ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ZALTOPROFEN AND PARACETAMOL IN COMBINED DOSAGE FORM BY ULTRAVIOLET SPECTROPHOTOMETRYHTML Full Text
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF ZALTOPROFEN AND PARACETAMOL IN COMBINED DOSAGE FORM BY ULTRAVIOLET SPECTROPHOTOMETRY
- K. Zahira * and P. N. Prasannakumaran
College of Pharmaceutical Sciences, Medical College, Thiruvananthapuram, Kerala - 695011, India
ABSTRACT: Two spectrophotometric methods are presented for the simultaneous estimation of Zaltoprofen (ZLT) and Paracetamol (PAR) in laboratory prepared mixture and pharmaceutical dosage form without prior separation. Method (1) is simultaneous equation method (Vierodt’s method); which depends on the fact that absorbance of a mixture is the sum of the individual absorbance of the components. Method (2) is Q-Absorbance ratio method, which depends upon the fact that, the ratio of absorbance at any two wavelengths is a constant value independent of concentration or path length. Absorbances are measured at two wavelengths one being the λ max of ZLT (227.5 nm) and other being a wavelength of equal absorptivity of the two components (236 nm). Calibration curve of the two methods are linear over the concentration ranges of 4- 9μg/ml and 8-13μg/ml for PAR (247.5nm) and ZLT respectively. The two methods proved to be simple specific, accurate and precise. Solvent used is HPLC grade methanol. The two methods are validated as per ICH guidelines were accuracy, precision, repeatability and robustness are found to be within the acceptable limit.
Zaltoprofen: Paracetamol: Simultaneous determination: Isoabsorptive point
INTRODUCTION: Zaltoprofen (ZLT) is a non-steroidal anti-inflammatory drug1. The chemical name of ZLT is (±)-2-(10, 11-dihydro-10-oxodibenzo [b, f] thiepin-2-yl) propionic acid .CAS No is 747411-43-6. Zaltoprofen selectively inhibits cyclooxygenase-2 activity and prostaglandin E2 production. It is used in the treatment of Rheumatoid arthritis, Osteoarthritis, and other chronic inflammatory Pain conditions ZLT is a unique compound that also has anti-bradykinin activity 1, 2, 3. Paracetamol (PAR) Paracetamol or acetaminophen, chemically named as N-acetyl-p aminophenol, is a widely used analgesic and antipyretic (fever reducer).
FIG 1: STRUCTURAL FORMULA OF ZALTOPROFEN
FIG 2: STRUCTURAL FORMULA OF PARACETAMOL
Zaltoprofen is marketed in combination with Paracetamol under the Trade Name REDUCIN A by JB Chemicals &Pharmaceuticals Ltd, Mumbai. Literature reveals that there are many methods for the individual determination of Zaltoprofen and Paracetamol; but to the best of my knowledge no methods are cited for the determination of Zaltoprofen and Paracetamol in combined dosage. So, it was proposed to develop an economical, rapid and simple Ultraviolet spectrophotometric method for the simultaneous estimation of these drugs in combined dosage forms 4, 5,
MATERIALS AND METHODS:
Spectrophotometric analysis was carried out on JASCO V 560 double beam with a fixed slit width (5nm) using a pair of 1 cm matched quarts cell.
Microsoft Excel was used for handling data and calculation
Zaltoprofen RS was kind gift from Intas Pharmaceuticals, Assam. Paracetamol RS was purchased from Indian Pharmacopoeia Commission, Ghaziabad. Purity of the compounds was checked according to the pharmacopoeia specification.
Methanol HPLC grade obtained from S D Fine - Chemicals Limited, Mumbai
Pharmaceutical dosage form
REDUCIN A (Paracetamol 325mg and Zaltoprofen 80 mg) Manufactured by JB Chemicals and Pharmaceuticals (P) Ltd, Mumbai was obtained from the local market
Stock and working standard solution
Stock standard solution
ZLT and PAR stock standard solution (1 mg/ml) prepared by dissolving 100mg of ZLT and PAR respectively in few millilitres of methanol into a 100 ml volumetric flask by sonicating, then completing the volume with methanol.
Working standard solution
10 ml of stock standard solution of both ZLT and PAR (1mg/ml) is diluted to 100 ml with methanol to get working standard solution (0.1 mg/ml).
