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1 Impact factor: 3.958/ICV: 4.10 ISSN: Pharma Science Monitor 8(3), Jul-Sep 2017 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR SIMULTANEOUS ESTIMATION OF CEFOPERAZONE AND TAZOBACTAM IN CEGAVA-TZ INJECTION DOSAGE Ekta D. Patel*, Tejendra J. Patel, Dhwani A. Shah, Kunjal L. Vegad, Yogesh K. Patel, Kaushik Sharda School of Pharmacy, Gandhinagar, Pethapur. R. Patel ABSTRACT Antimicrobial drugs are effective in the treatment of infections because of their selective toxicity means they have the ability to injure or kill an invading microorganism without harming the cells of the host. Cefoperazone (CEFO) inhibits bacterial cell wall formation, peptido glycan synthesis and inhibit enzyme transpeptidase. Tazobactam (TAZO) bind with beta lactamase enzyme and inactive them results in preventing the destruction of beta lactam antibiotics. A reversed phase high performance liquid chromatographic method was developed and validated for simultaneous estimation of Cefoperazone and Tazobactam in injection dosage form. The chromatographic separation was achieved using BDS hypersil C18 column (250 mm, 4.6 mm i.d., 5 μm) as stationary phase & Phosphate Buffer ph 4.0 : ACN (75:25 V/V). Detection was carried out at 260 nm. The average retention time for CEFO and TAZO were found to be 3.60 and min. Linearity for CEFO and TAZO were observed in the concentration range from μg/ml (r2 = ) and μg/ml (r2 = ). Accuracy of the method was studied by the recovery studies at three different levels 80%, 100% and 120% level. The recovery was found to be within the limits of acceptance criteria with average recovery of % for CEFO and % for TAZO. The high precision of proposed method is confirmed by % RSD below 2.0 for repeatability. The proposed simple, accurate and pricise RP-HPLC method was successfully applied for determination of CEFO and TAZO from Cegava-Tz injection dosage form for routine analysis. KEYWORDS: Cefoperazone, Tazobactam, Linearity, Accuracy, Cegava-Tz. INTRODUCTION Cefoperazone Cefoperazone (CEFO), is an anti -Bacterial, Βeta Lactam Antibiotic, is commonly used for Septicaemia, Lower respiratory tract, upper and lower urinary tract infections, Pneumonia, Meningitis, Biliary tract infection. Structure of Cefoperazone shown in figure: 1 [1-5]. official in Official in Indian Pharmacopoiea, British pharmacopoeia and United state pharmacopoeia/national formulary [6,7,8]. It is
2 Impact factor: 3.958/ICV: 4.10 ISSN: Figure: 1 Structure of Cefoperazone Mechanism of Action: Cefoperazone inhibit bacterial cell wall formation. Peptidoglycan is a component of the cell wall and provide mechanical stability by virtue of its highly cross linked latticework structure. Cefoperazone inhibit enzyme transpeptidase that brings about cross-linking between 5 th glycine of the existing Peptidoglycan in the cell wall and 4 th amino acid of the newly formed Peptidoglycan. It inhibit the peptido glycan synthesis [1,2]. Tazobactam Tazobactam (TEZO) is a Anti microbial, β lactamase inhibitors. It is an effective anti-microbial agent, It is used in Abdominal infection, Respiratory tract infection, septicaemia, skin infection, urinary tract infection. Structure of Tazobactam shown in figure: 2 [1,2,3,4,9]. It is official in United state pharmacopoeia / National formulary [8]. Figure: 2 Structure of Tazobactam Mechanism of Action: They bind with beta lactamase enzyme and inactive them. Hence they prevent the destruction at beta lactam antibiotics [1,2]. Introduction to dosage form Cegava-Tz Injection is used as antibiotic formulation. Cegava-Tz Injection contains Cefoperazone and Tazobactam as active ingredients. The emergence and subsequent widespread dissemination of bacterial resistance to a variety of beta-lactam antibiotics by the elaboration of ESBL (Extended Spectrum Beta Lactamase) poses a serious threat to the effective u se of beta lactam antibiotics and cephalosporins. Cefoperazone combination with the beta lactamase inhibitor tazobactam would be a strong basis for rational therapeutics when dealing with ESBL producing pathogen mediated infections. The beta lactamase inhibitor tazobactam in improving
