GB 5009.97-2023 (GB5009.97-2023) & related versions
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GB 5009.97-2023: PDF in English
GB 5009.97-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standards -- Determination of
cyclohexyl sulfamate in food
ISSUED ON. SEPTEMBER 06, 2023
IMPLEMENTED ON. MARCH 06, 2024
Issued by. National Health Commission of the People's Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
2 Principle... 4
3 Reagents and materials... 4
4 Instruments and equipment... 5
5 Analysis steps... 6
6 Expression of analysis results... 8
7 Precision... 9
8 Other... 9
9 Principle... 9
10 Reagents and materials... 10
11 Instruments and equipment... 11
12 Analysis steps... 11
13 Expression of analysis results... 13
14 Precision... 13
15 Other... 13
16 Principle... 14
17 Reagents and materials... 14
18 Instruments and equipment... 15
19 Analysis steps... 16
20 Expression of analysis results... 19
21 Precision... 20
22 Other... 20
Annex A Gas chromatogram of cyclohexylaminosulfonic acid derivatives... 21
Annex B Liquid chromatogram of cyclohexylsulfamic acid derivatives... 22
Annex C Cyclohexylsulfamic acid multiple reaction monitoring (MRM) chromatogram
... 23
National food safety standards -- Determination of
cyclohexyl sulfamate in food
1 Scope
This Standard specifies the method for the determination of cyclohexyl sulfamate in
food.
Method One -- Gas Chromatography is applicable to the determination of cyclohexyl
sulfamate in food (except distilled wine, fermented wine, prepared wine, cooking wine
and other ethanol-containing foods).
Method Two -- Liquid Chromatography is applicable to the determination of cyclamate
in food.
Method Three -- Liquid Chromatography-Mass Spectrometry/Mass Spectrometry is
applicable to the determination of cyclamate in food.
Method One -- Gas Chromatography
2 Principle
The cyclamate in the specimen is extracted with water. It reacts with sodium nitrite in
sulfuric acid medium to generate cyclohexanol nitrite and cyclohexanol. After
extraction with n-heptane, use gas chromatography-hydrogen flame ionization detector
to determine. Use external standard method to quantify.
3 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure, and the
water is grade one water specified in GB/T 6682.
3.1 Reagents
3.1.1 n-Heptane [CH3(CH2)5CH3]. chromatographically pure.
3.1.2 Petroleum ether. Boiling range is 30℃~60℃.
3.1.3 Sulfuric acid (H2SO4). 95.0%~98.0% (mass fraction); density. 1.84 g/mL.
3.1.4 Sodium nitrite (NaNO2).
3.1.5 Amylase. enzyme activity ≥1500 U/g.
3.1.6 Absolute ethanol (CH3CH2OH).
3.2 Preparation of reagents
3.2.1 Sodium nitrite solution (50 g/L). Weigh 50 g of sodium nitrite, dissolve in water
and dilute to 1000 mL. Mix well.
3.2.2 Sulfuric acid solution (200 g/L). Measure 108 mL of sulfuric acid and slowly add
it to 800 mL of water. Stir constantly to avoid localized overheating. After cooling, add
water to dilute to 1000 mL. Mix well.
3.3 Standard product
Sodium cyclohexyl sulfamate standard (C6H12NSO3Na). purity ≥99%, or a standard
certified by the country and awarded a reference material certificate.
3.4 Preparation of standard solution
3.4.1 Cyclohexylsulfamic acid standard stock solution (6 mg/mL). Accurately weigh an
appropriate amount of sodium cyclamate standard (accurate to 0.1 mg) into a 100 mL
volumetric flask. Dissolve in water and bring the volume to the mark. Mix well (the
conversion factor of sodium cyclohexylsulfamate into cyclohexylsulfamate is 0.8907).
Transfer the solution to a brown glass container. Store at 4°C away from light. It is valid
for 12 months.
3.4.2 Cyclohexylsulfamic acid standard intermediate solution (1200 μg/mL).
Accurately pipette 10 mL of cyclamate standard stock solution into a 50 mL volumetric
flask. Use water to bring the volume to the mark. Mix well. Transfer the solution to a
brown glass container. Store at 4°C away from light. It is valid for 6 months.
3.5 Materials
3.5.1 Centrifuge tube. 50 mL graduated centrifuge tube with screw cap.
3.5.2 Organic phase microporous filter membrane. 0.45 μm in pore size.
4 Instruments and equipment
4.1 Gas chromatograph. equipped with hydrogen flame ionization detector (FID).
4.2 Analytical balance. division is 1 mg, 0.1 mg.
4.3 Vortex mixer.
Vortex for 5 min. Perform ultrasonic extraction for 30 min. Mix well and bring to room
temperature.
