1. INTRODUCTION:

AZITHROMYCIN (Zithromax)[1-3] is (2R,3S,4R,5R,8R,10R,11R,12S,13R,14R)-13-[2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribohexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo- hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. It is a macrolide antibiotic related to erythromycin.It fights bacteria in the body. Zithromax is used to treat many different types of infections caused by bacteria, such as respiratory infections, skin infections, ear infections, and sexually transmitted diseases. In children, it is used to treat middle ear infection, pneumonia, tonsillitis, and strep throat.

Azithromycin prevents bacteria from growing by interfering with their protein synthesis. Azithromycin binds to the 50S subunit of the 70S bacterial ribosomes, and therefore inhibits RNA-dependent protein synthesis in bacterial cells.

CEFIXIME [4-7] is 5,6,7-[2-(2-Aminothiazol-4-yl)2(carboxymethoxyamino)acetoamido]3vinyl--3cephem-4-Carboxlic acid. It is a third generation cephalosporin anti bacterial drug given by oral route in the treatment of susceptible infections including gonorrhea, otitis media, pharynsitis and urinary tract infection. It works by fighting bacteria in the body. Cefixime is used to treat many different types of infections caused by bacteria. The bactericidal action of cephalosporin due to the inhibition of cell wall synthesis.

Reason for the combination of Cefixime with Azithromycin:

Neisseria Gonnorrhoeahe, one of the main pathogens of sexually transmitted male urethritis and female endocervicitis has been developing resistance to multiple antimicrobial agents extremely rapidly. Studies have shown that activity of cefixime against N.gonorrhoeae can be significantly enhanced in combination with Azithromycin[8].

The combination also shows a broad spectrum of activity, therby effective against a number of micro organisms.

Available formulations:

1. CEF-A KIT (Azithromycin 1000mg, Cefixime 400mg.)

2. ZiFi-AZ (Azithromycin 250mg, Cefixime 200mg)

Several analytical procedures have been proposed for the quantitative estimation of Azithromycin and Cefixime separately and in combination with other drugs. HPLC and UV methods for estimation of Azithromycin alone[13-18] and in combination with other drugs like Ambroxol Hydrochloride[19] are available. Similarly Cefixime alone[20-25] and in combination with other drugs like cefdinir[26], cefuroxime axetil[27], ofloxacin[28], cloxacillin[29] is estimated by UV and HPLC have also been reported.

To our knowledge simple, rapid and stability indicating analytical method for simultaneous determination of Azithromycin and Cefixime has not been reported so far. So attempt was taken to develop and validate a rapid stability indicating reversed-phase high performance liquid chromatographic method for the quality control of Azithromycin and Cefixime in pharmaceutical preparations with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time. The method was validated and found to be accurate, precise and reproducible.

MATERIALS AND METHOD:

Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC.

Electronic balance, Sonicator, 0.45µ membrane filter

Reagents and chemicals

Pharmaceutical grade Azithromycin and Cefixime were kindly supplied as a gift sample by Dr.Reddy’s Laboratory, Hyderabad, Andhra Pradesh, India. Methanol was of HPLC grade and collected from E. Merck, Darmstadt, Germany. Dipotassium hydrogen phosphates were analytical reagent grade supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from a Milli-QRO water purification system.

Commercial formulation

Azithromycin and Cefixime Tablets available in the market as ZiFi-AZ in composition of Azithromycin(250mg), Cefixime(200mg). The samples were properly checked for their manufacturing license numbers, batch numbers, production, expiry dates and stored properly.

Preparation and selection of mobile phase

The preliminary isocratic studies on a reverse phase C8 column with different mobile phase combination of Dipotassium hydrogen phosphate buffer pH 8.0 and Methanol were studied for simultaneous separation of both the drugs. The optimal composition of mobile phase determined to be Buffer:Methanol (60:40 v/v) and filtered through 0.45µ membrane filter.

Preparation of standard solution

250mg Azithromycin and 200mg Cefixime was dissolved in 100 ml of Diluent (1:1, K2HPO4: Methanol) and was further diluted to get stock solution of Azithromycin and Cefixime (To get 500µg/ml and 400 µg/ml Respectively). This is taken as a 100% concentration. Solution containing mixture of Azithromycin and Cefixime of five different concentrations (50%, 75%, 100% 125%, and 150% of target concentration) were prepared in the same way.

Preparation of sample solution

Sample solution containing both the drugs was prepared by dissolving tablet powder into Diluent (Methanol). Ten tablets were weighed separately. Their average weights were determined. Powder of tablets equivalent to one tablet weight were weighed and taken in a 100 ml volumetric flask, dissolved in diluents, shaken and sonicated for about 20 minutes then filtered through 0.45µ membrane filter. The filtered solution was further diluted in the diluent to make the final concentration of working sample equivalent to 100% of target concentration.

