11/9/2020 USP-NF Chlorhexidine Gluconate Topical Gel

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Printed by: Shruti Kharidia
O cial Status: Currently O cial on 09-Nov-2020
O cial Date: O cial as of 1-Aug-2019
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© 2020 USPC

Chlorhexidine Gluconate Topical Gel

DEFINITION
Chlorhexidine Gluconate Topical Gel is prepared from Chlorhexidine Gluconate Solution. It contains NLT 90.0% and NMT 110.0% of the
labeled amount of chlorhexidine gluconate (C22H30Cl2N10 · 2C6H12O7). [NOTE—The U.S. Food and Drug Administration has not reviewed
the safety and e cacy of Chlorhexidine Gluconate Topical Gel and it is not approved for marketing in the United States.]

IDENTIFICATION

Change to read:
• A. ▲The UV spectrum of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the
Assay.▲ (GH 1-Aug-2019)

• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

Delete the following:

▲• C. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST 〈201〉

Diluent: Acetonitrile and water (1:1)

Standard solution: 10 mg/mL of USP Potassium Gluconate RS in Diluent
Sample solution: Nominally 20 mg/mL of chlorhexidine gluconate from Topical Gel, prepared as follows. Transfer a suitable amount
of Topical Gel, equivalent to 500 mg of chlorhexidine gluconate, to a 25-mL volumetric ask. Add a su cient quantity of Diluent,

Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica gel
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sonicate with intermittent shaking for 30 min, and dilute with Diluent to volume. Centrifuge the solution for 5 min at 3000 rpm.
IA
Application volume: 10 µL
Developing solvent system: Alcohol, ethyl acetate, ammonium hydroxide, and water (5:1:1:3)
Spray reagent: Dissolve 2.5 g of ammonium molybdate in 50 mL of 2 N sulfuric acid in a 100-mL volumetric ask. Add 1.0 g of ceric
sulfate, swirl to dissolve, and dilute with 2 N sulfuric acid to volume.
IC

Analysis
Samples: Standard solution and Sample solution
Develop the chromatogram in the Developing solvent system until the solvent front has moved 10 cm from the point of spotting.
Remove the plate from the chamber and dry at 110° for 20 min. Allow to cool and spray with Spray reagent. Heat the plate at
110° for 10 min.
FF

Acceptance criteria: The principal spot of the Sample solution corresponds in color, size, and RF value to that of the Standard
solution.▲ (GH 1-Aug-2019)

ASSAY

Change to read:
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• PROCEDURE
Buffer: Dissolve 27.6 g of sodium dihydrogen phosphate and 10 mL of triethylamine in 1.5 L of water. Adjust with phosphoric acid to a
pH of 3.0 and dilute with water to 2 L.
Solution A: Acetonitrile and Buffer (30:70)
Solution B: Acetonitrile
Mobile phase: See Table 1.

Table 1

Time (min) Solution A (%) Solution B (%)

0 100 0

9 100 0

10 45 55

15 45 55

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11/9/2020 USP-NF Chlorhexidine Gluconate Topical Gel

Time (min) Solution A (%) Solution B (%)

16 100 0

21 100 0

Standard solution: 50 µg/mL of USP Chlorhexidine Acetate RS in Solution A

Sample stock solution: Nominally 0.4 mg/mL of chlorhexidine gluconate from Topical Gel, prepared as follows. Transfer a suitable
amount of Topical Gel, equivalent to 40 mg of chlorhexidine gluconate, to a 100-mL volumetric ask. Add about 70 mL of Solution A,
sonicate with intermittent shaking for 30 min, and dilute with Solution A to volume.
Sample solution: Nominally 80 µg/mL of chlorhexidine gluconate from the Sample stock solution in Solution A
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 239 nm. ▲For Identi cation A, use a diode array detector in the range of 200–400 nm.▲ (GH 1-Aug-2019)
Column: 4.6-mm × 25-cm; 5-µm packing L1
Column temperature: 40°
Flow rate: 1.5 mL/min
Injection volume: 50 µL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution

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Calculate the percentage of the labeled amount of chlorhexidine gluconate (C22H30Cl2N10 · 2C6H12O7) in the portion of Topical Gel
taken:
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Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU = peak response of chlorhexidine from the Sample solution

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rS = peak response of chlorhexidine from the Standard solution

CS = concentration of USP Chlorhexidine Acetate RS in the Standard solution (µg/mL)

CU = nominal concentration of chlorhexidine gluconate in the Sample solution (µg/mL)

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Mr1 = molecular weight of chlorhexidine gluconate, 897.76

