Methylene Blue Compounded Injection, Veterinary

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

Methylene Blue Compounded Injection, Veterinary, 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 (C₁₆H₁₈CIN₃S). Prepare Methylene Blue Compounded Injection, Veterinary, 10 mg/mL, as follows (see Pharmaceutical Compounding-Sterile Preparations (797)).

Dissolve an accurately weighed quantity of Methylene Blue in Sterile Water for Injection or Sodium Chloride Injection (0.9%), and dilute to volume with mixing. Sterilize by a suitable means, such as sterile filtration or autoclaving.

2 IDENTIFICATION

A. The UV absorption spectrum of the Sample solution exhibits maxima and minima at the same wavelengths as those of the Standard solution, as obtained in the Assay.

B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST (201)

Diluent: Methanol and water (1:1)

Standard solution: 5 mg/mL of USP Methylene Blue RS in Diluent

Sample solution: Dilute a portion of Injection with an equal volume of methanol.

Application volume: 1 µL

Developing solvent system: Alcohol, acetic acid, and water (3:3:4)

Analysis

Samples: Standard solution and Sample solution

Allow the spots to dry, and develop until the solvent front has moved about 10 cm above the line of application. Remove the plate from the chamber, and allow the solvent to evaporate.

Acceptance criteria: The R value of the principal spot from the Sample solution corresponds to that from the Standard solution.

3 ASSAY

3.1 PROCEDURE

All solutions containing methylene blue are recommended to 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 (C₁₆H₁₈CIN₃S) 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, 319.86

Acceptance criteria: 95.0%-105.0%

4 SPECIFIC TESTS

PH (791): 3.0-4.5

BACTERIAL ENDOTOXINS TEST (85): NMT 0.17 USP Endotoxin Units/mg of methylene blue

STERILITY TESTS (71): Meets the requirements

OTHER REQUIREMENTS: It meets the requirements in Labeling (7), Labels and Labeling for Injectable Products.

5 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Package in single-dose containers, preferably of Type I glass. Store at controlled room temperature, protected from light.

Change to read:

BEYOND-USE DATE: In the absence of performing and completing a sterility and endotoxin test, the storage conditions in Pharmaceutical

Compounding - Sterile Preparations (797), 14.3 Establishing a BUD for a CSP apply. (CN 1-Nov-2023)

LABELING: Label it to indicate that it is to be discarded after its Beyond-Use Date and to indicate the nominal content of methylene blue in the Injection and whether it was prepared in Sterile Water for Injection or in Sodium Chloride Injection (0.9%). Label it to indicate that it is for veterinary use only and to state the Beyond-Use Date.

USP REFERENCE STANDARDS (11)

USP Methylene Blue RS