GB 5009.97-2016 English PDF

GB 5009.97-2016: Determination of sodium cyclamate in foods
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GB 5009.97-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Sodium Cyclamate in Foods
ISSUED ON: AUGUST 31, 2016
IMPLEMENTED ON: MARCH 01, 2017
Issued by: National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 4 
1 Scope ... 5 
2 Principle ... 5 
3 Reagents and materials ... 6 
4 Instruments and equipment ... 7 
5 Analytical procedures ... 7 
6 Expression of analysis results ... 10 
7 Precision ... 10 
8 Other ... 10 
9 Principle ... 11 
10 Reagents and materials ... 11 
11 Instruments and equipment ... 12 
12 Analytical procedures ... 12 
13 Expression of analysis results ... 14 
14 Precision ... 15 
15 Other ... 15 
16 Principle ... 15 
17 Reagents and materials ... 15 
18 Instruments and equipment ... 16 
19 Analytical procedures ... 16 
20 Expression of analysis results ... 18 
21 Precision ... 18 
22 Other ... 19 
Appendix A Reference conditions of liquid chromatography-mass
spectrometry/mass spectrometry ... 20 
Appendix B Chromatograms of sodium cyclamate standard solution ... 22 
National Food Safety Standard -
Determination of Sodium Cyclamate in Foods
1 Scope
This Standard specifies three methods for the determination of sodium
cyclamate in foods - gas chromatography, liquid chromatography, and liquid
chromatography-mass spectrometry/mass spectrometry. [Translator note:
There is additional 1 nick-name of sodium cyclamate in Chinese]
Gas chromatography of this Standard applies to the determination of sodium
cyclamate in beverages, preserved fruits, fruit leather, pickled fruits, shelled and
unshelled cooked nuts and seeds, canned fruits, jams, pastries, bread, biscuits,
frozen drinks, jelly, compound seasonings, pickled vegetables, and fermented
bean curd foods.
Gas chromatography of this Standard does not apply to the determination of
this compound in white spirits.
Liquid chromatography of this Standard applies to the determination of sodium
cyclamate in beverages, preserved fruits, fruit leather, pickled fruits, shelled and
unshelled cooked nuts and seeds, compound wine, canned fruits, jams,
pastries, bread, biscuits, frozen drinks, jelly, compound seasonings, pickled
vegetables, and fermented bean curd foods.
Liquid chromatography-mass spectrometry/mass spectrometry of this Standard
applies to the determination of sodium cyclamate in white spirit, wine, yellow
rice wine, and cooking wine.
Method 1 -- Gas chromatography
2 Principle
The sodium cyclamate in the food is extracted using water. In a sulfuric acid
medium, sodium cyclamate is reacted with nitrous acid, to form cyclohexanol
nitrite. USE gas chromatograph-hydrogen flame ionization detector for
separation and analysis. USE the retention time for qualitation, external
standard method to quantify.
Accurately WEIGH 0.5612 g of sodium cyclamate standard; USE water to
dissolve and dilute to 100 mL; MIX well. 1.00 mL of this solution is equivalent
to 5.00 mg of cyclohexylsulfamic acid (The conversion factor of sodium
cyclamate and cyclohexylsulfamic acid is 0.8909). STORE in a refrigerator at
1 °C~4 °C. It can be stored for 12 months.
3.4.2 Standard use solution of cyclohexylsulfamic acid (1.00 mg/mL):
Accurately PIPETTE 20.0 mL of standard stock solution of cyclohexylsulfamic
acid; USE water to dilute to 100 mL; MIX well. STORE in a refrigerator at
1 °C~4 °C. It can be stored for 6 months.
4 Instruments and equipment
4.1 Gas chromatograph: Equipped with a hydrogen flame ionization detector
(FID).
4.2 Vortex mixer.
4.3 Centrifuge: Speed≥4000 r/min.
4.4 Ultrasonic oscillator.
4.5 Sample pulverizer.
4.6 10 μL micro syringe.
4.7 Thermostat water bath.
4.8 Balance: The sensitivity is 1 mg, 0.1 mg.
5 Analytical procedures
5.1 Preparation of sample solutions
5.1.1 Liquid sample processing
5.1.1.1 After shaking the ordinary liquid sample well, WEIGH 25.0 g of the
sample (may be filtered if necessary); USE water to dilute to 50 mL for use.
5.1.1.2 Carbon dioxide-containing sample: WEIGH 25.0 g of the sample in a
beaker; HEAT it in a 60 °C water bath for 30 min to remove carbon dioxide; LET
cool; USE water to dilute to 50 mL for use.
5.1.1.3 Alcohol-containing sample: WEIGH 25.0 g of the sample in a beaker;
USE sodium hydroxide solution (3.2.1) to adjust to a weak alkaline pH of 7~8;
low temperature for 20 min to clear stratification, the supernatant is taken and
stored in a refrigerator at 1 °C~4 °C for sample injection.
