Dorzolamide Hydrochloride and Timolol Maleate Ophthalmic Solution
DEFINITION
Dorzolamide Hydrochloride and Timolol Maleate Ophthalmic Solution is a sterile, isotonic, buffered, slightly viscous, aqueous solution of the combination of Dorzolamide Hydrochloride and Timolol Maleate. It contains an amount of Dorzolamide Hydrochloride (C10H16N2O4S3·HCl) equivalent to NLT 90.0% and NMT 110.0% of the labeled amount of dorzolamide (C10H16N2O4S3), and also contains an amount of Timolol Maleate (C13H24N4O3S·C4H4O4) equivalent to NLT 90.0% and NMT 110.0% of the labeled amount of timolol (C13H24N4O3S). It may contain a suitable preservative.
IDENTIFICATION
• A. Thin-Layer Chromatographic Identification Test 
201
201
Standard solution A:
1.5 mg/mL of USP Timolol Maleate RS in methanol
Standard solution B:
5.0 mg/mL of USP Dorzolamide Hydrochloride RS in methanol
Sample solution:
Equivalent to 5.0 mg/mL of dorzolamide from Ophthalmic Solution in methanol
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture or equivalent
Application volume:
20 µL
Developing solvent system:
Methylene chloride, methanol, and ammonium hydroxide (80:20:1)
Analysis:
Spot the Standard solution and Sample solution approximately 2 cm from the bottom of the plate. Evaporate the solvent using a current of air. Saturate the developing tank with the Developing solvent system, and equilibrate for approximately 1 h prior to use. Develop the chromatogram in the Developing solvent system until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing tank, and allow the plate to air-dry in a fume hood. Examine the plate under short-wavelength UV light at 254 nm, or expose to iodine vapors.
Acceptance criteria:
The RF values of the principal spots from the Sample solution correspond to the principal spots obtained from Standard solution A and Standard solution B.
• B.
The retention time of the major peak in the Sample solution correspond to that in the Standard solution, as obtained in the Assay for Dorzolamide.
• C.
The retention time of the major peak in the Sample solution corresponds to that in the Standard solution, as obtained in the Assay for Timolol.
ASSAY
• Dorzolamide
Solution A:
Acetonitrile
Solution B:
0.2% (v/v) of phosphoric acid in water
Mobile phase:
See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
|---|---|---|
| 0 | 5 | 95 |
| 15.0 | 5 | 95 |
| 15.1 | 95 | 5 |
| 20.0 | 95 | 5 |
| 20.1 | 5 | 95 |
| 30.0 | 5 | 95 |
Diluent:
Acetonitrile and Solution B (5:95)
Standard solution:
0.11 mg/mL of USP Dorzolamide Hydrochloride RS and 0.5 µg/mL each of USP Dorzolamide Related Compound B RS and USP Dorzolamide Related Compound D RS in Diluent
Sample solution:
Equivalent to 0.1 mg/mL of dorzolamide from Ophthalmic Solution in Diluent
Chromatographic system
Mode:
LC
Detector:
UV 253 nm
Column:
4.6-mm × 25-cm; 5-µm packing L7
Flow rate:
1.2 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
[Note—See Table 2 for the relative retention times. ]
Suitability requirements
Resolution:
NLT 3.0 between dorzolamide and dorzolamide related compound D; and NLT 3.0 between dorzolamide and dorzolamide related compound B
Relative standard deviation:
NMT 2.0% for the dorzolamide peak
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of the labeled amount of dorzolamide (C10H16N2O4S3) in the portion of Ophthalmic Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak response of dorzolamide from the Sample solution |
| rS | = | = peak response of dorzolamide from the Standard solution |
| CS | = | = concentration of USP Dorzolamide Hydrochloride RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of dorzolamide in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of dorzolamide, 324.44 |
| Mr2 | = | = molecular weight of dorzolamide hydrochloride, 360.90 |
Acceptance criteria:
90.0%–110.0%
• Timolol
Buffer:
Transfer 22 g of monobasic sodium phosphate into a 2-L volumetric flask. Dissolve in 1995 mL of water, and adjust with phosphoric acid to a pH of 2.8. Dilute with water to volume.
