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GENTAMICIN SULFATE

Category Antibacterial.

      Gentamicin Sulfate is the sulfate salt, or a mixture of such salts, of the antibiotic substances produced by the growth of Micromonospora purpurea. It has a potency equivalent to not less than 590 μg of gentamicin per mg, calculated on the dried basis.

Description White or almost white powder; hygroscopic.

Solubility Freely soluble in water; insoluble in acetone, in chloroform and in ether.

Contra-indication It is contra-indicated in patients with a history of hypersensitivity reactions to any member of aminoglycosides.

Warning

      1. It should not be used in pregnant women.

      2. It is potentially ototoxic and nephrotoxic, especially in elderly patients, infants and neonates or in patients with renal function impairment.

      3. It should be used with caution in patients with myasthenia gravis, parkinsonism, renal function impairment or eighth-cranial nerve impairment.

      4. Concurrent and/or sequential use of two or more aminoglycosides by any routes; or concurrent use of capreomycin, methoxyflurane, parenteral polymyxins, other nephrotoxic drugs, or neuromuscular blocking agents with aminoglycosides should be avoided. 

      5. It may cause neuromuscular blockade. 

      6. Hypersensitivity reactions of different degrees may occur.

Precaution

      1. Renal and vestibular function determinations, and audiogram may be required prior to, periodically during, and following prolonged therapy.

      2. Serum aminoglycoside concentrations should be monitored in all patients. 

Additional information

      1. Subcutaneous administration is not recommended and may be painful. 

      2. The initial symptoms of ototoxicity may be reversible; however, deafness may occur several weeks after therapy is discontinued.

Packaging and storage Gentamicin Sulfate shall be kept in tightly closed containers, protected from light.

Labelling The label on the container states (1) the number of μg of activity per mg; (2) storage condition; (3) parenteral or non-parenteral grade. 

Identification

      A. The infrared absorption spectrum is concordant with the spectrum obtained from Gentamicin Sulfate RS or with the reference spectrum of Gentamicin Sulfate (Appendix 2.1).

      B. Carry out the test as described in the “Thin-layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and as the mobile phase the lower layer obtained by shaking together 1 volume of chloroform, 1 volume of methanol and 1 volume of strong ammonia solution and allowing to separate. Apply separately to the plate, 1 μl of each of two solutions containing (A) 20 mg per ml of the test substance and (B) 20 mg per ml of Gentamicin Sulfate RS. After removal of the plate, allow to dry in air, spray with a 0.25 per cent w/v solution of ninhydrin in a mixture of equal volumes of pyridine and acetone, and heat at 105º for 2 minutes: the three principal spots in the chromatogram obtained from solution (A) correspond with those obtained from solution (B).

      C. It yields the reactions characteristic of sulfates (Appendix 5.1).

pH 3.5 to 4.5, in a 4.0 per cent w/v solution (Appendix 4.11).

Specific rotation +107º to +121º, calculated on the dried basis, determined in a 10.0 per cent w/v solution (Appendix 4.8).

Loss on drying Not more than 18.0 per cent w/w after drying at 110º at a pressure not exceeding 0.7 kPa (about 5 Torr) for 3 hours (Appendix 4.15).

Sulfate Not less than 32.0 per cent w/w and not more than 35.0 per cent w/w (Appendix 5.2). Dissolve about 250 mg of the test substance, accurately weighed, in 100 ml of distilled water and adjust the solution to pH 11 using strong ammonia solution. Add 10.0 ml of 0.1 M barium chloride VS and about 0.5 mg of phthalein purple. Titrate with 0.1 M sodium edetate VS, adding 50 ml of ethanol when the colour of the solution begins to change and continue the titration until the violet-blue colour disappears. Perform a blank determination (Residual Titration, Appendix 6.17). Each ml of 0.1 M barium chloride is equivalent to 9.606 mg of SO4.

Sulfated ash Not more than 1.0 per cent w/w (Appendix 5.3).

Methanol Not more than 1.0 per cent. Carry out the test as described in the “Gas Chromatography” (Appendix 3.4).

      Internal standard solution Transfer 2.5 ml of 1- propanol to a 500-ml volumetric flask, dilute with water to volume, and mix. This solution contains 0.50 per cent v/v of 1-propanol.

      Standard solution Transfer 1.25 ml of methanol and 1.25 ml of 1-propanol to a 500-ml volumetric flask, dilute with water to volume, and mix to obtain a Standard solution containing 0.25 per cent v/v of methanol and 0.25 per cent v/v of 1-propanol.

      Control solution Dissolve 0.50 g of the test substance in 2.0 ml of water.

      Test solution Dissolve 0.50 g of the test substance in 1.0 ml of Internal standard solution, add 1.0 ml of water, and mix.

