Ampicillin Injection

Penicillin antibacterial.

Ampicillin Injection is a sterile solution of Ampicillin Sodium in Water for Injections. It is prepared by dissolving Ampicillin Sodium for Injection in the requisite amount of Water for Injections immediately before use.

The injection complies with the requirements stated under Parenteral Preparations.

Ampicillin Injection should be used immediately after preparation.

Ampicillin Sodium for Injection is a sterile material consisting of Ampicillin Sodium, with or without excipients. It is supplied in a sealed container.

The contents of the sealed container comply with the requirements for Powders for Injections or Infusions stated under Parenteral Preparations and with the following requirements.

95.0 to 105.0% of the stated amount.

A. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of ampicillin sodium (RS 366). If the spectra are not concordant carry out the following procedure. Dissolve a quantity of the contents of the sealed container containing the equivalent of 0.25 g of ampicillin in 5 mL of water, add 0.5 mL of 2m acetic acid, mix and allow to stand for 10 minutes in ice. Filter through a sintered-glass filter (ISO 4793, porosity grade 3, is suitable), wash the residue with 2 to 3 mL of a mixture of 9 volumes of acetone and 1 volume of water, dry at 60° for 30 minutes and prepare a new spectrum of the residue. The spectrum of the residue is concordant with the reference spectrum of ampicillin trihydrate (RS 013).

B. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.

(1) Dissolve a quantity of the contents of the sealed container in sufficient sodium hydrogen carbonate solution to produce a solution containing the equivalent of 0.25% w/v of ampicillin.

(2) 0.25% w/v of ampicillin trihydrate BPCRS in sodium hydrogen carbonate solution.

(3) 0.25% w/v of each of ampicillin trihydrate BPCRS and amoxicillin trihydrate BPCRS in sodium hydrogen carbonate solution.

(a) Use a TLC silica gel F₂₅₄ silanised plate (Merck silanised silica gel 60 F_254s (RP-18) plates are suitable).

(b) Use the mobile phase 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 it to iodine vapour until spots appear and examine in daylight.

A mixture of 10 volumes of acetone and 90 volumes of a 15.4% w/v solution of ammonium acetate adjusted to pH 5.0 with glacial acetic acid.

The test is not valid unless the chromatogram obtained with solution (3) shows two clearly separated spots.

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).

C. Yield the reactions characteristic of sodium salts, Appendix VI.

pH of a solution containing the equivalent of 10.0% w/v of ampicillin, 8.0 to 10.0, Appendix V L, measured within 10 minutes of preparing the solution.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Shake a quantity of the contents of the sealed container containing the equivalent of 0.3 g of ampicillin with 80 mL of mobile phase A with the aid of ultrasound for 15 minutes, add sufficient mobile phase A to produce 100 mL and filter through a 0.4-µm filter.

(2) Dilute 1 volume of solution (1) to 100 volumes with mobile phase A.

(3) Add 1 mL of water to 0.2 g of anhydrous ampicillin BPCRS. Heat the solution at 60° for 1 hour and dilute 0.5 mL to 50 mL with mobile phase A.

(4) 0.025% w/v of anhydrous ampicillin BPCRS and 0.002% w/v of cefradine BPCRS in mobile phase A.

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Nucleosil C18 is suitable).

(b) Use gradient elution and the mobile phase described below. Equilibrate the column with a mobile phase ratio A:B of 85:15 and use this mobile phase for the system suitability solution (4). Inject solutions (1), (2) and (3) and start the elution isocratically with the chosen mobile phase. Immediately after elution of the ampicillin peak start the linear gradient elution.

(c) Use a flow rate of 1 mL per minute.

(d) Use an ambient column temperature.

(e) Use a detection wavelength of 254 nm.

(f) Inject 50 µL of each solution.

Mobile phase A  Dilute a mixture of 1 volume of dilute acetic acid, 100 volumes of 0.2m potassium dihydrogen orthophosphate and 100 volumes of acetonitrile to 2000 volumes with water.

Mobile phase B  Dilute a mixture of 1 volume of dilute acetic acid, 100 volumes of 0.2m potassium dihydrogen orthophosphate and 800 volumes of acetonitrile to 2000 volumes with water.

Inject mobile phase A and use the same elution gradient to obtain a blank.

The chromatogram obtained with solution (3) shows a peak due to ampicillin and a peak due to ampicillin dimer which has a retention time relative to ampicillin of about 2.8.

The test is not valid unless, in the chromatogram obtained with solution (4), the resolution factor between the peaks due to ampicillin and cefradine is at least 3.0. If necessary, adjust the composition of the mobile phase.

In the chromatogram obtained with solution (1):

the area of any peak corresponding to ampicillin dimer is not greater than 4.5 times the area of the principal peak in the chromatogram obtained with solution (2) (4.5%);

the area of any other secondary peak is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (2%).

Not more than 2.0% w/w, Appendix IX C. Use 0.3 g.

Carry out the test for bacterial endotoxins, Appendix XIV C. Dissolve the contents of the sealed container in water BET to give a solution containing the equivalent of 9.5 mg of ampicillin per mL (solution A). The endotoxin limit concentration of solution A is 1.5 IU per mL.

Determine the weight of the contents of 10 containers as described in the test for uniformity of weight, Appendix XII C1, Powders for Parenteral Administration.

Carry out the method for liquid chromatography, Appendix III D, using the following solutions.

(1) Dissolve a quantity of the mixed contents of the 10 containers in sufficient of solution A to produce a solution containing the equivalent of 0.006% w/v of ampicillin.

(2) 0.006% w/v of anhydrous ampicillin BPCRS in solution A.

(3) 0.025% w/v of anhydrous ampicillin BPCRS and 0.002% w/v of cefradine BPCRS in solution A.

(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm) (Nucleosil C18 is suitable).

(b) Use isocratic elution and the mobile phase described below.

(c) Use a flow rate of 1 mL per minute.

(d) Use an ambient column temperature.

(e) Use a detection wavelength of 254 nm.

(f) Inject 50 µL of each solution.

15 volumes of solution B and 85 volumes of solution A.

Solution A  Dilute a mixture of 1 volume of dilute acetic acid, 100 volumes of 0.2m potassium dihydrogen orthophosphate and 100 volumes of acetonitrile to 2000 volumes with water.

Solution B  Dilute a mixture of 1 volume of dilute acetic acid, 100 volumes of 0.2m potassium dihydrogen orthophosphate and 800 volumes of acetonitrile to 2000 volumes with water.

The assay is not valid unless, in the chromatogram obtained with solution (3), the resolution factor between the peaks due to ampicillin and cefradine is at least 3.0. If necessary, adjust the composition of the mobile phase to achieve the required resolution.

Calculate the content of C₁₆H₁₉N₃O₄S in a container of average content weight using the declared content of C₁₆H₁₉N₃O₄S in anhydrous ampicillin BPCRS.

The label of the sealed container states the quantity of Ampicillin Sodium contained in it in terms of the equivalent amount of ampicillin.