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d-Streptamine, O-3-amino-3-deoxy--d-glucopyranosyl-(1®6)-O-[6-amino-6-deoxy--d-glucopyranosyl(1®4)]-N1-(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-, (S)-.
» Amikacin has a potency of not less than 900 µg of C22H43N5O13 per mg, calculated on the anhydrous basis.
Packaging and storage Preserve in tight containers.
USP Reference standards 11
A: Thin-Layer Chromatographic Identification Test 201
Test solution: 6 mg per mL, in water. Apply 3 µL.
Standard solution: 6 mg per mL, in water. Apply 3 µL.
Mixed solution: a mixture of the Test solution and the Standard solution (1:1). Apply 3 µL.
Developing solvent system: a mixture of methanol, ammonium hydroxide, and chloroform (60:35:25).
Spray reagent: a 1 in 100 solution of ninhydrin in a mixture of butyl alcohol and pyridine (100:1).
Procedure Proceed as directed in the chapter, except to develop the chromatogram by continuous flow for 5.5 hours. Remove the plate from the chamber, allow the solvent to evaporate, and heat the plate at 110 for 15 minutes. Spray the plate with Spray reagent, and immediately locate the spots: amikacin appears as a pink spot, and the spots obtained from the Test solution and the Mixed solution correspond in distance from the origin to that obtained from the Standard solution.
B: The retention time of the peak for amikacin in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Specific rotation 781S: between +97 and +105.
Test solution: 20 mg per mL, in water.
Crystallinity 695: meets the requirements.
pH 791: between 9.5 and 11.5, in a solution containing 10 mg per mL.
Water, Method I 921: not more than 8.5%.
Residue on ignition 281: not more than 1.0%, the charred residue being moistened with 2 mL of nitric acid and 5 drops of sulfuric acid.
Mobile phase Use 0.115 N sodium hydroxide. Make adjustments if necessary (see System Suitability under Chromatography 621).
System suitability solution Prepare a solution in water containing about 0.02 mg of USP Amikacin RS per mL and 0.008 mg of USP Kanamycin Sulfate RS per mL.
Standard preparation Quantitatively dissolve an accurately weighed quantity of USP Amikacin RS in water to obtain a solution having a known concentration of about 0.02 mg per mL.
Assay preparation Transfer about 50 mg of Amikacin, accurately weighed, to a 250-mL volumetric flask, dilute with water to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with an electrochemical detector, a gold working electrode, and a pH silver-silver chloride reference electrode, a guard column that contains packing L47, and a 4-mm × 25-cm analytical column that contains packing L47. The electrochemical detector is used in the integrated amperometric mode with a range of 300 nC, an output of 1 V full scale, a rise time of 0.5 second, positive polarity, potential E = 0.04 V; t1 = 200 ms; E2 = 0.8 V; t2 = 190 ms; E3 = 0.8 V; t3 = 190 ms. The flow rate is about 0.5 mL per minute. Chromatograph the System suitability solution, and measure the peak areas as directed for Procedure: the relative retention times are about 0.8 for kanamycin and 1.0 for amikacin; and the resolution, R, between kanamycin and amikacin is not less than 3. Chromatograph the Standard preparation, and measure the peak areas as directed for Procedure: the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 3%.
Procedure Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in µg, of C22H43N5O13 in each mg of Amikacin taken by the formula:
2500(CE / W)(rU / rS)in which C is the concentration, in mg per mL, of USP Amikacin RS in the Standard preparation; E is the designated amikacin content, in µg per mg, of USP Amikacin RS; W is the weight, in mg, of Amikacin taken to prepare the Assay preparation; and rU and rS are the amikacin peak areas obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2159Pharmacopeial Forum: Volume No. 37(4)