroethylene, equipped with a threaded outer cover, and lined with polytetrafluoroethylene sealing gasket.

10.5.2 Microporous filter membrane: 0.22 um, aqueous phase.

11 Instruments and equipment

11.1 High-performance liquid chromatograph: equipped with a fluorescence detector.

11.2 Analytical balance: The sensitivities are 0.01 g, 0.001 g, and 0.0001 g, respectively.

11.3 Electric drying oven: 110 degrees C+/-0.5 degrees C.

11.4 Ultrasonic generator.

11.5 Vortex mixer.

11.6 pH meter: The accuracy is +/-0.01.

12 Analysis steps

12.1.1 Sample preparation Formula foods for infants and young children and formula foods for special medical purposes shall be mixed evenly. Cereal supplementary foods for infants and young children shall be crushed in advance and mixed.

12.1.2 Sample extraction Accurately weigh 0.2 g of the sample (accurate to 0.001 g) into the hydrolysis tube, add

0.5 mL of internal standard working solution and 5 mL of lithium hydroxide solution