3 Impact factor: 3.958/ICV: 4.10 ISSN: the spectrum of activity and efficacy of cefoperazone. The Combination is approved by CDSCO in 4/11/2009 [10]. Available marketed formulations: Brand Name: Cegava-Tz Manufacturer: Biocon Pharma Limited Ratio of Drug: Cefoperazone : Tazobactam ( 1000 mg : 125 mg) The review of literature revealed that couple of analytical methods including UV- Spectrophotometry and HPLC have been reported for Cefoperazone and Tazobactam individually or with other combinations. But there is no HPLC validation method was reported for this combination of drugs. So, there is cospicuous regerance of interest to develop simple and cost effective RP-HPLC method. MATERIALS AND METHODS Determination of wavelength for maximum absorbance Cefoperazone and Tazobactam are soluble in methanol therefore methanol is selected as solvent. 50 µg/ml solution of Cefoperazone and 6.25 µg/ml solution of Tazobactam were separately prepared in mobile phase. Each solution was scanned between nm in Double beam UV-visible spectrophotometer (Shimadzu, model 1800). Wavelength was selected from the overlay spectra of Cefoperazone and Tazobactam. Both the components show reasonably good response at 260 nm (Figure 3). Figure 3 : Overlay spectra of Cefoperazone (50 µg/ml) and Tazobactam (6.25 µg/ml) for determination of wavelength for maximum absorbance
4 Impact factor: 3.958/ICV: 4.10 ISSN: Chromatographic conditions The column used for chromatographic separations was C 18 (250 mm x 4.6 mm i.d., 5 µm particle size). The analytical wavelength was set at 260 nm and samples 20 µl were injected. The chromatographic separations were accomplished using mobile phase comprised of 0.02 M potassium dihydrogen phosphate Buffer (ph adjusted to 4.0 ± 0.1 using 1% orthophosphoric acid) and Acetonitrile (ACN) in the proportion of 75 : 25 ( %v/v) filtered through 0.45 µm filter (Millipore) and deaerated in ultrasonic bath. Mobile phase was pumped at a flow rate of 1.0 ml/min at ambient temperature. Preparation of mobile phase Buffer Solution : 2.72 g of Potassium dihydrogen phosphate was accurately weighed an dissolved in 1000 ml of HPLC water (0.02 M potassium dihydrogen phosphate) then ph 4.0 ± 0.1 was adjusted with 1% Ortho Phosphoric Acid. Mobile Phase : Phosphate Buffer : ACN (75 : 25 %v/v) Preparation of stock solution: Cefoperazone (50 µg/ml) and Tazobactam (6.25 µg/ml) An accurately weighed quantity of standard Cefoperazone (50 µg/ml) and Tazobactam (6.25 µg/ml) were transferred to 100 ml volumetric flasks and volumes were made up to mark with methanol to get Cefoperazone (50 µg/ml) and Tazobactam (6.25 µg/ml). Preparation of calibration curve Make the Series dilution with methanol to get final concentration of Cefoperazone having concentration range µg/ml (25, 37.5, 50, 62.5,75 µg/ml) and Tazobactam µg/ml (3.125, 4.687, 6.251, 7.812, μg/ml). The plot of peak area against concentration was plotted. Correlation coefficient and regression line equations for Cefoperazone and Tazobactam were calculated. TABLE 1: SYSTEM SUITABILITY PARAMETERS System Suitability Parameters Cefoperazone Tazobactam Retention Time (min) Tailing factor Theoretical plate Resolution 9.036
5 Impact factor: 3.958/ICV: 4.10 ISSN: Validation of the developed method To be done by using following parameter as per International Conference on Harmonization (ICH Guidelines Q2R1) [11, 12]. 1. Specificity 2. Linearity 3. Range 4. Accuracy 5. Precision 6. Detection limit 7. Quantitation limit 8. Robustness 9. System suitability testing Specificity: Specificity of an analytical method is its ability to measure the analyte accurately and specifically in the presence of component that may be expected to be present in the sample matrix. Chromatograms of standard and sample solutions of Cefoperazone and Tazobactam were compared. Linearity and range The linearity response was determined by analyzing 5 independent levels of calibration curve in the range of µg/ml (25, 37.5, 50, 62.5,75 μg/ml) for Cefoperazone and µg/ml (3.125, 4.687, 6.251, 7.812, μg/ml) for Tazobactam. The calibration curves of Cefoperazone and Tazobactam shown in Figure and respectively indicate that the response