Validated HPTLC Method for Simultaneous Quantitation of Esomeprazole and Domperidone in Combined Capsule Dosage Form
P. Asha*, I. Ponnilavarasan, Ch. Sunil Narendara Kumar and J. Dharuman
Dept. of Pharmaceutical Analysis, K.M.C.H College of Pharmacy, Kalapatti Road,Coimbatore-641048. Tamilnadu.
*Corresponding Author E-mail: ponn75@gmail.com
ABSTRACT:
A simple, precise, rapid, selective and economical high performance thin layer chromatographic (HPTLC) method has been established for simultaneous analysis of Esomeprazole and Domperidone in capsule dosage forms. The chromatographic separation was performed on precoated silica gel 60 GF254 plates with Acetonitrile: Chloroform: Ammonia (12:7. 5:0. 5)(v/v/v)as a mobile phase. The drugs on the plate were scanned at 222 nm. The drugs were satisfactorily resolved with Rf 0. 31 for domperidone and 0. 48 for esomeprazole. The method was validated for linearity, accuracy, precision, and specificity. The calibration plot was linear between 200-1000 ng/spot for esomeprazole and domperidone. The limit of detection and quantification for esomeprazole was 85. 07 and 255. 21ng/spot and domperidone was 79. 07 and 237. 23 ng/spot. The percent recovery study was done by standard addition method found in the range of 98. 7% and 99. 1% respectively. The proposed HPTLC method is economic, sensitive, and less time consuming than other chromatographic procedures.
KEYWORDS: Denisitometry, Quantification, Esomeprazole, Domperidone.
INTRODUCTION:
Esomeprazole is a proton pump inhibitor drug used for short-term treatment of erosion and ulceration of the esophagus caused by gastroesophageal reflux disease1.Chemically esomeprazole is 6-methoxy-2-[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole.The chemical structure of esomeprazole are shown in the figure 1.Domperidone chemically 5-chloro-1-{1-(3-(2,3-dihydro2-oxo1H-benzimidazol-yl)Propyl)-4-piperdinyl)-1-3-dihydro-2H-benzimiazole-2-one. It is a peripheral dopamine-2-receptor antagonist.It is a unique gastrokinetic and antiemetic drug.Combination of these two is used for the treatment of gastric esophagus reflux disease1-7. The chemical structure of domperidone as shown in the figure 2.
Literature survey reveals that Potentiometry, Absorption ratio method7 and more extensively HPLC have been reported for the individual and simultaneous determination of an esomeprazole8-12.Several analytical techniques such as RP-HPLC in tablet formulation and in human plasma, LC-MS in human plasma and Spectraphotometry were available for the determination of domperidone13-19.
MATERIALS AND METHODS:
Working standards of pharmaceutical grade domperidone and esomeprazole were obtained as generous gifts from Microlabs Pharmaceuticals limited, Bangalore, India. Fixed dose combination capsules (ESOZ-D 20) containing 30mg of Domperidone and 20mg of Esomeprazole were procured from private pharmacy Coimbatore, India.
INSTRUMENTATION:
The samples were spotted in the form of bands width 8mm with a Camag 100 µl sample (Hamilton Bonaduz, Switzerland) syringe on silicagel precoated aluminum plate 60 GF254 plates (20x10 cm with 250µm thickness) using a camag Linomat V sample applicator. The plates were prewashed with methanol and activated at 110˚c for 5min prior to chromatography. The constant application rate of 150 nl/sec was used and the space between two bands was 14.5 mm. The slit dimension was kept at 5.00 X 0.45 mm and the scanning speed was 10 mm per second. The monochromator band width was set at 20 nm, each track was scanned three times and base line correction was carried out. The mobile phase consist of Chloroform: Acetonitrile: Ammonia (12:5:3) and 20 ml of mobile phase was used per chromatography run. Linear ascending development was carried out in a 20 x 10 cm twin trough glass chamber saturated with a mobile phase. The optimized chamber saturation time for the mobile phase was 10 min at the room temperature (25o c) at relative humidity of 60 % and length of each chromatogram run was 8 cm. Following the development, the TLC plate was dried in a current of air with the help of an air dryer in wooden chamber with adequate ventilation. Densitometric scanning was performed using a Camag TLC scanner 3 in the reflectance – absorbance mode at 254 nm and operated by Wincats software. The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 200 – 400 nm. Evaluation was determined by peak area with linear regression.
PREPARATION OF STANDARD STOCK SOLUTION:
Accurately weighed quantity of esomeprazole and domperidone (10mg each) was dissolved in methanol and chloroform (1:1) and made upto 10ml to obtain a stock solution of 1mg/ml of Esomeprazole and Domperidone.
PREPARATION OF SAMPLE SOLUTION:
The powder of 20 capsules was weighed, mixed and accurately a quantity of the powder equivalent to about 20mg of esomeprazole and 30mg of domperidone is transferred into 100ml measuring flask. The solution was filtered through Whatman filter paper No.41 and the residue was washed thoroughly with methanol. The filtrate and washings were combined in a 100 ml volumetric flask and diluted to the mark with methanol. Transfer 1ml of the extract into 10ml volumetric flask and dilute to the mark with methanol to get a concentration of 20μg/ml of esomeprazole and 30μg/ml of domperidone.The results of formulation are shown in the table1.
