GB/T 35800-2018 English PDF (GB/T35800-2018)

GB/T 35800-2018: Determination of Hexamidine, Chlorhexidine and Their Salts as Preservatives in Cosmetics - High Performance Liquid Chromatography
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GB/T 35800-2018
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 71.100.70
Y 42
Determination of Hexamidine, Chlorhexidine and
Their Salts as Preservatives in Cosmetics - High
Performance Liquid Chromatography
ISSUED ON. FEBRUARY 6, 2018
IMPLEMENTED ON. SEPTEMBER 1, 2018
Issued by. General Administration of Quality Supervision, Inspection and
Quarantine;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative References ... 4 
3 Principle ... 4 
4 Reagents and Materials ... 4 
5 Instruments and Equipment ... 5 
6 Steps of Determination ... 6 
7 Result Calculation ... 8 
8 Detection Limit and Quantitation Limit ... 8 
9 Recovery Rate and Precision ... 8 
10 Allowable Difference ... 8 
Appendix A (Informative) Basic Information of Hexamidine Diisethionate
(isethionate) and Chlorhexidine Standards ... 9 
Appendix B (Informative) Liquid Chromatogram of Hexamidine and
Chlorhexidine ... 10 
Appendix C (Informative) Confirmation Test... 11 
Determination of Hexamidine, Chlorhexidine and
Their Salts as Preservatives in Cosmetics - High
Performance Liquid Chromatography
1 Scope
This Standard specifies the method of high performance liquid chromatography of
determining hexamidine, chlorhexidine and their salts as preservatives in cosmetics.
This Standard is applicable to the determination of hexamidine, chlorhexidine and their
salts as preservatives in aqueous, creamy, shampoo, powdered and lipstick cosmetics.
The detection limit and the limit of quantitation of this Standard. the detection limit of
hexamidine, chlorhexidine and their salts. 0.002%; the limit of quantitation. 0.004%.
2 Normative References
The following documents are indispensable to the application of this Standard. In terms
of references with a specified date, only versions with a specified date are applicable
to this Standard. The latest version (including all the modifications) of references
without a specified date is applicable to this Standard.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
3 Principle
Sample shall receive solvent extraction, centrifugation and microporous membrane
filtration, then, be determined through high performance liquid chromatography; it shall
be quantified through external standard method and confirmed through liquid
chromatography-mass spectrometry.
4 Reagents and Materials
Unless it is otherwise stipulated, all reagents shall be analytical purity. Water shall be
Grade-1 water that is stipulated in GB/T 6682.
4.1 Acetonitrile. chromatographic purity.
4.2 Methanol. chromatographic purity.
4.3 Tetrahydrofuran.
6 Steps of Determination
6.1 Sample Processing
6.1.1 Aqueous, creamy, shampoo and powdered sample
Weigh-take 0.5 g (accurate to 0.001 g) of sample, place it into a 50 mL PTFE centrifuge
tube with a plug (5.7); add 1 mL of saturated sodium chloride solution (4.6), then, start
vortex mixing. Add 9 mL of water; after vortex mixing, start centrifugation at 5,000 r/min
for 10 min. Transfer the supernatant into another 50 mL PTFE centrifuge tube with a
plug. In the sample residue, continue to add 10 mL of methanol (4.2); in ultrasonic
water bath (5.3), start ultrasonic extraction for 20 min. Start centrifugation at 5,000
r/min for 10 min; combine the supernatant, then, filter it through 0.45 μm microporous
membrane (5.8) for the determination with high performance liquid chromatography.
