Action and use
Penicillin antibacterial.
DEFINITION
Procaine Benzylpenicillin Injection is a sterile suspension of Benzylpenicillin (Procaine) Monohydrate in Water for Injections.
The injection complies with the requirements stated under Parenteral Preparations and with the following requirements.
Content of total penicillins, calculated as C13H20N2O2,C16H18N2O4S, H2O
90.0 to 110.0% of the stated amount of Procaine Benzylpenicillin.
Content of procaine, C13H20N2O2
36.0 to 44.0% of the stated amount of Procaine Benzylpenicillin.
CHARACTERISTICS
A white suspension.
IDENTIFICATION
A. Dilute a volume of the well-shaken suspension containing 10 mg of Procaine Benzylpenicillin to 10 mL with water and add 0.5 mL of neutral red solution. Add sufficient 0.01M sodium hydroxide to produce a permanent orange colour and then
add 1 mL of penicillinase solution. A red colour is produced rapidly.
B. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Shake a volume of the well-shaken suspension containing 50 mg of Procaine Benzylpenicillin with 5 mL of methanol, add a small quantity of water to dissolve any residue and dilute to 10 mL with water.
(2) 0.5% w/v of procaine benzylpenicillin BPCRS in acetone.
CHROMATOGRAPHIC CONDITIONS
(a) Use a TLC silica gel silanised plate (Merck silanised silica gel 60 plates are suitable).
(b) Use the mobile phase as described below.
(c) Apply 1 μL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, allow it to dry in air, expose to iodine vapour until spots appear and examine in daylight.
MOBILE PHASE
30 volumes of acetone and 70 volumes of a 15.4% w/v solution of ammonium acetate adjusted to pH 7.0 with 10M ammonia.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (2) shows two clearly separated spots.
CONFIRMATION
The two principal spots in the chromatogram obtained with solution (1) are similar in position, colour and size to those in the chromatogram obtained with solution (2).
C. Yields the reaction characteristic of primary aromatic amines, Appendix VI, producing a bright orange-red precipitate.
TESTS
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) To a quantity of the well-shaken suspension containing 70 mg of Procaine Benzylpenicillin add sufficient mobile phase to produce 50 mL, mix, filter and use the filtrate.
(2) Mix 1 mL of solution (1) and 1 mL of a 0.007% w/v solution of 4-aminobenzoic acid and add sufficient mobile phase to produce 100 mL.
(3) Dissolve 4 mg of 4-aminobenzoic acid in 25 mL of a solution containing 0.070% w/v of procaine benzylpenicillin BPCRS in the mobile phase.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 μm) (Lichrospher ODS is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.5 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 225 nm.
(f) Inject 20 μL of each solution.
(g) For solution (1) allow the chromatography to proceed for 1.5 times the retention time of the peak due to benzylpenicillin.
MOBILE PHASE
250 volumes of acetonitrile, 250 volumes of water and 500 volumes of a freshly prepared solution containing 1.4% w/v of potassium dihydrogen orthophosphate and 0.65% w/v of tetrabutylammonium hydroxide, adjusted to pH 7.0 with 1M potassium hydroxide. Adjust the pH of the mixture to 7.2 with 2M orthophosphoric acid, if necessary.
For solution (3), when the chromatogram is recorded under the prescribed conditions the substances elute in the following order: 4-aminobenzoic acid, procaine, benzylpenicillin.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3), the resolution factor between the peaks due to 4-aminobenzoic acid and procaine is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase.
LIMITS
In the chromatogram obtained with solution (1):
the area of any peak due to 4-aminobenzoic acid is not greater than 10 times the area of the corresponding peak in the chromatogram obtained with solution (2) (0.5%);
the area of any other secondary peak is not greater than the area of the peak corresponding to benzylpenicillin in the chromatogram obtained with solution (2) (1%).
Bacterial endotoxins
Carry out the test for bacterial endotoxins, Appendix XIV C, Method C. Dilute a quantity of the well-shaken suspension, if necessary, with water BET to produce a solution containing 3 mg of Procaine Benzylpenicillin per mL (solution A). The endotoxin limit concentration of solution A is 0.3 IU per mL. Carry out the test using a suitable dilution of solution A as described under Method C.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Add to a quantity of the well-shaken suspension containing 70 mg of Procaine Benzylpenicillin sufficient mobile phase to produce 100 mL, mix, filter and use the filtrate.
(2) 0.07% w/v of procaine benzylpenicillin BPCRS in the mobile phase.
(3) Dissolve 4 mg of 4-aminobenzoic acid in 25 mL of solution (2).
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used.
For solution (3), when the chromatogram is recorded under the prescribed conditions the substances elute in the following order: 4-aminobenzoic acid, procaine, benzylpenicillin.
SYSTEM SUITABILITY
The Assay is not valid unless, in the chromatogram obtained with solution (3), the resolution factor between the peaks due to 4-aminobenzoic acid and procaine is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobil phase.
DETERMINATION OF CONTENT
Calculate the content of C13H20N2O2 and of C13H20N2O2,C16H18N2O4S, H2O in the injection from the chromatograms obtained and using the declared content of C13H20N2O2 and of C13H20N2O2,C16H18N2O4S, H2O in procaine benzylpenicillin BPCRS.
3 g of Procaine Benzylpenicillin is approximately equivalent to 2 g of benzylpenicillin.
