Int J Pharm Pharm Sci, Vol 6, Issue 10, ??-??Original Article
SIMULTANEOUS QUANTITATIVE ESTIMATION OF MEBEVERINE HYDROCHLORIDE AND CHLORDIAZEPOXIDE IN CAPSULES USING SPECTROPHOTOMETRY
T. RAJESH, N. LAKSHMI PRASANNA, A. ASHOK KUMAR*
Department of Pharmaceutical analysis and Quality Assurance, Vijaya College of pharmacy, Munaganur (village), Hayathnagar (Mandal), Hyderabad 501511, India.
Email: ashok576@gmail.com
Received: 05 Jul 2014 Revised and Accepted: 10 Aug 2014
ABSTRACT
Objective: To develop a simple and cheap UV spectrophotometric method for the simultaneous quantitative estimation of Mebeverine hydrochloride and Chlordiazepoxide in MEVA C Capsules and validate as per ICH guidelines.
Methods: The optimized method uses triethylammonium phosphate buffer (pH 4.0) as a solvent for the estimation of assay of Mebeverine hydrochloride and Chlordiazepoxide in capsules at detection wavelengths of 263 nm and 245 nm respectively.
Results: The developed method exhibited linearity in the range of 7.5-22.5μg/ml for Mebeverine hydrochlorideand 2.5-7.5μg/ml for Chlordiazepoxide. Precision for Mebeverine hydrochloride and Chlordiazepoxide is exemplified by relative standard deviation of 0.589% and 0.332 respectively. Percentage Mean recovery for Mebeverine hydrochloride and Chlordiazepoxide was found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Mebeverine hydrochloride and Chlordiazepoxide was found to be 528ng/ml and 192ng/ml respectively, while limit of quantitiation (LOQ) for Mebeverine hydrochloride and Chlordiazepoxide was found to be 1.6µg/ml and 583ng/ml respectively.
Conclusion: A simple and a cheap UV spectrophotometric method were developed and validated for the simultaneous quantitative estimation of Mebeverine hydrochloride and Chlordiazepoxide in capsules as per ICH guidelines and hence it can be used for the routine analysis in various pharmaceutical industries.
Keywords: UV, Mebeverine hydrochloride, Chlordiazepoxide, Method development, Validation.
INTRODUCTION
Mebeverine hydrochloride (Figure 1) is a white crystalline powder having a molecular formula C25H35NO5HCl, molecular weight 466 and melting point 105-107ºC. It is freely soluble in water and ethanol (96%), while practically insoluble in diethyl ether [1]. IUPAC name of Mebeverine hydrochloride is 3,4-Dimethoxybenzoic acid 4-[ethyl[2-(4-methoxy phenyl)-1-methylethyl]amino]-butyl ester. It is a direct antispasmodic acting mainly on the smooth muscles of the gastrointestinal tract and particularly effective against the colonic spasm [2]. Mebeverine hydrochloride is widely used as a relaxant agent for the treatment of gastrointestinal spasmodic disorders such as irritable bowel syndrome [3].
Fig. 1: Structure of Mebeverine hydrochloride
Chlordiazepoxide (Figure 2) is a first among the class of benzodiazepines to be used clinically as anti-anxiety drug[4]. Chlordiazepoxide is a white crystalline powder possessing solubility in water, whose IUPAC name is (7-chloro-2(methylamino)-5-phenyl-3-H-1,4 benzodiazepine 4-oxide). Chlordiazepoxide mainly acts on limbic system and ascending reticular formation in the central nervous system. It binds to stereospecific benzodiazepine binding sites on GABA receptor complexes at several sites within the central nervous system including the limbic system and reticular formation. The binding will facilitates GABA mediated chloride channel opening and produce hyperpolarisation. This will increase the concentration of inhibitory neurotransmitter GABA and chloride ions in the CNS and decreases firing rate of neurons[5]. Mebeverine hydrochloride (135mg) and Chlordiazepoxide (5mg) is commercially available as capsules (trade name: MEVA C).