Spectral characteristics and wavelength selection
The absorption spectra of 8μg/ml ZLT and 8μg/ml PAR were recorded over the spectral wavelength range 210-300nm using methanol as blank
Linearity and construction of calibration curves
Accurately measured aliquots equivalent to 1-30 μg PAR and 1-15μgZLT are transferred from their working standard solution in to two series of 10 ml volumetric flasks and complete to volume with methanol. The spectra of prepared standard solution are canned from 210-300nm
For simultaneous equation method
Calibration curves are constructed relating the absorbance of zero order spectra of ZLT at 227.5(λmax of ZLT) nm and 247.5 nm(λmax of PAR) and PAR at 247 nm and 227.5 nm versus the corresponding concentrations and regression equations are computed
For Q-Absorbance ratio method
Overlay spectra of 8μg/ml ZLT and PAR 8μg/ml were used to determine isoabsorptive point. Calibration curves are constructed relating the absorbance of zero order spectra of ZLT at 227.5nm (λmax of ZLT) and 235.5(isoabsorptive point) and PAR at 227.5 and 235.5nm versus the corresponding concentrations and regression equations are computed.
Application of simultaneous equation method and Q absorbance ratio method for the determination of ZLT and PAR in laboratory prepared mixture.
Solutions containing different ratio of ZLT and PAR were prepared by transferring accurately measured aliquots from their standard working solution in to a series of 10 ml volumetric flasks and the volume was made with methanol. The final concentration range were 8-13μg/ml ZLT and 4-9μg/ml PAR. Zero order absorption spectra of these different laboratory prepared mixture were recorded from 210-300nmusing methanol as blank and procedure under linearity for each method was then followed. Concentrations of ZLT and PAR in the prepared sample were calculated from the corresponding computed regression equations.
Application to pharmaceutical dosage form
To determine the content of ZLT and PAR in commercial tablets, twenty tablets of REDUCIN A (B.NO:XRX2001, MFG AUG 2012, EXPJUL2014) marketed by JB Chemicals and Pharmaceuticals (P) Ltd, Mumbai were taken. Each tablet labelled to contain 325 mg PAR and 80 mg ZLT. Tablets were weighed and finely powdered and coating was removed. A portion of powder equivalent to 50mg PAR was weighed accurately and transferred in to a 50 ml volumetric flask.10 ml methanol was added and sonicated for 10 mints, filtered through Whitman filter paper in to a 50 ml volumetric flask. The residue was washed three times each with 10 ml methanol and the solution was completed to the mark with the same solvent. The general procedure described above under each method was followed to determine the concentration of both drugs in prepared dosage form solution. The analysis was carried out 6 times. Concentrations of ZLT and PAR in the prepared sample were calculated from the corresponding computed regression equations.
RESULTS AND DISCUSSION:
The main task of this work was to establish simple, sensitive and accurate analytical methods for the simultaneous estimation of ZLT and PAR in their pharmaceutical dosage form with satisfactory precision for good analytical practice (GAP). The overlapped absorption spectra of ZLT and PAR (Fig 3) hinder their determination by direct spectrophotometry especially in the high concentration of PAR as in the dosage form
FIG 3: OVERLAY SPECTRA OF ZLT ( ) AND PAR ( ) IN METHANOL.
Method 1: Simultaneous equation method (Vierodt’s method)
The simultaneous equation method was applied to solve the mixture of PAR (X) and ZLT (Y) of overlapping spectra by constructing two equations based upon the fact that at λ1(λmax of PAR) and λ2 (λmax of ZLT) the absorbance of the mixture is the sum of the individual absorbance of PAR and ZLT.ie,
A1= 0.104332CX+0.059002CY ........... (1)
A2=0.052835 CX+0.077388 CY .......... (2)
From Vierodt’s equation,
Cx= (A1ax2 – A2ax1) / (ax2ay1 – ax1ay2).
CY= (A2ay1 – A1ay2) / (ax2ay1 – ax1ay2).
