ABOUT AUTHORS:
Harshil R. Patel*, Sejal K. Patel
Department of Quality Assurance,
S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India.
*harshil285@gmail.com
ABSTRACT
The present manuscript describes simple, sensitive, rapid, accurate, precise and economical derivative spectroscopic methodfor the simultaneous determination of Eperisone Hydrochloride(EPE) and Lornoxicam (LOR) in synthetic mixture. Derivative spectroscopy offers a useful approach for the analysis of drugs in mixtures. In this study a first-derivative spectroscopic method was used for simultaneous determination of Eperisone Hydrochloride and Lornoxicam using the zero-crossing technique. The measurements were carried out at wavelengths of 264 nm and 225.2 nm for Eperisone Hydrochloride and Lornoxicam respectively. The method was found to be linear (r2>0.998) in the range of 2- 30 μg/ml for Eperisone Hydrochloride at 264 nm. The linear correlation was obtained (r2>0.996) in the range of 2-14 μg/ml for Lornoxicam at 225.2 nm. The limit of detection was 0.2565 and 0.235 μg/ml for Eperisone Hydrochloride and Lornoxicam respectively. The limit of quantification was 0.7774 and 0.7121 μg/ml respectively. The method was successfully applied for simultaneous determination of Eperisone Hydrochloride and Lornoxicam in synthetic mixture.
REFERENCE ID: PHARMATUTOR-ART-1763
INTRODUCTION
Eperisone Hydrochloride (EPE) is a well known antispasmodic drug1. Chemically it is 4-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride (Figure 1). It is official in Japanese Pharmacopoeia (JP)2. JP describes potentiometric titration method for its estimation. Literature survey reveals liquid chromatography-ESI- tandem mass spectrometry for determination of eperisone in human plasma3. Literature survey also reveals Liquid Chromatography – Electrospray Ionization - Mass Spectrometry4, GC-MS5 method for the determination of Eperisone in human plasma. Lornoxicam (LOR) (3E)-6-chloro-3-[hydroxy (pyridin-2- ylamino) methylene]-2-methyl-2, 3-dihydro-4H-thieno [2,3-e] [1, 2] thiazin-4-one 1, 1-dioxide (Figure 2) is a novel non- steroidal anti-inflammatory drug (NSAID) with marked analgesic activity6. Various analytical methods, such as RPHPLC7, Extractionless HPLC8, UV spectroscopy9, HPTLC10, LC-MS-MS11 determination of Lornoxicam in dosage forms and human plasma. Lornoxicam in combination with other drug like Diacerin12 and Thiocolchicoside13 have been also detected. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of EPE and LOR in their combined synthetic mixture or dosage forms. Literature survey does not reveal any simple spectrophotometric method for simultaneous estimation of EPE and LOR in synthetic mixture or combined dosage forms .The present communication describes simple, sensitive, rapid, accurate, precise and cost effective spectrophotometric method based on first order derivative for simultaneous estimation of both drugs in synthetic mixture.
MATERIALS AND METHODS
Apparatus
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. Spectra were automatically obtained by UV-Probe system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India) was used in the study.
Reagents and materials
EPE bulk powder was kindly gifted by Sun Pharmaceuticals Ltd., Vadodara, Gujarat, India and LOR bulk powder was kindly gifted by Acme Pharmaceuticals Ltd., Mehsana, Gujarat, India. Methanol (AR Grade, S. D. Fine Chemicals Ltd., Mumbai, India) and Whatman filter paper no. 41 (Millipore, USA) were used in the study.
Preparation of standard stock solutions
An accurately weighed standard EPE and LOR powder (10 mg) were weighed and transferred to 100 ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 μg/ml of each EPE and LOR.
Determination of the zero crossing points
This method is based on first order derivative spectroscopy to overcome spectral interference from other drug. Zero order spectrums of both the drugs were converted to first order derivative spectra with the help of spectra manager software.