Mr2 = molecular weight of chlorhexidine acetate, 625.55

Acceptance criteria: 90.0%–110.0%

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IMPURITIES

Change to read:
• LIMIT OF p-CHLOROANILINE
Solution A, Solution B, Mobile phase, and Chromatographic system: Proceed as directed in the Assay.
System suitability solution: 50 µg/mL of USP Chlorhexidine Acetate RS and 1 µg/mL of USP p-Chloroaniline RS in Solution A
Standard solution: 1.0 µg/mL of USP p-Chloroaniline RS in Solution A
Sample solution: Nominally 0.4 mg/mL of chlorhexidine gluconate from Topical Gel, prepared as follows. Transfer a suitable amount
of Topical Gel, equivalent to 40 mg of chlorhexidine gluconate, to a 100-mL volumetric ask. Add about 70 mL of Solution A,
sonicate with intermittent shaking for 30 min, and dilute with Solution A to volume. Centrifuge the solution.
System suitability
Samples: System suitability solution and Standard solution
▲[NOTE—The relative retention times for chlorhexidine and p-chloroaniline are 1.0 and 1.3, respectively.]
▲ (GH 1-AUG-2019)
Suitability requirements
Resolution: NLT 3.0 between chlorhexidine and p-chloroaniline, System suitability solution
Relative standard deviation: NMT 5.0%, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of p-chloroaniline in the portion of Topical Gel taken:

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11/9/2020 USP-NF Chlorhexidine Gluconate Topical Gel

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of p-chloroaniline from the Sample solution

rS = peak response of p-chloroaniline from the Standard solution

CS = concentration of USP p-Chloroaniline RS in the Standard solution (mg/mL)

CU = nominal concentration of chlorhexidine gluconate in the Sample solution (mg/mL)

Acceptance criteria: NMT 0.35%

Add the following:

▲• Organic Impurities

Solution A: 0.1% (v/v) tri uoroacetic acid in acetonitrile

Solution B: 0.1% (v/v) tri uoroacetic acid in water
Solution C: Solution A and Solution B (20:80)
Solution D: Solution A and Solution B (90:10)
Mobile phase: See Table 2.

Table 2

Time (min) Solution C (%) Solution D (%)

0 100 0

2 100 0

32 80 20

37 80 L 20
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47 70 30

54 70 30

55 100 0
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60 100 0

Diluent 1: Acetonitrile, water, and tri uoroacetic acid (10:90:0.1)

FF

Diluent 2: Acetonitrile and water (50:50)

System suitability solution: 0.2 mg/mL of USP Chlorhexidine System Suitability Mixture RS in Diluent 1
Sensitivity solution: 0.07 µg/mL of USP Chlorhexidine Acetate RS in Diluent 1
Sample stock solution: Nominally 0.8 mg/mL of chlorhexidine gluconate in Diluent 2 from Topical Gel prepared as follows. Transfer a
suitable amount of Topical Gel, equivalent to 80 mg of chlorhexidine gluconate, to a 100-mL volumetric ask. Add about 50 mL of
O

Diluent 2, sonicate with intermittent shaking for 30 min, and dilute with Diluent 2 to volume.
Sample solution: Nominally 0.2 mg/mL of chlorhexidine gluconate from Sample stock solution in Diluent 1. Pass through a suitable
lter of 0.45-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm × 25-cm; base-deactivated 5-µm packing L1
Column temperature: 30°
Flow rate: 1 mL/min
Injection volume: 50 µL
System suitability
Samples: System suitability solution and Sensitivity solution
[NOTE—See Table 3 for relative retention times.]
Suitability requirements
Resolution: NLT 1.5 between any pair of eluting peaks, System suitability solution
Signal-to-noise ratio: NLT 10 for chlorhexidine, Sensitivity solution
Analysis
Sample: Sample solution

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11/9/2020 USP-NF Chlorhexidine Gluconate Topical Gel

Calculate the percentage of each degradation product in the portion of Topical Gel taken:

Result = (rU/rT) × F × 100

rU = peak area of each degradation product from the Sample solution

rT = total peak area, determined by summing the chlorhexidine peak and all other peaks above the Sensitivity solution level,
except p-chloroaniline

F = relative response factor (see Table 3)

Calculate the percentage of total degradation products as the sum of the percentage of each degradation product and p-
chloroaniline from the Limit of p-Chloroaniline test.
Acceptance criteria: See Table 3.

