GB 5009.289-2023 English PDF

GB 5009.289-2023: National food safety standard - Determination of galactooligosaccharides in foods
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GB 5009.289-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard - Determination of
Galactooligosaccharides in Foods
ISSUED ON. SEPTEMBER 6, 2023
IMPLEMENTED ON. MARCH 6, 2024
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope... 3
2 Principle... 3
3 Reagents and Materials... 3
4 Instruments and Equipment... 5
5 Analytical Procedures... 6
6 Expression of Analysis Results... 8
7 Precision... 8
8 Others... 9
Appendix A Method for the Identification of Other Oligomeric Reducing Substances
... 10
Appendix B Mobile Phase Elution Procedure... 14
Appendix C Chromatogram of Derivatives in Standard Solution and Specimen Solution
... 15
Appendix D Method of Determining Retention Time Periods of Sugars with Different
Degrees of Polymerization through Liquid Chromatography - Mass Spectrometry... 16
National Food Safety Standard - Determination of
Galactooligosaccharides in Foods
1 Scope
This Standard specifies the method of liquid chromatography for the determination of
galactooligosaccharides in foods.
This Standard is applicable to the determination of galactooligosaccharides in infant formula
foods (excluding infant formula foods for special medical purposes), infant auxiliary foods,
dairy products, beverages and baked foods.
This Standard is not applicable to the determination of galactooligosaccharides in foods that
contain other oligomeric reducing substances that may cause interference.
2 Principle
The galactooligosaccharides (n is 2 ~ 7) in the specimen is extracted by aqueous solution and
derivatized by 2-aminobenzamide, enzymatically hydrolyzed by amyloglucosidase to remove
the interference of maltodextrin and starch, and then, detected by high performance liquid
chromatograph - fluorescence detector. The collected chromatographic peaks are qualitatively
determined based on the retention time of galactooligosaccharides with the same degree of
polymerization determined by liquid chromatography - tandem mass spectrometry under the
same test conditions. Adopt the external standard method of maltose glycan reference material
corresponding to the degree of polymerization and relative molecular weight for quantitative
determination. After deducting the lactose and maltose content, the galactooligosaccharides
content is obtained.
3 Reagents and Materials
Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and
the water is Grade-1 water specified in GB/T 6682.
WARNING---sodium cyanoborohydride and 2-methylpyridine-N-borane are hazardous
materials, which will release flammable gases when exposed to water and are dangerous
to the environment. Please follow the instructions for use of the reagents. Please wear
personal protective equipment and perform all operations under a fume hood.
3.1 Reagents
3.1.1 Dimethyl sulfoxide [(CH3)2SO]. chromatographically pure.
3.1.2 2-aminobenzamide (C7H8N2O).
3.1.3 Sodium cyanoborohydride (NaBH3CN).
3.1.4 2-methylpyridine-N-borane (C6H7N  BH3).
3.1.5 Acetic acid (CH3COOH).
3.1.6 Ammonium acetate (CH3COONH4).
3.1.7 Ammonium formate (HCOONH4).
3.1.8 Formic acid (HCOOH).
3.1.9 Acetonitrile (CH3CN). chromatographically pure.
3.1.10 Amyloglucosidase (CAS. 9032-08-0). enzyme activity unit  60 U/mg.
NOTE. for the determination method of enzyme activity, see A.3 in GB 1866.174-2016.
3.2 Preparation of Reagents
3.2.1 Acetic acid-dimethyl sulfoxide solution (3 + 7, volume ratio). respectively measure-take
3 mL of acetic acid and 7 mL of dimethyl sulfoxide in a beaker, evenly mix it and reserve it for
later use.
3.2.2 2-aminobenzamide derivative solution (0.35 mol/L). accurately weigh-take 476 mg of 2-
amonibenzamide and 630 mg of sodium cyanoborohydride or 1,070 mg of 2-methylpyridine-
N-borane in a beaker, use 10 mL of acetic acid-dimethyl sulfoxide solution (3 + 7) to dissolve
it and evenly mix it.
3.2.3 Ammonium acetate solution (0.2 mol/L, pH 4.5  0.1). weigh-take 1.54 g of ammonium
acetate, use 80 mL of water to dissolve it, then, use acetic acid to adjust pH to 4.5  0.1, and
use water to dilute to 100 mL. Prepare it right before use.
3.2.4 Amyloglucosidase solution (about 180 U/mL). weigh-take amyloglucosidase equivalent
to about 900 U of activity and dissolve it in 5 mL of ammonium acetate solution (0.2 mol/L,
pH 4.5  0.1), stir, until it is completely dissolved. Prepare it right before use.
