Gentamicin Sulfate CAS 1405-41-0 Assay ≥590 IU/mg (Anhydrous) Factory EP 11.0 Specifications

GENTAMICIN SULFATE
[1405-41-0]
DEFINITION
Mixture of the sulfates of antimicrobial substances produced by Micromonospora purpurea, the main components being gentamicins C1, C1a, C2, C2a and C2b.
Content: minimum 590 IU/mg (anhydrous substance).
CHARACTERS
Appearance: white or almost white, hygroscopic powder.
Solubility: freely soluble in water, practically insoluble in ethanol (96 per cent).
IDENTIFICATION
First identification: B, C.
Second identification: A, C.
A. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 25 mg of the substance to be examined in water R and dilute to 5 mL with the same solvent.
Reference solution. Dissolve the contents of a vial of gentamicin sulfate CRS in water R and dilute to 5 mL with the same solvent.
Plate: TLC silica gel plate R.
Mobile phase: the lower layer of a mixture of equal volumes of concentrated ammonia R, methanol R and methylene chloride R.
Application: 10 μL.
Development: over 2/3 of the plate.
Drying: in air.
Detection: spray with ninhydrin solution R1 and heat at 110℃ for 5 min.
Results: the 3 principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to the 3 principal spots in the chromatogram obtained with the reference solution.
B. Examine the chromatograms obtained in the test for composition.
Results: the chromatogram obtained with test solution (b) shows 5 principal peaks having the same retention times as the 5 principal peaks in the chromatogram obtained with reference solution (a).
C. It gives reaction (a) of sulfates (2.3.1).
TESTS
Solution S. Dissolve 0.8 g in carbon dioxide-free water R and dilute to 20 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than intensity 6 of the range of reference solutions of the most appropriate colour (2.2.2, Method II).
pH (2.2.3): 3.5 to 5.5 for solution S.
Specific optical rotation (2.2.7): + 107 to + 121 (anhydrous substance).
Dissolve 2.5 g in water R and dilute to 25.0 mL with the same solvent.
Composition. Liquid chromatography (2.2.29): use the normalisation procedure taking into account only the peaks due to gentamicins C1, C1a, C2, C2a and C2b.
Test solution (a). Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 mL with the mobile phase.
Test solution (b). Dilute 5.0 mL of test solution (a) to 25.0 mL with the mobile phase.
Reference solution (a). Dissolve 5 mg of gentamicin for peak identification CRS (containing impurity B) in the mobile phase and dilute to 25 mL with the mobile phase.
Reference solution (b). Dissolve 20.0 mg of sisomicin sulfate CRS (impurity A) in the mobile phase and dilute to 20.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of reference solution (b) to 100.0 mL with the mobile phase.
Reference solution (d). To 1 mL of reference solution (b), add 5 mL of test solution (a) and dilute to 50 mL with the mobile phase.
Column:
- size: l = 0.25 m, Ø = 4.6 mm;
- stationary phase: octadecylsilyl silica gel for chromatography R (5 μm);
- temperature: 35℃.
Mobile phase. To 900 mL of carbon dioxide-free water R, add 7.0 mL of trifluoroacetic acid R, 250.0 μL of pentafluoropropanoic acid R and 4.0 mL of carbonate-free
sodium hydroxide solution R, allow to equilibrate and adjust to pH 2.6 using carbonate-free sodium hydroxide solution R diluted 1 to 25. Add 15 mL of acetonitrile R and dilute to
1000.0 mL with carbon dioxide-free water R.
Flow rate: 1.0 mL/min.
Post-column solution: carbonate-free sodium hydroxide solution R diluted 1 to 25, previously degassed, which is added pulse-less to the column effluent using a 375 μL polymeric mixing coil.
Flow rate of post-column solution: 0.3 mL/min.