It was observed that LOR showed dA/dλ zero at 264 nm in contrast to EPE that has considerable dA/dλ at this wavelength. Further, EPE has zero dA/dλ at 225.2 nm while at this wavelength LOR has significant dA/dλ. Therefore wavelengths 264 nm and 225.2 nm were employed for the determination of EPE and LOR respectively without interference of other drug. The calibration curves were plotted at these two wavelengths of concentrations against dA/dλ separately. Seven working standard solutions having concentration 2, 5, 10, 15, 20, 25 and 30 μg/ml for EPE and 2, 4, 6, 8, 10, 12 and 14 μg/ml for LOR were prepared in methanol and the absorbances at 256 nm (zero crossing point for LOR) and 225.2 nm (zero crossing point for EPE) were measured and the calibration curves were plotted at these wavelengths.
Validation of the proposed method
The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines14.
Linearity (Calibration curve)
The calibration curves were plotted over a concentration range of 2-30 μg/ml for EPE and 2-14 μg/ml for LOR. Accurately measured standard solutions of EPE (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0ml) along with LOR (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4ml) were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with methanol. The absorbances of the derivatised spectra were measured at 264 nm and 225.2 nm for EPE and LOR respectively against methanol as blank. Six replicate analysis were carried out. Absorbance Vs concentration were plotted to obtain the calibration graph. Both drugs obey the Beer‘s law with the above concentration range with R2 value of 0.998 and 0.996 for EPE and LOR, respectively.
Method precision (Repeatability)
The precision of the instrument was checked by repeated scanning and measurement of absorbance of solutions (n = 6) for EPE (10 µg/ml) and LOR (6 µg/ml) without changing the parameter of the proposed spectrophotometry method.
Intermediate precision (Reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of EPE and LOR (5, 15, 25 µg/ml for EPE and 4, 8, 12 µg/ml for LOR). The result was reported in terms of relative standard deviation (% RSD).
Accuracy (Recovery study)
The accuracy of the method was determined by calculating recovery of EPE and LOR by the standard addition method. Known amounts of standard solutions of EPE and LOR were added at 75, 100 and 125 % level to prequantified sample solutions of EPE and LOR (10 µg/ml for EPE and 6 µg/ml for LOR). The solutions were measured at 264 nm for EPE and 225.2 nm for LOR and % recovery of the sample were calculated. The experiment was repeated for three times.
Limit of detection and Limit of quantification
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, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines14.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where, σ = the standard deviation of the response and S = slope of the calibration curve
Where, σ = the standard deviation of the response and S = slope of the calibration curve
Analysis of Synthetic mixture
EPE (50 mg) and LOR (4 mg) standard drug powder were accurately weighed and then mixed with commonly used formulation excipients like starch, lactose, magnesium stearate and talc in appropriate proportion. The mixture was then transferred to 100 ml volumetric flask containing 80 ml methanol and sonicated for 30 min. The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with methanol. The above solution (0.5 ml) was transferred to 10 ml volumetric flask and diluted up to mark with methanol to obtain 25 µg/ml EPE and 2 µg/ml LOR and their first derivative spectra were recorded.From the derivative spectra, the absorbance at 264 nm and 225.2 nm were noted for the estimation of EPE and LOR, respectively. From these absorbance values, the concentrations of EPE and LOR were determined using calibration graph. The analysis procedure was repeated six times in synthetic mixture.
RESULTS AND DISCUSSION
Zero-order absorption spectra of EPE and LOR showed overlapping peaks that interfere with the simultaneous determination of this formulation (Fig. 3).
Derivative spectroscopy, based on a mathematical transformation of the spectra zero-order curve into the derivative spectra, allows a fast, sensitive and precise resolution of a multicomponent mixture and overcomes the problem of overlapping of a multicomponent system. Derivative spectroscopy on the basis of zero-crossing measurements involves measurement of the absolute value of the total derivative spectrum at an abscissa value corresponding to the zero-crossing wavelength of the derivative spectra of individual components, which should be only a function of the concentration of other component. The spectroscopic parameters including derivative order, wavelength and Δλ values should be optimized to obtain maximum resolution, sensitivity and reproducibility. In this study first derivative technique (D1) traced with Δλ= 8 nm was used to resolve the spectral overlapping. The optimums D1values without interference for EPE and LOR were 264 and 225.2 nm, respectively (figure 4).