Fig. 2: Structure of Chlordiazepoxide
A detailed literature survey reveals that there exists literature concerning analytical method development and validation for individual drugs Mebeverine[1,3,6-7] and Chlordiazepoxide[8-13] in various matrices. Also analytical methods are reported for Mebeverine with other drug combinations[2,14-19] and similarly Chlordiazepoxide with other drug combinations[4,20-23]. While there is only one literature reported on UV spectrophotometric method development and validation for the simultaneous quantitative estimation of Mebeverine and Chlordiazepoxide as drug combination in pharmaceutical dosage forms[24]. Hence we have explored in developing a new, accurate, precise and linear UV spectrophotometric method for the quantitative estimation of Mebeverine and Chlordiazepoxide in MEVA C capsules and validate as per ICH guidelines.
MATERIALS AND METHODS
Materials
Instrument
A double beam UV-visible spectrophotometer (Shimadzu, model 1800) having two matched quartz cells with 1 cm light path and loaded with UV probe software (version 2.41) was used for recording of spectra and measuring absorbance. An electronic weighing analytical balance (0.1mg sensitivity, Shimadzu AY 220), digital pH meter (DELUX model 101) and a sonicator (sonica, model 2200 MH) were used in this study.
Chemicals and Reagents
Analytically pure samples of Mebeverine hydrochloride and Chlordiazepoxide with purities greater than 99% were obtained as gift samples from Chandra labs, Hyderabad, India and tablet formulation [MEVA C] was procured from MEDPLUS, Hyderabad, India with labelled amount 135mg of Mebeverine hydrochloride and 5mg of Chlordiazepoxide. Triethylamine (AR Grade) and ortho phosphoric acid (AR Grade) were obtained from SD Fine chemicals (Hyderabad, India). 0.45μm Nylon membrane filters were obtained from Spincotech Private Limited, Hyderabad, India.
Methods
Solvent
Solvent used is prepared by adding 5 ml of triethylamine to 1000 ml of distilled water and later pH was adjusted to 4.0 using 30% v/v of ortho phosphoric acid in water.
Selection of suitable detection wavelength
Suitable wavelength for Mebeverine hydrochloride and Chlordiazepoxide for the total experiment was determined by recording UV spectrums in the range of 200-400 nm and suitable wavelength for Mebeverine hydrochloride and Chlordiazepoxide was considered as 263 nm and 245 nm respectively. (Figure 3 and 4).
Fig. 3: UV spectrum of standard Mebeverine hydrochloride.
Fig. 4: UV spectrum of standard Chlordiazepoxide.
Preparation of stock and working standard solution for mebeverine hydrochloride
10mg of Mebeverine hydrochloride was accurately weighed and taken in 100 ml clean and dry volumetric flask containing 80 ml of solvent and then the solution was made up to the mark using the solvent. This is considered as the standard stock solution (100µg/ml). 1.5 ml of the stock solution was pipetted out and made up to 10 ml to get a concentration 15µg/ml, treated as working standard, 100% target concentration.
Preparation of stock and working standard solution for chlordiazepoxide
10mg of Chlordiazepoxide was accurately weighed and taken in 100 ml clean and dry volumetric flask containing 80 ml of solvent and then the solution was made up to the mark using the solvent. This is considered as the standard stock solution (100µg/ml). 0.5 ml of the stock solution was pipetted out and made up to 10 ml to get a concentration 5µg/ml, treated as working standard, 100% target concentration.
Preparation of stock and working sample solution for mebeverine hydrochloride
Ten capsules were opened and only white powders in it were weighed separately and the average weight was determined. The average weight was transferred to a 100 ml volumetric flask containing 100 ml diluent and then stirred for 10 minutes, followed by filtration through 0.45µ nylon membrane filter to get sample stock solution of 1.35mg/ml. 0.11 ml of the above stock solution was pipetted out and made up to 10 ml to get working sample solution equivalent to a concentration of working standard of 15µg/ml.
Preparation of stock and working sample solution for chlordiazepoxide
Ten capsules were opened and only tablets in it were weighed separately and the average weight was determined. Ten tablets present in ten capsules were grinded in a pestle and mortar and the average weight was transferred to a 100 ml volumetric flask containing 100 ml diluent and then stirred for 10 minutes, followed by filtration through 0.45µ nylon membrane filter to get sample stock solution of 50µg/ml. 1 ml of the above stock solution was pipetted out and made up to 10 ml to get working sample solution equivalent to a concentration of working standard of 5 µg/ml.