5.2 Preparation and derivatization of standard solution series
Accurately PIPETTE 0.50 mL, 1.00 mL, 2.50 mL, 5.00 mL, 10.0 mL, 25.0 mL of
1.00 mg/mL standard solution of cyclohexylsulfamic acid in 50 mL volumetric
flasks; ADD water to dilute to the mark. Prepare the standard solution series,
with concentrations of 0.01 mg/mL, 0.02 mg/mL, 0.05 mg/mL, 0.10 mg/mL, 0.20
mg/mL, and 0.50 mg/mL. Prepare when used for derivatization.
Accurately PIPETTE 10.0 mL of the standard series solution. The derivatization
is same as (5.1.3).
5.3 Determination
5.3.1 Chromatographic conditions
5.3.1.1 Chromatographic column: Weakly polar quartz capillary column
(internally coated with 5% phenyl methyl polysiloxane, 30 m×0.53 mm×1.0 μm)
or an equivalent column.
5.3.1.2 Column temperature heating program: Initial temperature 55 °C is kept
for 3 min. At 10 °C/min, the temperature is raised to 90 °C and kept for 0.5 min.
At 20 °C/min, the temperature is raised to 200 °C and kept for 3 min.
5.3.1.3 Injection port: Temperature is 230 °C. Injection volume is 1 μL,
splitless/split injection. Split ratio is 1:5 (Split ratio and mode may be adjusted
according to chromatographic instrument conditions).
5.3.1.4 Detector: Hydrogen flame ionization detector (FID). Temperature is
260 °C.
5.3.1.5 Carrier gas: High-purity nitrogen. Flow rate is 12.0 mL/min. Make-up
flow rate is 20 mL/min.
5.3.1.6 Hydrogen: 30 mL/min; air 330 mL/min (The flow rate of the carrier gas,
hydrogen, air may be adjusted according to the instrument conditions).
5.3.2 Chromatographic analysis
PIPETTE 1 μL of the supernatant of derivatization-treated standard series
solutions of each concentration (5.2); inject them into the gas chromatograph.
The peak area of the response value of different concentrations of the analyte
can be measured. USE the concentration as the abscissa. USE the sum of the
areas of the two peaks of cyclohexanol nitrite and cyclohexanol as the ordinate.
DRAW the standard curve.
Method 2 -- High-performance liquid chromatography
9 Principle
After the sodium cyclamate in the food is extracted using water, in a strong
acidic solution, it is reacted with sodium hypochlorite, to form N,N-
dichlorocyclohexylamine. After using n-heptane to extract, USE high-
performance liquid chromatography to detect; USE the retention time for
qualitation, external standard method to quantify.
10 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure;
the water is the Grade 1 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 Sulfuric acid (H2SO4).
10.1.4 Sodium hypochlorite (NaClO).
10.1.5 Sodium bicarbonate (NaHCO3).
10.1.6 Zinc sulfate (ZnSO4 • 7H2O).
10.1.7 Potassium ferrocyanide {K4[Fe(CN)6] • 3H2O}.
10.1.8 Petroleum ether: Boiling range is 30 °C~60 °C.
10.2 Reagent preparation
10.2.1 Sulfuric acid solution (1+1): 50 mL of sulfuric acid is carefully and slowly
added to 50 mL of water and mixed.
10.2.2 Sodium hypochlorite solution: USE sodium hypochlorite (10.1.4) to dilute;
STORE in a brown bottle. KEEP the available chlorine content above 50 g/L;
MIX well. It is necessary to calibrate the commercial products in time. Prepare
when used.
10.2.3 Sodium bicarbonate solution (50 g/L): WEIGH 5 g of sodium bicarbonate;
USE water to dissolve and dilute to 100 mL; MIX well.
CENTRIFUGE (3000 r/min) for 20 min. TRANSFER the supernatant out; USE
water to wash the residue and dilute to 50 mL for use. For high-protein-
containing samples, when ultrasonically extracting, 2.0 mL of zinc sulfate
solution (10.2.4) and 2.0 mL of potassium ferrocyanide solution (10.2.5) may
be added. For high-fat-containing samples, before extraction, 25 mL of
petroleum ether (10.1.8) may be added; after shaking, DISCARD the petroleum
ether layer to remove fat.
12.1.2 Liquid sample processing
12.1.2.1 After shaking the ordinary liquid sample well, 25.0 g of the sample may
be directly weighed. USE water to dilute to 50 mL for use (may be filtered if
necessary).
12.1.2.2 Carbon dioxide-containing sample: WEIGH 25.0 g of the sample in a
beaker; HEAT it in a 60 °C water bath for 30 min to remove carbon dioxide; LET
cool; USE water to dilute to 50 mL for use.
12.1.2.3 Alcohol-containing sample: WEIGH 25.0 g of the sample in a beaker;
USE sodium hydroxide solution to adjust to a weak alkaline pH of 7~8; HEAT it
in a 60 °C water bath for 30 min to remove alcohol; LET cool; USE water to
dilute to 50 mL for use.
