A Comparison Of Microbiological Assay And Chemical Assay Using Hplc Or Spectrophotometric Assay Methods To Determine The Potency Of Macrolide Antibiotics

Abstract

Microbiological assay as one of the most important methods of quantification of the antibiotics and determination of the efficacy of antibiotics towards test microorganisms was used in this study for the assay of the macrolide antibiotics azithromycin, clarithromycin and erythromycin in their pharmaceutical dosage forms applying the agar diffusion method. Using a strain of Bacillus pumilusATCC No. 8241 as the test organism, azithromycin and clarithromycin at concentrations ranging from 1 to 4 µg/ml and erythromycin at concentrations ranging from 5 to 20 µg/ml could be measured in capsules and tablets. The method was linear (r 2 > 0.99), precise, reproducible (coefficient of variation less than 10%) and accurate, the content percent of the tested drugs was within the pharmacopoeial limit. The statistical analysis showed that there is no significant variation of the assays when the same batch was assayed several times within the day and between the days. The method was found satisfactory for the assay of the drugs in their pharmaceutical dosage forms and successfully applied for assessment of an in vitro antibacterial activityof the macrolide antibiotics. In addition to the microbiological assay, clarithromycin was assayed by the current pharmacopoeial reverse-phase HPLC with UV detection at 210 nm and C18 column method. The results obtained by the two methods were compared, and according to the statistical analysis of the results using the student t-test, there is no significant variation between the two methods of assay (probability > 0.05). The study developed a method for the assay of azithromycin in its pharmaceutical dosage forms using Lambda 2 UV/VIS spectrophotometric analysis at 225 nm band. Azithromycin at concentrations ranging from 0.25 to4 mg/ml and it is corresponding absorbance were used for calibration curve, and azithromycin samples at iv concentration of 0.5 mg/ml was measured in capsules. All samples were found to be within in the pharmacopoeial limit and the method was linear (r 2 = 0.9998). When the chemicalmethod was compared with microbiological assay statistically using the student t-test, it’s found that there was no significant difference between the two methods in the determination of azithromycin inits pharmaceutical dosage forms (probability > 0.05). The study tries to develop HPLC-method for the assay of erythromycin stereate tablets, but it’s found very difficult to analyze erythromycin stereate tablets by HPLCdue to interference of the peaks of the erythromycin and the excipients of the formulation.