Development and Validation of Derivative Spectroscopic Method for Simultaneous Estimation of Cefadroxil and Probenecid
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1 2350 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 7 Issue 1 January March 2014 Research Paper IJPSN MAHETA Development and Validation of Derivative Spectroscopic Method for Simultaneous Estimation of Cefadroxil and Probenecid Pratik S. Maheta*, Pratik R. Patel, Rajesh R. Parmar, M.M.K. Modasiya and Dushyant A. Shah APMC College of Pharmaceutical Education and Research, Himatnagar , Gujarat, India. Received April 25, 2013; accepted July 31, 2013 ABSTRACT A novel, simple, accurate, sensitive, precise and economical derivative spectroscopic method was developed and validated for the determination of cefadroxil and probenecid in synthetic mixture. First order derivative spectroscopy method was adopted to eliminate spectral interference. The method obeys Beer s Law in concentration ranges of 4-36 μg/ml for cefadroxil and of 5-25 μg/ml of probenecid. The zero crossing point for cefadroxil and probenecid was 260 nm and nm respectively in 0.1N HCl. The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation. This method has been successively applied to synthetic mixture and no interference from the synthetic mixture s excipients was found. KEYWORDS: Cefadroxil; Probenecid; UV spectroscopic method; first order derivative spectroscopy; Method validation. Introduction Cefadroxil (Figure 1) belongs to a class of first generation cephalosporin compounds, the β-lactam antibiotics that act as a bactericidal by disrupting the synthesis of the peptidoglycan layer of bacterial cell walls (Katzun, 2007).They are used for the treatment of skin and soft tissue infection, pharyngitis & UTI. Probenecid (Figure 1) is uricosuric agent and also act as adjuvant. Probenecid is used with cephalosporin as an adjuvant because it delays the rate of excretion of cephalosporin. Thus increase its effect in various infection like urinary tract infection, tonsillitis and pharyngitis, skin and soft tissue infection (Rang and Dale, 2003). Nowadays, the mixtures of these active components are present in pharmaceutical formulations as tablet forms. Both the drugs are official in Indian pharmacopoeia Several UV and chromatographic methods have been cited in the literature for the estimation of cefadroxil (Suddhasattya et al., 2010; Chetan et al., 2010; El-Gindy et al., 2000; Madhuri et al., 2012;Patil et al., 2011; Anjum, 2011; Dhoka and Chopadeet al., 2012) and probenecid (Chudhari et al., 2006; Jain et al., 1997; Jain et al., 1998; Sireesha et al., 2004; Shindeet al., 1994 Kurian et al., 2012) that seems to be no reports concerning methods for the simultaneous determination of both analytes (cefadroxil and probenecid) using UV in the commercial pharmaceutical preparations has been published. Derivative spectroscopic method involves the conversion of a normal spectrum to its first, second, or higher derivative spectrum. The normal absorption spectrum is referred to as the fundamental, zero order or Do spectrum. First order derivative spectra are also known as D1 spectra. Therefore in the present research work, our aim is to develop a novel, simple, accurate, sensitive, precise and economical derivative spectroscopic analytical method to estimate cefadroxil and probenecid in their synthetic mixture. Fig.1.Structure of cefadroxil (A) and probenecid (B). 2350