3.2.5 Ammonium formate solution (50 mmol/L, pH 4.4  0.1). weigh-take 3.15 g of ammonium
formate, use 900 mL of water to dissolve it, then, use formic acid to adjust pH to 4.4  0.1, and
use water to dilute it to 1,000 mL. Prepare it right before use.
3.3 Reference Materials
3.3.1 Maltose reference material (C12H22O11, CAS. 69-79-4), purity  98%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.3.2 Maltotriose reference material (C18H32O16, CAS. 1109-28-0), purity  98%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.3.3 Maltotetraose reference material (C24H42O21, CAS. 34612-38-9), purity  95%, or a
standard substance certified by the state and awarded a standard substance certificate.
3.3.4 Maltopentaose reference material (C30H52O26, CAS. 34620-76-3), purity  95%, or a
standard substance certified by the state and awarded a standard substance certificate.
3.3.5 Maltohexaose reference material (C36H62O31, CAS. 34620-77-4), purity  95%, or a
standard substance certified by the state and awarded a standard substance certificate.
3.3.6 Maltoheptaose reference material (C42H72O36, CAS. 34620-78-5), purity  95%, or a
standard substance certified by the state and awarded a standard substance certificate.
3.3.7 Lactose reference material (C12H22O11, CAS. 63-42-3), purity  98%, or a standard
substance certified by the state and awarded a standard substance certificate.
3.4 Preparation of Standard Solutions
3.4.1 Standard stock solutions (10.00 mg/mL). respectively and accurately weigh-take 100 mg
(accurate to 0.1 mg) of lactose, maltose, maltotriose, maltotetraose, maltopentaose,
maltohexaose and maltoheptaose reference materials, use water to dissolve them, reach a
constant volume of 10 mL, and evenly mix them. Transfer each standard stock solution into a
liquid storage bottle and store it at 4 C. It shall remain valid for 1 month.
3.4.2 Mixed standard intermediate solution (1.000 mg/mL). draw-take 1.00 mL of each standard
stock solution (10.00 mg/mL) into a 10 mL volumetric flask, add water to reach a constant
volume to the scale and evenly mix it. Prepare it right before use.
3.4.3 Mixed standard service solution (100.0 g/mL). draw-take 1.00 mL of the mixed standard
intermediate solution (1.000 mg/mL) in a 10 mL volumetric flask, add water to reach a constant
volume to the scale and evenly mix it. Prepare it right before use.
3.4.4 Mixed standard series of working solutions. respectively draw-take 1.00 mL, 2.50 mL and
5.00 mL of the mixed standard intermediate solution (1.000 mg/mL) in a 10 mL volumetric
flask, add water to dilute to the scale and evenly mix it. Respectively draw-take 0.10 mL, 1.00
mL and 5.00 mL of the mixed standard service solution (100.0 g/mL) in a 10 mL volumetric
flask, add water to reach a constant volume to the scale and evenly mix it. The mass
concentration of the mixed standard series of working solutions is respectively 1.000 g/mL,
10.00 g/mL, 50.00 g/mL, 100.0 g/mL, 250.0 g/mL and 500.0 g/mL. Prepare them right
before use.
4 Instruments and Equipment
4.1 High performance liquid chromatograph. equipped with fluorescence detector.
4.2 Balance. with a division value of 0.1 mg and 1 mg respectively.
following steps to process it simultaneously with the sample according to 5.1.3.
5.1.5 Derivatization of standard series working solution
Transfer 20 μL of standard series working solution into a 2 mL centrifuge tube with a screw
cap, add 200 μL of 2-aminobenzamide derivative solution; vortex and mix for 30 s; incubate in
a 60°C water bath for 120 min; take it out and place it to reach room temperature; add 1 mL of
ammonium acetate solution (0.2 mol/L, pH 4.5 ± 0.1) and vortex for 30 seconds. Then transfer
0.50mL of the mixed solution into a 2mL centrifuge tube; add 200μL of ammonium acetate
solution (0.2mol/L, pH4.5±0.1); incubate in a 50℃ water bath for 30min; take it out and place
it to reach room temperature; add 0.70mL of acetonitrile; mix well; centrifuge at 6000r/min for
5min; the supernatant is filtered with a 0.22μm organic microporous membrane for high-
performance liquid chromatography measurement.
5.2 Reference Conditions of Instrument
5.2.1 Chromatographic column. amide bonded surface porous silica gel column [150 mm  4.6
mm (inner diameter), particle size 2.7 m], or equivalent column.
