Chlorhexidine Gluconate Topical Solution
DEFINITION
Chlorhexidine Gluconate Topical Solution is prepared from Chlorhexidine Gluconate Solution. It contains NLT 90.0% and NMT 110.0% of the labeled amount of chlorhexidine gluconate (C22H30Cl2N10·2C6H12O7).
IDENTIFICATION
• A.
The retention time of the major peak for chlorhexidine from the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
• B. Thin-Layer Chromatographic Identification Test 
201
201
Standard solution:
10 mg/mL of USP Potassium Gluconate RS
Sample solution:
Nominally 20 mg/mL of chlorhexidine gluconate from the Topical Solution
Adsorbent:
0.25-mm layer of chromatographic silica gel
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 flask. Add 1.0 g of ceric sulfate, swirl to dissolve, and dilute with 2 N sulfuric acid to volume.
Analysis
Samples:
Standard solution and Sample solution
Develop the chromatogram in a 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.
for 20 min. Allow to cool, and spray with Spray reagent. Heat the plate at 110 for 10 min.
Acceptance criteria:
The principal spot from the Sample solution corresponds in color, size, and RF value to that from the Standard solution.
ASSAY
• Procedure
Solution A:
Dissolve 27.6 g of monobasic sodium 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 2000 mL. Prepare a mixture of the resulting solution and acetonitrile (70:30).
Solution B:
Acetonitrile
Mobile phase:
See the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 100 | 0 |
| 9 | 100 | 0 |
| 10 | 45 | 55 |
| 15 | 45 | 55 |
| 16 | 100 | 0 |
| 21 | 100 | 0 |
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:
50 µg/mL of USP Chlorhexidine Acetate RS in Solution A.
Sample solution:
Nominally about 80 µg/mL of chlorhexidine gluconate from the Topical Solution, prepared as follows. Transfer an amount of Topical Solution, equivalent to 40 mg of chlorhexidine gluconate, to a 100-mL volumetric flask, and dilute with methanol to volume. Further dilute a 10-mL portion of this solution with Solution A to 50 mL.
Chromatographic system
Mode:
LC
Detector:
UV 239 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
40
Flow rate:
1.5 mL/min
Injection size:
50 µL
System suitability
Sample:
System suitability solution
[Note—The approximate relative retention times for chlorhexidine and p-chloroaniline are about 1.0 and 1.3, respectively. ]
Suitability requirements
Resolution:
NLT 3.0 between chlorhexidine and p-chloroaniline
Relative standard deviation:
NMT 2.0% for the chlorhexidine peak, NMT 5.0% for the p-chloroaniline peak
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of C22H30Cl2N10·2C6H12O7 in the portion of Topical Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of chlorhexidine from the Sample solution |
| rS | = | = peak area 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) |
| Mr1 | = | = molecular weight of chlorhexidine gluconate, 897.76 |
| Mr2 | = | = molecular weight of chlorhexidine acetate, 625.55 |
Acceptance criteria:
90.0%–110.0%
IMPURITIES
Organic Impurities
• Procedure: Limit of p-Chloroaniline
Solution A, Solution B, Mobile phase, System suitability solution, and Chromatographic system:
Proceed as directed in the Assay.
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 the Topical Solution, prepared as follows. Transfer an amount of Topical Solution, equivalent to 40 mg of chlorhexidine gluconate, to a 100-mL volumetric flask, and dilute with Solution A to volume.
Analysis
Samples:
Standard solution and Sample solution
Acceptance criteria
The p-chloroaniline peak area from the Sample solution is NMT the p-chloroaniline peak area from the Standard solution (equivalent to NMT 500 ppm in the portion of Chlorhexidine Gluconate Solution used to prepare the Topical Solution).
SPECIFIC TESTS
• pH 791:
5.0–7.0
791:
5.0–7.0
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, protected from light. Store at controlled room temperature.
• USP Reference Standards 11
11
USP Chlorhexidine Acetate RS
USP p-Chloroaniline RS
USP Potassium Gluconate RS 
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Morgan Puderbaugh, B.S.
Associate Scientific Liaison 1-301-998-6833 |
(SM32010) Monographs - Small Molecules 3 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2626
Pharmacopeial Forum: Volume No. 36(1) Page 90