is linear over the concentration range by correlation coefficient (r) value for Cefoperazone and for Tazobactam. Accuracy (n = 3) It was carried out to determine the suitability and reliability of the proposed method. Accuracy was determined by calculating the % Recovery of Cefoperazone and Tazobactam from the marketed formulation by the standard addition method in which, known amounts of standards powders of Cefoperazone and Tazobactam at 80%, 100% and 120% levels were added to the preanalysed samples. The recovered amounts of Cefoperazone and Tazobactam were calculated at each level and % recovery was reported. Precision
6 Impact factor: 3.958/ICV: 4.10 ISSN: It provides an indication of random error in results and was expressed as% Relative standard deviation. a. Repeatability The repeatability was checked by repeatedly (n=6) injecting 50 μg/ml Cefoperazone and 6.25μg/ml Tazobactam, sample and recording the responses. b. Intraday Precision Intraday precision was determined by assay of sample solution three times in a day for three different concentrations (Combined standard samples of concentrations 25, 50 and 75 µg/ml for Cefoperazone and 3.125, 6.25 and µg/ml for Tazobactam). c. Interday Precision Interday precision was determined by an assay of sample solution on three different days for three different concentrations (Combined standard samples of concentrations 25, 50 and 75 µg/ml for Cefoperazone and 3.125, 6.25 and µg/ml for Tazobactam). Limit of Detection (LOD) and Limit of Quantitation (LOQ) According to the ICH recommendation, the approach based on the standard deviation (SD) of the response and slope was use for the determining the LOD and LOQ values. LOD=3.3 σ/s LOQ=10 σ/s Where, σ =Standard deviation of response and S= Slope of calibration curve. Assay of pharmaceutical dosage form Each bottle containing Cefoperazone and Tazobactam in ratio of 1000:125 mg respectively was weighed. Take Powder equivalent to 50 mg of Cefoperazone and 6.25 mg of Tazobactam was weighed and transferred in a 100 ml volumetric flask and methanol was added. This solution was sonicated for 15 minutes and final volume was made to the mark with methanol. The solution was filtered through Whatman filter paper No. 41. The filtrate 1 ml was transferred in a 10 ml volumetric flask and diluted to the mark with methanol to obtain Cefoperazone (50 µg/ml) and Tazobactam (6.25 µg/ml). Concentration was calculated by regression equation method and % Assay was calculated. Applicability of proposed method was tested by analyzing parental formulation. Results and Discussion
7 Impact factor: 3.958/ICV: 4.10 ISSN: Specificity No interference of peaks were found in the chromatogram indicating that excipients used in the dosage form did not interfere with the estimation of the drugs by the proposed method for the simultaneous estimation of Cefoperazone and Tazobactam in the combined dosage form, hence the method is specific (Figure 4-8). Figure: 4 Chromatogram of mobile phase Figure: 5 Chromatogram of standard CEFO(50 μg/ml) Figure: 6 Chromatogram of standard TAZO (6.25 μg/ml)
8 Impact factor: 3.958/ICV: 4.10 ISSN: Figure 7: Chromatogram of CEFO (50 μg/ml) and TAZO (6.25 μg/ml) from standard solution Figure 8: Chromatogram of sample CEFO (50 μg/ml) and TAZO (6.25 μg/ml) Linearity and Range It was found that Lambert-Beer s law was followed in the concentration ranges of μg/ml and μg/ml for CEFO and TAZO respectively. Calibration curve of CEFO and TAZO was obtained by plotting the mean peak area against concentration (μg/ml) showed in Fig. 9 and 10 respectively. The straight line equations and correlation coefficient for CEFO and TAZO are shown in Table 2 and 3. The calibration curves of Cefoperazone and Tazobactam shown in Figure and respectively indicate that the response is linear over the concentration range by correlation coefficient (r) value for Cefoperazone and for Tazobactam. TABLE 2: LINEARITY DATA FOR CEFOPERAZONE Conc. (μg/ml) Area
9 Impact factor: 3.958/ICV: 4.10 ISSN: Figure 9: Calibration curve of CEFO (25-75 μg/ml) TABLE 3: LINEARITY DATA FOR TAZOBACTAM Conc. (μg/ml) Area Figure 10: Calibration curve of Tazobactam( μg/ml)
