INTRODUCTION:
Cinitapride, chemically 4-amino-N-[3-(Cyclohexan-1-yl-methyl)-4-piperidinyl]-2-ethoxy-5-
Nitrobenzamide1. It has an empirical formula C21H30N4O4 and molecular weight 402.49 g.mol-1. Cinitapride is a drug that has against action to the serotoninergic 5-HT2 and D2 dopaminergic receptors that has been indicated in the gastroesophageal reflux and in the functional disorders of gastrointestinal motility treatment. The use of cinitapride is efficient and safe in treatment of patients with disorders in the gastric emptiness related to gastroesophageal reflux and functional dyspepsia as well as in individuals that present irritable bowel syndrome with constipation and abdominal pain2,3. Pantoprazole sodium is chemically Sodium 5-(difluoro methoxy)-2-[[(3,4-dimethoxy-2- pyridinyl) methyl] sulfinyl]-1H-benzimidazole sesquihydrate[4]. It has an empirical formula of C16H15F2N3O4S and molecular weight of 383.37 g mol-1. The combination of Cinitapride (3mg) and pantoprazole (40mg) is widely used to treat the patients suffering from non-ulcer dyspepsia or gastroesophageal reflux disease. It is also used for treating ulcers of the stomach and duodenum, and the Zollinger-Ellison Syndrome.
Introducing HPTLC into pharmaceutical analysis represents a major step in terms of quality assurance. Today HPTLC is rapidly becoming a routine analytical technique due to its advantages of low operating costs, high sample throughput and the need for minimum sample preparation. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase-unlike HPLC - thus reducing the analysis time and cost per analysis.
The review of the literature revealed that no method is yet reported for the simultaneous estimation of both the drugs in combined dosage forms but few methods for individual determination of cinitapride and pantoprazole was reported5-7. These includes determination of free levels of drug in human plasma by Liquid Chromatography - Tendem Mass Spectrometry, Simple extractive colorimetric, RP-HPLC in human plasma8-10 . To date, there have been no published reports about the simultaneous estimation of cinitapride and pantoprazole by HPTLC pharmaceutical dosage forms. This present study reports for the first time simultaneous estimation of cinitapride and pantoprazole by HPTLC in pharmaceutical dosage forms. The proposed method is validated as per ICH guidelines.
MATERIALS AND METHODS:
Instrument used:
· High Performance Thin Layer Chromatograph (HPTLC)
Ø Manufacturer: CAMAG, Muttenz, Switzerland
Ø Sample applicator: CAMAG Linomat V
Ø Scanner: CAMAG TLC scanner 3
Ø Syringe: 100-μL Hamilton (Reno, Nevada, USA)
Ø Detector: Deuterium
Ø TLC plate : Pre coated silica gel 60 F 254 (E. Merck KGaA, Darmstadt, Germany) plates
Ø Twin through chamber: CAMAG, Muttenz, Switzerland
Analytical Balance (Model: Sartorius CP 124S)
Reagents and Solutions:
Reference Standards (RS) of Cinitapride and Pantoprazole were kindly gifted by Cadila Pharmaceuticals Ltd., Ahmedabad and Astron Research Ltd., Ahmedabad respectively. The pharmaceutical preparation of combination of cinitapride and pantoprazole that is Cintodac (Zydus Adilac) contains 3 mg of Cinitapride and 40 mg of Pantoprazole purchased from local market. Methanol and Ethyl acetate of analytical reagent grade were purchased by Finar Chemical Ltd (India). All the solutions were protected for light and were analyzed on the day of preparations.
EXPERIMENTAL:
Instrumentation:
The samples were spotted in the form of bands of width 4 mm with a Camag 100 micro litre sample (Hamilton, Bonaduz, Switzerland) syringe on silica gel precoated aluminium plate 60 F – 254 plates, [10 cm × 20 cm with 250 μm thickness; E. Merck, Darmstadt, Germany)] using a Camag Linomat V (Switzerland) sample applicator. The plates were prewashed with methanol and activated at 110°C for 5 min prior to chromatography. A constant application rate of 5 μL.s-1 was used and the space between two bands was 6 mm. The monochromator bandwidth was set at 20 nm, each track was scanned three times and baseline correction was used. The mobile phase consisted of ethyl acetate: methanol (9:1 v/v) and 50 mL of mobile phase was used per chromatography run. Linear ascending development was carried out in a 20 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for the mobile phase was 60 min at room temperature. The length of each chromatogram run was 8.5 cm. Following the development the HPTLC plates were dried in a room temperature. The source of radiation used was deuterium lamp emitting a continuous UV spectrum between 200 and 300 nm. Concentrations of the compound chromatographed were determined from the intensity of the diffused light. Evaluation was carried out by peak areas with linear regression.
Preparation of standard stock solution of Cinitapride and Pantoprazole:
Standard stock solutions of concentration 1000 µg/mL of cinitapride and 1000 µg/mL of pantoprazole were prepared separately using methanol. From the standard stock solution, the mixed standard solution was prepared using the methanol to contain 20 µg/mL of cinitapride and 200 µg/mL of pantoprazole. The stock solution was stored and protected from light. The calibration curve was obtained by applying 5µL of this solution so it makes the range 100 – 700 ng/spot for cinitapride and 1000 -7000 ng/spot for pantoprazole which shown in Fig. 1 in that lane 1 -7 was for standard and lane 8 was for marketed solution.
