Amiloride Hydrochloride and Hydrochlorothiazide Tablets

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Amiloride Hydrochloride and Hydrochlorothiazide Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of amiloride hydrochloride

(C₆H₈ClN₇O · HCl) and hydrochlorothiazide (C₇H₈ClN₃O₄S₂).

2 IDENTIFICATION

A. The retention times of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.

B. The UV spectrum of the amiloride peak of the Sample solution exhibits maxima and minima at the same wavelengths as that of the Standard solution, as obtained in the Assay. The UV spectrum of the hydrochlorothiazide peak of the Diluted sample solution exhibits maxima and minima at the same wavelengths as that of the Diluted standard solution, as obtained in the Assay. (USP 1-Aug-2019)

3 ASSAY

Change to read:

3.1 Procedure

Buffer: Dissolve 136 g of monobasic potassium phosphate in 800 mL of water. Adjust with phosphoric acid to a pH of 3.0. Dilute with water to 1000 mL.

Mobile phase: Methanol, water, and Buffer (25:71:4)

Standard stock solution: 1.0 mg/mL of USP Amiloride Hydrochloride RS in methanol

Standard solution: 0.1 mg/mL of USP Amiloride Hydrochloride RS and 1 mg/mL of USP Hydrochlorothiazide RS, prepared as follows.

Transfer 10.0 mL of the Standard stock solution to a 100-mL volumetric flask containing 100 mg of USP Hydrochlorothiazide RS and 20.0mL of methanol. Add 4.0 mL of 1 N hydrochloric acid, and dilute with water to volume.

Diluted standard solution: 0.005 mg/mL of USP Amiloride Hydrochloride RS and 0.05 mg/mL of USP Hydrochlorothiazide RS, from the Standard solution, diluted with water (USP 1-Aug-2019)

Sample solution: Nominally 0.1 mg/mL of amiloride hydrochloride and 1 mg/mL of hydrochlorothiazide, prepared as follows. (USP 1-Aug-2019) Transfer an equivalent to 5 mg of amiloride hydrochloride and 50 mg of hydrochlorothiazide (USP 1-Aug-2019) from powdered Tablets (NLT 20) to a 50-mL volumetric flask. Add 15.0 mL of methanol and 2.0 mL of 1 N hydrochloric acid. Sonicate for 10 min, dilute with water to volume, sonicate for an additional 10 min, and filter.

Diluted sample solution: Nominally 0.005 mg/mL of amiloride hydrochloride and 0.05 mg/mL of hydrochlorothiazide, from the Sample solution, diluted with water (USP 1-Aug-2019)

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 286 nm. For Identification B, use a diode array detector in the range of 200–400 nm. (USP 1-Aug-2019)

Column: 3.9-mm × 30-cm; 10-μm (USP 1-Aug-2019) packing L1

Flow rate: 1 mL/min

Injection volume: 10 μL

Run time: NLT 2 times the retention time of amiloride (USP 1-Aug-2019)

3.3 System suitability

Sample: Standard solution

[Note-The relative retention times for hydrochlorothiazide and amiloride

(USP 1-Aug-2019) are about 0.7 and 1.0, respectively.]

3.4 Suitability requirements

Resolution: NLT 2.0 between hydrochlorothiazide and amiloride  (USP 1-Aug-2019)

Relative standard deviation: NMT 2.0% for hydrochlorothiazide and amiloride

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of amiloride hydrochloride (C₆H₈ClN₇O · HCl) in the portion of Tablets taken:

Result = (r_u/r_s) × (C_s/C_u)× 100

r_u = peak response of amiloride (USP 1-Aug-2019) from the Sample solution

r_s = peak response of amiloride (USP 1-Aug-2019) from the Standard solution

C_s = concentration of USP Amiloride Hydrochloride RS (USP 1-Aug-2019) in the Standard solution (mg/mL)

C_u = nominal concentration of amiloride hydrochloride in the Sample solution (mg/mL)

Calculate the percentage of the labeled amount of hydrochlorothiazide (C₇H₈ClN₃O₄S₂) in the portion of Tablets taken:

Result = (r_u/r_s) × (C_s/C_u) × 100

r_u = peak response of hydrochlorothiazide from the Sample solution

r_s = peak response of hydrochlorothiazide from the Standard solution

C_s = concentration of USP Hydrochlorothiazide RS in the Standard solution (mg/mL)