5.2.2 Specimens of jelly, candy, rice flour, starch products, etc.
Weigh 2 g of specimen (accurate to 0.001 g) into a centrifuge tube. Add 20 mL of water
(0.2 g of amylase is required for specimens such as rice flour and starch products). After
mixing evenly, heat in a water bath at 60℃±2℃ for 20 min. Vortex for 5 min. Perform
ultrasonic extraction for 10 min. Mix well and bring to room temperature.
5.2.3 Other solid and semi-solid specimens
Weigh 2 g of specimen (accurate to 0.001 g) into a centrifuge tube. Add 20 mL of water.
Vortex for 5 min. Perform ultrasonic extraction for 30 min. Mix well and bring to room
temperature.
5.2.4 Liquid specimen
Weigh 2 g of specimen (accurate to 0.001 g) into a centrifuge tube. Add water to 20 mL.
Vortex for 5 min. Perform ultrasonic extraction for 10 min. Mix well and bring to room
temperature.
5.3 Derivatization
Place the centrifuge tube containing the specimen extract solution in an ice bath for 10
min, then add 10 mL of n-heptane, 5 mL of sodium nitrite solution (50 g/L), and 5 mL
of sulfuric acid solution (200 g/L) in sequence. Mix well. Place in ice bath for 30 min.
Shake 3 to 5 times during this period. Remove and vortex for 3 min. Centrifuge at 9000
r/min for 3 min at 4°C (if emulsification occurs, slowly add absolute ethanol dropwise.
At the same time, shake the centrifuge tube gently until the emulsification breaks.
Centrifuge at 9000 r/min for 3 mins at 4°C). Pass the supernatant through the organic
phase microporous filter membrane for measurement on the machine.
5.4 Preparation and derivatization of standard series working solutions
Accurately pipette 0.05 mL, 0.10 mL, 0.25 mL, 0.50 mL, 1.00 mL, 2.5 mL, and 5.0 mL
of cyclohexylaminosulfamic acid standard intermediate solution (1200 μg/mL) into
centrifuge tubes. Dilute to 20 mL with water and derivatize according to the
determination step in 5.3.At this time, the concentrations of the analytes in n-heptane
are respectively equivalent to 6 μg/mL, 12 μg/mL, 30 μg/mL, 60 μg/mL, 120 μg/mL,
300 μg/mL, and 600 μg/mL. Prepare when needed.
5.5 Instrument reference conditions
5.5.1 Chromatographic column. medium polar capillary column internally coated with
50% phenyl-50% methylpolysiloxane, 30 m × 0.32 mm × 0.25 μm or a column with
equivalent performance.
5.5.2 Column temperature rising program. initial temperature is 50°C and maintain for
3 min. Raise temperature to 70°C at 10°C/min and hold for 0.5 min. Raise the
temperature to 220°C at 30°C/min and hold for 3 min.
5.5.3 Inlet temperature. 230℃.
5.5.4 Injection volume. 1 μL.
5.5.5 Injection method. split injection, with a split ratio of 1.5.
5.5.6 Detector. hydrogen flame ionization detector (FID), with a temperature of 260℃.
5.5.7 Carrier gas. high-purity nitrogen, with a flow rate of 2.0 mL/min.
5.5.8 Hydrogen. 32 mL/min. Air. 300 mL/min.
5.6 Preparation of standard curve
The derivatized standard series working solution is injected into the gas chromatograph.
The peak area of the corresponding derivative is measured. Draw a standard curve with
the concentration of cyclohexylsulfamic acid in the standard series working solution as
the abscissa and the sum of the peak areas of the cyclohexylsulfamic acid derivatives
cyclohexanol nitrite and cyclohexanol as the ordinate.
For the gas chromatogram of cyclohexylsulfamic acid derivatives, see Figure A.1 in
Annex A.
5.7 Determination of specimen solution
The derivatized sample solution is injected into the gas chromatograph. Use the
retention time of the two peaks of cyclohexyl sulfamic acid derivative cyclohexanol
nitrite and cyclohexanol to qualitatively determine. The sum of the measured peak areas
is calculated as the measured area. Obtain the concentration of the analyte in the
specimen solution according to the standard curve. Calculate the content of the analyte
in the specimen. If the response of the analyte in the derivatized sample solution
exceeds the linear range, dilute it with n-heptane before injecting the specimen for
analysis.