10 Impact factor: 3.958/ICV: 4.10 ISSN: Accuracy (%Recovery): Accuracy of the methods was assured, involving analysis of formulation samples to which certain amounts of authentic drugs were added. The resulting mixtures were assayed, and the results obtained for both drugs were compared to those expected. The good recoveries prove the good accuracy of the proposed methods. The results of the accuracy study are shown in Table 4 for CEFO and TAZO respectively. The results show that the percentage recoveries for CEFO and TAZO were found to be in the range of % and % respectively. TABLE 4: DETERMINATION OF ACCURACY OF CEFO AND TAZO (N = 3) Drug Amount Taken(µg/ml) Amount added (µg/ml) Amount found (µg/ml) % Recovery ± S.D (n=3) Cefoperazone ± ± ± Tazobactam ± ± ± Precision and Repeatability The precision of the method was demonstrated by inter-day and intra-day variation studies. In the intra-day studies, three repeated injections of standard solutions were made and the response factor of drug peaks and percentage RSD were calculated. In the inter-day variation studies, three injections of standard solutions were made for three consecutive days and response of drug peaks and percentage RSD were calculated. From the data obtained, the developed RP- HPLC method was found to be precise. The repeatability data for CEFO and TAZO are shown in Table 5. The % RSD was found to be 0.18% for CEFO and 0.15% for TAZO. TABLE 5 : REPEATABILITY STUDY OF CEFO AND TAZO Sr. No. Conc. of CEFO (50 μg/ml) Conc. of TAZO (6.25 μg/ml) Mean SD % RSD The data for Intraday and Interday precision for CEFO is shown in Table 6. The % RSD for Intraday and Interday precision was found to be and for CEFO. The % RSD for Intraday and Interday precision was found to be and for TAZO.
11 Impact factor: 3.958/ICV: 4.10 ISSN: TABLE 6 : INTERMEDIATE PRECISION STUDY OF CEFO AND TAZO Drug Conc. (μg/ml) Intra-day precision Inter-day precision Mean ± S.D % RSD Mean ± S.D % RSD (n = 3) (n = 3) Cefoperazone ± ± ± ± ± ± Tazobactam ± ± ± ± ± ± Robustness Robustness was carried by varying parameters from the optimized chromatographic conditions (Table 7). TABLE 7: DATA OF ROBUSTNESS OF CEFO AND TAZO Parameter Value Area Cefoperazone Tazobactam ph Mean ± SD ± ± % RSD Flow rate Mean ± SD ± ± % RSD : 27 (Buffer : ACN) : 25 (Buffer : ACN) Mobile phase 77 : 23 (Buffer : ACN) Mean ± SD ± ± % RSD LOD and LOQ LOD for CEFO and TAZO was found to be μg/ml and μg/ml respectively. Similarly LOQ for CEFO and TAZO was found to be μg/ml and μg/ml respectively (Table 9). Assay of formulation
12 Impact factor: 3.958/ICV: 4.10 ISSN: TABLE 8: ASSAY RESULT OF CEGAVA-TZ BY RP-HPLC METHOD Formulation Cefoperazone Tazobactam Cegava-Tz Amount Labelled (mg) Amount Found (mg) % Amount found SD (n = 3) Amount Labelled (mg) Amount Found (mg) % Amount found SD (n = 3) ± ± RESULTS AND DISCUSSION The objective of the proposed work was to develop and validate novel analytical method for simultaneous estimation of Cefoperazone and Tazobactam in pharmaceutical formulations according to ICH guidelines. A number of methods appeared in the literature, for estimation of individual drugs or combination with other drugs by UV and HPLC methods. So far there is no specific method for the simultaneous estimation of Paracetamol and Ondansetron. In view of the above fact, a simple RP- HPLC method was planned to develop with high sensitivity, accuracy, precision with costs effective. Various compositions of mobile phase were used. The best results were obtained with 0.02 M potassium dihydrogen phosphate (ph adjusted to 4.0 ± 0.1 using orthophosphoric acid) Buffer and ACN in the proportion of 75 : 25 (v/v) at 1.0 ml/min flow rate. The peaks were well resolved with a resolution factor of The estimation was carried out at 260 nm using a UV detector keeping the flow rate of 1.0 ml/min and injection volume 20 μl. The proposed HPLC method was validated for precision, accuracy studies and the results were within the range thus the method is precise and more accurate. There is no significant changes in the results, thus the method is more robust. The optical regression characteristics and validation parameters are shown in Table 9.
