Journal of Pharmaceutical and Pharmacological Sciences

Volume 2017; Issue 01
13 Aug 2017

Simultaneous Estimation of Chlorzoxazone Paracetamol Famotidine and Diclofenac Potassium in Their Combined Dosage Form by Thin Layer Chromatography

Research Article

Usmangani K Chhalotiya*, Dhruv B Patel, Dimal A Shah, Falgun A Mehta, Kashyap K Bhatt

Indukaka Ipcowala College of Pharmacy, Gujarat, India

*Corresponding author: UsmanganiKC, Indukaka Ipcowala College of Pharmacy,Department of Pharmaceutical Chemistry and Analysis; E- Mail:usmangani84@gmail.com

Received Date: 19 November, 2016; Accepted Date: 21 February, 2017: Published Date: 28 February, 2017

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Abstract

Introduction

References

Figures

Tables

Suggested Citation

Abstract

 

A sensitive, selective and precise high performance thin layer chromatographic method has been developed for the estimation of chlorzoxazone, paracetamol, diclofenac potassium and famotidine in the pharmaceutical dosage form. TLC aluminum plates pre-coated with silica gel 60F254used as the stationary phase, while chloroform: methanol: ethyl acetate: hexane: ammonia (10: 2.5: 1.5: 1: 0.1, v/v/v/v/v) used as mobile phase. The Rf values were observed 0.74± 0.01, 0.52 ± 0.01, 0.30 ± 0.01 and 0.14± 0.01 for chlorzoxazone, paracetamol, diclofenac potassium and famotidine, respectively. The densitometry analysis was carried out in absorbance mode at 282 nm. The method was linear in the range of 250-1500 ng/spot for chlorzoxazone, diclofenac potassium and famotidine and 500- 3000 ng/spot for paracetamoland method was validated as per ICH guideline. The limit of detection and limit of quantization were found to be 35.98ng/spot and 109.05 ng/spot, respectively for chlorzoxazone, 99.74ng/spot and 302.25 ng/spot, respectively for paracetamol, 58.63 ng/spot and 177.69 ng/spot, respectively for diclofenac,and 50.93 ng/spot and 154.35 ng/spot, respectively for famotidine. The proposed method was successfully applied to the estimation of chlorzoxazone, paracetamol, diclofenac potassium and famotidine in the pharmaceutical dosage form.

 

Keywords:Chlorzoxazone (CLZ); Paracetamol (PCM); Diclofenac potassium (DCL); Famotidine (FAM); HPTLC; Validation

Introduction

 

Chlorzoxazone (CLZ) is chemically 5-chloro-2,3-dihydro-1,3-benzoxazol-2-one. The empirical formula of CLZ is C7H4ClNO2and a molecular weight is 169.56 g/mol. It is NSAID. It inhibits multisynaptic reflex a.c. involved in producing and maintaining skeletal muscle spasm.Paracetamol (PCM) is chemically N-(4-hydroxyphenyl)acetamide. The empirical formula for PCM isC8H9NO2 and a molecular weight is 151.163 g/mol. It inhibiting both is forms of cyclooxygenase; COX-1, COX-2, and COX-3 enzymes involved in prostaglandin (PG) synthesis. Diclofenac potassium (DCL) is chemically 2-{2-[(2,6-dichlorophenyl)amino]phenyl} acetic acid and empirical formula of DCL is C14H11Cl2NO2and molecular weight is 318.13g/mol. It inhibition of leukocyte migration and the enzyme cylooxygenase (COX-1 and COX-2), leading to the peripheral inhibition of prostaglandin synthesis.Famotidine (FAM) is chemically3-[({2-(diaminomethylidene)amino]-1, 3-thiazol-4-yl}methyl)sulfanyl]-Nsulfamoylpropanimidamide and empirical formula for FAM is C8H15N7O2S3 and molecular weight is 337.44 g/mol. It is competitive

 

Histamine H2-receptor antagonist and inhibits many of the isoenzymes of the hepatic CYP450 enzyme system.