      Chromatographic system The chromatographic procedure may be carried out using (a) a glass column (1.5 m × 4 mm) packed with ethylvinylbenzene divinylbenzene copolymer having a nominal surface area of 500 to 600 m2 per g and an average pore diameter of 0.0075 μm (b) nitrogen as the carrier gas at a constant flow rate of 30 ml to 40 ml per minute, and (c) a flame-ionization detector. Maintain the column at a constant temperature between 120º and 140º and the injection port and the detector at a temperature at least 50º higher than that of the column.

      Chromatograph Standard solution, and measure the peak responses as directed under Procedure: the resolution factor between the 1-propanol and methanol peaks is not less than 1.0.

      Chromatograph Control solution, measure the peak responses as directed under Procedure, and examine the chromatogram: if any peak is observed at a retention time corresponding to that of 1-propanol, use the response of that peak to correct the 1-propanol peak response in the chromatogram obtained from Test solution.

      Procedure (Note Use peak areas where peak responses are indicated.) Using a syringe with a polyteftipped plunger, separately inject equal volumes (about 2 μl) of Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the 1-propanol and the methanol peak responses.

      Calculation Calculate the percentage of methanol in the test substance taken by the expression:

1.58(P/M)(RU/RS),

in which P is the percentage (v/v) of methanol in Standard solution, M is the quantity, in g, of the test substance taken to prepare Test solution, RU is the ratio of the methanol peak response to the 1-propanol peak response (corrected, if necessary, by subtracting the response of any peak at the locus of the 1-propanol peak observed in the chromatogram of Control solution) in the chromatogram obtained from Test solution, and RS is the ratio of the methanol peak response to the 1-propanol peak response in the chromatogram obtained from Standard solution.

Content of gentamicins Carry out the test as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).

      o-Phthalaldehyde solution Dissolve 1.0 g of ophthalaldehyde in 5 ml of methanol, and add 95 ml of 0.4 M boric acid, previously adjusted with 8 M potassium hydroxide to a pH of 10.4, and 2 ml of thioglycolic acid. Adjust the resulting solution with 8 M potassium hydroxide to a pH of 10.4.

      Mobile phase Mix 700 ml of methanol, 250 ml of water, and 50 ml of glacial acetic acid. Dissolve 5 g of sodium 1-heptanesulfonate in this solution. Make adjustments if necessary.

      Standard solution Prepare a solution of Gentamicin Sulfate RS in water containing about 0.65 mg per ml. Transfer 10 ml of this solution to a suitable test-tube, add 5 ml of 2-propanol and 4 ml of o-phthalaldehyde solution, mix, and add 2-propanol to obtain 25 ml of solution. Heat at 60º in a water-bath for 15 minutes, and cool.

     Test solution Using the test substance, proceed as directed for Standard solution.

     Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (10 cm × 5 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (5 μm), (b) Mobile phase at a flow rate of 1.5 ml per minute, and (c) an ultraviolet photometer set at 330 nm. Chromatograph Standard solution, and record the peak responses as directed under Procedure: the capacity factor determined from the gentamicin C1 peak is between 2 and 7, the column efficiency determined from the gentamicin C2 peak is not less than 1200 theoretical plates, the resolution factor between any two peaks is not less than 1.25, and the relative standard deviation for replicate injections is not more than 2.0 per cent.

      Procedure (Note Use peak areas where peak responses are indicated.) Separately inject equal volume (about 20 μl) of Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The elution order is gentamicin C1, gentamicin C1a, gentamicin C2a, and gentamicin C2.

      Calculation Calculate the percentage contents of gentamicin C1, gentamicin C1a, gentamicin C2a, and gentamicin C2 taken by the expression:

100rf /rs,

in which rf is the peak response corresponding to the particular gentamicin, and rs is the sum of the responses of all four peaks: the content of gentamicin C1 is between 25 and 50 per cent, the content of gentamicin C1a is between 10 and 35 per cent, and the sum of the contents of gentamicin C2a and gentamicin C2 is between 25 and 55 per cent.

Assay Carry out the microbiological assay of Gentamicin Sulfate according to the “Microbiological Assay of Antibiotics” (Appendix 6.10).

Other requirements Gentamicin Sulfate intended for parenteral administration complies with the following additional requirements.

      Bacterial endotoxins When tested as described in the “Test for Bacterial Endotoxins” (Appendix 8.5), it contains not more than 0.71 Endotoxin Unit per mg of gentamicin.

      Sterility Complies with the “Sterility Test” (Method I, Appendix 10.1).

MONOGRAPHS • GENTAMICIN SULFATE
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หมายเหตุ / Note : TP II 2011 PAGE 111-113