Where, Cx = Concentration of PAR and Cy = Concentration of ZLT.A1&A2 were absorbance of sample at 247.5 nm and 227.5 nm respectively.ax1 and ax2 were absorptivity of PAR at 247.5 nm and 227.5 nm respectively.ay1and ay2 were absorptivity of ZLT at 247.5 nm and 227.5 nm respectively.
FIG 4: OVERLAY SPECTRA OF PAR IN METHANOL
FIG5: OVERLAY SPECTRA OF ZLT IN METHANOL.
FIG6: CALIBRATION CURVE OF PAR AT 227.5NM IN METHANOL.
FIG7: CALIBRATION CURVE OF PAR AT 475.5NM IN METHANOL.
FIG8: CALIBRATION CURVE OF ZLT AT 227.5NM IN METHANOL.
FIG9: CALIBRATION CURVE OF ZLT AT 247.5NM IN METHANOL.
Method 2: Q - Absorbance Ratio Method.
The absorption spectra of PAR(X) and ZLT (Y) show severe overlapping that prevents the use of direct spectrophotometry for the analysis of either PAR or ZLT without preliminary separation. According to literature, the absorbance ratio method depends on the property that for a substance which obeys Beers Law at all wavelengths, the ratio of absorbance at any two wavelengths is a constant value independent of concentration or path length (Q value).
Absorbance are measured at two wavelengths one (λ2) being the λmax of ZLT (227.5nm) and the other (λ1) being the wavelength of the equal absorptivity of the two components, i.e., isoabsorptive point(236nm).
Cx = (Qm - Qy)/(Qx - Qy)×A1/ax1
Cy = (Qm – Qx)/(Qy - Qx)×A1/ay1
Where, Qm = Absorbance of sample at 227.5nm / Absorbance of the sample at 236 nm. Qx = Absorptivity of PAR at 227.5 nm / Absorptivity of ZLT at 236 nm Qy = Absorptivity of ZLT at 227.5 nm / Absorptivity of ZLT at 236 nmA1 = Absorbance of the sample of sample at 236nm (isoabsorptive point) and A2 is the absorbance of sample at 227.5nm.ax1= Absorptivity of PAR at 236 nm and ay1 = Absorptivity of ZLT at 236 nm.
FIG10: OVERLAY ABSORPTION SPECTRA OF PAR AND ZLT SHOWING ISOABSORPTIVE POINT.
FIG11: CALIBRATION CURVE OF PAR AT 236NM IN METHANOL.
FIG12: CALIBRATION CURVE OF ZLT AT 236 NM IN METHANOL.
TABLE1: ANALYTICAL PARAMETERS OF PROPOSED METHOD.
|Simultaneous equation method||Q absorption ratio method|
|Wavelength (λmax)||247.5nm||227.5nm||236 nm||236 nm|
|Linearity range||4-9μg/ml||8-13μg/ml||4-9 μg/ml||8-13μg/ml|
|SD of intercept||0.00012||0.00029||0.00032||0.00035|
|SD of slope||0.00016||0.00024||0.00012||0.00025|
N=6 replicates.SD=standard deviation.
Validation of the methods has been performed according to ICH recommendations
Linearity. The calibration range for PAR and ZLT was established through consideration of the practical range necessary according to Beers –Lamberts law. The linearity response was determined by analysing 6 independent levels of concentrations in the range of 4-9μg/ml at 247.5 nm for PAR and 8-13μg/ml at 227.5 nm for ZLT for Simultaneous equation method and at 236 nm for Q Absorbance Ratio method. Fig 6-9 shows the linearity .The values of correlation coefficients of
PAR and ZLT were close to unity indicating good linearity.The characteristic parameters for the constructed equations are summarized in Table 1 and Validation data are mentioned in Table 2.
Precision. The intraday precision was carried out through 3 replicate analyses of 4μg/ml, 6μg/m, 9μg/ml of PAR and 8μg/ml, 10μg/ml and 12μg/ml of ZLT respectively. The interday precision was also carried by using above mentioned concentrations of pure samples for 3 consecutive days. The developed method is found to be precise as the %RSD value for intraday and interday precision were less than the ICH limit.
TABLE 2: SUMMARY OF VALIDATION PARAMETERS FOR THE PROPOSED METHODS
|Method 1||Method 2||Method 1||Method 2|
a :The intraday (n = 3), average of concentrations;10,2.4615μg/ml PAR and ZLT respectively, repeated three times within the day.
b:The interday(n = 3), average of concentrations; 10 ,2.4615μg/ml PAR and ZLT respectively, repeated three times within the day.