 

The combined dosage form of CLZ, PCM, DCL and FAM is used as muscle relaxant.CLZ, PCM, DCL and FAM are official in United State Pharmacopoeia and British Pharmacopoeia. Official method has been reported for CLZ, PCM, DCL and FAM in United StatePharmacopoeia and British Pharmacopoeia. Some of UV, HPLC, TLC methods has been reported for the estimation of CLZ, PCM, DCL and FAM alone and with other drug combination. No method has been reported for the estimation of CLZ, PCM, DCL and FAM in their combined dosage form[1-27].In comparison to LC and LC-MS/MS methods, HPTLC method is considered to be a good alternative, and it should be widely explored as an important tool in routine drug analysis. A major advantage of HPTLC is its ability to analyze several samples simultaneously using a small quantity of mobile phase. This reduces the time and cost of analysis.

 

Experimental

 

HPTLC instrument

 

The samples were applied in the form of a bands of width 8 mm with a Camag 100μl sample syringe (Hamilton, Switzerland) using Camag Linomat 5 (Switzerland) sample applicator on pre-coated silica gel aluminum plate 60 F254(10 cm x 10 cm with 0.2 mm thickness, E. Merck, Germany). Camag TLC scanner4 was used for the densitometric scanning.

 

Chemicals and reagents

 

Analytically pure CLZ, PCM, DCL and FAM from Sun pharmaceutical industry ltd. Vadodara, India were obtained as gift samples. Methanol (AR grade) of SRL Private Ltd. and chloroform of Chemdyes Corporation (AR grade) were used. Ammonia and hexane of Chiti-Chem Corporation (AR grade) were used. Ethyl acetate of Astron Chemicals (AR grade)Tablet formulation fast ran MR(Horizon biocauticals Pvt. Ltd.) containing 500 mg of PCM, 250 mg CLZ, 50 mg DCL and 10 mg FAM was procured from local pharmacy.

 

Chromatographic system

 

Sample application

 

Standards and formulation samples of CLZ, PCM, DCL and FAM were applied on the HPTLC plates in the form of narrow bands of 6 mm length, 10 mm from the bottom and left edge, and with 9 mm distance between two bands. Samples were applied under a continuous stream of nitrogen gas.

 

Mobile phase and development

 

Plates were developed using a mobile phase consisting of chloroform: methanol: ethyl acetate: hexane: ammonia (10: 2.5: 1.5: 1: 0.1, v/v/v/v/v). Linear ascending development was carried out in a twin-trough glass chamber equilibrated with the mobile phase vapors for 30 min at 25 ± 20C. Ten milliliters of the mobile phase (5 ml in the trough containing the plate and 5 ml in the other trough) was used for each development and was allowed to migrate a distance of 80 mm, sample application rate is 200nl/sec. After development, the HPTLC plates were dried completely using continuous stream of nitrogen.

 

Densitometric analysis

 

Densitometric scanning was performed in the absorbance mode under control by winCATS planar chromatography software. The source of radiation was the deuterium lamp and bands were scanned at 282 nm. The slit dimensions were 6 mm length and 0.45 mm width, with a scanning rate of 20 mm/s.Concentrations of the compound were determined from the intensity of diffusely reflected light and evaluated as peak areas against concentrations using a linear regression equation.

 

Preparation of standard stock solution

 

PCM (10 mg),CLZ (5 mg), DCL (5mg) and FAM (5mg) were accurately weighed and transferred to 10 ml volumetric flasks and dissolved in few ml of methanol. Volumes were made up to the mark with methanol to yield a solution containing 1000mg/ml of PCM and 500 mg/ml of CLZ, DCL and FAM. Aliquot from the stock solutions of PCM, CLZ, DCL and FAM were appropriately diluted with mobile phase to obtain working standard of 100mg/ml of PCM and 50 mg/ml of CLZ, DCL and FAM respectively.

 

Validation

 

Validation of the developed HPTLC method was carried out according to International Conference on Harmonization (ICH) guidelines Q2 (R1) for specificity, sensitivity, accuracy, precision, repeatability, and robustness [28].

 

Linearity of calibration curves

 

Linearity of the method was evaluated by constructing calibration curves at six concentration levels over a range of 500–3000 ng/band for PCM and 250-1500 ng/band for CLZ, DCL and, FAM by applying 5ml to 30ml from stock solution has been applied on HPTLC plate using sample applicator.The calibration curves were developed by plotting peak area versus concentration (n = 6) with the help of the winCATS software.

