DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE HPLC METHOD FOR ASSAY OF AZITHROMYCIN IN POWDER FOR ORAL SUSPENSION

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About Author:
Swapna.G*
Department of pharmaceutical Pharmaceutical & Quality Assurance,
Nirmala College of Pharmacy, Mangalagiri, Atmakuru, Guntur -522 203.
*swapna.goday.gs@gmail.com

Abstract
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

Introduction
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 [3], columetric and amperometric detection [4], and fluorescence detection [5],and microbiological diffusion method on an 8 x 8 Latin Square to find out which of the  test microorganisms used show the highest sensitivity [6].The  methods  reported in literature  for the determination of azithromycin  is HPLC, using electrochemical detection [7,8] or  UV detector [9]. 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 [10].

Experimental

Materials
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.

Equipments
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 preparation
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.

Sample preparation
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.

Method validation
The HPLC method was validated in terms of precision, accuracy and linearity according to ICH guidelines [10]. 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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