C_u = nominal concentration of hydrochlorothiazide in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0% of the labeled amount of amiloride hydrochloride (C₆H₈ClN₇O · HCl) and hydrochlorothiazide (C₇H₈ClN₃O₄S₂)

4 PERFORMANCE TESTS

Change to read:

Dissolution 〈711〉

Medium: 0.1 N hydrochloric acid; 900 mL

Apparatus 2: 50 rpm

Time: 30 min

Determine the percentage of the labeled amount of amiloride hydrochloride and hydrochlorothiazide dissolved using the Spectrophotometric procedure or the Chromatographic procedure.

Spectrophotometric procedure (USP 1-Aug-2019)

Amiloride standard solution: 0.005 mg/mL of USP Amiloride Hydrochloride RS, prepared as follows. Transfer 52 mg of USP Amiloride Hydrochloride RS to (USP 1-Aug-2019) a 200-mL volumetric flask. Dissolve in and dilute with methanol to volume. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, and dilute with Medium to volume.

Hydrochlorothiazide standard solution: 0.01 mg/mL of USP Hydrochlorothiazide RS, prepared as follows. Transfer 100 mg of USP Hydrochlorothiazide RS to a 100-mL volumetric flask. Dissolve in and dilute with methanol to volume. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, and dilute with Medium to volume. Transfer 10.0 mL of the resulting solution to a 50-mL volumetric flask, and dilute with Medium to volume.

Sample solution A: Pass a portion of the solution under test through a glass fiber filter of 0.45-μm pore size.

Sample solution B: Transfer 5.0 mL of Sample solution A to a 25-mL volumetric flask, and dilute with Medium to volume.

Blank: Medium

Instrumental conditions

Mode: UV-Vis

Analytical wavelengths: 363 nm for amiloride hydrochloride; 270 nm for hydrochlorothiazide

4.1 Analysis

Samples: Amiloride standard solution, Hydrochlorothiazide standard solution, Sample solution A, and Sample solution B

Calculate the percentage of the labeled amount (USP 1-Aug-2019) of amiloride hydrochloride (C₆H₈ClN₇O · HCl) dissolved:

Result = [(A_u× C_s× V)/(A_s× L)] × 100

A_u = absorbance of Sample solution A

C_s = concentration of USP Amiloride Hydrochloride RS in (USP 1-Aug-2019) the Amiloride standard solution (mg/mL)

V = volume of Medium, 900 mL

A_s = absorbance of the Amiloride standard solution

L = label claim of amiloride hydrochloride (USP 1-Aug-2019) (mg/Tablet)

Calculate F, the ratio of absorbance of the Amiloride standard solution at 270 nm to that at 363 nm:

Result = A_s270/A_s363

A_s270 = absorbance of the Amiloride standard solution at 270 nm

A_s363 = absorbance of the Amiloride standard solution at 363 nm

Calculate A , the absorbance of Sample solution B at 270 nm, corrected for the interference of amiloride:

Result = A_u270 − [(A_u363 × F)/D]

A _u270= absorbance of Sample solution B at 270 nm

A_u363 = absorbance of Sample solution A at 363 nm

F = ratio of the absorbance of the Amiloride standard solution at 270 nm to that at 363 nm

D = dilution factor of Sample solution B, 5 (USP 1-Aug-2019)

Calculate the percentage of the labeled amount (USP 1-Aug-2019) of hydrochlorothiazide (C₇H₈ClN₃O₄S₂) dissolved:

Result = [(A_uc× C_s× V × D)/(A_s × L)] × 100

A_uc = corrected absorbance of Sample solution B (USP 1-Aug-2019) at 270 nm

C_s = concentration of USP Hydrochlorothiazide RS in (USP 1-Aug-2019) the Hydrochlorothiazide standard solution (mg/mL)

V = volume of Medium, 900 mL

D = dilution factor of Sample solution B, 5

A_s = absorbance of the Hydrochlorothiazide standard solution

L = label claim of hydrochlorothiazide (mg/Tablet)

Chromatographic procedure (USP 1-Aug-2019)

Buffer and Mobile phase: Prepare as directed in the Assay.

