Tuesday, 26 March 2013

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



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

Results and discussion
Optimization of the chromatographic conditions
During the analysis of basic drugs like azithromycin  one of the well known problem is peak tailing. Since these compounds strongly interact with polar ends of HPLC column packing materials, causing severe peak asymmetry and low separation efficiencies. High purity silica backbone and advances in bonding technology have alleviated the tailing problem of polar compounds in HPLC to a significant extent. During the optimization of the method, different columns (Inertsil C8, 250 mm × 4.6 mm, 5 μm; Zorbax C18 250 mm × 4.6 mm, 5 μm; Symmetry C18 250 mm × 4.6 mm, 5 μm) and two organic solvents (acetonitrile and methanol) were tested. The chromatographic conditions were also optimized by using different buffers like phosphate, acetate and citrate for mobile phase preparation. After a series of screening experiments, it was concluded that phosphate buffers gave better peak shapes than their acetate and citrate counterparts. The chromatographic separation was achieved on a  Asahipak ODP 4E (250 mm × 4.6 mm, 5 μm)  column, by using a mixture of ammonium dihydrogen phosphate–methanol (0.05M) (30:70, v/v) as mobile phase. Asahipak  columns are having high pH (9–13) and temperature (20–60 °C) stability. About mobile phase, due to the lack of other chromophore than the ester group in azithromycin and, therefore, the need to work at a low wavelength (210 nm), methanol was considered as organic solvent instead of acetonitrile . At 30°C column temperature and pH 9.0 of mobile phase, the peak shape of azithromycin was found symmetrical. The flow rate kept was 1.5mL/min to achieve adequate retention time of  azithromycin peak  (Fig. 1-2)
Validation of method
Specificity
The specificity of the HPLC method is illustrated in (Figs. 3-7) where complete separation of azithromycin was noticed in presence of impurities.  In addition there was no interference at  analysis with photo diode detector, purity angle was less than purity threshold for the analyte. he retention time of azithromycin in the chromatogram of placebo solution. In peak  purity This shows that the peak of analytes was pure and excipients in the formulation did not interfere the analyte
Selectivity
Neither formulation ingredients nor degradation products interfered with quantitation of azithromycin. All samples and placebo were analyzed using the assay chromatographiccondition described. No evidence of interactive  degradation products was seen during evaluation. However  azithromycin  was observed to be susceptible to acidic and oxidative condition. So avoid acidic condition during analysis.Mild degradation was observed in base , water, thermal, and UV degradation.Selectivity was demonstrated showing that azithromycin peak was free of interference of degradation products indicating that the proposed method is stability indicating.
Accuracy
Accuracy of the method was calculated by recovery studies at six levels for 50% and 150% level and three levels for 75%, 100%, and 125%. (Table 1).The mean percentage recovery obtained for azithromycin was found to be in between 99.90 and 101.0%  respectively.
Table 1
Results of the recovery analysis of  azithromycin
S.No.
% spike level
Amount added(mg)
Amount recovered (mg)
%Recovery
%Mean Recovery
% RSD
1.
50
93.71
93.60
99.9
100.2
0.4
2.
93.91
93.63
99.7
3.
93.79
94.32
100.6
4.
93.72
94.18
100.5
5.
93.65
94.04
100.4
6.
93.62
94.00
100.4
1.
75
148.01
147.96
100.0
99.9
0.2
2.
148.19
148.20
100.0
3.
148.16
147.74
99.7
1.
100
198.68
199.09
100.2
100.2
0.1
2.
198.73
199.42
100.3
3.
198.23
198.37
100.1
1.
125
243.69
245.84
100.9
101.0
0.2
2.
243.75
246.56
101.2
3.
243.57
245.87
100.9
1.
150
299.84
302.39
100.9
100.9
0.3
2.
299.48
301.45
100.7
3.
299.51
302.65
101.1
4.
299.80
302.24
100.8
5.
299.85
301.51
100.6
6.
299.84
303.82
101.3
 
Precision
The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing five replicate analyses of the same working solution. The relative standard deviation (R.S.D.) obtained for  azithromycin was  0.2. (Table2).
Table 2
 System suitability parameters
Parameters
Azithromycin
Theoritical Plates
8335
Peak Symmetry
0.9
%RSD
0.2
The intra- and inter-day variability or precision data are summarized in (Table3). The intra-day precision of the developed LC method was determined by preparing the samples of the same batch. 6. 4 g of sample weighed  into six separate 250 mL volumetric flask ,and made upto  the mark with diluent . Filtered   about  10 mL  of  above solution through 0.45µm nylon syringe filter by discarding first 4 mL solution. Injected  blank,  and six replicate injections of repeatability solutions.The %R.S.D,% assay of the assay results was used to evaluate the method precision. The inter-day precision was also determined by the same procedure. The results indicated the good precision of the developed method (Table3).
Table .3
Intra -and inter- day assay precision data(n=12)
Sample No.
Method Precision
Intermediate Precision
Over all % RSD (n=12)
1
98.3
101.8



1.0



2
100.2
101.3
3
100.1
98.6
4
99.9
100.2
5
99.9
100.3
6
100.5
101.1
Mean
99.8
100.6
% RSD
0.8
1.1
View Within Article Linearity
Linearity was determined for azithromycin in the range of 382–1208 μg/mL. The correlation coefficient (‘r’) value for the drug was >0.999. Typically, the regression equation for the calibration curve was found to bey = 966.6x − 8620.8 for azithromycin.
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
Robustness of the method was investigated under a variety of conditions including changes of pH of the mobile phase, flow rate, percentage of methanol in the mobile phase and column oven temperature. The  standard solution is injected in five replicates and sample solution of 100% concentration is prepared and injected  for every condition and % R.S.D. of assay was calculated for each condition. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 4)
Table 4
Results of robustness study.
Robustness parameter
Level
% RSD of Results
pH of mobile phase
8.8  ( Low pH)
0.1
9.2 ( High pH)
0.1
organic composition
High % organic strength
0.1
Low % organic strength
1.1
flow rate
1.4 mL/min
0.2
1.6 mL/min
0.1
column oven temperature
25
0.1
35
0.3

Conclusion
A simple, specific, linear, precise, accurate and stability indicating RP-HPLC method has been developed and validated for quantitative determination of azithromycin  in powder for oral suspension. Statistical analysis proves that method is repeatable and selective for the analysis of azithromycin in powder for oral suspension. As the method seperates the drug from its degradation products it can be employed as a stability indicating one. The developed method can be applicable for pharmaceutical dosage forms and in process testing.
References
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[2] J.E.Kapusnik-under,M.a. sande, H.F.chambers,Farmacos antimicrobianos,in J.Hardman, L.Limbird (Eds.), Goodman&gilman. As Bases Farmacologicas da Terapeutica, ninth ed., Mc Graw Hill, Santiago,1996.
[3] H.G. Fouda, R.P.Schneider (1995) Ter.drug Mon 17:183.
[4] I. Kanfer, M.F. Skinner, R.B. Walker (1998) J Chromatogr A  812:286
[5] J.S. Torano, H.P. Guchelaar, J Chromatogr. B  (1998) 720:910.
[6] T. Turcinov, S. Pepeljnjak, J. Pharm.Biom.  Anal (1998) 17:97.
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[8] The United States Pharmacopoeia, 24 ed., Rockville: United states Pharmacopeial convention , 2000.
[9] N.Kovacic, J.Marincel, J.Chromatographia (1988) 25:1003.
[10] Validation of analytical methods and procedures :Text and Methodology Q2 (R1):ICH Harmonised Tripartite Guidline. 2007 Nov.

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