 

Accuracy

 

Accuracy is closeness of the test results obtained by the method to the true value and should be established across specified range of analytical Procedure. The accuracy of the method was determined by calculating recoveries of PCM, CLZ, DCL and FAM by method of standard additions.Known amount of PCM(0, 500, 1000, 1500 ng/spot) and CLZ, DCL and FAM(0, 250, 500, 750 ng/spot) were taken from the working standard solutions (1000ng/spot of PCM and 500ng/spot of CLZ, DCL and FAM respectively).It was added to a pre quantified sample and the amount of PCM, CLZ, DCL and FAM were estimated by measuring the peak area and by fitting these values to the straight-line equation of calibration curve.The proposed acceptance criteria for the accuracy studies are ranges from 95-105 %.

 

Precision

 

Precision is closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample.Precision was evaluated in terms of intraday and interday precisions. Standard solutions of 100 mg/ml of PCM and 50 mg/ml of CLZ, DCL,and FAM, were prepared and used for the precision study. Intraday precision was determined by analyzing sample solutions of PCM (500 ng/spot, 1000 ng/spot, and 3000 ng/spot), CLZ, DCL and FAM(250 ng/spot, 750 ng/spot, 1500 ng/spot) at three levels covering low, medium, and high concentrationsof the calibration curve three times on the same day. Interday precision was determined by analyzing sample solutions of PCM, CLZ, DCL and FAM at three levels covering low, medium, and high concentrations over a period of 3 days. The peak areas obtained were used to calculate mean and RSD values. Less than 5 % RSD values indicate that the method is precise.

 

Specificity

 

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. The specificity of the method was ascertained by analyzing PCM, CLZ, DCL and FAMin presence of excipients commonly used for tablet formulations. The bands of PCM, CLZ, DCL and FAMwere confirmed by comparing Rf values and respective spectra of sample with those of standards. The peak purity of PCM, CLZ, DCL and FAMwas assured by comparing the spectra at three different levels, that is, peak start, peak apex and peak end positions.Selectivity describes the ability of an analytical method to differentiate various substances in a sample. The proposed method shows well resolution of all four molecules from their pharmaceutical dosage form.

 

Sensitivity

 

Sensitivity of the method was determined with respect to LOD and LOQ. Noise was determined by scanning a blank band (methanol) six times. LOD was calculated as 3 times the noise level, and LOQ was calculated as 10 times the noise level.

 

Robustness

 

Small changes in the chamber saturation time, solvent migration distance and mobile phase composition were introduced and the effects on the results were examined. Robustness of the method was determined in triplicate at a concentration level of 2000 ng/band for PCM and 1000 ng/spot for DCL and 750ng/spot for CLZ and FAM. The mean and RSD of peak areas were calculated.

 

Analysis of marketed formulations

 

Twenty tablets were weighed accurately and finely powdered. Tablet powder equivalent to 500 mg of PCM, 250 mg of CLZ, 50 mg of DCL and 10mgof FAM was accurately weighed and transferred to a 100 ml volumetric flask. A few ml (40 ml) of methanol was added to the above flask and flask was sonicated for 15 min. The solution was filtered using what man filter paper No. 41 in another 100 ml volumetric flask and make up the volume up to the mark with the methanol.

 

A solution containing 300 ng/band FAM and 1500 ng/band DCLwere injected as per the above chromatographic conditions and peak areas were recorded.Appropriate volume of the aliquot was transferred to a 10 ml volumetric flask and the volume was made up to the mark with the mobile phase to obtain a solution containing 750 ng/band CLZ and 1500 ng/band PCM. The quantifications were carried out by keeping these values to the straight line equation of calibration curve.

 

Results and Discussion

 

Optimization of the Mobile Phase

 

To develop the HPTLC method for analysis of PCM, CLZ, DCL and FAM in the pharmaceutical dosage form for routine analysis, selection of the mobile phase was carried out on the basis of polarity. A mobile phase that would give a dense and compact band with an appropriate Rf value for PCM, CLZ, DCL and FAM was desired. Various mobile phases such as acetone-methanol, methanol-chloroform acetic acid, methanol-toluene-ammonia,methanol-toluene-glacialaceticacid, toluene-ethylacetate-methanol, methanol-acetonitrile-glacial acetic acid were evaluated in different proportions.A mobile consisting of chloroform: methanol: ethyl acetate: hexane: ammonia (10: 2.5: 1.5: 1: 0.1, v/v/v/v/v)gave good separation of PCM, CLZ, DCL and FAM from its matrix. It was also observed that chamber saturation time and solvent migration distance were crucial in the chromatographic separation. Therefore, chloroform: methanol: ethyl acetate: hexane : ammonia (10: 2.5: 1.5: 1: 0.1, v/v/v/v/v) mobile phase with a chamber saturation time of 30 min at 25 0C and solvent migration distance of 80 mm was used. Densitogram of PCM, CLZ, DCL and FAM,

