GB 5009.309-2025National food safety standard - Determination of asparagine and glutamine in foods (English PDF)

1 Scope

1 This standard lays down the pre-column derivatization high performance liquid chromatographic method and the liquid chromatography tandem mass spectrometric method for the determination of asparagine and glutamine in foods. In the first method the direct extraction procedure serves for the determination of free asparagine and free glutamine in foods for special dietary uses, and the extraction after enzymatic digestion serves for the determination of total asparagine and total glutamine in foods for special dietary uses. The second method serves for the determination of total asparagine and total glutamine in foods for special dietary uses.

2 First method - principle

2 The sample is either extracted directly or digested with Streptomyces griseus protease; asparagine and glutamine then undergo a derivatization reaction with dansyl chloride, the derivatives are separated on a C18 column and detected with an ultraviolet detector, and quantification is against an external standard.

3 First method - reagents and materials

3 Unless otherwise stated the reagents used in this method are of analytical grade and the water is grade one water as specified in GB/T 6682.

3.1 The reagents listed are methanol and acetonitrile of chromatographic grade; dansyl chloride; anhydrous sodium carbonate; anhydrous disodium hydrogen phosphate; methylamine hydrochloride; tris(hydroxymethyl)aminomethane, that is Tris base; Streptomyces griseus protease of type XIV, from Streptomyces griseus, with an enzyme activity of at least 3.5 U/mg; phosphoric acid of chromatographic grade with a content of at least 85 %; and concentrated hydrochloric acid.

3.2.1 The dansyl chloride solution of 2.0 g/L is prepared by weighing 0.2 g of dansyl chloride, dissolving it in acetonitrile and making up to 100 mL; it is prepared just before use.

3.2.2 The hydrochloric acid solution of 1.0 mol/L is prepared by taking 8.3 mL of concentrated hydrochloric acid and diluting it with water to 100 mL.

3.2.3 The Tris buffer of 0.05 mol/L is prepared by weighing 6.06 g of tris(hydroxymethyl)aminomethane into a 1000 mL beaker, dissolving it in 800 mL of water, adjusting the pH to 8.0 +/- 0.1 with 1.0 mol/L hydrochloric acid solution and diluting with water to 1000 mL. It is kept at 4 °C and used within 1 month.

3.2.4 The protease solution of 52.5 U/mL is prepared by weighing 0.15 g of Streptomyces griseus protease and dissolving it in Tris buffer to 10 mL; it is prepared just before use.

3.2.5 The sodium carbonate buffer solution of 60 mmol/L is prepared by weighing 0.318 g of anhydrous sodium carbonate, dissolving it in 40 mL of water, adjusting the pH to 9.5 +/- 0.1 with 1.0 mol/L hydrochloric acid solution and making up with water to 50 mL; it is prepared just before use.

3.2.6 The methylamine hydrochloride solution of 20 g/L is prepared by weighing 2.0 g of methylamine hydrochloride and dissolving it in water to 100 mL; it is kept at 4 °C and used within 3 months.

3.2.7 The disodium hydrogen phosphate solution of 10 mmol/L is prepared by weighing 1.42 g of anhydrous disodium hydrogen phosphate, dissolving it in 800 mL of water, adjusting the pH to 6.5 +/- 0.1 with phosphoric acid and diluting with water to 1000 mL.

3.2.8 The 20 % methanol solution is prepared by taking 20 mL of methanol and diluting it with water to 100 mL.

3.3 The standards are asparagine, CAS number 70-47-3, and glutamine, CAS number 56-85-9, each of purity not less than 99 %, or a certified reference material with a national certificate.

3.4.1 and 3.4.2 The asparagine and the glutamine standard stock solutions of 2.00 mg/mL are each prepared by weighing exactly 100 mg of the standard to 0.0001 g, dissolving it in 20 % methanol solution and making up to 50 mL. They are kept at 4 °C in the dark and used within 3 months.

3.4.3 The mixed standard intermediate solution of 20.0 µg/mL is prepared by taking exactly 1.00 mL of each of the asparagine and glutamine stock solutions into a 100 mL volumetric flask, making up to the mark with water and mixing; it is prepared just before use.