RESULTS AND DISCUSSION
Method development
Various buffers were explored (inorganic and organic based) as solvents, including Potassium dihydrogen orthophosphate, triethylammonium phosphate and ammonium acetate buffers varying pH in the ranges of 2-7. Mebeverine hydrochloride and Chlordiazepoxide were found to be soluble and stable for minimum of 1 hour at room temperature using pH 4.0 triethylammonium phosphate buffer and hence this buffer was used for the determination of suitable detection wavelength and working concentration of both drugs. In order to test the applicability of the developed method to a commercial formulation, MEVA C was studied at working concentration. Assay at working concentration for both the samples in the formulation was in acceptance limits (98-102%) during extraction of drugs in the samples using the solvent for 10 minutes. The protocol affords reproducible quantification of the drugs in the samples ranging between 98 and 102%, which is the standard level in any pharmaceutical quality control. Hence the method is optimized. Figures 5 and 6 represent UV spectrums of the formulation, mebeverine hydrochloride and chlodiazepoxide respectively.
Fig. 5: UV spectrum of Mebeverine hydrochloride (white powder) of the sample.
Fig. 6: UV spectrum of Chlordiazepoxide (tablet) of the sample.
Method validation
Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. UV spectrophotometric method developed was validated according to International Conference on Harmonization (ICH) guidelines [25] for validation of analytical procedures. The method was validated for the parameters like linearity, accuracy, system precision, intra-day precision, inter-day precision/ intermediate precision/ ruggedness, robustness, limit of detection (LOD) and limit of quantitiation (LOQ).
Precision
System precision
Six replicate recording of absorbances for both the drugs at working concentration showed % RSD (Relative Standard Deviation) less than 2, which indicates the acceptable reproducibility and thereby the precision of the system. System precision results are tabulated in Table 1.
Method precision
Method precision was determined by performing assay of both the drugs in the formulation under the test of repeatability (Intra day precision) at working concentration.
Repeatability (Intra day precision)
Six replicate recording of absorbances for both the samples in the formulation from the same homogeneous mixture at working concentration showed % RSD less than 2 concerning % assay for the drugs which indicate that the method developed is method precise by the test of repeatability and hence can be understood that the method gives consistently reproducible results (Table 2).
Table 1: System precision results (UV absorbance) of Mebeverine and Chlordiazepoxide
| n | Mebeverine hydrochloride | Chlordiazepoxide |
| 263 nm | 245 nm | |
| 1 | 0.407 | 0.431 |
| 2 | 0.411 | 0.430 |
| 3 | 0.413 | 0.428 |
| 4 | 0.409 | 0.430 |
| 5 | 0.412 | 0.432 |
| Average | 0.410 | 0.4302 |
| SD | 0.0024 | 0.0014 |
| % RSD | 0.585 | 0.3254 |
Table 2: Intra day precision results of Mebeverine hydrochloride and Chlordiazepoxide
| n | Mebeverine
HCl (263 nm) |
Chlordiazepoxide
245 nm |
| % Assay | % Assay | |
| 1 | 100.79 | 99.00 |
| 2 | 99.81 | 99.23 |
| 3 | 99.32 | 99.69 |
| 4 | 100.29 | 99.23 |
| 5 | 99.56 | 99.77 |
| Average | 99.95 | 99.38 |
| S. D. | 0.589 | 0.330 |
| % RSD | 0.589 | 0.332 |
Linearity
Standard solutions of Mebeverine hydrochloride and Chlordiazepoxide at different concentrations level (50%, 75%, 100%, 125%, and 150%) were prepared in triplicates. Calibration curves were constructed by plotting the concentration level versus corresponding absorbance for both the drugs. The results show an excellent correlation between absorbance and concentration level within the concentration range of 7.5-22.5µg/ml for Mebeverine hydrochloride and 2.5-7.5µg/ml for Chlodiazepoxide (Tables 3 and 4). The correlation coefficients were greater than 0.99 for both the drugs, which meet the method validation acceptance criteria and hence the method is said to be linear for both the drugs.