 

photograph of TLC plate andthree dimensional overlays of HPTLC densitograms of calibration bands of PCM, CLZ, DCL and FAM are depicted in figures (Figure 1-3).

 

Validation

 

Linearity and calibration curves.

 

The method was found to be linear for PCMin concentration range of 500-3000 ng/band (n= 6) and for CLZ, DCL and FAM250-1500 ng/band (n= 6), respectively. Figure-3 displays a three-dimensional overlay of HPTLC densitograms of the calibration bands of PCM, CLZ, DCL and FAM at 282 nm. The regression data shown in(Table1) reveal a good linear relationship over the concentration range studied, demonstrating the suitability of the method for analysis.

 

Accuracy

 

Accuracy was determined by the application of analytical procedure to recovery studies, where a known amount of standard is spiked into preanalyzed samples solutions. Results of the accuracy studies from excipients matrix are shown in (Table4). Recovery values demonstrated the accuracy of the method in the desired range.

 

Precision

 

In all instances, RSD values were less than 2%, confirming the precision of the method. Repeatability of the scanning device was studied by applying and analyzing sample seven times. RSD was less than 2%, which was well below the instrumental specifications.Summary of validation parameters are shown in (table2).

 

The RSD values obtained were less than 2%, which was under the acceptance criteria of ICH method validation guideline (<2%). The results indicated that the method is repeatable and reproducible.

 

Limit of detection and limit of quantification

 

Under the experimental conditions used, the lowest amount of drug that could be detected LOD was found to be 35.98ng/band,99.74 ng/band, 58.63 ng/band and 50.93 ng/band for PCM, CLZ, DCL and FAM, respectively and LOQ was found to be109.05 ng/band 302.25 ng/band, 177.69 ng/band and 154.35 ng/band for PCM, CLZ, DCL and FAM, respectively.It indicate that the nanogram quantity of all the drugs can be estimated accurately and precisely which means that the method is sensitive.

 

Specificity

 

There was no interfering peak at the Rf value of PCM, CLZ, DCL and FAM from excipients added in the synthetic formulation. In addition, there was no interference from excipients present in the commercial formulation, thereby confirming the specificity of the method.

 

Robustness

 

The low values of RSD obtained after introducing small, deliberate changes in parameters of the developed HPTLC method confirmed its robustness. The robustness data of the proposed method are shown in(table3).

 

Analysis of marketed formulation

 

Marketed formulation was analyzed using proposed method which gave percentage recovery of 98.46%, 98.26%, 98.09% and 99.01% for PCM, CLZ, DCL and FAM, respectively. No interference from the excipients present in the marketed tablet formulation was observed.

 

Conclusions

 

A selective, sensitive, accurate and precise high performance thin layer chromatography method has been developed for the simultaneous identification and quantification of chlorzoxazone, paracetamol, and famotidine and diclofenac potassiumin their combined pharmaceutical dosage form. The method was successfully validated in accordance with ICH guidelines. It can be conveniently used for routine quality control analysis of chlorzoxazone, paracetamol, and famotidine and diclofenac potassium in marketed tablet without any interference from excipients.

 

Acknowledgement

 

The authors are thankful to Mercury pharmaceuticals Ltd., Baroda and Blue Cross Laboratory, Mumbai, Indiafor providing gift sample of CLZ, PCM, DCL and FAM respectively. The authors are very thankful to Principal, Indukaka Ipcowala College of Pharmacy, and New Vallabh Vidyanagar for providing necessary facilities to carry out research work.

References

 

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Figures

 

 

Figure1-3:Densitogram of PCM, CLZ, DCL and FAM, photograph of TLC plate and three dimensional overlays of HPTLC densitograms of calibration bands of PCM, CLZ, DCL and FAM are depicted in figures (Figure 1-3).

