Nevirapine Oral Suspension

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

Nevirapine Oral Suspension contains NLT 90.0% and NMT 110.0% of the labeled amount of nevirapine (C₁₅H₁₄N₄O).

2 IDENTIFICATION

2.1 A. THIN-LAYER CHROMATOGRAPHY (201)

Standard solution: 5 mg/mL of USP Nevirapine Anhydrous RS in chloroform

Sample solution: Transfer a volume of Oral Suspension, equivalent to 10 mg of nevirapine, to an 8-mL glass stoppered tube. Pipet 2.0 mL of chloroform into the tube. Shake the solution and allow the two phases to separate; then, using a disposable glass Pasteur pipet, remove some of the organic layer from the bottom, and transfer it to another container.

Chromatographic system

(See Chromatography (621). Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel 60 F254

Application volume: 5 µL

Developing solvent system: Ethyl acetate, isopropanol, and concentrated ammonium hydroxide (18:2:0.1)

Spray reagent: 1.35 g of ferric chloride in 25 mL of water and 1.64 g of potassium ferricyanide in 25 mL of water. Mix the two solutions immediately before use.

Analysis

Samples: Standard solution and Sample solution

Develop in a chamber saturated with a solvent system until the solvent front has moved 6-7 cm from the point of application. Remove the plate from the chamber, mark the solvent front, and dry. Examine under UV light at 254 nm, and outline the spots with a soft pencil. Spray the plate with Spray reagent.

Acceptance criteria: The R_F value (approximately 0.4-0.5) of the principal blue spot, under UV and after spraying, from the Sample solution, corresponds to that from the Standard solution.

2.2 B.

The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

PROCEDURE

Diluent: Methanol and water (1:4)

Solution A: 13.6 g of monobasic potassium phosphate in 1900 mL of water. Adjust with phosphoric acid to a pH of 3.0, and dilute with water to 2000 mL.

Solution B: Acetonitrile and Solution A (3:97)

Solution C: Acetonitrile and Solution A (24:76)

Mobile phase: See the gradient table below.

Standard stock solution: Dissolve 50 mg of USP Nevirapine Anhydrous RS in 20 mL of methanol in a 50-mL volumetric flask. Sonicate with intermittent swirling until the sample dissolves. Add water to 1 cm below the meniscus, cool to room temperature, and dilute with water to volume. The concentration is 1 mg/mL of nevirapine.

Standard solution: 0.3 mg/ml, of nevirapine from the Standard stock solution diluted with Diluent

Stock impurity solution: 3 mg of USP Nevirapine Related Compound A RS and 3 mg of USP Nevirapine Related Compound B RS in 20 mL of methanol in a 100-mL volumetric flask. Sonicate to dissolve. Add water to 1 cm below the meniscus, cool to room temperature, and dilute with water to volume.

System suitability solution: Transfer 15.0 mL of Standard stock solution and 2.0 mL of Stock impurity solution to a 50-mL volumetric flask, and dilute with Diluent to volume.

Weight determination: Using a 1-to 10-ml suitable pipet and a positive displacement tip, withdraw 5.0 mL of Oral Suspension. The sample should be free of air bubbles. Dispense into a tared vial, and record the weight of the Oral Suspension to ±0.1 mg.

Sample solution: Using a 1-to 10-ml suitable pipet and a positive displacement tip, withdraw Oral Suspension equivalent to 60 mg of nevirapine. The sample should be free of air bubbles. Remove the excess Oral Suspension by wiping the outside of the tip carefully so as not to touch the opening of the tip, and deliver the sample into a 200-mL tared volumetric flask. Record the sample weight to the nearest ±0.1 mg. Add 40 mL of methanol, and sonicate for 5 min with intermittent swirling. Add water to 1 cm below the meniscus. Do not shake the flask. Allow the solution to attain room temperature, and dilute with water to volume. Shake the flask gently, and allow to stand for 5 min.

Chromatographic system

(See Chromatography (621). System Suitability.)

Mode: LC

Detector: UV 254 nm

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

Guard column: 4.6 - mm * 12.5 - mm 5-µm packing L10

Column temperature: 35°

Flow rate: 1.5 mL/min

Injection size: 20 μL.

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 3.0 between nevirapine and nevirapine related compound A; NLT 1.7 between nevirapine and nevirapine related compound B, System suitability solution

Tailing factor: NMT 1.5 for the nevirapine peak, System suitability solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Measure the responses for the nevirapine peak. Calculate the percentage of C₁₅H₁₄N₄O in the portion of Oral Suspension taken:

                         Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution 

C_S = concentration of USP Nevirapine Anhydrous RS in the Standard solution (mg/mL) 

C_U = nominal concentration of the Sample solution (mg/mL) 

Acceptance criteria: 90.0%-110.0%

4 PERFORMANCE TESTS

DISSOLUTION (711)

Medium: 0.1 N hydrochloric acid; 900 mL

Apparatus 2: 25 rpm

Time: 45 min

Analysis: Determine the amount of C₁₅H₁₄N₄O dissolved by using the following method.