Accuracy: Accuracy was assured by applying standard addition technique where good percentage recoveries were obtained establishing the accuracy of the proposed method. The recovery studies were carried out by adding known amount of reference standard to samples at 80%, 100% and 120 %level and analysed.
Sensitivity: The limit of detection (LOD) and limit of quantitation (LOQ) were determined from y-intercept and slope of calibration curve according to ICH guidelines.
Robustness: Solutions of both the drugs in
methanol were studied for their stability at ambient temperature for 24 h. Absorbance variation was found to be less than 1%.
Analysis of commercial tablet:
The proposed methods; simultaneous equation method and Q absorbance ratio method was applied for the simultaneous determination of ZLT and PAR in combined tablet dosage form and amount of PAR and ZLT were found to be 98.77% and 99.34% respectively for simultaneous equation method and 99.98 % and 98.69% for Q absorbance ratio method. Results are tabulated below.
TABLE 3: RESULTS – ANALYSIS OF COMMERCIAL TABLETS
|Brand name||Company||Formulation||Label claim||Amount found(mg)|
|Method 1||Method 2|
|REDUCIN A||JB Chemicals and Pharmaceuticals (P) Ltd||Tablet
|PAR 325 mg||321.0025||324.935|
|ZLT 80 mg||79.472||78.952|
CONCLUSIONS: Simple, rapid and sensitive method is proposed for the simultaneous analysis of ZLT and PAR with overlapping spectra. The proposed method does not require any sophisticated mathematical treatment for the absorption data. The ZLT and PAR were well resolved and quantified without prior separation by simple mathematical treatment. The application of the proposed method was validated by the determination of both the drugs by in several laboratory prepared mixture and in pharmaceutical tablets with good accuracy and precision, therefore the developed method is adequate for routine quality control analysis of the formulation.
ACKNOWLEDGEMENTS: The authors are thankful to Dr. Joyamma varkey, HOD of college of Pharmaceutical sciences, Medical College, Thiruvananthapuram for providing all necessary facilities, and also thankful to Intas Pharmaceuticals Pvt. Ltd. Assam, for providing the gift sample of Zaltoprofen .
- Boovizhikannan Thangabalan, Palanirajan Vijayaraj Kumar: RP-HPLC Method Development and Validation Of Zaltoprofen In Pure Form and In Pharmaceutical Formulation .Int. J. Drug Dev. & Res., October-December 2012; 4: 275-278.
- Manish Kumar et al: Development and Validation of HPLC-UV Method for the Estimation of Zaltoprofen in Human Plasma. Journal of Pharmacy Research 2011;4:3753-3755
- Kiran B. Aher, Girija B. Bhavar, Hemant P. Joshi, and Sanjay R. Chaudhari: Economical Spectrophotometric Method for Estimation of Zaltoprofen in Pharmaceutical Formulations. Pharm Methods. 2011; 2: 152–156.
- Gowramma B, Meyyanathan S N: Development and Validation of LC-MS/MS Method for The Estimation of Zaltoprofen Enantiomers In Pharmaceutical Formulation. Int J Pharm Bio Sci 2013; 4:883 – 889.
- Lee HW, Seo JH, Kim YW, Jeong SY, Lee KT: Determination of zaltoprofen in human plasma by liquid chromatography with electrospray tandem mass spectrometry: application to a pharmacokinetic study. Rapid Communications in Mass Spectrometry.2006; 20:2675-2680.
How to cite this article:
Zahira NK and Prasannakumaran PN: Analytical Method Development and Validation of Zaltoprofen and Paracetamol in Combined Dosage Form by Ultraviolet Spectrophotometry. Int J Pharm Sci Res 2015; 6(2): 682-87.doi: 10.13040/IJPSR.0975-8232.6 (2).682-87.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
N. K. Zahira * and P. N. Prasannakumaran
College of Pharmaceutical Sciences, Medical College, Thiruvananthapuram, Kerala - 695011, India
18 June, 2014
28 August, 2014
17 October, 2014
01 February, 2015