Tables

 

PARAMETERS CLZ PCM DCL FAM
Linearity range (ng/spot) 250-1500 500-3000 250-1500 250-1500
Slope

 

14.278 5.682 10.902 10.808
Standard deviation of slope 0.2094 0.03033 0.2148 0.294737
Intercept

 

4619.6 4953.4 9033.6 4310.6
Standard deviation of intercept 155.7122 171.73 193.72 166.82
Correlation coefficient 0.995 0.997 0.995 0.996

Table 1:The regression data shown

 

Amount of Sample Set Amount drug Average amount recovered (ng/spot) % Recovery
(ng/spot) Spiked (ng/spot)
CLZ PCM CLZ PCM CLZ PCM CLZ PCM
500 1000 1 0 0 495.47 994.47 99.09 99.44
2 0 0
3 0 0
500 1000 1 250 500 747.89 1498.8 99.58 99.88
2 250 500
3 250 500
500 1000 1 500 1000 997.13 1985.6 99.42 98.55
2 500 1000
3 500 1000
500 1000 1 750 1500 1244.1 2489.8 98.81 98.93

 

Table 4:Recovery values demonstrated the accuracy of the method in the desired range.

 

Amount of Sample Set Amount drug Average amount recovered (ng/spot) % Recovery
(ng/spot) Spiked (ng/spot)
FAM DCL FAM DCL FAM DCL FAM DCL
500 500 1 0 0 496.73 504.38 99.01 100.3
2 0 0
3 0 0
500 500 1 250 250 744.73 749.28 999.6 99.85
2 250 250
3 250 250
500 500 1 500 500 997.75 998.99 98.94 99.79
2 500 500
3 500 500
500 500 1 750 750 1245.07 1246.1 99.34 99.22

 

Table 4:Recovery values demonstrated the accuracy of the method in the desired range

 

PARAMETERS CLZ PCM DCL FAM
Rf 0.74 0.52 0.29 0.14
Detection limit (ng/spot) 35.98 99.74 58.63 50.93
Quantization limit (ng/spot) 109.05 302.25 177.69 154.35
Accuracy (%) 98.81-99.57 98.55-99.88 99.22-100.27 98.94-99.55
Intra-day (n=3) (% RSD) 1.18-1.249 0.94-1.31 1.33-1.60 0.82-1.06
Inter-day (n=3) (% RSD) 1.71-1.89 1.45-1.68 1.44-1.83 1.57-1.93
Repeatability study (n=6) (% RSD) 1.77-1.96 1.77-1.86 1.71-1.85 1.58-1.83

 

Table 2:Summary of validation parameters

 

Parameters Amt of CLZ Amt of ClZ Recovered ± SD Amt of PCM Amt of PCM Recovered ± SD Amt of DCL Amt of DCL Recovered ± SD Amt of FAM Amt of FAM Recovered ± SD
Chamber saturation time : 20 min 750 741±22.9 2000 1960.3±38.07 1000 962.7±31.89 750 766.8±21.65
Chamber saturation time : 40 min 750 753.6±29.50 2000 1941.3±48.22 1000 1014.1±36.90 750 774.9±6.24
Wave length 280 750 762.6±22.85 2000 1943.4±32.71 1000 953.5±28.53 750 780.3±17.92
Wave length 280 750 768.6±17.38 2000 1977.6±12.01 1000 949.6±22.03 750 757.5±10.40
chloroform:methanol:ethayl acetate:hexane:ammonia 750 732.6±10.40 2000 1978.4±34.58 1000 940.2±30.55 750 771.6±23.62
(9: 3.5: 1.5: 1: 0.1v/v/v/v/v)
chloroform:methanol:ethayl acetate:hexane:ammonia 750 771.2±24.57 2000 1952.6±36.115 1000 956.7±19.73 750 748.7±14.9
(11: 2: 1: 1: 0.1 v/v/v/v/v)

 

 

Table 3:The robustness data of the proposed method

Suggested Citation

 

Citation: Chhalotiya UK, Patel DB, Shah DA, Mehta FA, Bhatt KK(2017)Simultaneous Estimation of Chlorzoxazone Paracetamol, Famotidine and Diclofenac Potassium in Their Combined Dosage Form by Thin Layer Chromatography. J Pharmaceut Pharma Sci 2017: G111

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