FIGURE:1 Chemical Structure of Esomeprazole
FIGURE: 2 Chemical structure of Domperidone
RESULTS OF FORMULATION:
Table 1: Analysis of formulation
|
Formulation (ESOZ-D) |
Amount (mg/capsule) |
Percentage Assay |
% R.S.D |
|
|
Labelled amount (mg) |
Amount Found (mg) |
|||
|
Domperidone |
30 |
29.94 |
99.4% |
0.8682 |
|
Esomeprazole |
20 |
19.82 |
99.8% |
0.5806 |
RESULTS AND DISCUSSION:
VALIDATION OF THE METHOD:
Validation of the optimized HPTLC method was carried out with respect to the following parameters.
Linearity and Regression:
The drug response was linear over the concentration range between 200-1000 ng/spot for domperidone and esomeprazole.The linear regression data showed a regression coefficient of 0.9980 for both esomeprazole and domperidone.The standard chromatogram peak as shown in the figure 3.
FIGURE: 3 typical chromatogram obtained from domperidone and esomeprazole standard
(Rf 0.31 for domperidone and Rf 0.48 for esomeprazole)
Precision:
Precision was measured by analysis of sample solutions at three different concentrations.Intra-day precision was found by analysis of standard drug at three times on the same day.Inter-day precision was carried out using at three different days, and percentage relative standard deviation (%RSD) was calculated. The RSD was found to be less than 2 for both intra-day and inter-day precision. Repeatability of sample application was carried out by spotting 1µl of drug solution for six times. From the peak areas, the percentage RSD was determined.The results from the study of precision as shown in the table2.
Limit of detection and quantification:
Signal to noise ratios of 3:1 and 10:1 were obtained. For the LOD and LOQ respectively. The LOD and LOQ were found to be 79.07 and 237.23 ng/spot for domperidone and 85.07 and 255.21 ng/spot for esomeprazole respectively.
Table 2: Results from determination of precision
|
Domperidone Concentration ( ng/spot ) |
(% RSD) Inter-day |
(% RSD) Intra-day |
Esomeprazole Concentration (ng/spot) |
(% RSD) Inter-day |
(% RSD) Intra-day |
|
2 |
1.83 |
1.38 |
2 |
0.75 |
1.81 |
|
4 |
1.46 |
1.54 |
4 |
1.90 |
1.33 |
|
6 |
1.11 |
1.69 |
6 |
1.65 |
1.74 |
Table 3: Results from determination of Robustness
|
Parameter |
Modification |
Domperidone (Recovery %) |
Esomeprazole (Recovery %) |
|
Mobile phase ratio |
9:7:4 10:6:4 |
97.35 98.56 |
97.56 98.84 |
|
Detection wavelength (nm) |
229 nm |
98.96 |
98.52 |
|
Slit dimension |
5.00x 0.3m micro |
98.65 |
97.99 |
Table.4. Recovery Data for the Proposed Method
|
Drug |
Level |
Label claim (50 mg/capsule) |
Amount added |
Total amount (mg) |
Amount Recovered (mg) |
Recovery (%) |
(% RSD) |
|
Domperidone |
1 2 3 |
30 30 30 |
2 4 6 |
4.0038 7.8403 11.9012 |
2.0049 3.9753 5.9898 |
99.9% 98.3% 99.2% |
0.3806 0.5757 0.4809 |
|
Esomeprazole |
1 2 3 |
20 20 20 |
2 4 6 |
4.0102 8.0100 11.8402 |
2.0170 4.0195 5.9468 |
99.8% 99.8% 99.2% |
0.3015 0.8159 0.3653 |
FIGURE:4 Typical Chromatogram obtained from Domperidone and Esomeprazole Sample
Robustness of the method:
The standard deviation of peak areas were calculated for each parameter and the % RSD was found to be less than 2%.The results from the study of robustness as shown in the table 3.
Ruggedness:
The ruggedness of the method was checked by using different analysts and instruments. The relative standard deviation of the results obtained by different analysts using instruments was <1.0%.
RECOVERY STUDIES:
The recovery study was carried out by mixing a known quantity of drug with pre-analysed sample and contents were reanalyzed by the proposed method was found to be 99.1% and 99.5% respectively. The values were given in the table 4.The sample chromatogram peak as shown in the figure 4.
CONCLUSION:
This HPTLC method for simultaneous analysis of domperidone and esomeprazole in capsule dosage form is simple, economic, accurate, precise, specific, robust, and rapid. It does not suffer interference from excipents present in the pharmaceutical preparation and can be conviently adopted for quality control analysis.
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Received on 01.12.2010 Modified on 15.01.2011
Accepted on 27.01.2011 © AJRC All right reserved
Asian J. Research Chem. 4(6): June, 2011; Page 928-931