5.8 Blank test
Except that no specimen is added, the measurement steps in 5.2 and 5.3 are followed.
6 Expression of analysis results
The cyclohexylsulfamic acid content in the specimen is calculated according to formula
(1).
10 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure, and the
water is grade one water specified in GB/T 6682.
10.1 Reagents
10.1.1 n-Heptane [CH3(CH2)5CH3]. chromatographically pure.
10.1.2 Acetonitrile (CH3CN). chromatographically pure.
10.1.3 Petroleum ether. boiling range is 30℃~60℃.
10.1.4 Sulfuric acid (H2SO4). 95.0%~98.0% (mass fraction); density. 1.84 g/mL.
10.1.5 Sodium hypochlorite (NaClO).
10.1.6 Sodium bicarbonate (NaHCO3).
10.1.7 Absolute ethanol (CH3CH2OH).
10.2 Preparation of reagents
10.2.1 Sulfuric acid solution (1+1). Measure 250 mL of sulfuric acid. Slowly add 250
mL of water. Stir constantly to avoid local overheating and mix well.
10.2.2 Sodium hypochlorite solution. Dilute sodium hypochlorite with water. Mix well.
Keep the available chlorine content above 120 g/L. Store in a brown bottle. Prepare
when needed. Commercially available products can also be used, requiring calibration.
10.2.3 Sodium bicarbonate solution (50 g/L). Weigh 25 g of sodium bicarbonate.
Dissolve in water and dilute to 500 mL. Mix well.
10.3 Standard product
Same as 3.3.
10.4 Preparation of standard solution
10.4.1 Cyclohexylsulfamic acid standard stock solution (6 mg/mL). Same as 3.4.1.
10.4.2 Cyclohexylaminosulfonic acid standard intermediate solution (1200 μg/mL).
Same as 3.4.2.
10.5 Materials
10.5.1 Centrifuge tube. 50 mL graduated centrifuge tube with screw cap.
10.5.2 Organic phase microporous filter membrane. 0.45 μm in pore size.
11 Instruments and equipment
11.1 Liquid chromatograph. equipped with UV detector or diode array detector.
11.2 Analytical balance. sensitivity is 1 mg, 0.1 mg.
11.3 Vortex mixer.
11.4 High speed centrifuge.
11.5 Ultrasonic generator.
11.6 Crusher.
11.7 Grinder.
11.8 Homogenizer.
11.9 Constant temperature water bath device.
12 Analysis steps
12.1 Preparation of specimen
Same as 5.1.
12.2 Specimen extraction
Same as 5.2.
12.3 Derivatization
Slowly add 4 mL of sulfuric acid solution (1+1), 10 mL of n-heptane, and 2 mL of
sodium hypochlorite solution to the centrifuge tube containing the specimen extract
solution. Vortex for 3 min. Centrifuge at 9000 r/min for 3 min (if emulsification occurs,
add absolute ethanol slowly and shake the tube gently until the emulsification breaks.
Centrifuge at 9000 r/min for 3 min at 4°C). Immediately transfer 3 mL of supernatant
to another centrifuge tube. Add 15 mL of sodium bicarbonate solution (50 g/L). Vortex
for 5 min. Centrifuge at 9000 r/min for 3 min. Pass the supernatant through the organic
phase microporous filter membrane for measurement on the machine.
12.4 Preparation and derivatization of standard series working solutions
Accurately pipette 0.05 mL, 0.10 mL, 0.25 mL, 0.50 mL, 1.0 mL, 2.5 mL, and 5.0 mL
of cyclohexylaminosulfonic acid standard intermediate solution (1200 μg/mL) into
centrifuge tubes. Dilute to 20 mL with water. Derivatize according to the determination
procedure in 12.3.At this time, the concentrations of the analytes in n-heptane are
16 Principle
After the cyclohexyl sulfamate in the specimen is extracted with the extraction solution,
it is measured by liquid chromatography-mass spectrometry/mass spectrometry,
qualitatively determined by retention time and relative ion abundance, and quantified
by the isotope dilution internal standard method.
17 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure, and the
water is grade one water specified in GB/T 6682.
17.1 Reagents
17.1.1 Acetonitrile (CH3CN). chromatographically pure.
17.1.2 Formic acid (HCOOH). chromatographically pure.
17.1.3 Methanol (CH3OH). chromatographically pure.
17.1.4 Dichloromethane (CH2Cl2).
17.2 Preparation of reagents
17.2.1 Acetonitrile water solution containing 0.1% formic acid. Pipette 1.0 mL of
formic acid. Add 300 mL of acetonitrile. Dilute with water to 1000 mL. Mix well.
