Detection
PH An aqueous solution of 25mg per ml, PH 3.0~4.5( Appendix Ⅵ H).
Clarity and colour of solution Dissolve 1.0g of the substance in 10 ml of water, the solution is clear; any colour produced is not more intense than that of reference solution Y4 or YG4(Appendix Ⅸ A).
Related substance
Dissolve a accurately weighed quantity of the substance in mobile phase A and dilute to obtain a test solution containing 0.5mg per ml; dilute a quantity of Ciprofloxacin Hydrochloride CRS with mobile phase A to obtain a reference solution containing 1g per ml. Dissolve 15 mg of Impurity A RS in a mixture of 0.6ml of 6 mol/L ammonia solution and a quantity of water in a 100 ml flask, dilute to volume with water and mix well. Dilute 1 ml of the solution to100ml with mobile phase A and mix well, take it as the Impurity A CRS. Carry out the method for HPLC (Appendix ⅤD), using a column packed with octadecylsilane bonded silica, a mixture of 0.025mol/L phosphoric acid solution- acetonitrile(87:13) ( adjusted to PH 3.0±0.1 with triethylamine) as the mobile phase A, acetonitrile, gradiently elute as mobile phase B with flow rate of 1.5ml per min according to the table below.
Dissolve a quantity of Ofloxacin CRS, Ciprofloxacin Hydrochloride CRS and Impurity Ⅰ CRS in mobile phase A and dilute to obtain a mixture of 5g of Ofloxacin CRS, 0.5mg of Ciprofloxacin Hydrochloride CRS and 10g of Impurity Ⅰ CRS. Inject 20l of the mixture solution into the chromatograph, take 278nm as detection wavelength, and record the chromatogram, the retention time for the peak of Ciprofloxacin Hydrochloride is about 12 minutes. The resolution factor between the peaks of Ofloxacin, Ciprofloxacin Hydrochloride and Impurity Ⅰ complies with the related requirements. Inject 20l of the reference solution into the chromatograph, take 278nm as detection wavelength, adjust the detection sensitivity to make the peak of main ingredient be about 20% of the sum of all the peaks.
Inject 20l of test solution, the reference solution and the impurity A CRS into the chromatograph, take 278nm and 262nm as detection wavelength, and record the chromatogram, the retention time for the peak of Ciprofloxacin Hydrochloride is 1, for impurity E, B, C, I, and D are separately 0.3, 0.6, 0.7, 1.1 and 1.2. The area of the peak in the chromatogram obtained with impurity A CRS (detected under 262nm) is not more than 0.3% calculated on peak area by external standard method.
The areas of the peak in the chromatogram obtained with Impurity B, C, D and E (detected under 262nm) should not more than those of the principal peak obtained with reference solution(0.2%) calculated on peak area after correction (multiply correction factor of 0.7, 0.6, 1.4 and 6.7). The area of the peak in the chromatogram obtained with any other impurity (detected under 278nm) should not more than that of the peak obtained with reference solution (0.2%). The sum of all the impurity area after correction is not more than 3.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.7%). Disregard any peak obtained with the test solution that is less than 0.1 time the area of the principal peak in the chromatogram obtained with the reference solution.
Time( minute) Mobile phase A (%) Mobile phase B (%)
0 100 0
16 100 0
53 40 60
54 100 0
65 100 0