Chromatographic conditions

The mobile phase, a mixture of Dipotassium hydrogen phosphate buffer and methanol (60:40v/v) pumped at a flow rate of 1 ml/min through the column (Agilant Zorbax C8; 5μ, 150 X 4.6 mm) at 45ºC.The mobile phase was degassed prior to use under vacuum by filtration through a 0.45μ membrane filter. Both drugs showed good absorbance at 230 nm, which was selected as wavelength for further analysis.

Validation of hplc method

Method Validation:

Method validation [9-12] is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH) guidelines for parameters like system suitability, linearity and range, precision (repeatability), intermediate precision (ruggedness), specificity, accuracy and robustness.

System suitability

System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of Azithromycin and Cefixime. Various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters.

Specificity

The specificity of the method was determined by checking the interference of any of the possible degradation products generated during the forced degradation study of the drugs. The forced degradation of the drug was carried out with 0.1 N HCl, 0.1 N NaOH, 3% v/v hydrogen peroxide, heat (60°C) and photolysis (365 nm) for determining the stability nature of the drugs. The degradation samples were prepared by taking suitable aliquots of the drug and drug product solutions, and then undertaking the respective stress testing procedures for each solution. After the fixed time period the treated test solutions were diluted up to the mark with mobile phase. The specific stress conditions are described as follows.

Acidic degradation condition:

Acidic degradation was carried out by adding 2 ml of 0.1N HCl, and after 30 minutes neutralizing the mixture by adding 0.1N NaOH.

Alkali degradation condition.

Alkali degradation was carried out by adding 2 ml of 0.1N NaOH, and after 30 minutes neutralizing the mixture by adding 0.1N HCl.

Oxidative degradation condition:

Oxidative degradation was performed by exposing the drug to 2 ml of 3% (v/v) H2O2 for 30 minutes.

Thermal degradation condition:

Thermal degradation was performed by heating the drug content at 60ºC on a thermostatically controlled water bath for 30 minutes.

Photolytic degradation condition:

Photolytic degradation was carried out by exposing the drug content to UV light (365nm) inside a UV chamber for 180 minutes.

Linearity

Linearity of the method was determined by constructing calibration curves. Standard solutions of Azithromycin and Cefixime of different concentrations level (50%, 75%, 100%, 125%, and 150%) were used for this purpose. Each measurement was carried out in six replicates and the peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

Accuracy(Recovery studies)

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 50%, 100% and 150%. Known amounts of standard Azithromycin and Cefixime were added to pre-analyzed samples and were subjected to the proposed HPLC method.

Precision

Precision was evaluated by carrying out six independent sample preparation of a single lot
of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise.

Robustness of method

To evaluate the robustness of the developed RP-HPLC method, flow and temperature variations were done. The effect of change in the flow and temperature were studied. The method was found to be unaffected by flow and temperature variation.

RESULTS AND DISCUSSION:

Optimization of the Chromatographic conditions

Optimization of mobile phase was carried out based on resolution, tailing factor and theoretical plates obtained for Azithromycin and Cefixime. During the trial runs both the drugs were tested with different mobile phase compositions like methanol: water, buffer : acetonitrile, buffer : methanol at various compositions and flow rates. The mobile phase consisting of buffer : methanol (60:40, v/v) at a flow rate of 1.0 ml/min was selected which gave sharp, well-resolved peaks for Azithromycin and Cefixime. The retention times for Azithromycin and cefixime were 2.7 and 4.6minutes, respectively. Both the drugs absorbed UV radiations appreciably at 230 nm, so the same was selected as the detection wavelength. The separation was carried out at room temperature.

Fig.1, 2, 3 represents the chromatograms of standard drugs, drugs in combined tablet formulation and blank mobile phase run, respectively

Figure 1 Typical Chromatogram of standard Azithromycin and Cefixime

	Name	Retention Time	Area	USP Resolution	s/n	USP Tailing	USP Plate Count
1	Azithromycin	2.753	2363603		398.473065	1	3942
2	Cefixime	4.676	3301853	8.632	352.370760	1	4693

Figure 2 Typical chromatogram of Azithromycin and Cefixime in marketed formulation.

	Name	Retention Time	Area	USP Resolution	s/n	USP Tailing	USP Plate Count
1	Azithromycin	2.753	2344777		515.346544	1	3792
2	Cefixime	4.678	3280524	8.481	473.236679	1	4543

Figure 3 Typical chromatogram of Mobile phase blank run

System Suitability

Results of system suitability study are summarized in Table 1. Six consecutive injections of the standard solution showed uniform retention time, theoretical plate count, tailing factor and resolution for both the drugs which indicate a good system for analysis.

