11/27/2020 USP-NF Acetazolamide

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O cial Status: Currently O cial on 27-Nov-2020
O cial Date: O cial as of 1-May-2020
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© 2020 USPC

Acetazolamide

C4H6N4O3S2 222.25
Acetamide, N-[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]-;
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide [59-66-5]; UNII: O3FX965V0I.

DEFINITION
Acetazolamide contains NLT 98.0% and NMT 102.0% of acetazolamide (C4H6N4O3S2), calculated on the anhydrous basis.

IDENTIFICATION
Change to read:
• A. ▲SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉, Infrared Spectroscopy: 197K▲ (CN 1-MAY-2020)
• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

ASSAY
• PROCEDURE
Mobile phase: Dissolve 4.1 g of anhydrous sodium acetate in 950 mL of water, add 20 mL of methanol and 30 mL of acetonitrile, and
mix. Adjust with glacial acetic acid to a pH of 4.0.
L
Standard solution: 0.1 mg/mL of USP Acetazolamide RS prepared as follows. Transfer USP Acetazolamide RS into a suitable
IA
volumetric ask, add 0.5 N sodium hydroxide equivalent to 10% of the nal volume, and dilute with water to volume.
Sample solution: 0.1 mg/mL of Acetazolamide prepared as follows. Transfer Acetazolamide into a suitable volumetric ask, add 0.5 N
sodium hydroxide equivalent to 10% of the nal volume, and dilute with water to volume.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
IC

Mode: LC
Detector: UV 254 nm
Column: 4.6-mm × 25-cm; packing L1
Flow rate: 2 mL/min
FF

Injection volume: 20 µL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 0.73%
O

Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of acetazolamide (C4H6N4O3S2) in the portion of Acetazolamide taken:

Result = (rU/rS) × (CS/CU) × 100

rU = peak response of acetazolamide from the Sample solution

rS = peak response of acetazolamide from the Standard solution

CS = concentration of USP Acetazolamide RS in the Standard solution (mg/mL)

CU = concentration of Acetazolamide in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the anhydrous basis

IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 0.1%
• CHLORIDE AND SULFATE 〈221〉, Chloride
Sample solution: Digest 1.5 g with 75 mL of water at about 70° for 5 min. Cool to room temperature, and lter.

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11/27/2020 USP-NF Acetazolamide

Acceptance criteria: A 25-mL portion of the ltrate shows no more chloride than corresponds to 0.10 mL of 0.020 N hydrochloric acid
(0.014%).
• CHLORIDE AND SULFATE 〈221〉, Sulfate
Sample solution: A 25-mL portion of the ltrate prepared in the test for Chloride and Sulfate, Chloride
Acceptance criteria: It shows no more sulfate than corresponds to 0.20 mL of 0.020 N sulfuric acid (0.04%).
• SELENIUM 〈291〉
Sample: 200 mg
Acceptance criteria: NMT 30 ppm
Delete the following:
▲• SILVER-REDUCING SUBSTANCES

Sample: 5 g
Analysis: Thoroughly wet the Sample with alcohol. Add 125 mL of water, 10 mL of nitric acid, and 5.0 mL of 0.1 N silver nitrate VS. Stir
with a mechanical stirrer for 30 min. Filter, add 5 mL of ferric ammonium sulfate TS to the ltrate, and titrate with 0.1 N ammonium
thiocyanate VS to a reddish-brown endpoint.
Acceptance criteria: NLT 4.8 mL of 0.1 N ammonium thiocyanate is required.▲ (USP 1-Aug-2019)

Delete the following:

▲• ORDINARY IMPURITIES 〈466〉

Standard solution: Acetone and methanol (1:1)

Test solution: Acetone and methanol (1:1)
Eluant: n-Propyl alcohol and 1 N ammonium hydroxide (88:12)
Visualization: 1▲ (USP 1-Aug-2019)
Add the following:
▲• ORGANIC IMPURITIES