Table 3: Calibration data for Mebeverine hydrochloride (263 nm)
| % Level | Concentration
(µg/ml) |
Absorbance
1 |
Absorbance
2 |
Absorbance
3 |
| 50 | 07.50 | 0.204 | 0.210 | 0.209 |
| 75 | 11.25 | 0.282 | 0.286 | 0.278 |
| 100 | 15.00 | 0.413 | 0.410 | 0.412 |
| 125 | 18.75 | 0.495 | 0.490 | 0.489 |
| 150 | 22.50 | 0.598 | 0.591 | 0.588 |
| Regression equation | y=0.026x-0.002 | y=0.025x+0.011 | y=0.025x+0.0076 | |
| Regression coefficient | 0.994 | 0.995 | 0.991 |
Table 4: Calibration data for Chlordiazepoxide (245 nm)
| % Level | Concentration
(µg/ml) |
Absorbance
1 |
Absorbance
2 |
Absorbance
3 |
| 50 | 2.5 | 0.209 | 0.214 | 0.209 |
| 75 | 3.75 | 0.327 | 0.322 | 0.325 |
| 100 | 5 | 0.432 | 0.430 | 0.435 |
| 125 | 6.25 | 0.527 | 0.531 | 0.536 |
| 150 | 7.5 | 0.630 | 0.645 | 0.653 |
| Regression equation | y=0.083x-0.0101 | y=0.085x-0.0098 | y=0.086x-0.001 | |
| Regression coefficient | 0.999 | 0.997 | 0.999 |
Accuracy
Accuracy was determined by means of recovery experiments, by the determination of % mean recovery of both the drugs in the formulation at three different levels (50-150%). At each level, three determinations were performed. Percent mean recovery and %RSD between recoveries are calculated as shown in Table 5. The accepted limits of mean recovery are 98% -102% and %RSD not more than 2% and all observed data were within the required range, which indicates good recovery values and hence the accuracy of the method developed.
Table 5: Results of Accuracy studies for Mebeverine hydrochloride and Chlordiazepoxide
| Concentration level (%) | % Mean Recovery Mebeverine HCl | % Mean Recovery Chlordiazepoxide |
| 50 | 100.67 | 98.97 |
| 100 | 99.95 | 99.38 |
| 150 | 101.02 | 99.78 |
Robustness
The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is concluded that the method is robust as it is found that the % RSD is less than 2 regarding the determination of assays of both the drugs in the formulation, despite deliberate variations done concerning pH ± 0.2 and detection wavelength ± 2 nm (Tables 6 and 7).
Sensitivity
The sensitivity of measurement of Mebeverine hydrochloride and Chlordiazepoxide by use of the proposed method was estimated in terms of the limit of quantitation (LOQ) and limit of detection (LOD). LOQ and LOD were calculated by the use of the equations LOD = 3.3 x N / B and LOQ = 10x N / B where N is the standard deviation of intercepts of calibration plots and B is the average of the slopes of the corresponding calibration plot.
Table 6: Robustness results of Mebeverine hydrochloride sample
| Variation parameter | Variation | % mean Assay | %RSD |
| pH(± 0.2) | 2.8 | 100.02 | 0.588 |
| 3.0 | 99.65 | 0.643 | |
| 3.2 | 98.99 | 0.410 | |
| Wave length (± 2 nm) | 261 | 100.7 | 0.956 |
| 263 | 101.2 | 1.045 | |
| 265 | 99.54 | 1.120 |
Table 7: Robustness results of Chlordiazepoxide sample
| Variation parameter | Variation | % mean Assay | %RSD |
| pH(± 0.2) | 2.8 | 99.63 | 0.342 |
| 3.0
3.2 |
99.36
100.76 |
0.998
0.682 |
|
| Wave length (± 2 nm) | 243
245 247 |
98.63
99.2 100.7 |
0.482
0.396 0.421 |
The limit of detection (LOD) for Mebeverine hydrochloride and Chlordiazepoxide were found to be 528ng/ml and 192ng/ml respectively, while limit of quantitiation (LOQ) for Mebeverine hydrochloride and Chlordiazepoxide were found to be 1.6µg/ml and 583ng/ml respectively. Optical characteristics and validation parameters results are summarized in Table 8.