2 Maheta et al: Spectroscopic Method for Estimation of Cefadroxil and probenecid 2351 Materials and Methods Instrument A Shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Chemicals and Reagents Reference standard of cefadroxil was obtained from Sehat Pharma Pvt. Ltd, Himatnagar and reference standard of probenecid was obtained from Geno Pharmaceutical Ltd, Goa. Methanol (A.R.) grade, 0.1N HCl and calibrated glassware were employed throughout the work. Preparation of Standard and Sample solutions Preparation of 0.1N hydrochloric acid (0.1N HCl) 0.1N HCl can be prepared by adding hydrochloric acid (8.5 ml) in 1000 ml volumetric flask and diluting up to the mark with Distilled water. (0.1N HCl) Preparation of standard stock solution An accurately weighed quantity of cefadroxil (25 mg) and probenecid (25 mg) were transferred to a separate 25 ml volumetric flask. Add 10 ml methanol, sonicate and diluted to the mark with methanol (1000 μg/ml) each. From the above solutions prepare solutions having concentration cefadroxil and probenecid (100 μg/ml) each with methanol. Preparation of standard solution From the standard stock solutions prepare solutions having concentration cefadroxil (10 μg/ml) and probenecid (10 μg/ml) with 0.1N HCl. Preparation of synthetic mixture The synthetic mixture of cefadroxil and probenecid was prepared in the ratio of 1:1. Cefadroxil (250 mg) and probenecid (250 mg) were taken. Then common excipients, which are used in the tablet formulation, were added in this mixture and total 1 gm of mixture was prepared and it was used in further study. Preparation of sample solution From synthetic mixture, powder equivalent to 25 mg of cefadroxil and 25 mg of probenecid was transferred to a 25 ml volumetric flask. Methanol (10 ml) was added to it and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with methanol. From the above stock solution 10 ml of solution is taken and diluted to the mark in 100 ml volumetric flask. The above solution was suitably diluted with 0.1N HCl to get a final concentration of 10 μg/ml of cefadroxil and 10 μg/ml of probenecid. Method development Derivative conditions The derivative spectra were recorded by using software UV PROBE (Transformation: derivative, Derivative order: 1, Delta lamda: 8, Scaling factor 1, Wavelength range: nm). Determination of analytical wavelength The standard solutions of cefadroxil (10 μg/ml) and probenecid (10 μg/ml) were scanned separately in the UV range of nm. The zero order spectra thus obtained was then processed to obtain first derivative spectrum. It appeared that (Figure 2) cefadroxil showed zero crossing at nm while probenecid showed zero crossing at nm and nm. At 260 nm cefadroxil showed zero absorbance and probenecid showed reasonable absorbance, while at nm probenecid showed zero absorbance and cefadroxil showed reasonable absorbance so these two wavelengths were selected for further measurement. Validation of the method The method was validated according to ICH guidelines (Q2-R1, 2005). Fig.2. First order derivative spectra of cefadroxil (A) and probenecid (B).
3 2352 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 Linearity and range The calibration curves were plotted over a concentration range of 4-36 μg/ml and 5-25 μg/ml for cefadroxil and probenecid, respectively. Accurately measured standard solutions of cefadroxil (100 μg/ml) (4, 12, 20, 28 and 36 ml) and probenecid (100 μg/ml) (5, 10, 15, 20 and 25 ml) were transferred to a series of 100 ml of volumetric flasks and diluted to the mark with 0.1N HCl. First derivative absorbance (D1) was measure at nm for cefadroxil and nm for probenecid against 0.1N HCl as blank. The calibration curve was constructed by plotting absorbance Vs cefadroxil and probenecid, respectively. Figure 3 shows the linearity spectra of cefadroxil and probenecid. (A) Fig.3.Linearity of cefadroxil (A) and probenecid (B). (B)
4 Maheta et al: Spectroscopic Method for Estimation of Cefadroxil and probenecid 2353 Limit of detection (LOD) and limit of quantification (LOQ) The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal-to-noise ratio (S/N) using the following equations designated by International Conference on Harmonization (ICH) guidelines. LOD = 3.3 σ/s LOQ = 10 σ/s Where, σ = the standard deviation of the response and S = slope of the calibration curve. Method precision (% Repeatability) The precision of the instrument was checked by repeated scanning and measurement of absorbance of solution of (n = 6) of cefadroxil (20 μg/ml) and probenecid (15 μg/ml) without changing