A: Infrared Absorption á197Kñ.

B: It responds to the tests for Sulfate á191ñ.

Test solution: 10mg per mL,in water.

Internal standard solution— Transfer 2.5mLof n-propyl alcohol to a 500-mLvolumetric flask,dilute with water to volume,and mix.This solution contains 0.50%(v/v)of n-propyl alcohol.

Standard preparation— Transfer 1.25mLof methanol and 1.25mLof n-propyl alcohol to a 500-mLvolumetric flask,dilute with water to volume,and mix to obtain a Standard preparationcontaining 0.25%(v/v)of methanol and 0.25%(v/v)of n-propyl alcohol.

Control solution— Dissolve 0.50g of Gentamicin Sulfate in 2.0mLof water.

Test preparation— Dissolve 0.50g of Gentamicin Sulfate in 1.0mLof Internal standard solution,add 1.0mLof water,and mix.

Chromatographic system (see Chromatography á621ñ)—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 120and 140,and the injection port and detector block are maintained at a constant temperature at least 50higher than the column temperature.Nitrogen is used as the carrier gas at a constant flow rate of between 30and 40mLper 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 2µL)of the Standard preparationand the Test preparationinto 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: in which Pis the percentage (v/v)of methanol in the Standard preparation,Mis the quantity,in g,of Gentamicin Sulfate taken to prepare the Test preparation,RUis 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 RSis 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.

o-Phthalaldehyde solution— Dissolve 1.0g of o-phthalaldehyde in 5mLof methanol,and add 95mLof 0.4Mboric acid,previously adjusted with 8Npotassium hydroxide to a pHof 10.4,and 2mLof thioglycolic acid.Adjust the resulting solution with 8Npotassium hydroxide to a pHof 10.4.

Mobile phase— Mix 700mLof methanol,250mLof water,and 50mLof glacial acetic acid.Dissolve 5g of sodium 1-heptanesulfonate in this solution.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Prepare a solution of USP Gentamicin Sulfate RSin water containing about 0.65mg per mL.Transfer 10mLof this solution to a suitable test tube,add 5mLof isopropyl alcohol and 4mLof o-Phthalaldehyde solution,mix,and add isopropyl alcohol to obtain 25mLof solution.Heat at 60in a water bath for 15minutes,and cool.

Test preparation— Using Gentamicin Sulfate,proceed as directed for Standard preparation.

Chromatographic system (see Chromatography á621ñ)—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 1.5mLper 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 C1peak is between 2and 7,the column efficiency determined from the gentamicin C2peak is not less than 1200theoretical plates,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Test preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.The elution order is gentamicin C1,gentamicin C1a,gentamicin C2a,and gentamicin C2.Calculate the percentage contents of gentamicin C1,gentamicin C1a,gentamicin C2a,and gentamicin C2taken by the formula: in which rfis the peak area response corresponding to the particular gentamicin,and rsis the sum of the responses of all four peaks area:the content of gentamicin C1is between 25%and 50%,the content of gentamicin C1ais between 10%and 35%,and the sum of the contents of gentamicin C2aand gentamicin C2is between 25%and 55%.