Table 3

Acceptance Criteria, NMT

Name Relative Retention Time Relative Response Factor (%)

p-Chloroanilinea 0.15 — —

Chlorhexidine oxazinone
analogb 0.23 1.0 0.2

Chlorhexidine aminec 0.25 1.0 0.6

Chlorhexidine guanidined 0.35 1.0 2.0

Chlorhexidine ureae 0.36 1.0 0.2

p-Chlorophenyl ureaf 0.50 L 1.34 0.3

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Chlorhexidine nitrileg 0.60 1.0 0.4

Chlorhexidine dimerh 0.85 1.0 0.5

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o-Chlorhexidinei 0.90 1.0 0.4

Chlorhexidine glucityl triazinej 0.96 1.0 0.7

Chlorhexidine 1.0 — —
FF

Oxochlorhexidinek 1.4 1.0 0.7

Any unspeci ed degradation —

product 1.0 0.2
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Total degradation products — — 4.0▲ (GH 1-Aug-2019)

a  Do not include this peak in the sum of the total peak areas for the calculation of degradation products.
b  (5R,6S)-2-[(4-Chlorophenyl)amino]-5-hydroxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]-5,6-dihydro-4H-1,3-oxazin-4-one.
c  1-(6-Aminohexyl)-5-(4-chlorophenyl)biguanide.
d  1-[6-(Carbamimidoylamino)hexyl]-5-(4-chlorophenyl)biguanide.
e
  N-{[6-({[(4-Chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]carbamimidoyl}urea.
f
  1-(4-Chlorophenyl)urea.
g
  1-(4-Chlorophenyl)-5-[6-[(cyanocarbamimidoyl)amino]hexyl]biguanide.
h  1,5-Bis[5-(4-chlorophenyl)biguanidylhexyl]biguanide.
i  1-(2-Chlorophenyl)-5-[6-({[(4-chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]biguanide.
j  1-(4-Chlorophenyl)-5-[6-({4-[(4-chlorophenyl)amino]-6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-1,3,5-triazin-2-
yl}amino)hexyl]biguanide.
k  N-(4-Chlorophenyl)-N′-{[6-({[(4-chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]carbamimidoyl}urea.

SPECIFIC TESTS
• PH 〈791〉

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11/9/2020 USP-NF Chlorhexidine Gluconate Topical Gel

Sample solution: Nominally 1% of chlorhexidine gluconate from Topical Gel in water

Acceptance criteria: 5.0–7.0

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed containers, protected from light. Store at controlled room temperature.

Change to read:
• USP REFERENCE STANDARDS 〈11〉
USP Chlorhexidine Acetate RS
▲ USP Chlorhexidine System Suitability Mixture RS

This is a mixture of chlorhexidine and the following impurities (other impurities may also be present).
Chlorhexidine gluconate;
1,1′-Hexamethylenebis[5-(p-chlorophenyl)biguanide] di-D-gluconate.
C22H30Cl2N10 · 2C6H12O7 897.76
Chlorhexidine nitrile;
1-(4-Chlorophenyl)-5-[6-[(cyanocarbamimidoyl)amino]hexyl]biguanide. C16H24ClN9 377.88
Chlorhexidine urea;
N-{[6-({[(4-Chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]carbamimidoyl}urea. C16H26ClN9O 395.89
p-Chlorophenyl urea;
1-(4-Chlorophenyl)urea. C7H7ClN2O 170.60
Chlorhexidine amine;
1-(6-Aminohexyl)-5-(4-chlorophenyl)biguanide. C14H23ClN6 310.83
Chlorhexidine dimer;
1,5-Bis[5-(4-chlorophenyl)biguanidylhexyl]biguanide. C30H47Cl2N15 688.70
Chlorhexidine glucityl triazine;
1-(4-Chlorophenyl)-5-[6-({4-[(4-chlorophenyl)amino]-6-[(1S,2R,3R,4R)-1,2,3,4,5-pentahydroxypentyl]-1,3,5-triazin-2-
yl}amino)hexyl]biguanide. C28H38Cl2N10O5 665.57
Oxochlorhexidine;
N-(4-Chlorophenyl)-N′-{[6-({[(4-
chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]carbamimidoyl}urea. C22H29Cl2N9O 506.43
Chlorhexidine oxazinone analog;

one. C13H15ClN2O6 330.72

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(5R,6S)-2-[(4-Chlorophenyl)amino]-5-hydroxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]-5,6-dihydro-4H-1,3-oxazin-4-
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Chlorhexidine guanidine;
1-[6-(Carbamimidoylamino)hexyl]-5-(4-chlorophenyl)biguanide. C15H25ClN8 352.87
o-Chlorhexidine;
1-(2-Chlorophenyl)-5-[6-({[(4-chlorophenyl)carbamimidoyl]carbamimidoyl}amino)hexyl]biguanide. C22H30Cl2N10 505.45▲
IC

(GH 1-Aug-2019)
USP p-Chloroaniline RS
▲
▲ (GH 1-Aug-2019)
FF

Auxiliary Information- Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

CHLORHEXIDINE GLUCONATE TOPICAL Sujatha Ramakrishna SM32020 Small Molecules 3

O

GEL Principal Scienti c Liaison

Page Information:

USP43-NF38 - 4741
USP42-NF37 1S - 8847
USP42-NF37 - 4699

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