The linearity of the method was established from first-derivative spectra by measurement of the absorbance of standard solutions containing varying concentrations of each compound in the presence of constant concentration of the other one. The calibration curves were constructed by plotting the D1value against EPE and LOR concentration at the zero-crossing wavelength of LOR (264 nm) and EPE (225.2 nm), respectively.
Linear correlation was obtained between absorbances and concentrations of EPE and LOR in the concentration ranges of 2-30 µg/ml and 2-14 µg/ml, respectively. The linearity of the calibration curve was validated by the high values of correlation coefficient of regression. Relative standard deviation was less than 2 %, which indicates that proposed method is repeatable. The low % RSD values of interday (0.2977 – 1.1549 and 0.9762 – 1.3378 for EPE at 264 nm and LOR at 225.2 nm, respectively) and intraday (0.3645 –0.5629 and 0.6806 – 1.1190for EPE at 264 and LOR at 225.2 nm, respectively).Low % RSD values for EPE and LOR, reveal that the proposed method is precise. LOD and LOQ values for EPE were found to be 0.2565 and 0.7774 µg/ml at 264 nm. LOD and LOQ values for LOR were found to be 0.235 and 0.7121 µg/ml at 225.2 nm. These data show that method is sensitive for the determination of EPE and LOR. The regression analysis data and summary of validation parameters for the proposed method is summarized in Table 1.
Table 1: Regression analysis data and summary of validation parameters for the proposed method
|
PARAMETERS |
EPE |
LOR |
|
|
Wavelength range (nm) |
264 |
225.2 |
|
|
Beer’s law limit (µg/ml) |
2 - 30 |
2 - 14 |
|
|
Regression equation (y = a + bc)
Slope (b) Intercept (a) |
y = 0.0027x + 0.0013 0.0027 0.0013 |
y = 0.00066x + 0.00024 0.00066 0.00024 |
|
|
Correlation Coefficient (r2) |
0.998 |
0.996 |
|
|
Accuracy (Recovery) (n = 3) |
|
98.87±0.36 |
100.43± 0.69 |
|
Method precision (Repeatability) (% RSD, n = 6), |
1.1217 |
1.2864 |
|
|
Interday (n = 3) (% RSD) |
0.2977 – 1.1549 |
0.9762 – 1.3378 |
|
|
Intraday(n = 3) (% RSD) |
0.3645 –0.5629 |
0.6806 – 1.1190 |
|
|
LOD(µg/ml) |
0.2565 |
0.2350 |
|
|
LOQ (µg/ml) |
0.7774 |
0.7121 |
|
|
Assay ± S. D. (n = 6) |
99.63 ± 0.78 |
101.13 ± 1.64 |
|
RSD = Relative standard deviation. LOD = Limit of detection. LOQ = Limit of quantification. S. D. is standard deviation
The recovery experiment was performed by the standard addition method. The mean recoveries were 98.87 ± 0.3575 and 100.438 ± 0.6893for EPE and LOR, respectively (Table 2).
Table 2: Recovery data of proposed method
|
Drug |
Level |
Amount taken (µg/ml) |
Amount added (%) |
% Mean recovery ± S.D. (n = 3) |
|
EPE |
I |
10 |
75 |
98.38 ± 0.23 |
|
II |
10 |
100 |
99.08 ± 0.51 |
|
|
III |
10 |
125 |
99.14 ± 0.33 |
|
|
LOR |
I |
6 |
75 |
100.62 ± 0.51 |
|
II |
6 |
100 |
100.63 ± 1.28 |
|
|
III |
6 |
125 |
100.07 ± 0.28 |
S. D. is Standard deviation and n is number of replicate
The resultsof recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine EPE and LOR in their combined dosage form. The results obtained for EPE and LOR were comparable with the corresponding labeled amounts (Table 3). No interference of the excipients with the absorbance of interest appeared; hence the proposed method is applicable for the routine simultaneous estimation of EPE and LOR in mixture.