the parameter of proposed method. Intermediate precision (Reproducibility) The intraday and interday precision of the proposed method was determined by analysing the corresponding responses 3 times on the same day and on 3 different days of different concentrations of standard solutions of cefadroxil and probenecid. Accuracy (% Recovery) The accuracy of the method was determined by calculating recoveries of cefadroxil and probenecid by the standard addition method. Known amounts of standard solution of cefadroxil and probenecid were added at 80%, 100% and 120% levels to prequantified sample solutions of cefadroxil and probenecid. The amounts of cefadroxil and probenecid were calculated by applying obtained values to the regression equations. Analysis of drugs in synthetic mixture probenecid, respectively. The amounts of the cefadroxil and probenecid present in the sample solution were calculated by fitting the responses into the regression equation for cefadroxil and probenecid in the proposed method. Results and Discussion The methods discussed in the present work provide a convenient and accurate way for simultaneous analysis of cefadroxil and probenecid. In first order derivative spectroscopy, wavelengths selected for quantitation were nm for cefadroxil (zero cross for probenecid) and 260 nm for probenecid (zero cross for cefadroxil). Both the drugs obey the Beer s law with the concentration range (cefadroxil: 4-36 μg/ml, probenecid: 5-25 μg/ml) with R 2 value of and for cefadroxil and probenecid, respectively (n=3). Linearity data for cefadroxil and probenecid is shown in (Table 1). The limit of detection (LOD) and limit of quantitation (LOQ) value were found to be and μg/ml for cefadroxil and and μg/ml for probenecid, respectively. Table 2 shows the intraday data for estimation of cefadroxil and probenecid. The mean percentage RSD for cefadroxil and probenecid was found and 1.15 respectively for intra-day precision. Table 3 shows the interday data for estimation of cefadroxil and probenecid. The mean percentage RSD for cefadroxil and probenecidwas found 1.39 and 1.30 respectively for inter-day precision. The mean % recovery was found to be % and % for cefadroxil and probenecid, respectively (Table 4) (n=3). The mean % assay was found to be % and % for cefadroxil and probenecid, respectively (n=6) (Table 5). The overall results of various validation parameters were summarized in Table 6. The response of the sample solution was measured at nm and 260 nm for quantification of cefadroxil and TABLE 1 Linearity data for cefadroxil andprobenecid nm* ± SD %RSD PROBENECID 260 nm* ± SD %RSD ± ± ± ± ± ± ± ± ± ± *Average of three determination TABLE 2 Intraday precision data for estimation of cefadroxil andprobenecid nm* ± SD (μ g/ml) PROBENECID 260 nm* ± SD ± ± ± ± ± ± ± ± ± ± *Average of three determination
5 2354 Int J Pharm Sci Nanotech Vol 7; Issue 1 January March 2014 TABLE 3 Interday precision data for estimation of cefadroxil andprobenecid nm* ± SD Probenecid 260 nm* ± SD ± ± ± ± ± ± ± ± ± ± *Average of nine determination TABLE 4 Statistical analysis of recovery data. Level Amt. of sample Amt. of drug added Amt. recovered % Mean recovery* ± SD PROBENECID PROBENECID PROBENECID PROBENECID 80% ± ± % ± ± % ± ± 1.48 *Average of three determination TABLE 5 Analysis of synthetic mixture. Syntheticmixture Label claim(mg) Assay (content in mg)* % of label claim*± % SD ± PROBENECID 250 PROBENECID ± *Average of six determinations. TABLE 6 Summary of validation parameter. First order derivative Parameters spectroscopic Method PROBENECID Zero crossing points 260 nm nm Concentration range Slope Intercept Correlation coefficient LOD LOQ Repeatability (, n = 6) Precision (%RSD) Interday (n = 9) Intraday (n = 3) Accuracy(n=3) % % % Assay(n=6) Conclusions The developed method was novel, simple, accurate, precise and economical which would be used to estimate cefadroxil and probenecid in their synthetic mixture in routine analysis. The method was fully validated showing