6.1.2 Creamy sample
Weigh-take 0.5 g (accurate to 0.001 g) of sample, place it into a 50 mL PTFE centrifuge
tube with a plug (5.7); add 2 mL of tetrahydrofuran (4.3), start vortex mixing. Add 8 mL
of water; after vortex mixing, start centrifugation at 5,000 r/min for 10 min. Transfer the
supernatant into another 50 mL PTFE centrifuge tube with a plug. In the sample
residue, continue to add 10 mL of methanol (4.2); in ultrasonic water bath (5.3), start
ultrasonic extraction for 20 min. Start centrifugation at 5,000 r/min for 10 min; combine
the supernatant, then, filter it through 0.45 μm microporous membrane (5.8) for the
determination with high performance liquid chromatography.
6.2 Conditions of Determination
The determination with high performance liquid chromatography shall be based on the
following reference conditions.
a) Chromatographic column. XTerra MS C18, 5 μm, 250 mm x 4.6 mm (internal
diameter), or others with equivalent performance;
b) Mobile phase. please refer to Table 1;
c) Flow rate. 1.0 mL/min;
d) Column temperature. 30 °C;
e) Detection wavelength. 260 nm;
f) Injection volume. 10 μL.
6.5 Blank Test
Follow the above determination conditions and steps, other than the step of weighing-
taking sample.
7 Result Calculation
The result shall be calculated in accordance with Formula (1). The calculation result
shall retain 2 decimals (the calculation result shall deduct the blank value).
Where,
W---the percentage content of hexamidine, chlorhexidine and their salts in the sample,
expressed in %;
c---the concentration of hexamidine and chlorhexidine in the sample, which is obtained
through the standard working curve, expressed in (μg/mL);
V---the ultimate constant volume of the sample solution, expressed in (mL);
m---sample mass, expressed in (g).
8 Detection Limit and Quantitation Limit
The detection limit of hexamidine, chlorhexidine and their salts shall be 0.002%; the
quantitation limit shall be 0.004%.
9 Recovery Rate and Precision
Within the concentration range of 0.004% ~ 0.3 %, the recovery rate of hexamidine,
chlorhexidine and their salts shall be between 84.1% ~ 99.7%, the relative standard
deviation shall be 1.1% ~ 7.8%.
10 Allowable Difference
The absolute difference of two independent determination results obtained under
repeatability conditions shall not exceed 10% of arithmetic mean value.
GB/T 35800-2018
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 71.100.70
Y 42
Determination of Hexamidine, Chlorhexidine and
Their Salts as Preservatives in Cosmetics - High
Performance Liquid Chromatography
ISSUED ON. FEBRUARY 6, 2018
IMPLEMENTED ON. SEPTEMBER 1, 2018
Issued by. General Administration of Quality Supervision, Inspection and
Quarantine;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative References ... 4 
3 Principle ... 4 
4 Reagents and Materials ... 4 
5 Instruments and Equipment ... 5 
6 Steps of Determination ... 6 
7 Result Calculation ... 8 
8 Detection Limit and Quantitation Limit ... 8 
9 Recovery Rate and Precision ... 8 
10 Allowable Difference ... 8 
Appendix A (Informative) Basic Information of Hexamidine Diisethionate
(isethionate) and Chlorhexidine Standards ... 9 
Appendix B (Informative) Liquid Chromatogram of Hexamidine and
Chlorhexidine ... 10 
Appendix C (Informative) Confirmation Test... 11 
Determination of Hexamidine, Chlorhexidine and
Their Salts as Preservatives in Cosmetics - High
Performance Liquid Chromatography
1 Scope
This Standard specifies the method of high performance liquid chromatography of
determining hexamidine, chlorhexidine and their salts as preservatives in cosmetics.
This Standard is applicable to the determination of hexamidine, chlorhexidine and their
salts as preservatives in aqueous, creamy, shampoo, powdered and lipstick cosmetics.
The detection limit and the limit of quantitation of this Standard. the detection limit of
hexamidine, chlorhexidine and their salts. 0.002%; the limit of quantitation. 0.004%.