Diluent: Dehydrated alcohol and water (1:1)

Mobile phase: Acetonitrile and water (23:77)

System suitability solution: Transfer 10 mg of USP Nevirapine Anhydrous RS and 15 mg of methylparaben to a 250-mL volumetric flask, dissolve with 2 mL of Diluent, and dilute with Medium to volume.

Standard solution: Transfer 28 mg of USP Nevirapine Anhydrous RS to a 500-ml volumetric flask, add 2 mL of Diluent, and sonicate for 1 min. The Standard will not be completely dissolved at this point. Dilute with Medium to volume, and visually examine the solution to ensure that the Standard is completely dissolved. The final concentration is 0.056 mg/mL of nevirapine.

Sample solution: For sample mixing, gently shake the bottle for approximately 10 s by inverting it slowly and rotating it from side to side. The sample should be free of air bubbles. Do not sonicate the sample. Using a 1-to 10-mL suitable positive displacement pipet set at 5 mL, withdraw the equivalent of 50 mg of nevirapine. Remove excess Oral Suspension by wiping the outside of the tip carefully so as not to touch the opening of the tip. Introduce the sample into the dissolution vessel over a period of 1-2 s by immersing the tip of the pipet midway between the paddle and the side of the vessel, approximately 1 cm below the meniscus. Similarly dispense the Oral Suspension into the other vessels. At 45 min, withdraw 5 mL of the solution under test, and pass through a nylon filter of 0.45-µm pore size, discarding the first 2 mL.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 214 nm

Column: 3.9-mm x 15-cm; 5-um packing L1

Guard column: 3.9-mm x 20-mm packing L1

Flow rate: 1 mL/min

Injection size: 10 µL

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 5.0 between nevirapine and methylparaben, System suitability solution

Tailing factor: NMT 1.8, System suitability solution

Relative standard deviation: NMT 2.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Record the chromatograms for at least 14 min, and measure the responses for the nevirapine peaks..

Calculate the percentage of C₁₅H₁₄N₄O dissolved:

                         Result = (r_U x C_S x V₁)/(r_S x V₂ x L) x 100

r_U = peak response from the Sample solution

C_S = concentration of USP Nevirapine Anhydrous RS in the Standard solution (mg/mL)

V₁ = volume of the Mediurn, 900 mL

r_S = peak response from the Standard solution

V₂ = volume of Oral Suspension taken (mL)

L = label claim (mg/mL)

Tolerances: NLT 80% (Q) of the labeled amount of C₁₅H₁₄N₄O is dissolved. 15

5 IMPURITIES

ORGANIC IMPURITIES

PROCEDURE

Diluent, Solution A, Solution B, Solution C, and Mobile phase: Prepare as directed in the Assay.

Standard stock solution: Use the Standard stock solution, prepared as directed in the Assay.

Standard solution: 0.3 µg/mL of nevirapine from the Standard stock solution diluted with Diluent

System suitability solution: Prepare as directed in the Assay.

Weight determination: Use the weight obtained as directed for Weight determination in the Assay.

Sample solution: Prepare as directed in the Assay.

Chromatographic system

Mode: LC

Detector: UV 254 nm

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

Column temperature: 35°

Flow rate: 1.5 mL/min

Injection size: 20 μl.

System suitability

Samples: Standard solution and System suitability solution

Suitability requirements

Resolution: NLT 3.0 between nevirapine and nevirapine related compound A and NLT 1.7 between nevirapine and nevirapine related compound B, System suitability solution

Tailing factor: NMT 1.5 for nevirapine, System suitability solution

Relative standard deviation: NMT 10.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each unknown impurity in the portion of Oral Suspension taken:

                         Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response for each impurity from the Sample solution

r_S = peak response for nevirapine from the Standard solution

C_S = concentration of USP Nevirapine Anhydrous RS in the Standard solution (mg/mL) 

C_U = nominal concentration of nevirapine in the Sample solution (mg/mL)

Acceptance criteria

Individual unknown impurities: NMT 0.1%

Total unknown impurities: NMT 0.2%

[NOTE-The excipients and their degradation products should not be included in the determination of impurities.]

6 SPECIFIC TESTS

MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECIFIED MICROORGANISMS (62): It meets the requirements of the tests for absence of Escherichia coli. The total aerobic microbial count does not exceed 100 cfu/ml, and the total combined molds and yeasts count does not exceed 50 cfu/mL.

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in well-closed containers. Store at 25, excursions permitted between 15° and 30°.

USP REFERENCE STANDARDS (11)

USP Neviragine Anhydrous BS

USP Neviragine Related Compound A RS

5,11-Dihydro-6H-11-ethyl-4-methyl- dipyrido[3,2-b:2',3'-e] [1,4]diazepin-6-one.

C₁₄H₁₄N₄O         254.29

USP Nevirapine Related Compound B. RS

5,11-Dihydro-4-methyl-6H-dipyrido[3,2-b:2',3'-e][1,4]diazepin-6-one.

C₁₂H₁₀N₄O          226.23