Table 8: Optical characteristics and validation parameters
| Parameters | Mebeverine hydrochloride | Chlordiazepoxide |
| Detection wavelength (nm) | 263 | 245 |
| Beer’s Law limits (µg/ml) | 7.5-22.5 | 2.5-7.5 |
| Sandell’s sensitivity (µg/cm2/0.001 absorbance unit) | 0.1 | 0.01 |
| Regression equation (y = mx+c) | (y y=0.025x+0.0055 | Y=0.084x+0.0069 |
| Correlation coefficient (r2) | 0.993 | 0.999 |
| Slope (m) | 0.025 | 0.084 |
| Intercept (c) | 0.0055 | 0.0069 |
| (% RSD) System precision | 0.585 | 0.325 |
| (% RSD) Intra-day precision | 0.589 | 0.332 |
| (% RSD) Inter-day precision | ≤2 | ≤2 |
| Accuracy (% Mean Recovery) | ||
| 50 % Level | 100.67 | 98.97 |
| 100 % Level | 99.95 | 99.38 |
| 150 % Level | 101.02 | 99.78 |
| LOD (µg/ml) | 0.528 | 0.192 |
| LOQ (µg/ml) | 1.6 | 0.583 |
| Robustness | ||
| pH(± 0.2) (% RSD) | ≤2 | ≤2 |
| Wavelength (± 2 nm) (% RSD) | ≤2 | ≤2 |
CONCLUSION
A cheap and a rapid UV spectrophotometric method was developed and validated for the quantitative estimation of Mebeverine hydrochloride and Chlordiazepoxide in capsules as per ICH guidelines. The developed method exhibited linearity in the range of 7.5-22.5μg/ml for Mebeverine hydrochloride and 2.5-7.5μg/ml for Chlordiazepoxide.
The precision for Mebeverine hydrochloride and Chlordiazepoxide is exemplified by relative standard deviation of 0.589% and 0.322 respectively.
Percentage Mean recovery for Mebeverine hydrochloride and Chlordiazepoxide were found to be in the range of 98‐102, during accuracy studies. The limit of detection (LOD) for Mebeverine hydrochloride and Chlordiazepoxide were found to be 528ng/ml and 192ng/ml respectively, while limit of quantitiation (LOQ) for Mebeverine hydrochloride and Chlordiazepoxide were found to be 1.6µg/ml and 583ng/ml respectively. Accordingly it is concluded that the developed UV spectrophotometric method is accurate, precise, linear, rugged and robust and therefore the method can be used for the routine analysis of Mebeverine hydrochloride and Chlordiazepoxide in capsules in various pharmaceutical industries.
CONFLICT OF INTERESTS
Declared None
ACKNOWLEDGEMENT
The authors would like to thank the management of Vijaya college of pharmacy (VJYH), Hyderabad, for providing the necessary facilities to carry out of this research work. The authors are grateful to Chandra labs, Hyderabad for providing drugs in form of gift sample.
REFERENCES
- Arayne MS, Sultana N, Siddiqui FA. A new RPHPLC method for analysis of mebeverine hydrochloride in raw materials and tablets. Pak J Pharm Sci 2005;18(2):11-4.
- Walash MI, Sharaf El-din MMK, El-enany NM, Eid MI, Shalan SM. Simultaneous determination of sulpiride and mebeverine by HPLC method using fluorescence detection: application to real human plasma. Chem Cent J 2012;6(13):1-12.
- Souri E, Aghdami AN, Adib N. A stability indicating HPLC method for determination of mebeverine in the presence of its degradation products and kinetic study of its degradation in oxidative condition. Res Pharm Sci 2014;9(3):199-206.
- Gupta SP, Upmanyu N, Garg G. Development and validation of spectrophotometric, HPTLC and HPLC methods for the determination of Imipramine and Chlordiazepoxide in pharmaceutical dosage forms. Pharm Sin 2012;3(2):185-92.
- Sujatha N, Pavani KH. Analytical method development and validation of amitriptyline hydrochloride and chlordiazepoxide in tablet by RP-HPLC. Indian J Res Pharm Biotechnol 2013;1(5):655-9.
- De Schutter JA, De Croo F, Vander Weken G, Venden Bossche W, De Moerloose P. Stability study and quantitative determination of mebeverine hydrochloride in tablets by means of reversed-phase high-performance liquid chromatography. Chromatographia 1985;20:185-92.