Standard stock solution: Use the Standard solution from the Assay.

Standard solution: 0.005 mg/mL of USP Amiloride Hydrochloride RS and 0.05 mg/mL of USP Hydrochlorothiazide RS, from the Standard stock solution, diluted with Medium

Sample solution: Pass a portion of the solution under test through a filter of 0.45-μm pore size.

4.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 286 nm

Column: 4.6-mm × 15-cm; 5-μm (USP 1-Aug-2019) packing L1

Flow rate: 1 mL/min

Injection volume: 50 μL

4.3 System suitability

Sample: Standard solution

4.4 Suitability requirements

Resolution: NLT 2.0 (USP 1-Aug-2019) between hydrochlorothiazide and amiloride

Relative standard deviation: NMT 2.0%

4.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled (USP 1-Aug-2019) amount of amiloride hydrochloride (C₆H₈ClN₇O · HCl) and hydrochlorothiazide (C₇H₈ClN₃O₄S₂) dissolved:

Result = (r_u/r_s) × (C_s/L) × V × 100

r_u = peak response of amiloride or hydrochlorothiazide from the Sample solution

r_s = peak response of amiloride or hydrochlorothiazide from the Standard solution

C_s = concentration of USP Amiloride Hydrochloride RS or USP Hydrochlorothiazide RS (USP 1-Aug-2019) in the Standard solution (mg/mL)

L = label claim of amiloride hydrochloride or hydrochlorothiazide (mg/Tablet)

V = volume of Medium, 900 mL

Tolerances: NLT 80% (Q) of the labeled amount of amiloride hydrochloride (C₆H₈ClN₇O · HCl) and NLT 75% (Q) of the labeled amount of hydrochlorothiazide C₇H₈ClN₃O₄S₂) are dissolved.

Change to read:

Uniformity of Dosage Units 〈905〉, Content Uniformity: Meet the requirements  (USP 1-Aug-2019)

5 IMPURITIES

Change to read:

Limit of Benzothiadiazine Related Compound A (USP 1-Aug-2019)

Buffer, Mobile phase, and Sample solution: Prepare as directed in the Assay.

System suitability solution: Use the Standard solution from the Assay. (USP 1-Aug-2019)

Standard solution: 0.01 mg/mL  (USP 1-Aug-2019) of USP Benzothiadiazine Related Compound A RS in Mobile phase

Chromatographic system: Proceed as directed in the Assay, except for the Injection volume.(USP 1-Aug-2019)

Injection volume: 20 μL. For System suitability, use 10 μL. (USP 1-Aug-2019)

5.1 System suitability

Sample: System suitability solution (USP 1-Aug-2019)

[Note-The relative retention times for hydrochlorothiazide and amiloride (USP 1-Aug-2019) are about 0.7 and 1.0, respectively.]

5.2 Suitability requirements

Resolution: NLT 2.0 between hydrochlorothiazide and amiloride (USP 1-Aug-2019)

Relative standard deviation: NMT 2.0% for hydrochlorothiazide and amiloride (USP 1-Aug-2019)

5.3 Analysis

Samples: Sample solution and Standard solution

Calculate the percentage of benzothiadiazine related compound A in the portion of Tablets taken:

Result = (r_u/r_s) × (C_s/C_u)  (USP 1-Aug-2019) × 100

r_u = peak response of benzothiadiazine related compound A from the Sample solution

r_s = peak response of benzothiadiazine related compound A from the Standard solution

C_s = concentration of USP Benzothiadiazine Related Compound A RS in the Standard solution (mg/mL) (USP 1-Aug-2019)

C_u = nominal concentration of hydrochlorothiazide (USP 1-Aug-2019) in the Sample solution (mg/mL) (USP 1-Aug-2019)

Acceptance criteria: NMT 1.0%

6 ADDITIONAL REQUIREMENTS

Change to read:

Packaging and Storage: Preserve in well-closed containers. Store at controlled room temperature. Protect from light. (USP 1-Aug-2019)

USP Reference Standards 〈11〉

USP Amiloride Hydrochloride RS

USP Benzothiadiazine Related Compound A RS

4-Amino-6-chloro-1,3-benzenedisulfonamide.

C₆H₈ClN₃O₄S₂ 285.73