3.4.4 The series of mixed working standard solutions is prepared by taking suitable volumes of the mixed standard intermediate solution of 20.0 µg/mL into 10 mL volumetric flasks and making up to the mark with water, which gives mass concentrations of 0 µg/mL, 0.500 µg/mL, 1.00 µg/mL, 2.00 µg/mL, 5.00 µg/mL, 10.0 µg/mL and 20.0 µg/mL; the series is prepared just before use.

3.5 The materials are screw-capped glass bottles of 20 mL and organic microporous filter membranes of 0.22 µm.

5 First method - analytical procedure

5.1 A solid sample that is not a powder is crushed and mixed until uniform; a powdered solid sample or a liquid sample is shaken until uniform.

5.2.1 In the direct extraction procedure, 5.0 g of a solid sample is weighed to 0.001 g into a dry 50 mL beaker, dissolved in a suitable amount of warm water at 40 °C to 45 °C, cooled to room temperature, then transferred and made up to 25 mL. Exactly 1.00 mL of this is transferred to a 150 mL conical flask, about 25 mL of water is added and the flask is mixed on a vortex mixer, the pH is adjusted to 4.5 +/- 0.1 with 1.0 mol/L hydrochloric acid solution, the liquid is transferred to a 250 mL volumetric flask and made up to the mark with water and shaken, and the solution to be derivatized is obtained by centrifuging or filtering. For a liquid sample, 1.0 g to 5.0 g is weighed to 0.001 g into a 150 mL conical flask and the later steps are the same as for a solid sample.

5.2.2 In the enzymatic extraction procedure, 5.0 g of a solid sample is weighed to 0.001 g into a dry 50 mL beaker, dissolved in a suitable amount of warm water at 40 °C to 45 °C, cooled to room temperature, then transferred and made up to 25 mL. Exactly 1.00 mL of this is transferred to a 20 mL screw-capped glass bottle, and 0.5 mL of protease solution, 3.0 mL of Tris buffer and 0.2 mL of methanol are added in that order; the bottle is mixed on a vortex mixer and digested for 16 h in a thermostatic water bath at 37 °C. It is then taken out and cooled to room temperature, the whole digest is transferred to a 250 mL volumetric flask and made up to the mark with water and shaken, and the solution to be derivatized is obtained by centrifuging or filtering. For a liquid sample, 0.5 g to 1.0 g is weighed to 0.001 g into a 20 mL screw-capped glass bottle and the later steps are the same as for a solid sample.

5.3 For the derivatization reaction, exactly 1.00 mL of the solution to be derivatized is taken into a 15 mL centrifuge tube, 1.00 mL of sodium carbonate buffer solution and 1.00 mL of dansyl chloride solution are added, the tube is mixed thoroughly and left to react for 2 h at room temperature in the dark, being taken out and shaken to homogeneity after about 1 h. Then 0.10 mL of methylamine hydrochloride solution is added and mixed on a vortex mixer to stop the reaction, the tube is left to stand in the dark until precipitation is complete, and the liquid is filtered through a 0.22 µm membrane ready for measurement. Exactly 1.00 mL of each of the series of mixed working standard solutions is taken and derivatized at the same time as the sample solutions.

5.4 The reference chromatographic conditions are as follows: a C18 column of 250 mm by 4.6 mm and 5 µm particle size, or a column of equivalent performance; mobile phase A of 10 mmol/L disodium hydrogen phosphate solution and mobile phase B of methanol, with the gradient elution conditions of Table 1; a flow rate of 1.0 mL/min; a column temperature of 35 °C; a detection wavelength of 247 nm; and an injection volume of 10 µL. Table 1 gives the gradient elution conditions as the proportions of mobile phase A and mobile phase B against time: at 0.0 min and 17.0 min they are 70 % and 30 %, at 17.1 min and 19.0 min they are 20 % and 80 %, and at 19.1 min and 24.0 min they are again 70 % and 30 %.

5.5 The derivatized mixed working standard solutions are injected into the liquid chromatograph in turn, and a calibration curve is drawn for each analyte with the mass concentration of asparagine or of glutamine in the working standard solutions on the horizontal axis and the peak area of the asparagine derivative or of the glutamine derivative on the vertical axis. The chromatogram of the derivatized standard solution is shown in Figure A.1 of Annex A.

