Methylene Blue 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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DEFINITION

Methylene Blue Injection is a sterile solution of Methylene Blue in Water for Injection. It contains NLT 95.0% and NMT 105.0% of the labeled amount of methylene blue trihydrate (USP 1-Aug-2022) (C₁₆H₁₈ClN₃S . 3H₂O).

1 IDENTIFICATION

A. The UV absorption spectra of the major peak of the Sample solution exhibit maxima and minima at the same wavelengths as those of the corresponding peak of the Standard solution, as obtained in the Assay.

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

2 ASSAY

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2.1 PROCEDURE

All solutions containing methylene blue should (USP 1-Aug-2022) be freshly prepared prior to analysis.

Solution A: 0.1% trifluoroacetic acid in water

Solution B: Acetonitrile

Diluent: Solution A and Solution B (70:30)

Mobile phase: Solution A and Solution B (75:25)

Standard solution: 100 µg/mL of USP Methylene Blue RS in Diluent

Sample solution: Nominally 100 µg/mL of methylene blue in Diluent, from Injection

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 246 nm. For Identification A, use a diode array detector in the range of 200–400 nm.

Column: 4.6-mm × 10-cm; 3.5-µm packing L11

Temperatures

Autosampler: 5°

Column: 30°

Flow rate: 1 mL/min

Injection volume: 5 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of methylene blue trihydrate (USP 1-Aug-2022) (C₁₆H₁₈ClN₃S . 3H₂O) in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of methylene blue from the Sample solution

r_S = peak response of methylene blue from the Standard solution

C_S = concentration of USP Methylene Blue RS in the Standard solution (µg/mL)

C_U = nominal concentration of methylene blue in the Sample solution (µg/mL)

M_r1 = molecular weight of methylene blue trihydrate, 373.90

M_r2 = molecular weight of methylene blue anhydrous, 319.85

Acceptance criteria: 95.0%–105.0%

3 IMPURITIES

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3.1 ORGANIC IMPURITIES

Solution A, Solution B, Diluent, and Chromatographic system: Proceed as directed in the Assay.

Mobile phase: (USP 1-Aug-2022) See Table 1.

Table 1

System suitability solution: 1 mg/mL of USP Methylene Blue RS and 0.03 mg/mL of USP Azure B RS in Diluent

Standard solution: 1 µg/mL of USP Methylene Blue RS in Diluent

Sensitivity solution: 0.25 µg/mL of USP Methylene Blue RS in Diluent from the Standard solution

Sample solution: Nominally (USP 1-Aug-2022) 500 µg/mL of methylene blue (USP 1-Aug-2022) in Diluent from Injection

System suitability

Samples: System suitability solution, Standard solution, and Sensitivity solution

Suitability requirements

Resolution: NLT 3.5 between the methylene blue and azure B peaks, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each specified degradation product (USP 1-Aug-2022) in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of each specified degradation product (USP 1-Aug-2022) from the Sample solution

r_S = peak response of methylene blue from the Standard solution

C_S = concentration of USP Methylene Blue RS in the Standard solution (µg/mL)

C_U = nominal concentration of methylene blue in the Sample solution (µg/mL)

Calculate the percentage of any unspecified degradation product (USP 1-Aug-2022) in the portion of Injection taken:

Result = (r_U/r_T) × 100

r_U = peak response of any unspecified degradation product (USP 1-Aug-2022) from the Sample solution

r_T = sum of all the peak responses from the Sample solution

Acceptance criteria: See Table 2. The reporting threshold is (USP 1-Aug-2022) 0.05%.

Table 2

^a 3-Amino-7-(methylamino)phenothiazin-5-ium chloride.

^b 3-Amino-7-(dimethylamino)phenothiazin-5-ium chloride.

^c Azure B is quantitated using the test for Limit of Azure B and is included in the table for identification purposes.

^d Total degradation products include the sum of the results from the tests for Organic Impurities and Limit of Azure B.

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3.2 LIMIT OF AZURE B

Diluent, Mobile phase, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Standard solution: 3 µg/mL of USP Methylene Blue RS in Diluent

System suitability: Proceed as directed in the Assay using the Standard solution from the Assay.

[Note—The relative retention times for azure B and methylene blue are 0.70 and 1.00, respectively.]

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of azure B in the portion of Injection taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of azure B from the Sample solution

r_S = peak response of methylene blue from the Standard solution

C_S = concentration of USP Methylene Blue RS in the Standard solution (µg/mL) (USP 1-Aug-2022)

C_U = nominal concentration of methylene blue in the Sample solution (µg/mL) (USP 1-Aug-2022)

Acceptance criteria: NMT 3.0%

4 SPECIFIC TESTS

pH 〈791〉: 3.0–4.5

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BACTERIAL ENDOTOXINS TEST 〈85〉: Meets the requirements (USP 1-Aug-2022)

Add the following:

STERILITY TESTS 〈71〉: Meets the requirements (USP 1-Aug-2022)

OTHER REQUIREMENT: It meets the requirements in Injections and Implanted Drug Products 〈1〉.

5 ADDITIONAL REQUIREMENTS

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PACKAGING AND STORAGE: Preserve in single-dose containers, preferably of Type I glass. Store at controlled (USP 1-Aug-2022) room temperature, protected from light. Do not refrigerate or freeze.

Add the following:

LABELING: The label to indicate for "Intravenous use only" (USP 1-Aug-2022)

USP REFERENCE STANDARDS (11)

USP Azure B RS

3-(Dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride.

C₁₅H₁₆CIN₃S  305.82

USP Methylene Blue RS