13 Impact factor: 3.958/ICV: 4.10 ISSN: TABLE 9: OPTICAL REGRESSION CHARACTERISTICS AND VALIDATION PARAMETERS Parameter Cefoperazone Tazobactam Calibration Range (μg/ml) μg/ml μg/ml Regression Equation y = x y = x Slop (m) Intercept (c) Correlation co-efficient(r2) Repeatability (n=6) Intraday ( % RSD, n = 3 ) Interday ( % RSD, n = 3 ) Detection limit (LOD) (μg/ml) μg/ml μg/ml Quantification (μg/ml) Limit (LOQ) μg/ml μg/ml CONCLUSION The proposed method was found to be simple, precise, accurate, linear, robust and rapid for simultaneous determination of Cefoperazone and Tazobactam in injection dosage form labeled Cegava-Tz. The developed method gave good resolution between Cefoperazone and Tazobactam with short analysis time (10 min). Results are in good agreement with claim which indicates there is no interference of routinely used excipients. The proposed RP-HPLC method was easily and conveniently applied for determination of Cefoperazone and Tazobactam from combined dosage form for regular monitoring, pharmaceutical manufacturing and research. The percentage of Cefoperazone and Tazobactam was found to be satisfactory, which is comparable with the corresponding label claim. Conflict of interest The author declared that there is no conflict of interest. ACKNOWLEDGEMENT It is very great pleasure and proud sense of reverence that I express my gratitude and thanks to Dr. Yogis K. Patel principal of the Sharda School of pharmacy. Author is also thankful to Ms. Dhwani Shah for supporting every time. REFERENCES 1. Gilman AG., Hardman JG. and Limbard LE: Goodman and Gilman s The Pharmacological basis of therapeutics. McGraw Hill Publishers, New York, Edition 10, 2002:
14 Impact factor: 3.958/ICV: 4.10 ISSN: Goyal RK: Derasari and Gandhi S Elements of pharmacology. B. S. Shah prakashan, Edition 17, 2008: Sweet man Sean C: Martindale the complete drug reference. Pharmaceutical Press, Edition 36, 2009: 227, Neil O, M.J: The Merck Index An encyclopedia of chemicals, drugs and biological. The Royal Society of Chemistry, USA, Edition 14, 2004: Drug bank Drug Profile of Cefoperazone [cited 2013 Sep]; Available from : 6. Indian Pharmacopoeia, Government of India, Ministry of health and family welfare, The Indian Pharmacopoeia Commission, Ghaziabad, 2010: British Pharmacopoeia, The Department of Health, Social Services & Public Safety, London, 2010: United States Pharmacopoeia 35, Rockville M.D., United States pharmacopoeial Convention Inc., 2010: 2554,2555,4755, Drug bank Drug Profile Of Tazobactam [cited 2013 Sep]; Available from: Medindia.net, Introduction Of Dosage Form, [cited 2013 Sep]; Available from: ICH Steering Committee, ICH Q2B Validation of Analytical Procedures; 1996 methodology. London (CPMP/ICH/281/95): European Agency for th e Evaluation of Medicinal Products, International Commission on Harmonization, Switzerland. 12. ICH Topic Q2(R1) Validation of Analytical Procedures: Text and Methodology.
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