5.6 The derivatized test solution is injected into the liquid chromatograph, the corresponding peak area is obtained and the mass concentration of asparagine or glutamine in the sample solution is read from the calibration curve. The response of the target compound in the test solution shall fall within the linear range of the calibration curve; where it exceeds that range, the solution to be derivatized shall be diluted and the derivatization and analysis repeated before injection.

5.7 In the blank test the sample is omitted and all other operations are the same as for the sample.

6 First method - expression of the analytical result

6.1 For the direct extraction procedure, the contents of free asparagine and free glutamine in the sample are calculated by formula (1) from the mass concentration of asparagine or glutamine in the sample solution read from the calibration curve, in micrograms per millilitre, the final volume to which the sample was made up in millilitres, the dilution factor, the mass of the sample in grams and a unit conversion coefficient of 1000. The result is expressed in milligrams per gram and is kept to three significant figures.

6.2 For the enzymatic extraction procedure, the total asparagine and total glutamine in the sample are calculated by formula (2) from the same quantities with the mass concentration of the blank test solution subtracted from that of the sample solution. The result is expressed in milligrams per gram and is kept to three significant figures.

8 First method - precision and other

7 The absolute difference between two independent determinations obtained under repeatability conditions shall not exceed 10 % of their arithmetic mean.

8 With a test portion of 5.0 g of a solid sample, the detection limit of the method is 0.2 mg/g and the quantification limit is 0.6 mg/g for both analytes; with a test portion of 1.0 g of a liquid sample, the detection limit is 0.04 mg/g and the quantification limit is 0.12 mg/g for both analytes.

9 Second method - principle and reagents

9 The sample is digested with Streptomyces griseus protease, diluted and measured by liquid chromatography tandem mass spectrometry, and quantification is by the stable isotope internal standard method.

10 Unless otherwise stated the reagents used in this method are of analytical grade and the water is grade one water as specified in GB/T 6682. The reagents listed are formic acid, methanol and acetonitrile of chromatographic grade; tris(hydroxymethyl)aminomethane; Streptomyces griseus protease of type XIV, from Streptomyces griseus, with an enzyme activity of at least 3.5 U/mg; and concentrated hydrochloric acid.

10.2 The 0.02 % formic acid solution is prepared by taking 0.2 mL of formic acid and dissolving it in 1 L of water. The hydrochloric acid solution of 1.0 mol/L, the Tris buffer of 0.05 mol/L, the protease solution of 52.5 U/mL and the 20 % methanol solution are as prepared in the first method.

10.3 The standards are asparagine and glutamine as in the first method, together with the stable isotope internal standards asparagine hydrate labelled with two nitrogen-15 atoms, CAS number 287484-32-6, and glutamine labelled with two nitrogen-15 atoms, CAS number 204451-48-9, each of purity not less than 98 %, or a certified reference material with a national certificate.

10.4.3 and 10.4.4 The internal standard stock solutions of 2.00 mg/mL are prepared by weighing exactly 114 mg of the labelled asparagine hydrate standard, or 100 mg of the labelled glutamine standard, to 0.0001 g, dissolving in 20 % aqueous methanol and making up to 50 mL; they are kept at 4 °C in the dark and used within 3 months.

10.4.5 The mixed internal standard solution is prepared by taking exactly 1.00 mL of each of the two labelled internal standard stock solutions into a 1000 mL volumetric flask and making up to the mark with water; it is prepared just before use.

10.4.6 The mixed standard intermediate solution of 1.00 µg/mL is prepared by taking exactly 2.50 mL of the mixed standard solution of 20.0 µg/mL of 3.4.3 into a 50 mL volumetric flask, making up to the mark with water and mixing; it is prepared just before use.

10.4.7 The series of mixed working standard solutions is prepared by taking suitable volumes of the mixed standard intermediate solution of 1.00 µg/mL together with 1.00 mL of the mixed internal standard solution into 10 mL volumetric flasks and making up to the mark with water, which gives mass concentrations of 0 ng/mL, 10.0 ng/mL, 20.0 ng/mL, 50.0 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL and 1000 ng/mL; the series is prepared just before use. A note states that the range of the calibration curve may be chosen according to the actual mass concentration in the samples, but that it shall contain at least six concentration points including the zero point.

