DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE HPLC METHOD FOR ASSAY OF AZITHROMYCIN IN POWDER FOR ORAL SUSPENSION
Department of pharmaceutical Pharmaceutical & Quality Assurance,
Nirmala College of Pharmacy, Mangalagiri, Atmakuru, Guntur -522 203.
A simple, precise and accurate reversed phase liquid chromatographic method has been developed for the assay of azithromycin in powder for oral suspension. The chromatographic separation was achieved on a Asahipak ODP 40 E(250 mm × 4.6 mm, 5 μm) analytical column. A mixture of methanol–ammonium dihydrogen phosphate (0.05M) (30:70, v/v) (pH 9.0) was used as the mobile phase, at a flow rate of 1.5 mLmin-1 and detector wavelength at 210 nm. The retention time of azithromycin was found to be at 8.0 min. The validation of the proposed method was carried out for specificity, linearity, accuracy, precision, robustness and stability indicating assay. The linear dynamic range is from 382–1208 μgmL-1 for azithromycin. The percentage recovery obtained for azithromycin is 101.0%. The developed method can be used for pharmaceutical dosage form and in process testing.
REFERENCE ID: PHARMATUTOR-ART-1711
Azithromycin [9-de-oxy-9a-aza-9a-methyl-9a-homoerythromycin A dihydrate] is an azalide, a subclass of macrolide antibiotics as shown in Fig.1. It is derived from erythromycin; however it differs chemically from erythromycin in that a methyl substituted nitrogen atom is incorporated into the lactone ring, thus making the lactone ring 15 membered. Azithromycin powder for oral suspension is indicated for the treatment of the following infections when caused by microorganisms sensitive to azithromycin particularly those of the respiratory tract, such as pharyngitis / tonsilities, uncomplicated skin and soft tissue infections. It is also effective against certain urinary tract infections and veneral diseases, such as non-gonococcal urithrities, Chlamydia,gonorrhea and cervicities [1-2]. The assay is performed for azithromycin in powder for oral suspension 200mg/5mL. Assays reported in the literature for the determination of azithromycin in biological fluids include HPLC using atmospheric pressure chemical ionization , columetric and amperometric detection , and fluorescence detection ,and microbiological diffusion method on an 8 x 8 Latin Square to find out which of the test microorganisms used show the highest sensitivity .The methods reported in literature for the determination of azithromycin is HPLC, using electrochemical detection [7,8] or UV detector . The present manuscript describes a simple, rapid, precise and accurate isocratic reversed phase HPLC method for assay, impurity interference and stability indicating assay of azithromycin in powder for oral suspension dosage form.The developed method was validated as per the International Conference on Harmonization (ICH) guideline .
Azithromycin standard and impurity-F(Azithromycin related compound), impurity-I(N-Demethylazithromycin),impurity-J(Desosaminylazithromycin),impurity-L(AzithromycinN-oxide) was supplied by Aziant Drug Research Solutions,Hyderabad, India. Ammonium dihydrogen phosphate, Hydrogen peroxide, Sodium hydroxide , HPLC methanol, Hydrochloric acid were purchased from E. Merck (India) Ltd. Worli,Mumbai, India. The 0.45µm nylon filters and PVDF filters were purchased from Advanced Micro Devices Pvt.Ltd.Chandigarh, India. MilliQ water was used throughout the experiment.
Analysis was performed on a chromatographic system of Agilent 1200 series with variable wave length detector and photodiode array detector. A chromatographic separation was achieved on Asahipak ODP 4E (250 mm × 4.6 mm, 5 μm) analytical column. Data acquisition was made with EZchrom elite software .The peak purity was checked with the photodiode array detector.
Standard stock solution of azithromycin (0.8 mg mL-1) was prepared in diluent which was a mixture of methanol and water (50:50,v/v). Filtered about 10mL of above solution through 0.45µ nylon syringe filter by discarding first 4 mL of solution.
Standard solution and calibration graph
Standard stock solution of azithromycin (106.45 mg/mL) was prepared in diluent which was a mixture of methanol and water (50:50,v/v). To study the linearity range serial dilutions were made by adding this standard stock solution in the range of 50 to150% (382-1208 mg mL-1). A graph was plotted as concentration of drug versus peak area response. It was found to be linear. The system suitability test was performed from five replicate injections of standard solution.
The azithromycin powder for oral suspension container was tapped initially to loosen the powder. The powder was reconstituted by adding the required quantity of water .The reconstituted container was shaked for approximately 15 minutes. The container was allowed to stand for about 30 minutes. The shaking procedure of the container was repeated for an additional 15 minutes. 6.4 g of the reconstituted suspension was taken in 250 mL volumetric flask through 10mL syringe. Made upto the mark with diluent. Filtered about 10mL of above solution through 0.45µm nylon syringe filter by discarding first 4 mL solution.
The HPLC method was validated in terms of precision, accuracy and linearity according to ICH guidelines . Assay method precision was determined using six-independent test solutions. The intermediate precision of the assay method was also evaluated using different analyst on different days. The accuracy of the assay method was evaluated by spiking the azithromycin drug substance on placebo in the range of about 50 to 150% level. The sample was extracted as described in Section 2.4 and were analyzed using the developed HPLC method. Linearity test solutions were prepared as described in Section 2.3. The degradation of azithromycin and placebo was performed under different stress conditions (hydrolysis, oxidation,UV,humidity,alkaline, acidic, and thermal stress).To determine the robustness of the method, the final experimental conditions were altered and the results were examined. The flow rate was varied by (±) 0.1 mLmin-.1 The percentage of organic modifier was varied by (±) 5%. Column temperature was varied by (±) 5 °C and pH of mobile phase was varied by (±) 0.1.
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