Mobile phase:
Methanol and Buffer (40:60)
System suitability solution:
Transfer 88 mg of USP Timolol Maleate RS into a 50-mL volumetric flask. Transfer 8 mL of 0.1 M sodium hydroxide into the flask, mix, and heat at 70
for 15 h. Dilute with Mobile phase to volume, and mix well. Transfer 5 mL of this solution into a 25-mL volumetric flask, and add 28 mg of USP Dorzolamide Hydrochloride RS into the same flask. Dilute with Mobile phase to volume. [Note—The preparation generates timolol impurity G and timolol impurity B. ]
for 15 h. Dilute with Mobile phase to volume, and mix well. Transfer 5 mL of this solution into a 25-mL volumetric flask, and add 28 mg of USP Dorzolamide Hydrochloride RS into the same flask. Dilute with Mobile phase to volume. [Note—The preparation generates timolol impurity G and timolol impurity B. ]
Standard solution:
0.35 mg/mL of USP Timolol Maleate RS in Mobile phase
Sample solution:
Equivalent to 0.25 mg/mL of timolol from Ophthalmic Solution in Mobile phase
Chromatographic system
Mode:
LC
Detector:
UV 295 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Column temperature:
40
Flow rate:
1 mL/min
Injection volume:
20 µL
System suitability
Sample:
System suitability solution
[Note—See Table 3 for the relative retention times. ]
Suitability requirements
Resolution:
NLT 1.5 between timolol impurity G and timolol impurity B
Relative standard deviation:
NMT 2.5% for timolol for five replicate injections
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of the labeled amount of timolol (C13H24N4O3S) in the portion of Ophthalmic Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak response from the Sample solution |
| rS | = | = peak response from the Standard solution |
| CS | = | = concentration of USP Timolol Maleate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of timolol in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of timolol, 316.42 |
| Mr2 | = | = molecular weight of timolol maleate, 432.49 |
Acceptance criteria:
90.0%–110.0%
IMPURITIES
• Organic Impurities: Dorzolamide Hydrochloride
Mobile phase, Standard solution, Sample solution, and Chromatographic system:
Proceed as directed in the Assay for Dorzolamide.
System suitability
Sample:
Standard solution
Suitability requirements
Resolution:
NLT 3.0 between dorzolamide and dorzolamide related compound D; and NLT 3.0 between dorzolamide and dorzolamide related compound B
Relative standard deviation:
NMT 2.0% for dorzolamide
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of dorzolamide related compound D in the portion of Ophthalmic Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of dorzolamide related compound D from the Sample solution |
| rS | = | = peak area of dorzolamide related compound D from the Standard solution |
| CS | = | = concentration of USP Dorzolamide Related Compound D RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of dorzolamide in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of dorzolamide, 324.44 |
| Mr2 | = | = molecular weight of dorzolamide related compound D, 332.85 |
Calculate the percentage of dorzolamide related compound B in the portion of Ophthalmic Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of dorzolamide related compound B from the Sample solution |
| rS | = | = peak area of dorzolamide related compound B from the Standard solution |
| CS | = | = concentration of USP Dorzolamide Related Compound B RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of dorzolamide in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of dorzolamide, 324.44 |
| Mr2 | = | = molecular weight of dorzolamide related compound B, 360.90 |
Calculate the percentage of any individual unspecified impurity in the portion of Ophthalmic Solution taken:
Result = (rU/rT) × 100
| rU | = | = peak area of any individual unspecified impurity from the Sample solution |
| rT | = | = sum of the all peak areas from the Sample solution |
Acceptance criteria
Individual impurities:
See Table 2. Disregard any impurity peaks less than 0.10%.