Table 1: Result of system suitability tests of Azithromycin and Cefixime

PARAMETERS	AZITHROMYCIN	CEFIXIME
Linearity range	250-750µg/mL	200-600µg/mL
Correlation coefficient	0.999	0.999
Slope	23427x+5164	32502x+8561
Retention time	2.7	4.6
Resolution Factor		8.632
USP plate count	3942	4693
Tailing factor*	1	1
Limit of Detection (LOD)	3.7 µg/mL	3.4 µg/mL
Limit of quantification (LOQ)	12.56µg/mL	11.36µg/mL

*=%Mean

Specificity

Chromatograms shown in figure1 and figure 2 explain that retention time for standard sample and commercial product of Azithromycin and Cefixime are same. This proves that, excipients have no effect on the analytical method. On the other hand, blank peak did not overlap drug peak. So the method is highly selective. After forced degradation also, there are no interferences, hence the method is specific. A linear relationship between peak areas (average peak areas of six replicates) versus concentrations was observed for Azithromycin and Cefixime in the range of 50% to 150% of nominal concentration. Correlation coefficient was 0.999 for both the drugs which prove that the method is linear. Calibration curve of Azithromycin and Cefixime are shown in Fig 6 and 7

Linearity:

Figure 6 Linearity of Azithromycin

Figure 7 Linearity of Cefixime

Accuracy

Table 2: Accuracy (%recovery) results of Azithromycin and Cefixime

Sample no.	Azithromycin
Sample no.	Spiked Amount (mg)	Recovered Amount (mg)	% Recovered		%Average recovery
1 2 3	12.5mg 25mg 37.5mg	12.25 mg 24.75 mg 38.2 mg	98 99 102		99.66%
	Cefixime
1 2 3	10mg 20mg 30mg	10.2 mg 19.6 mg 30.3 mg	102 98 101	100.33%

Results of accuracy study are presented in table 2. The measured value was obtained by recovery test. Spiked amount of both the drug were compared against the recovery amount.

% Recovery was 99.66% for Azithromycin and 100.33% Cefixime. All the results indicate that the method is highly accurate.

Precision

Results of Intraday and inter day variability were summarized in table 3. Intraday variability was done from 9.00 am to 6.00 pm on the same day. % RSD of peak areas was calculated for various run .The method is precise as % RSD of peak area was less than 2% in all tests

Table3: Intra day and inter day precision result of Azithromycin and Cefixime

Drug

%RSD (intra-day)

%RSD (Inter-day)

Azithromycin

Cefixime

1.07

1011

1.15

1.32

Robustness

The results of robustness of the present method showed that changes were made in the flow rate and temperature did not produce significant changes in analytical results which are presented in Table 4 . As the changes are not significant we can say that the method is robust

Table 4: Results for robustness test of Azithromycin and Cefixime

Parameters	Changes	RT	USP Tailing	USP Plate count
AZITHROMYCIN
Flow rate (ml/min)	0.5	3.5	1	3787
	1.5	2.3	1	3784
Temperature	40ºc	2.9	1	3727
	50ºc	2.5	1	3746
CEFIXIME
Flow rate (ml/min)	0.5	5.5	1	4370
	1.5	3.8	1	4378
Temperature	40ºc	5.1	1	4315
	50ºc	4	1	4276

CONCLUSION:

A validated stability-indicating RP-HPLC method has been developed for determination of Azithromycin and Cefixime in their bulk and combined tablet dosage forms. The results obtained by the stress degradation conditions of the drugs show that the method is specific and stability-indicating. The method was found to be simple, accurate, precise and sensitive. The method was successfully applied for the determination of both drugs in combined tablet dosage form. In the future, this method may be applied for routine analysis of both the drugs in API, formulations, dissolution studies, bioavailability and pharmacokinetic studies.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Reddy’s Laboratories, Hyderabad, Andhra Pradesh, India for providing the gift samples and to the Rainbow Pharma Training Lab, Kukatpally, Hyderabad, Andhra Pradesh, India for providing the research facilities.

REFERENCES:

1. http://www.drugs.com/zithromax.html

2. http://www.drugbank.ca/drugs/DB00207

3. Indian Pharmacopoeia 2007, Govt. of India, The Indian pharmacopoeia commission, Ghaziabad, Ministry of Health and Family welfare.

4. http://www.drugs.com/mtm/cefixime.html

5. http://www.drugbank.ca/drugs/DB00671

6. Goodman and Gilman’s the pharmacological basis of therapeutics-11th edition (2006) by kaball.

7. Martindale The complete drug reference.

8. http://www.springerlink.com/content/f1p727694750p035/

9. Sharma SK: Validation of pharmaceutical products and process. The Eastern Pharmacist 2001: 21-23.

10. Linda LN: Reviewer guidance-Validation of chromatographic methods, Center for drug evaluation and research. 1994: 1-30.

