BP 2025 (Ph. Eur. 11.6 update)
Action and use
Penicillin antibacterial.
DEFINITION
Benzylpenicillin Infusion is a sterile solution containing Benzylpenicillin Sodium and a suitable buffer. It is supplied as a ready-to-use solution.
The infusion complies with the requirements stated under Parenteral Preparations and with the following requirements. Where appropriate, the infusion also complies with the requirements stated under Unlicensed Medicines.
Content of benzylpenicillin, C16H18N2O4S
90.0 to 105.0% of the stated amount.
CHARACTERS
A clear, colourless solution.
IDENTIFICATION
A. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions in water.
(1) Dilute a volume of the infusion to produce a solution containing the equivalent of 0.6% w/v of benzylpenicillin.
(2) 0.64% w/v of benzylpenicillin sodium EPCRS.
(3) 0.5% w/v of each of benzylpenicillin sodium EPCRS and phenoxymethylpenicillin EPCRS.
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 the 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 5.0 with glacial acetic acid.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (3) shows two clearly separated spots.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) is similar in position, colour and size to that in the
chromatogram obtained with solution (2).
B. In the Assay, the retention time of the principal peak in the chromatogram obtained with solution (1) is similar to that of
the principal peak in the chromatogram obtained with solution (2).
TESTS
Acidity or alkalinity
pH, 6.0 to 7.5, Appendix V L.
Related substances
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in water, prepared immediately before use.
(1) Dilute a volume of the infusion to produce a solution containing the equivalent of 0.6% w/v of benzylpenicillin.
(2) Dilute 1 volume of solution (1) to 100 volumes.
(3) Dissolve 5 mg of benzylpenicillin for system suitability EPCRS in 0.35 mL of methanol R1 and add 0.65 mL of water.
(4) Dilute 1 volume of solution (2) to 5 volumes.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (3 μm) (YMC-Pack Pro is suitable).
(b) Use gradient elution and the mobile phase described below.
(c) Use a flow rate of 1.5 mL per minute.
(d) Use a column temperature of 50°.
(e) Use a detection wavelength of 225 nm.
(f) Inject 20 μL of each solution.
MOBILE PHASE
Mobile phase A 10 volumes of a 6.8% w/v solution of potassium dihydrogen orthophosphate adjusted to pH 3.4 with a 50% w/v solution of orthophosphoric acid, 30 volumes of methanol R1 and 60 volumes of water.
Mobile phase B 10 volumes of a 6.8% w/v solution of potassium dihydrogen orthophosphate adjusted to pH 3.4 with a 50% w/v solution of orthophosphoric acid, 35 volumes of water and 55 volumes of methanol R1.
| Time (min) |
Mobile Phase A (% V/V) |
Mobile Phase B (% V/V) |
Comment |
| 0 – 7 | 70 | 30 | isocratic |
| 7 – 17 | 70 → 0 | 30 → 100 | linear gradient |
| 17 – 22 | 0 | 100 | isocratic |
When the chromatograms are recorded under the prescribed conditions, the relative retentions with reference to benzylpenicillin (retention time, about 7 minutes) are: impurity A, about 0.22; impurity D, about 0.33; impurity C, about 0.35; impurity E, about 0.48 and 0.55; impurity B, about 0.62; impurity F, about 0.81 and 0.83; impurity G, about 1.47; impurity H, about 1.90.
SYSTEM SUITABILITY
The test is not valid unless, in the chromatogram obtained with solution (3):
the resolution between the peaks due to the epimers of impurity F is at least 1.2;
the resolution between the peaks due to impurities D and C is at least 1.5.
LIMITS
Identify any peaks in the chromatogram obtained with solution (1) corresponding to impurities A, D, E and F using the chromatogram obtained with solution (3) and multiply the areas of these peaks by the following correction factors: impurity A, 1.3; impurity D, 0.6; impurity E, 2.0; impurity F, 1.7.
In the chromatogram obtained with solution (1);
the sum of the areas of any peaks corresponding to the isomers of impurity E is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (2.0%);
the sum of the areas of any peaks corresponding to the epimers of impurity F is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (2.0%);
the area of any peak corresponding to impurity B is not greater than half the area of the principal 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 principal peak in the chromatogram obtained with solution (4) (0.2%);
the sum of the areas of all the secondary peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with solution (2) (3.0%).
Disregard any peak with an area less than 0.75 times the area of the principal peak in the chromatogram obtained with solution (4) (0.15%).
Bacterial endotoxins
Dilute the infusion with water BET to contain the equivalent of 10 mg of benzylpenicillin per mL (solution A). The endotoxin
limit concentration of solution A is 1.6 IU per mL, Appendix XIV C.
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions in water prepared immediately before use.
(1) Dilute a volume of the infusion to produce a solution containing the equivalent of 0.12% w/v of benzylpenicillin.
(2) 0.13% w/v of benzylpenicillin sodium EPCRS.
CHROMATOGRAPHIC CONDITIONS
The chromatographic conditions described under Related substances may be used but using isocratic elution, a flow rate of 1.2 mL per minute and an injection volume of 10 μL.
MOBILE PHASE
30 volumes of mobile phase B and 70 volumes of mobile phase A as described under Related substances.
DETERMINATION OF CONTENT
Calculate the content of C16H18N2O4S in the infusion using the declared content of C16H17N2NaO4S in benzylpenicillin sodium EPCRS. Each mg of C16H17N2NaO4S is equivalent to 0.9383 mg of C16H18N2O4S .
LABELLING
The quantity of active ingredient is stated in terms of the equivalent amount of benzylpenicillin.
IMPURITIES
The impurities limited by the requirements of this monograph include those listed under Benzylpenicillin Sodium.