- Al-Deeb Q, Al-Hadiya BM, Foda NH. Quantitative analysis of mebeverine in dosage forms by HPLC. Chromatographia 1997;44:427-30.
- Dubois JG, Atassi G, Hanocq M. High-performance liquid chromatographic determination of chlordiazepoxide, its metabolises and oxaziridines generated after UV irradiation. J Chromatogr A 1994;662(2):255-62.
- Roberts SE, Delaney MF. Determination of chlordiazepoxide, its hydrochloride and related impurities in pharmaceutical formulations by reversed-phase high-performance liquid chromatography. J Chromatogr 1984;283:265-72.
- Greizerstein HB, Mclaughlin IG. The High-Pressure Liquid Chromatographic Determination of Chlordiazepoxide and Its N-Demethyl Metabolite in Mouse Brain. J Liq Chromatogr 1980;3(7):1023-30.
- Puglisi CV, Desilva JAF. Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography. Anal Lett 1978;11(2):135-60.
- Kohlhof K. Determination of chlordiazepoxide in mouse plasma by gas chromatography— negative-ion chemical ionization mass spectrometry. J Chromatogr B Biomed Appl 1994;660(1):95-101.
- Sun SR. Quantitative determination of chlordiazepoxide and its metabolites in serum by fluorescence TLC-densitometry. J Pharm Sci 1978;67(5): 639-41.
- El Walily AFM, El Gindy A, Bedair MF. Application of first-derivating UV spectrophotometry, TLC-densitometry and liquid chromatography for the simultaneous determination of mebeverine hydrochloride and sulpiride. J Pharm Biomed Anal 1999;21:535-48.
- Shama SA, Amin AS. Spectrophotomertric microdetermination of nefopam, mebeverine and phenylpropanolamine hydrochloride in pharmaceutical formulations using alizarins. Spectrochim Acta A Mol Biomol Spectrosc 2004;60:1969-74.
- Zayed SI. Simultaneous determination of mebeverine hydrochloride and sulpiride using the first derivatives of ratio spectra and chemometric methods. Anal Sci 2005;21:985-89.
- El-Didamony AM. Spectrophotometric determination of benzydamine HCl, levamisole HCl andmebeverine HCl through ion-pair complex formation with methyl orange. Spectrochim Acta A Mol Biomol Spectrosc 2008;69:270-5.
- Abdelaleem EA, Abdel wahah NS. Validated chromatographic and spectrophotometric methods for analysis of some amoebicide drugs in their combined pharmaceutical preparation. Pak J Pharm Sci 2013:26(1):175-83.
- Naouib RA, Abdelkauy M. Development and validation of stability indicating HPLC and HPTLC method for determination of sulpiride and mebeverine hydrochloride in combination. Eur J Med Chem 2010;45(9):3719-25.
- Patel S, Patel NJ. Spectrophotometric and chromatographic simultaneous estimation of amitriptyline hydrochloride and chlordiazepoxide in tablet dosage form. Indian J Pharm Sci 2009;71(4):472-6.
- Patel SK, Patel NJ. Simultaneous RP-HPLC Estimation of Trifluoperazine Hydrochloride and Chlordiazepoxide in Tablet Dosage Forms. Indian J Pharm Sci 2009;71(5):545-7.
- Patel S, Patel NJ, Patel SA. Simultaneous Spectrophotometric Estimation of Imipramine Hydrochloride and Chlordiazepoxide in Tablets. Indian J Pharm Sci 2009;71(4):468-72.
- Venisetty RK, Kamarapu SK. RP-HPLC Method development and validation for simultaneous estimation of clidinium bromide, chlordiazapoxide and dicyclomine hydrochloride in bulk and combined tablet dosage forms. Int J Ad Biomed Pharm Res 2013;2(1):35-40.
- Patel DJ, Patel JK, Patel VP. Simultaneous spectrophotometric estimation of mebeverine hydrochloride and chlordiazepoxide in tablet dosage form. Inventi Rapid. Pharm Anal Qual Assur 2011;78(11).
- International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. Validation of Analytical Procedures: Text and Methodology ICH Q2 (R1). 2005.