11. ICH-Q2B, Validation of Analytical Procedures: Methodology. ICH Harmonized Tripartite Guideline, Geneva, 1996: 1-8.

12. ICH-Q2A, Text on Validation of Analytical Procedures. ICH Harmonized Tripartite Guideline, Geneva, 1995: 2-3.

13. BN Suhagia et al. Determination of Azithromycin in pharmaceutical dosage forms by Spectrophotometric method. IJPS. 68(2); 2006: 242-245.

14. Al-Rimawi F, Kharoaf M. Analysis of azithromycin and its related compounds by RP-HPLC with UV detection. J Chromatogr Sci. 48(2); 2010: 86-90.

15. Yang ZY et al. Determination of azithromycin by ion-pair HPLC with UV detection. J Pharm Biomed Anal. 49(3); 2009: 811-815. http://www.ncbi.nlm.nih.gov/pubmed/19162426

16. [16] N. Kovačić-Bošnjak et al. Reversed-phase HPLC analysis of the semisynthetic macrolide antibiotic azithromycin Chromatographia. 25(11); 1988: 999-1003.

17. El-Gindy A et al. Optimization and validation of a stability-indicating RP-HPLC method for determination of azithromycin and its related compounds. J AOAC Int. 94(2); 2011: 513-522. http://www.ncbi.nlm.nih.gov/pubmed/21563685

18. [18] Patricia Zubata et al. A new HPLC method for azithromycin quantitation. J Pharm Biomed Anal. 27(5); 2002: 833-836.http:// www.sciencedirect.com/science/article/pii/S0731708501005544

19. Pisarev VV et al. Development and validation of a reversed-phase HPLC method for simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet dosage form. J Pharm Biomed Anal. 48(5); 2008: 1481-1484. http://www.ncbi.nlm. nih.gov/ pubmed/18993009

20. SP Gandhi, SJ Rajput. Study of degradation profile and development of stability indicating methods for cefixime trihydrate IJPS. 71(4); 2009: 438-442.

21. Shaikh KA et al. Determination of cefixime blood plasma levels by HPLC. Antibiot Khimioter. 54(7-8); 2009: 37-40. http://www.ncbi.nlm.nih.gov/pubmed/20201402

22. Rolando Gonzalez-Hernandezet al. Reversed phase high performance liquid chromatographic determination of cefixime in bulk drugs. J Liq Chrom and Rel Tech. 24(15); 2001: 2315-2324. http://www.tandfonline.com/doi/abs/10.1081/JLC-100105143

23. Andrew J. Falkowski, Zee M. Look. Determination of cefixime in biological samples by reversed-phase high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 422; 1987: 145-152. http://www.sciencedirect.com/ science/ article/pii/0378434787804474

24. Ahmed E. M. Saeed et al. Development and validation of a high performance liquid chromatography method for determination of cefixime trihydrate and it’s degraded products formed under stress condition of uv light. IJPSR, 3(2); 2012: 469-473. http://www.ijpsr.com/V3I2/22%20Vol.%203,%20Issue%202,%20Feb.%202012,%20IJPSR-1021,%20Paper%2022.pdf

25. Paresh B¹ et al. Difference Spectroscopic, and High-Performance Liquid Chromatographic Methods for the Determination of Cefixime in Pharmaceutical Formulations. J of AOAC International. 89(4); 2006: 987-994. http://www.ingenta connect.com/content/aoac/jaoac/2006/00000089/00000004/art00010

26. Khan A et al. Simultaneous determination of cefdinir and cefixime in human plasma by RP-HPLC/UV detection method: Method development, optimization, validation, and its application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 879(24); 2011: 2423-2429. http://www.ncbi. nlm.nih.gov/pubmed/21782531

27. K. Azhagesh Raj et al. Determination of cefixime trihydrate and cefuroxime axetil in bulk drug and pharmaceutical dosage forms by hplc. International J of ChemTech Research. 2(1); 2010: 334-336. http://sphinxsai.com/sphinxsaiVol_2No.1/ChemTech_Vol_ 2No.1/ChemTech_Vol_2No.1PDF/CT=55%20%28334-336%29. pdf

28. Santosh V. Gandhi et al. A simple and sensitive rp hplc method for simultaneous estimation of cefixime and ofloxacin in combined tablet dosage form. IJPPS. 3(1); 2011: 920-923. http://www.ijppsjournal.com/Vol3Issue1/920.pdf

29. Ajit R Wankhede. Development and Validation of RP-HPLC Method For Simultaneous Estimation of Cefixime and Cloxacillin in Tablet Dosage Form. Ijpba. 1(3); 2010: 105-109. http://www.ijpba.info/ijpba/index.php/ijpba/article/view/105

Received on 12.06.2012 Modified on 09.07.2012

Asian J. Research Chem. 5(8): August, 2012; Page 1067-1073