12.1.2.4 Milk-containing beverage: WEIGH 25.0 g of the sample in a 50 mL
centrifuge tube; ADD 3.0 mL of zinc sulfate solution (10.2.4) and 3.0 mL of
potassium ferrocyanide solution (10.2.5); MIX well. After centrifuging for
stratification, TRANSFER the supernatant out; USE water to wash the residue
and dilute to 50 mL for use.
12.1.3 Derivatization
Accurately PIPETTE 10 mL of the prepared sample solution (12.1.1 or 12.1.2);
ADD 2.0 mL of sulfuric acid solution (10.2.1), 5.0 mL of n-heptane (10.1.1), and
1.0 mL of sodium hypochlorite solution (10.2.2). Vigorously oscillate for 1 min;
LET it stand for stratification. After removing the aqueous layer, ADD 25 mL of
sodium bicarbonate solution (10.2.3) to the n-heptane layer; oscillate for 1 min.
LET it stand; the upper organic phase is filtered through a 0.45 μm microporous
organic-phase filter membrane. The filtrate is used for injection.
12.2 Reference conditions of instrument
12.2.1 Chromatographic column: C18 column, 5 μm, 150 mm×3.9 mm (i, d), or
chromatographic column with equivalent performance.
12.2.2 Mobile phase: Acetonitrile + water (70+30).
m - Sample mass, in grams (g);
1000 - Conversion factor for conversion of μg/g to g/kg.
The calculation result is expressed as the arithmetic mean of two independent
determination results obtained under repeated conditions. The result retains
three significant digits.
14 Precision
The absolute difference between the two independent determination results,
obtained under repeated conditions, shall not exceed 10% of the arithmetic
mean.
15 Other
When the sampling amount is 5 g, the detection limit of this method is 0.010
g/kg; the limit of quantification is 0.030 g/kg.
Method 3 -- Liquid chromatography-mass
spectrometry/mass spectrometry
16 Principle
The wine sample is heated in a water bath to remove ethanol; then USE water
to dilute to the mark. USE liquid chromatography-mass spectrometry/mass
spectrometry to determine the sodium cyclamate therein. USE the external
standard method to quantify.
17 Reagents and materials
Unless otherwise stated, the reagents used in this method are analytically pure;
the water is the Grade 1 water specified in GB/T 6682.
17.1 Reagents
17.1.1 Methanol (CH3OH): Chromatographically pure.
17.1.2 Ammonium acetate (CH3COONH4).
19.2 Reference conditions of instrument
19.2.1 Chromatographic column: C18 column, 1.7 μm, 100 mm×2.1 mm (i, d),
or chromatographic column with equivalent performance.
19.2.2 Mobile phase: Methanol, 10 mmol/L ammonium acetate solution.
19.2.3 Gradient elution: See Table A.1 in Appendix A.
19.2.4 Flow rate: 0.25 mL/min.
19.2.5 Injection volume: 10 μL.
19.2.6 Column temperature: 35 °C.
19.3 Operating conditions of mass spectrometry
19.3.1 Ion source: Electrospray ionization source (ESI).
19.3.2 Scanning mode: Multiple-reaction monitoring (MRM) scanning.
19.3.3 The mass spectrometry tuning parameters shall be optimized to the
optimum conditions, to ensure that the sensitivity of sodium cyclamate in
positive ion mode is optimal. Adjust the relative abundance of qualitative ions
in positive and negative modes to be close. For mass spectrometry tuning
parameters and qualitative and quantitative ions, see Appendix A.
19.4 Making of standard curve
The prepared standard series solution (17.3.4), in ascending order of
concentration, is injected for determination. The chromatographic peak area of
the quantitative ion of sodium cyclamate is plotted against the corresponding
concentration, to obtain a standard curve regression equation. For typical
sodium cyclamate standard solution selecting reaction monitoring mass
chromatogram, see Appendix B.
19.5 Qualitative determination
Under the same test conditions, determine the sample solution (19.1). If the
retention time of sodium cyclamate in the mass chromatogram of the sample
solution is consistent with the standard solution (The range of variation is within
±2.5%); and the deviation BETWEEN the relative abundance of the qualitative
ions of the sample AND the relative abundance of the qualitative ions in the
standard solution of equivalent concentration does not exceed the provisions of
Table 1, it can be determined that there is sodium cyclamate in the sample.
GB 5009.97-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Sodium Cyclamate in Foods
ISSUED ON: AUGUST 31, 2016
IMPLEMENTED ON: MARCH 01, 2017
Issued by: National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 4 
1 Scope ... 5 
2 Principle ... 5 
3 Reagents and materials ... 6 
4 Instruments and equipment ... 7 
5 Analytical procedures ... 7 
6 Expression of analysis results ... 10