Table 2
| Name | Relative Retention Time |
Acceptance Criteria, NMT (%) |
|---|---|---|
| Maleic acid | 0.33 | Disregard |
| Dorzolamide related compound Da | 0.83 | 0.5 |
| Dorzolamide | 1.00 | — |
| Dorzolamide related compound Bb | 1.17 | 2.0 |
| Any individual unspecified impurity | — | 0.5 |
| Total impurities | — | 3.0 |
|
a
(4S,6S)-4-Amino-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
b
(4RS,6SR)-4-(Ethylamino)-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
|
||
• Organic Impurities: Timolol Maleate
Mobile phase, System suitability solution, and Sample solution:
Proceed as directed in the Assay for Timolol.
Standard stock solution:
Use the Standard solution as prepared in the Assay.
Standard solution:
3.5 µg/mL of USP Timolol Maleate RS from the Standard stock solution in Mobile phase
Chromatographic system
Prepare as directed in the Assay. In addition the run time is at least two times the retention time of timolol.
System suitability
Sample:
System suitability solution
Suitability requirements
Resolution:
NLT 1.5 between timolol impurity G and timolol impurity B
Relative standard deviation:
NMT 2.5% for timolol for five replicate injections
Analysis
Samples:
Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Ophthalmic Solution taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100
| rU | = | = peak area of each impurity from the Sample solution |
| rS | = | = peak area of timolol from the Standard solution |
| CS | = | = concentration of USP Timolol Maleate RS in the Standard solution (mg/mL) |
| CU | = | = nominal concentration of timolol in the Sample solution (mg/mL) |
| Mr1 | = | = molecular weight of timolol, 316.42 |
| Mr2 | = | = molecular weight of timolol maleate, 432.49 |
Acceptance criteria
Individual impurities:
See Table 3. Disregard any impurity peaks less than 0.10%.
Table 3
| Name | Relative Retention Time |
Acceptance Criteria, NMT (%) |
|---|---|---|
| Dorzolamide and maleic acid | 0.49 | Disregard |
| Timolol impurity Ga | 0.58 | 0.5 |
| Timolol impurity Bb | 0.70 | 1.0 |
| Timolol | 1.00 | — |
| Timolol impurity Dc | 1.51 | 0.5 |
| Any individual unspecified impurity | — | 0.6 |
| Total impurities | — | 2.0 |
|
a
4-Morpholino-1,2,5-thiadiazol-3-ol 1-oxide.
b
3-(tert-Butylamino)-2-(4-morpholino-1,2,5-thiadiazol-3-yloxy)propan-1-ol.
c
4-Morpholino-1,2,5-thiadiazol-3-ol.
|
||
SPECIFIC TESTS
• Sterility Tests 71:
Meets the requirements
71:
Meets the requirements
• pH 791:
5.4–5.9, at 22
791:
5.4–5.9, at 22
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight containers protected from light, at controlled room temperature.
• USP Reference Standards 11
11
USP Dorzolamide Hydrochloride RS 
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USP Dorzolamide Related Compound B RS
(4R,6S)-4-(Ethylamino)-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
C10H16N2O4S3·HCl 360.90
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(4R,6S)-4-(Ethylamino)-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
C10H16N2O4S3·HCl 360.90
USP Dorzolamide Related Compound D RS
(4S,6S)-4-Amino-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
C8H12N2O4S3·HCl 332.85
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(4S,6S)-4-Amino-6-methyl-5,6-dihydro-4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide hydrochloride.
C8H12N2O4S3·HCl 332.85
USP Timolol Maleate RS
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Feiwen Mao, M.S.
Senior Scientific Liaison (301) 816-8320 |
(SM32010) Monographs - Small Molecules 3 |
| 71 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison (301) 816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 3204
Pharmacopeial Forum: Volume No. 38(3)