satisfactory data for all the method validation parameters tested. The developed method can be used by quality control department to determine the assay of pharmaceutical preparations. Acknowledgements The authors are grateful to Sehat Pharma Ltd, Himatnagar and Geno Pharmaceutical Ltd, Goa for providing free gift sample of cefadroxil and probenecid, respectively. We acknowledge the APMC college of Pharmaceutical Education and Research, Himatnagar for providing the facilitites to complete the work. References AnjumA (2011). Development and validation of new analytical methods for the estimation of cefadroxil in bulk and pharmaceutical dosage form. M.Pharm Thesis. Rajiv gandhi university. Chetan P, Kamlesh P, Sen DJ, Badmanaban R and Ashish P (2010). Development and validation of spectrophotometric methods for the estimation of cefadroxil in tablet dosage forms. J Chem Pharm Res. 2(2): Chudhari SV, Karnik A, Adhikary A, Tandale RS and Vavia PR (2006). Simultaneous UV spectrophotometric method for estimation of cefuroxime axetil and probenecid from solid dosage form.ind J Pharm Sci. 8(1): Dhoka MVand Chopade SS (2012). Method development & comparative statistical evaluation of HPLC & HPTLC method
6 Maheta et al: Spectroscopic Method for Estimation of Cefadroxil and probenecid 2355 for simultaneous estimation of cefadroxilmonohydrate & ambroxol hydrochloride. Indo Global J Pharm Sci. 2(2): El-Gindy A, El-Walily AF and Bedair MF (2000). First-derivative spectrophotometric and LC determination of cefuroxime and cefadroxil in urine. J Pharm Biomed Anal. 23(2-3): ICH Q2 (R1)(2005). Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use, ICH Harmonised Tripartite Guideline. IndianPharmacopoeia (2010).TheIndianPharmacopoeia Commission, Ghaziabad, Govt. of India, Ministry of Health and Family Welfare, Volume-II, pp IndianPharmacopoeia (2010). The IndianPharmacopoeia Commission, Ghaziabad, Govt. of India, Ministry of Health and Family Welfare, Volume-III, pp Jain D, Jain DK and Trivedi PK (1998).Simultaneous spectrophotometric determination of amoxicillin and probenecid in tablet dosage form. Ind J Pharm Sci. 1: Jain D, Jain DK and Trivedi PK (1997).Derivative spectrophotometric method for simultaneous estimation of cephalexin and probenecid in two component solid dosage form. Ind J Pharm Sci. 1: Katzung BG (2007). Basic & Clinical Pharmacology, 10 th ed. The Mc Graw Hill Publishing Company Limited, New Delhi, pp Kurian T, Kurian J, William H and Bijumol C (2012).Simultaneous multicomponent analysis in pharmaceutical formulation by liquid chromatography. Hygeia J D Med. 4(2): Madhuri TR, Spandana M,Aruna D and Susmitha K (2012). Development and validation of new analytical methods for the quantitative estimation of cefadroxil in bulk and pharmaceutical dosage forms. Int J Pharm World Res. 3(2): Patil SV, Patil SD, Patil BF, Patil SP, Tarade VA and Zahid AMJ (2011). Reverse phase HPLC method for analysis of cefadroxil in pharmaceutical dosage form.int J Pharm Res Dev. 3(6): RangHP, DaleMM, RitterJM and Flower RJ (2003). Rang and Dale Pharmacology, 5 th ed. Elsevier Publication, pp Shinde VM, Desai BS and Tendolkar NM (1994). Simultaneous determination of cephalexin & probenecid from tablets by RP- HPLC.Ind J Pharm Sci. 56(2): Sireesha KR, Mhaske DV, Kadam SS and Dhaneshvar SR (2004). Development and validation of a HPTLC method for simultaneous estimation of cefuroxime axetil and probenecid. Ind J Pharm Sci. 66(3): Suddhasattya D, Kalyani K, Samyuktha B, Sahoo SK, Mohapatra S, Murthy PN and dirham K (2010). Development and validation of a UV-VIS spectrophotometric method for the estimation and degradation monitoring of cefadroxil in bulk and pharmaceutical dosage forms. Int J Chem Res. 1(1): Address correspondence to:pratik S. Maheta, Department of Quality assurance, APMC College of Pharmaceutical Education and Research, College campus, Motipura, Himatnagar , Gujarat, India. Mob: mehtapratik2@yahoo.com
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