Midazolam Injection

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

Midazolam Injection is a sterile solution of Midazolam Hydrochloride in Water for Injection or of Midazolam in Water for Injection prepared with the aid of Hydrochloric Acid. It contains the equivalent of NLT 90.0% and NMT 110.0% of the labeled amount of midazolam (C₁₈H₁₃ClFN₃). It may contain Sodium Chloride, Benzyl Alcohol, and/or a chelating agent.

2 IDENTIFICATION

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

[Note-Protect all prepared Standard and sample solutions from light.]

3.1 Procedure

Buffer: 6.7 g/L of dibasic sodium phosphate heptahydrate in water. Adjust with phosphoric acid to a pH of 5.0 ± 0.1.

Solution A: Prepare a filtered and degassed mixture of acetonitrile, methanol and Buffer (8:3:9).

Solution B: Acetonitrile and Buffer (3:1)

Mobile phase: See the gradient table below.

Standard solution: Dissolve USP Midazolam RS in about 2 mL of methanol, and dilute quantitatively, and stepwise if necessary, with Solution A to obtain a 0.2-mg/mL solution.

Sample solution: [Note-The midazolam present in the Injection converts from the open-ring form to the closed-ring form when diluted with Solution A. The midazolam potency is determined based on the peak area of the closed-ring form. It takes approximately 60 min at 40° or 2–3 h at room temperature to complete the conversion. The Standard solution is not subject to this conversion process.] Transfer a volume of Injection to a suitable volumetric flask, and dilute with Solution A to obtain a solution containing about 0.2 mg/mL of midazolam.

Transfer the resulting solution into suitable crimp top vials, seal tightly, and heat at about 40° for 60 min. Allow this solution to cool to room temperature before injection.

Chromatographic system

• (See Chromatography 〈621〉, System Suitability.)
• Mode: LC
• Detector: UV 254 nm
• Column: 4.6-mm × 25-cm; packing L1
• Flow rate: 1.0 mL/min
• Injection size: 50 µL

System suitability

• Sample: Standard solution
• Suitability requirements
• Column efficiency: NLT 5500 theoretical plates
• Tailing factor: NMT 2.5
• Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of labeled amount of C₁₈H₁₃ClFN₃ in the portion of Injection taken:

Result = (rᵤ/rₛ) × (Cₛ/Cᵤ) × 100

rᵤ = peak response from the Sample solution

rₛ = peak response from the Standard solution

Cₛ = concentration of USP Midazolam RS in the Standard solution (mg/mL)

Cᵤ = nominal concentration of Midazolam in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–110.0%

4 IMPURITIES

4.1 Organic Impurities

[Note-Protect all prepared Standard and sample solutions from light.]

4.1.1 Procedure

Buffer, Solution A, Solution B, Mobile phase, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Standard stock solution: Use Standard solution in the Assay.

Standard solution: 0.5 µg/mL USP Midazolam RS in Solution A from Standard stock solution

Control solution: 0.1 µg/mL USP Midazolam RS in Solution A from Standard solution

System suitability

Samples: Standard solution and Control solution

Suitability requirements

• Tailing factor: NMT 2.5 for midazolam peak, Standard solution
• Column efficiency: NLT 5500 theoretical plates, Standard solution
• Signal-to-noise ratio: NLT 10, Control solution
• Relative standard deviation: NMT 8.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Injection taken:

Result = (rᵤ/rₛ) × (Cₛ/Cᵤ) × (1/F) × 100

rᵤ = peak response of the individual impurity from the Sample solution

rₛ = peak response of midazolam from the Standard solution

Cₛ = concentration of USP Midazolam RS in the Standard solution (mg/mL)

Cᵤ = nominal concentration of Midazolam in the Sample solution (mg/mL)

F = relative response factor; 0.51 for the peak eluting at a relative retention between 0.79 and 0.97 with respect to midazolam; 1.0 for all other peaks

Acceptance criteria

• Individual known impurity: NMT 0.5%
• Individual unknown impurity: NMT 0.1%
• Total impurities: NMT 1.0%
• [Note-Disregard all solvent- and excipient-related peaks.]

5 SPECIFIC TESTS

5.1 Benzyl Alcohol Content (if present)

Buffer: 3.4 g/L of monobasic sodium phosphate in water. Adjust with phosphoric acid to a pH of 3.5.

Mobile phase: Acetonitrile and Buffer (7:13)

System suitability solution: 0.05 mg/mL of USP Midazolam RS and 0.5 mg/mL of USP Benzyl Alcohol RS in Mobile phase

Standard solution: 0.5 mg/mL of USP Benzyl Alcohol RS in Mobile phase

Sample solution: Transfer a measured volume of Injection to a suitable volumetric flask. Dilute with Mobile phase to obtain a concentration of about 0.5 mg/mL of benzyl alcohol, based on the labeled content of benzyl alcohol in the Injection.

Chromatographic system

• (See Chromatography 〈621〉, System Suitability.)
• Mode: LC
• Detector: UV 254 nm
• Column: 4.6-mm × 25-cm; L1 packing
• Flow rate: 1.0 mL/min
• Injection size: 50 µL

System suitability

• Sample: System suitability solution
• Suitability requirements
• Resolution: NLT 6.0 between benzyl alcohol and midazolam
• Tailing factor: NMT 2.0 for benzyl alcohol
• Relative standard deviation: NMT 2.0% for benzyl alcohol

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of benzyl alcohol in the volume of Injection taken:

Result = (rᵤ/rₛ) × (Cₛ/Cᵤ) × 100

rᵤ = peak response of benzyl alcohol from the Sample solution

rₛ = peak response of benzyl alcohol from the Standard solution

Cₛ = concentration of USP Benzyl Alcohol RS in the Standard solution (mg/mL)

Cᵤ = nominal concentration of benzyl alcohol in the Sample solution (mg/mL)

Acceptance criteria: The content of benzyl alcohol meets the requirements in Injections and Implanted Drug Products 〈1〉, Specific Tests, Vehicles and added substances.

5.2 Particulate Matter in Injections 〈788〉

Meets the requirements for small-volume injections

5.3 Bacterial Endotoxins Test 〈85〉

It contains NMT 8.33 USP Endotoxin Units/mg of midazolam.

5.4 pH 〈791〉

2.5–3.7

5.5 Sterility Tests 〈71〉

Meets the requirements

5.6 Other Requirements

It meets the requirements for Injections and Implanted Drug Products 〈1〉.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in single-dose containers, preferably of Type 1 glass. Store between 15° and 30°.

Labeling: Label to indicate the vehicle used and the names and concentrations of any added preservatives. Indicate if the product is preservative free.

USP Reference Standards 〈11〉

USP Benzyl Alcohol RS

USP Midazolam RS