Preparation of test sample solutions:
The amount of cinitapride and pantoprazole in tablets, expressed as a percentage of label claim were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present in tablets. The spectra of marketed formulation was shown in Fig. 2. The drug content was found to be 100.40 ± 1.01% and 99.19 ± 0.99% respectively was shown in Table 1.
Optimization of the HPTLC method:
The TLC procedure was optimized with a view to develop a simultaneous assay method for cinitapride and pantoprazole respectively. The mixed standard stock solution (20 µg/mL of cinitapride and 200 µg/mL of pantoprazole) spotted on to HPTLC plates and run in different solvent systems. Initially, toluene, ethyl acetate and methanol were tried in different ratios. Toluene was used to impart the necessary non-polarity to mobile phase to obtain a suitable Rf value. Initially, toluene and methanol in the ratio of 7: 2 v/v was selected but the cinitapride spot was not moved from application position where as for pantoprazole 0.35 Rf obtained. Finally ethyl acetate: methanol (9:1) is used in different ratio to separate the both the drugs. Finally, the mobile phase consisting of ethyl acetate: methanol (9:1 v/v) was found optimum. The mobile phase was run up to a distance of 8.5 cm; which takes approximately 20 min for complete development of the HPTLC plate.
Validation of the method:11
Validation of the optimized HPTLC method was carried out with respect to the following parameters.
Linearity and range:
From the mixed standard stock solution 20 µg/mL of cinitapride and 200 µg/mL of pantoprazole, 5 to 35 μl solution spotted on HPTLC plate to obtain final concentration 100-700 ng/spot for cinitapride and 1000-7000 ng/spot for pantoprazole. The plate was then developed using the mobile phase ethyl acetate: methanol (9:1) and the peak areas were plotted against the corresponding concentrations to obtain the calibration curves.
Accuracy:
Accuracy of the method was carried out by applying the method to drug sample (cinitapride and pantoprazole combination tablet ) to which know amount of cinitapride and pantoprazole standard powder corresponding to 80, 100 and 120% of label claim had been added (Standard addition method), mixed and the powder was extracted and analyzed by running chromatogram in optimized mobile phase.
Precision:
The precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analysis of three different concentrations (200, 400, 600 ng/spot for cinitapride and 2000, 4000, 6000 ng/spot) of the drug in six times on the same day. The intermediate precision of the method was checked by repeating studies on three different days.
Limit of detection and limit of quantitation:
The limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions. It is usually expressed as the concentration of analyte in the sample.
Where, s = standard deviation of the response,
S = slope
The limit of quantification (LOQ) is the lowest amount of analyte in a sample that can be determined with acceptable precision and accuracy. It is usually expressed as the concentration of analyte in the sample.
Where, s = standard deviation of the response
S = slope
Robustness of the method:
Following the introduction of small changes in the mobile phase composition (± 0.1 mL for each component), the effects on the results were examined. Mobile phases having different compositions, e.g. ethyl acetate: methanol (9.1: 0.9 v/v), (8.9: 1.1 v/v), were tried and chromatograms were run. The amount of mobile phase was varied over the range of ± 5 %.The robustness of the method was determined at three different concentration levels 200, 400 and 600 ng/spot and 2000, 4000 and 6000 ng/spot for cinitapride and pantoprazole respectively.
Specificity:
The specificity of the method was determined by analyzing standard drug and test samples. The spot for cinitapride and pantoprazole in the samples was confirmed by comparing the Rf and spectrum of the spot with that of a standard.
RESULTS AND DISCUSSION:
The results of validation studies on simultaneous estimation method developed for cinitapride and pantoprazole in the current study involving ethyl acetate: methanol (9:1) as the mobile phase for HPTLC are given below.
Linearity:
The linearity range was found to be r 2 = 0.9987 for cinitapride and r 2 = 0.9948 for pantoprazole over the concentration range between 100-700 ng/spot for cinitapride and 1000-7000 ng/spot for pantoprazole.
Accuracy:
As shown from the data in Table 2 good recoveries of the cinitapride and pantoprazole in the range from 98.3 – 99.9 % and 98 – 99.8 % were obtained at various added concentrations respectively.
Precision:
The results of the repeatability and intermediate precision experiments are shown in Table 2. The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were < 2 %, respectively as recommended by ICH guidelines.
LOD and LOQ:
The LOD was found to be 48.13 ng/ml and 168.84 ng/ml, for cinitapride and pantoprazole respectively. LOQ was found to be 145.87 ng/ml and 511.66 ng/ml, for cinitapride and pantoprazole respectively.
Robustness of the method:
The standard deviation of peak areas was calculated for each parameter and the % RSD was found to be less than 2 %.
Specificity:
The specificity for cinitapride and pantoprazole was found to be 99.33 ± 0.11% and 99.75 ± 0.35% respectively.
Fig 1: Overlay spectra of cinitapride and pantoprazole
Table 1: Assay results of Marketed formulations
|
Capsule formulation |
Labeled claim (mg) |
Amount found (mg) |
% Recovery ± S.D* |
|
Cintodac |
Cinitapride (3mg) |
3.01 |
99.33 ± 0.11% |
|
Pantoprazole (40mg) |
39.67 |
99.75 ± 0.35% |
*Average of five determination