Table 3: Analysis of EPE and LOR in Synthetic mixture
|
Sample
|
Label claim (mg) |
Amount found (mg) |
% Label claim ± S. D. (n = 6) |
|||
|
EPE |
LOR |
EPE |
LOR |
EPE |
LOR |
|
|
I |
50 |
4 |
49.816 |
4.045 |
99.63± 0.78 |
101.13 ± 1.64 |
S. D. is standard deviation and n is number of replicate
CONCLUSION
The proposed spectrophotometric method was found to be simple, sensitive, accurate and precise for determination of EPE and LOR in synthetic mixture. The method utilizes easily available and cheap solvent for analysis of EPE and LOR hence the method was also economic for estimation of EPE and LOR from mixture. The common excipients and additives are usually present in the mixture do not interfere in the analysis of EPE and LOR in method; hence it can be conveniently adopted for routine quality control analysis of the drugs in mixture.
ACKNOWLEDGEMENT
The authors are grateful to Sun Pharmaceuticals Ltd. Vadodara, Gujarat, India and Acme Pharmaceuticals Ltd. Ahmedabad, Gujarat, India for providing gift samples of Eperisone Hydrochloride and Lornoxicam and also to Department of Quality Assurance, S.K. Patel College of Pharmaceutical Education and Research, Ganpat University, Mehsana, Gujarat, India for providing the facilities to carry the research work.
REFERENCE:
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4.Ding L, Wei X, Zhang S, Sheng J, and Zhang Y. Rapid and Sensitive Liquid Chromatography–Electrospray Ionization- Mass Spectrometry Method for the Determination of Eperisone in Human Plasma. Journal of Chromatographic Science, 42(5). 2004, p.254-258.
5.Ding L, Wang X, Yang Z, and Chen Y. The use of HPLC/MS, GC/MS, NMR, UV and IR to Identify Degradation product of Eperisone hydrochloride in the tablets, Journal of pharm and Bio Anal, 46, 2008, p.282-287.
6.Maryadele J O Neil. The Merck Index: An Encyclopedia of chemicals, drugs and biologicals, 14th edition. New Jersey: Published by Merck Research Laboratories, Division of Merck and Co., Inc. Whitehouse station, 2006, p.5582
7.Devanaboyina N, Satyanarayana T, Rao B. A novel RP-HPLC method for the quantification of lornoxicam in formulation. Journal of Pharmacy Research, 4(12), 2011, p.4741-4743.
8.Shital B, Nikhil K. Extractionless High-Performance Liquid Chromatographic method for determination of lornoxicam in human plasma. Asian Journal of Pharmaceutical and Clinical Research, 5(1), 2012, p.122-124.
9.Sunit S, Ranjit G, Sachin P, Amulya B, Ranjit M. Development of Ultraviolet Spectrophotometric Method for Analysis of Lornoxicam in Solid Dosage Forms. Tropical Journal of Pharmaceutical Research, 11(2), 2012, p.269-273.
10.Shital B, Santosh G, Padmanabh D, Navjot G. High performance thin layer chromatographic determination of lornoxicam in human plasma. Journal of Chemical, Biological and Physical Sciences, 2(1), 2011-2012, p.279-283.
11.Zeng L, Chen Y, Zhang F, Zhong F. Determination of Lornoxicam in human plasma by LC/MS/MS. Pubmed,39(2), 2004, p.132-135.
12.Rele V, Warkar B. Estimation of Lornoxicam and Diacerin in dosage form by Simultaneous equation & Q-analysis method using UV Spectroscopic technique. International Journal of Pharma and Bio Sciences, 2(2), 2011, p.124-129.
13.Pankaj K, Shubhanjali S, B Subudhia, Ashok G. Bioanalytical Method development & Validation for the Simultaneous estimation of Thiocolchicoside &Lornoxicam in Human plasma and in Pharmaceutical dosage form by RP-HPLC. International Journal of Pharmacy and Pharmaceutical Sciences, 4(3), 2012, p.252-259.
14.The International Conference on Harmonization, Q2 (R1), Validation of Analytical Procedure, Text and Methodology, 2005.
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