2 Normative References
The following documents are indispensable to the application of this Standard. In terms
of references with a specified date, only versions with a specified date are applicable
to this Standard. The latest version (including all the modifications) of references
without a specified date is applicable to this Standard.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
3 Principle
Sample shall receive solvent extraction, centrifugation and microporous membrane
filtration, then, be determined through high performance liquid chromatography; it shall
be quantified through external standard method and confirmed through liquid
chromatography-mass spectrometry.
4 Reagents and Materials
Unless it is otherwise stipulated, all reagents shall be analytical purity. Water shall be
Grade-1 water that is stipulated in GB/T 6682.
4.1 Acetonitrile. chromatographic purity.
4.2 Methanol. chromatographic purity.
4.3 Tetrahydrofuran.
6 Steps of Determination
6.1 Sample Processing
6.1.1 Aqueous, creamy, shampoo and powdered sample
Weigh-take 0.5 g (accurate to 0.001 g) of sample, place it into a 50 mL PTFE centrifuge
tube with a plug (5.7); add 1 mL of saturated sodium chloride solution (4.6), then, start
vortex mixing. Add 9 mL of water; after vortex mixing, start centrifugation at 5,000 r/min
for 10 min. Transfer the supernatant into another 50 mL PTFE centrifuge tube with a
plug. In the sample residue, continue to add 10 mL of methanol (4.2); in ultrasonic
water bath (5.3), start ultrasonic extraction for 20 min. Start centrifugation at 5,000
r/min for 10 min; combine the supernatant, then, filter it through 0.45 μm microporous
membrane (5.8) for the determination with high performance liquid chromatography.
6.1.2 Creamy sample
Weigh-take 0.5 g (accurate to 0.001 g) of sample, place it into a 50 mL PTFE centrifuge
tube with a plug (5.7); add 2 mL of tetrahydrofuran (4.3), start vortex mixing. Add 8 mL
of water; after vortex mixing, start centrifugation at 5,000 r/min for 10 min. Transfer the
supernatant into another 50 mL PTFE centrifuge tube with a plug. In the sample
residue, continue to add 10 mL of methanol (4.2); in ultrasonic water bath (5.3), start
ultrasonic extraction for 20 min. Start centrifugation at 5,000 r/min for 10 min; combine
the supernatant, then, filter it through 0.45 μm microporous membrane (5.8) for the
determination with high performance liquid chromatography.
6.2 Conditions of Determination
The determination with high performance liquid chromatography shall be based on the
following reference conditions.
a) Chromatographic column. XTerra MS C18, 5 μm, 250 mm x 4.6 mm (internal
diameter), or others with equivalent performance;
b) Mobile phase. please refer to Table 1;
c) Flow rate. 1.0 mL/min;
d) Column temperature. 30 °C;
e) Detection wavelength. 260 nm;
f) Injection volume. 10 μL.
6.5 Blank Test
Follow the above determination conditions and steps, other than the step of weighing-
taking sample.
7 Result Calculation
The result shall be calculated in accordance with Formula (1). The calculation result
shall retain 2 decimals (the calculation result shall deduct the blank value).
Where,
W---the percentage content of hexamidine, chlorhexidine and their salts in the sample,
expressed in %;
c---the concentration of hexamidine and chlorhexidine in the sample, which is obtained
through the standard working curve, expressed in (μg/mL);
V---the ultimate constant volume of the sample solution, expressed in (mL);
m---sample mass, expressed in (g).
8 Detection Limit and Quantitation Limit
The detection limit of hexamidine, chlorhexidine and their salts shall be 0.002%; the
quantitation limit shall be 0.004%.
9 Recovery Rate and Precision
Within the concentration range of 0.004% ~ 0.3 %, the recovery rate of hexamidine,
chlorhexidine and their salts shall be between 84.1% ~ 99.7%, the relative standard
deviation shall be 1.1% ~ 7.8%.
10 Allowable Difference
The absolute difference of two independent determination results obtained under
repeatability conditions shall not exceed 10% of arithmetic mean value.