17.2.2 Aqueous solution containing 0.1% formic acid. Pipette 1.0 mL of formic acid.
Dilute to 1000 mL with water. Mix well.
17.2.3 Methanol solution containing 0.1% formic acid. Pipette 1.0 mL of formic acid.
Dilute to 1000 mL with methanol. Mix well.
17.3 Standard product
17.3.1 Same as 3.3.
17.3.2 Sodium cyclohexyl sulfamate-D4 (C6H8D4NSO3Na) isotope internal standard
product. purity ≥98%, or standard certified by the country and awarded a reference
material certificate.
17.4 Preparation of standard solution
17.4.1 Cyclohexylsulfamate standard stock solution (5 mg/mL). Accurately weigh an
appropriate amount of sodium cyclohexylsulfamate standard (accurate to 0.1 mg) into
a 100 mL volumetric flask. Dissolve in water and bring the volume to the mark. Mix
well (the conversion coefficient of sodium cyclohexylsulfamate into
cyclohexylsulfamate is 0.8907). Transfer the solution to a brown glass container. Store
at 4°C away from light. It is valid for 12 months.
17.4.2 Cyclohexylsulfamate isotope internal standard stock solution (100 μg/mL).
Accurately weigh an appropriate amount of sodium cyclohexylsulfamate-D4 (accurate
to 0.1 mg) in a 100 mL volumetric flask. Dissolve in water and bring the volume to the
mark. Mix well (the conversion factor of sodium cyclohexylsulfamate-D4 into
cyclohexylsulfamate-D4 is 0.8928). Transfer the solution to a brown glass container.
Store at 4°C away from light. It is valid for 12 months.
17.4.3 Cyclohexylsulfamic acid standard intermediate solution (10 μg/mL). Accurately
pipette 0.20 mL of cyclohexylsulfamic acid standard stock solution into a 100 mL
volumetric flask. Bring the volume to the mark with water. Mix well. Transfer the
solution to a brown glass container. Store at 4°C away from light. It is valid for 1 month.
17.4.4 Cyclohexylsulfamic acid isotope internal standard intermediate solution (5
μg/mL). Accurately pipette 5.0 mL of cyclohexylsulfamic acid isotope internal standard
stock solution into a 100 mL volumetric flask. Bring the volume to the mark with water.
Mix well. Transfer the solution to a brown glass container. Store at 4°C away from light.
It is valid for 1 month.
17.4.5 Cyclohexylsulfamic acid standard series working solution. Accurately pipette 10
μL, 20 μL, 50 μL, 100 μL, 200 μL, 500 μL, and 1 000 μL of cyclohexylsulfamic acid
standard intermediate solution into 10 mL volumetric flasks. Then add 100 μL of
cyclohexylsulfamic acid isotope internal standard intermediate solution, respectively.
Dilute the volume to the mark with acetonitrile aqueous solution containing 0.1%
formic acid. The concentrations of cyclamate are 10 ng/mL, 20 ng/mL, 50 ng/mL, 100
ng/mL, 200 ng/mL, 500 ng/mL, and 1 000 ng/mL. The concentration of cyclohexyl
sulfamic acid isotope internal standard in the standard series working solution is 50
ng/mL. Prepare when needed.
17.5 Materials
17.5.1 Centrifuge tube. 50 mL graduated centrifuge tube with screw cap.
17.5.2 Organic phase microporous filter membrane. 0.22 μm in pore size.
18 Instruments and equipment
18.1 Liquid chromatography-mass spectrometer/mass spectrometer equipped with
electrospray (ESI) ion source.
18.2 Analytical balance. sensitivity is 1 mg, 0.1 mg.
18.3 Vortex mixer.
......
| Standard ID | GB 5009.97-2023 (GB5009.97-2023) | | Description (Translated English) | (National food safety standards Determination of myo-inositol in food) | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 14,133 | | Date of Issue | 2023-09-06 | | Date of Implementation | 2024-03-06 | | Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation | | Summary | This standard specifies the method for the determination of myo-inositol (myo-inositol) in food. The first gas chromatography method is suitable for the determination of myo-inositol in infant formulas, formulas for special medical purposes, milk and dairy products, and beverages. The second microbiological method is suitable for the determination of myo-inositol in food. |
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