5.2.2 Mobile phase. mobile phase A is ammonium formate solution (50 mmol/L, pH 4.4  0.1);
mobile phase B is acetonitrile.
5.2.3 Mobile phase flow rate. 1.0 mL/min. For the mobile phase gradient elution procedure, see
B.1 in Appendix B.
5.2.4 Detector. fluorescence detector, with an excitation wavelength of 355 nm and an emission
wavelength of 430 nm.
5.2.5 Column temperature. 30 C.
5.2.6 Injection volume. 20 L.
5.3 Drawing of Standard Curve
Respectively inject the mixed standard series of working solution derivative solutions into the
high performance liquid chromatograph to determine the peak areas of the chromatograms of
maltose reference materials and lactose derivatives with different degrees of polymerization.
Take the concentration of maltose reference materials and lactose reference material in the
mixed standard series of working solutions as the x-coordinate, and the peak area of the maltose
reference material derivatives with different degrees of polymerization as the y-coordinate to
draw a standard curve of maltose reference materials and lactose derivatives with different
degrees of polymerization. The chromatogram of maltose reference materials and lactose
derivative solutions with different degrees of polymerization is shown in Figure C.1 in
Appendix C.
5.4 Determination of Specimen Solution
Inject the specimen solution into the high performance liquid chromatograph. Refer to
Appendix D to determine the retention time periods of sugars with different degrees of
polymerization, respectively record the sum of the peak areas of sugar derivatives with each
degree of polymerization, and the peak areas of lactose and maltose derivatives. In accordance
with the standard curve of the reference material derivatives with the corresponding
polymerization degree, respectively calculate the concentration of sugars with different degrees
of polymerization in the specimen solution. In accordance with the standard curve of lactose
and maltose reference material derivatives, respectively calculate the concentration of lactose
and maltose in the specimen solution. For the chromatogram of the specimen after
derivatization, see Figure C.2 in Appendix C.
6 Expression of Analysis Results
The content X of galactooligosaccharides in the specimen is calculated in accordance with
Formula (1).
Where,
X---the content of galactooligosaccharides in the specimen, expressed in (g/kg);
ρDPi---the concentration of sugar derivatives with different degrees of polymerization obtained
through the standard curve, expressed in (g/mL) (i = 2 ~ 7);
ρ0---the concentration of derivatives in the blank specimen solution, expressed in (g/mL);
ρlactose---the concentration of lactose derivatives in the specimen solution, expressed in (g/mL);
ρmaltose---the concentration of maltose derivatives in the specimen solution, expressed in
(g/mL);
V---the constant volume of the specimen, expressed in (mL);
1,000---conversion factor;
m---the mass of the specimen, expressed in (g).
The calculation results, which are expressed as the arithmetic mean of the results of two
independent determinations obtained under repeatability conditions, shall retain 3 significant
figures.
7 Precision
The absolute difference between the results of two independent determinations obtained under
repeatability conditions shall not exceed 15% of the arithmetic mean.
lactose maltose
Appendix A
Method for the Identification of Other Oligomeric Reducing Substances
A.1 Principle
In the specimen solution, amyloglucosidase is used to remove maltodextrin and starch
interference, the lactose and galactooligosaccharides in the sample are hydrolyzed by -
galactosidase, then, derivatized by 2-aminobenzamide. The high performance liquid
chromatograph - fluorescence detector is used for detection. The collected signals are
segmented in accordance with the retention time of the polymerization degree, then, the peaks
are summed. The external standard method of the maltose glycan reference material with the
corresponding degree of polymerization is adopted to respectively perform the quantitative
determination. Then, the total derivative content is calculated, and after deducting the blank
solution and maltose derivative content, the total content of other oligomeric reducing
substances is calculated. If the total content of other oligomeric reducing substances in the
specimen is greater than the quantitation limit, then, the standard method is not applicable to
the determination of galactooligosaccharides in the specimen.
A.2 Reagents
A.2.1 -galactosidase solution. enzyme activity  4,000 U/mL.
NOTE. the determination method for enzyme activity refers to GB/T 33409-2016.For different
batches of -galactosidase solutions, before use, they need to be subject to quality
verification.
A.2.2 Acetonitrile solution (75%, volume fraction). measure-take 75 mL of acetonitrile and
add water to dilute to 100 mL.
The others are the same as Chapter 3.
A.3 Instruments and equipment
Same as Chapter 4.
A.4 Analytical procedures
A.4.1 Verification of -galactosidase...