Limit of methanol
Internal standard solution
Transfer 2.5 mL of n-propyl alcohol to a 500-mL volumetric flask, dilute with water to volume, and mix. This solution contains 0.50% (v/v) of n-propyl alcohol.
Standard preparation
Transfer 1.25 mL of methanol and 1.25 mL of n-propyl alcohol to a 500-mL volumetric flask, dilute with water to volume, and mix to obtain a Standard preparation containing 0.25% (v/v) of methanol and 0.25% (v/v) of n-propyl alcohol.
Control solution
Dissolve 0.50 g of Gentamicin Sulfate in 2.0 mL of water.
Test preparation
Dissolve 0.50 g of Gentamicin Sulfate in 1.0 mL of Internal standard solution, add 1.0 mL of water, and mix.
Chromatographic system
(see
Chromatography 621)The gas chromatograph is equipped with a flame-ionization detector and a 4-mm × 1.5-m column packed with support S3. The column temperature is maintained at a constant temperature between 120
and 140
, and the injection port and detector block are maintained at a constant temperature at least 50
higher than the column temperature. Nitrogen is used as the carrier gas at a constant flow rate of between 30 and 40 mL per minute. Chromatograph the
Standard preparation, and measure the peak responses as directed under
Procedure: the resolution,
R, between the
n-propyl alcohol peak and the methanol peak is not less than 1.0. Chromatograph the
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
n-propyl alcohol, use the response of that peak to correct the
n-propyl alcohol peak response in the chromatogram obtained from the
Test preparation.
Procedure
Using a syringe with a polytef-tipped plunger, separately inject equal volumes (about 2 µL) of the
Standard preparation and the
Test preparation into the chromatograph, record the chromatograms, and measure the
n-propyl alcohol and the methanol peak area responses. Calculate the percentage of methanol in the Gentamicin Sulfate taken by the formula:
1.58(P/ M)(RU / RS),
in which
P is the percentage (v/v) of methanol in the
Standard preparation; M is the quantity, in g, of Gentamicin Sulfate taken to prepare the
Test preparation; RU is the ratio of the methanol peak area response to the
n-propyl alcohol peak area response (corrected, if necessary, by subtracting the response of any peak at the locus of the
n-propyl alcohol peak observed in the chromatogram of the
Control solution) in the chromatogram obtained from the
Test preparation; and
RS is the ratio of the methanol peak area response to the
n-propyl alcohol peak area response in the chromatogram obtained from the
Standard preparation: not more than 1.0% of methanol is found.
Content of gentamicins
o-Phthalaldehyde solution
Dissolve 1.0 g of o-phthalaldehyde in 5 mL of methanol, and add 95 mL of 0.4 M boric acid, previously adjusted with 8 N potassium hydroxide to a pH of 10.4, and 2 mL of thioglycolic acid. Adjust the resulting solution with 8 N 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 (see
System Suitability under
Chromatography 621).
Standard preparation
Prepare a solution of
USP 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 isopropyl alcohol and 4 mL of
o-Phthalaldehyde solution, mix, and add isopropyl alcohol to obtain 25 mL of solution. Heat at 60
in a water bath for 15 minutes, and cool.
Test preparation
Using Gentamicin Sulfate, proceed as directed for Standard preparation.
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 330-nm detector and a 5-mm × 10-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the resolution,
R, between any two peaks is not less than 1.25, the capacity factor determined from the gentamicin C
1 peak is between 2 and 7, the column efficiency determined from the gentamicin C
2 peak is not less than 1200 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Test preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. The elution order is gentamicin C
1, gentamicin C
1a, gentamicin C
2a, and gentamicin C
2. Calculate the percentage contents of gentamicin C
1, gentamicin C
1a, gentamicin C
2a, and gentamicin C
2 in the portion of Gentamicin Sulfate taken by the formula:
100rf / rs,
in which
rf is the peak area response corresponding to the particular gentamicin; and
rs is the sum of the area responses of all four peaks: the content of gentamicin C
1 is between 25% and 50%, the content of gentamicin C
1a is between 10% and 35%, and the sum of the contents of gentamicin C
2a and gentamicin C
2 is between 25% and 55%.
Other requirements
Where the label states that Gentamicin Sulfate is sterile, it meets the requirements for
Sterility Tests 71 and for
Bacterial endotoxins in
Gentamicin Injection. Where the label states that Gentamicin Sulfate must be subjected to further processing during the preparation of injectable dosage forms, it meets the requirements for
Bacterial endotoxins in
Gentamicin Injection.