Solution A: 13.61 g/L of monobasic potassium phosphate in water

Mobile phase: Methanol and Solution A (10:90)

L
Standard stock solution: 0.4 mg/mL of USP Acetazolamide RS in Mobile phase prepared as follows. Transfer a weighed amount of
USP Acetazolamide RS to a suitable volumetric ask and add methanol to ll 20% of the nal volume. Sonicate for 15 min with
occasional shaking to dissolve. Dilute with Mobile phase to volume.
IA
Standard solution: 0.005 mg/mL of USP Acetazolamide RS in Mobile phase from Standard stock solution
Sensitivity solution: 0.5 µg/mL each of USP Acetazolamide RS from Standard solution in Mobile phase
Sample solution: 1 mg/mL of Acetazolamide in Mobile phase prepared as follows. Transfer a weighed amount of Acetazolamide to a
suitable volumetric ask and add methanol to ll 20% of the nal volume. Sonicate for 15 min with occasional shaking to dissolve.
Dilute with Mobile phase to volume.
IC

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 254 nm
FF

Column: 4.6-mm × 15-cm; 3.5-µm packing L10

Column temperature: 30°
Flow rate: 1.0 mL/min
Injection volume: 30 µL
Run time: NLT 9.5 times the retention time of acetazolamide
O

System suitability
Samples: Standard solution and Sensitivity solution
Suitability requirements
Tailing factor: NMT 2.0, Standard solution
Relative standard deviation: NMT 5.0%, Standard solution
Signal-to-noise ratio: NLT 14, Sensitivity solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Acetazolamide taken:

Result = (rU/rS) × (CS/CU) × (1/F) × 100

rU = peak area of each impurity from the Sample solution

rS = peak area of acetazolamide from the Standard solution

CS = concentration of USP Acetazolamide RS in the Standard solution (mg/mL)

CU = concentration of Acetazolamide in the Sample solution (mg/mL)

F = relative response factor for each individual impurity (see Table 1)

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11/27/2020 USP-NF Acetazolamide

Acceptance criteria: See Table 1.

Table 1

Acceptance Criteria, NMT

Name Relative Retention Time Relative Response Factor (%)

Desacetyl acetazolamidea 0.45 0.48 0.3

Acetazolamide acid analogb 0.54 1.0 0.5

Acetamidothiadiazolec 0.82 1.5 0.5

Acetazolamide 1.0 1.0 —

Mercaptothiadiazole analogd 1.6 0.46 0.5

Chlorothiadiazole analoge 1.9 1.4 0.5

Acetazolamide dimerf 6.3 1.0 0.5

Any unspeci ed impurity — 1.0 0.1

Total impurities — — 1.0

a
  5-Amino-1,3,4-thiadiazole-2-sulfonamide.
b
  5-Acetamido-1,3,4-thiadiazole-2-sulfonic acid.
c  N-(1,3,4-Thiadiazol-2-yl)acetamide.
d 

f
N-(5-Mercapto-1,3,4-thiadiazol-2-yl)acetamide.
  N-(5-Chloro-1,3,4-thiadiazol-2-yl)acetamide.
L
  N,N′-{5,5′-[(Hydrosulfonylamino)sulfonyl]bis(1,3,4-thiadiazole-5,2-diyl)}diacetamide.
IA
▲ (USP 1-Aug-2019)

SPECIFIC TESTS
• WATER DETERMINATION 〈921〉, Method I: NMT 0.5%
IC

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and store at room temperature.
• USP REFERENCE STANDARDS 〈11〉
USP Acetazolamide RS
FF

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

Topic/Question Contact Expert Committee

O

ACETAZOLAMIDE Devarshi Narendra Thaker SM32020 Small Molecules 3

Scienti c Liaison

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 46(5)

Page Information:

USP43-NF38 - 73
USP42-NF37 1S - 8621
USP42-NF37 - 75

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