Chlorhexidine Gluconate Solution
C22H30Cl2N10·2C6H12O7 897.76
2,4,11,13-Tetraazatetradecanediimidamide, N,N¢¢-bis(4-chlorophenyl)-3,12-diimino-, di-d-gluconate;
1,1¢-Hexamethylenebis[5-(p-chlorophenyl)biguanide] di-d-gluconate
[18472-51-0].
C22H30Cl2N10·2C6H12O7 897.76
2,4,11,13-Tetraazatetradecanediimidamide, N,N¢¢-bis(4-chlorophenyl)-3,12-diimino-, di-d-gluconate;
1,1¢-Hexamethylenebis[5-(p-chlorophenyl)biguanide] di-d-gluconate
[18472-51-0].DEFINITION
Chlorhexidine Gluconate Solution is an aqueous solution of chlorhexidine gluconate. It contains NLT 19.0% and NMT 21.0% of C22H30Cl2N10·2C6H12O7 (w/v).
IDENTIFICATION
• A. Infrared Absorption 
197K
197K
Standard solution:
5 mg/mL of USP Chlorhexidine RS in 70% alcohol. Recrystallize this solution, and dry the crystals at 105
for 1 h.
for 1 h.
Sample solution:
To 1 mL of Solution add 40 mL of water, and cool in ice. Add 10 N sodium hydroxide, dropwise with stirring, until the solution produces a red color on thiazol yellow paper, and add 1 mL in excess. Filter, wash the precipitate with water until the washings are free from alkali, recrystallize the residue from 70% alcohol, and dry the crystals at 105 for 1 h.
for 1 h.
• B. Thin-Layer Chromatographic Identification Test 201
201
Standard solution:
20 mg/mL of USP Potassium Gluconate RS
Sample solution:
Dilute 10 mL of Solution with water to 50 mL. This solution contains 40 mg/mL of chlorhexidine gluconate.
Adsorbent:
0.25-mm layer of chromatographic silica gel
Application volume:
5 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
Diluent:
27.6 g of monobasic sodium phosphate in 1.5 L of water. Adjust with phosphoric acid to a pH of 3.0, and dilute with water to 2000 mL.
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. Mix 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 stock solution:
Transfer 5.0 mL of Solution to a 250-mL volumetric flask, and dilute with water to volume.
Sample solution:
Transfer 5.0 mL of the Sample stock solution to a 250-mL volumetric flask, and dilute with Solution A.
Chromatographic system
Mode:
LC
Detector:
UV 239 nm
Column:
4.6-mm × 25-cm; base-deactivated 5-µm packing L1
Column temperature:
40
Flow rate:
1.5 mL/min
Injection size:
50 µL
System suitability
[Note—The approximate relative retention times for chlorhexidine and p-chloroaniline are about 1.0 and 1.3, respectively. ]
Sample:
System suitability solution
Suitability requirements
Resolution:
NLT 3.0 between chlorhexidine and p-chloroaniline
Relative standard deviation:
NMT 2.0% from the chlorhexidine peak, and NMT 5.0% from the p-chloroaniline peak
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage (w/v) of C22H30Cl2N10·2C6H12O7 in the portion of Solution taken:
Result = (rU/rS) × (0.25 × CS) × (Mr1/Mr2)
| rU | = | = peak area response of chlorhexidine from the Sample solution |
| rS | = | = peak area response of chlorhexidine from the Standard solution |
| CS | = | = concentration of USP Chlorhexidine Acetate RS in the Standard solution (µg/mL) |
| Mr1 | = | = molecular weight of chlorhexidine gluconate, 897.76 |
| Mr2 | = | = molecular weight of chlorhexidine acetate, 625.55 |
Acceptance criteria:
19.0%–21.0% (w/v)
IMPURITIES
Organic Impurities
• Procedure 1
Diluent, Solution A, Solution B, and Mobile phase:
Proceed as directed in the Assay.
Sample stock solution:
Transfer 5.0 mL of Solution to a 100-mL volumetric flask, and dilute with water to volume.
Sample solution:
Transfer 5.0 mL of the Sample stock solution to a 25-mL volumetric flask, and dilute with Diluent to volume. This solution contains 2 mg/mL of chlorhexidine gluconate.
Reference solution A:
Transfer 3.0 mL of the Sample solution to a 100-mL volumetric flask, and dilute with Diluent to volume. This solution contains 0.06 mg/mL of chlorhexidine gluconate.
Reference solution B:
Transfer 2.0 mL of Reference solution A to a 100-mL volumetric flask, and dilute with Diluent to volume. This solution contains 0.0012 mg/mL of chlorhexidine gluconate.
System suitability solution:
Transfer 10 mg of USP Chlorhexidine Related Compounds RS to a 10-mL volumetric flask. Dissolve in 2 mL of acetonitrile, and dilute with Diluent to volume.
Chromatographic system:
Proceed as directed in the Assay, except the Injection size and chromatograph are programmed as shown in the gradient table below.
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 100 | 0 |
| 15 | 100 | 0 |
| 16 | 45 | 55 |
| 21 | 45 | 55 |
| 22 | 100 | 0 |
| 27 | 100 | 0 |
Injection size:
20 µL
System suitability
Sample:
System suitability solution
[Note—The relative retention times for the main related compound peak and chlorhexidine are 0.6 and 1.0, respectively. ]
Suitability requirements
Resolution:
The two peaks between the main related compound peak and the chlorhexidine peak should be at least partially resolved from each other and completely resolved from the chlorhexidine peak.
Analysis
Samples:
Sample solution, Reference solution A, and Reference solution B
Examine the chromatogram from the Sample solution.
Acceptance criteria:
The sum of the peak areas, other than chlorhexidine and any peak areas less than that obtained for chlorhexidine in the chromatogram from Reference solution B, is NMT the peak area for chlorhexidine in the chromatogram from Reference solution A (3.0%).
• Procedure 2: Limit of p-Chloroaniline
Diluent, 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 Diluent
Sample stock solution:
Transfer 5.0 mL of Solution to a 100-mL volumetric flask, and dilute with water to volume.
Sample solution:
Transfer 10.0 mL of Sample stock solution to a 250-mL volumetric flask, and dilute with Diluent to volume. This solution contains 0.4 mg/mL of chlorhexidine gluconate.
Analysis
Samples:
Standard solution and Sample solution
Acceptance criteria:
NMT 500 µg/mL
The p-chloroaniline peak area response of the Sample solution is NMT the p-chloroaniline peak area response of the Standard solution.
SPECIFIC TESTS
• Specific Gravity 841:
1.06–1.07
841:
1.06–1.07
• pH 791:
5.5–7.0, when diluted 1 in 20 with water
791:
5.5–7.0, when diluted 1 in 20 with water
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers, protected from light, at controlled room temperature.
• USP Reference Standards 11
11
USP Chlorhexidine RS 
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USP Chlorhexidine Acetate RS
USP Chlorhexidine Related Compounds 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 2624
Pharmacopeial Forum: Volume No. 36(1) Page 88