12 Second method - analytical procedure

12.1 A solid sample that is not a powder is crushed and mixed until uniform; a powdered solid sample or a liquid sample is shaken until uniform.

12.2 The digestion of the sample follows 5.2.2. For the dilution, exactly 1.00 mL of the solution obtained is transferred to a 10 mL volumetric flask, 1.00 mL of the mixed internal standard solution is added, and the flask is made up to the mark with water. After mixing, the solution is filtered through a 0.22 µm membrane ready for measurement. A note states that the volume transferred may be adjusted according to the actual concentration in the sample.

12.3.1 The reference chromatographic conditions are as follows: a hydrophilically modified C18 column of 150 mm by 4.6 mm and 2.6 µm particle size, or a column of equivalent performance; mobile phase A of 0.02 % formic acid solution and mobile phase B of acetonitrile, with the gradient elution conditions of Table 2; a flow rate of 0.3 mL/min; a column temperature of 35 °C; and an injection volume of 5 µL. Table 2 gives the gradient elution conditions as the proportions of mobile phase A and mobile phase B against time: at 0.0 min and 0.5 min they are 80 % and 20 %, at 6.0 min they are 50 % and 50 %, at 6.1 min and 7.0 min they are 20 % and 80 %, and at 7.1 min and 10.0 min they are again 80 % and 20 %.

12.3.2 The reference mass spectrometric conditions are as follows: positive electrospray ionization; multiple reaction monitoring as the scan mode; a nebulizing gas temperature of 300 °C; an ion source temperature of 250 °C; a spray voltage of 3500 V; a sheath gas flow rate of 12.6 L/min; an auxiliary gas flow rate of 3.0 L/min; and the quantifier and qualifier ions, the collision energies and the lens voltages of asparagine, glutamine and their internal standards as given in Table 3. Table 3 lists for each of the four compounds the precursor ion, the product ions, the collision energy in electronvolts and the lens voltage in volts, marks the quantifier ion with an asterisk, and states that the qualifier and quantifier ions may be adjusted to suit the instrument.

12.4 The series of mixed working standard solutions is injected into the liquid chromatograph tandem mass spectrometer in turn, and a calibration curve is drawn for each analyte with the mass concentration of asparagine or of glutamine in the working standard solutions on the horizontal axis and the ratio of the peak area of asparagine or glutamine to that of the corresponding stable isotope internal standard on the vertical axis. The multiple reaction monitoring chromatogram of the standard solution is shown in Figure A.2.

12.5.1 For the qualitative judgement, the retention time of the asparagine and glutamine peaks in the sample, measured under the same test conditions, shall deviate from that of the working standard solution by no more than +/- 2.5 %, and the relative ion ratio of the ions detected shall agree with the relative ion ratio of a working standard solution of comparable concentration. The permitted deviation shall meet Table 4, which allows a relative deviation of +/- 20 % where the relative ion ratio is above 50 %, of +/- 25 % where it is above 20 % and up to 50 %, of +/- 30 % where it is above 10 % and up to 20 %, and of +/- 50 % where it is 10 % or less.

12.5.2 For the quantitative determination the test solution is injected into the liquid chromatograph tandem mass spectrometer, the corresponding peak area ratio is obtained, and the mass concentration of asparagine or glutamine in the sample solution is read from the calibration curve.

12.6 In the blank test the sample is omitted and all other operations are the same as for the sample.

15 Second method - result, precision and other

13 The total asparagine and total glutamine of the sample are calculated by formula (3) from the mass concentration of asparagine or glutamine in the sample solution and in the blank test solution read from the calibration curve, in nanograms per millilitre, the final volume to which the sample was made up in millilitres, the dilution factor, the mass of the sample in grams and two unit conversion coefficients of 1000. The result is expressed in milligrams per gram and is kept to three significant figures.

14 The absolute difference between two independent determinations obtained under repeatability conditions shall not exceed 10 % of their arithmetic mean.

15 With a test portion of 5.0 g of a solid sample, the detection limit of the method is 0.04 mg/g and the quantification limit is 0.12 mg/g for both analytes; with a test portion of 1.0 g of a liquid sample, the detection limit is 0.008 mg/g and the quantification limit is 0.024 mg/g for both analytes.