1. INTRODUCTION:
Pantoprazole (PNP), 2-((4,5-dimethoxy pyridine-2-yl)methylsulfinyl)-6-(difluromethoxy)-1H-benzo[d] imidazole, is a proton pump inhibitors acts through the inhibition of hydrogen-potassium adenosine triphosphatase (H+/ K+ - ATPase) in gastric parietal cells and widely used as anti-ulcer drugs1,2 (Fig. 1). Cinitapride (CNT), 4-amino-N-(1-((cyclohex-3-enyl) methyl) piperidin-4-yl)-2-ethoxy-5-nitrobenzamide, is used as gastroprokinetic and anti-ulcer agent. It acts via inhibition of serotonergic 5-HT2 and dopaminergic D2 receptors and stimulation of serotonergic 5-HT4 receptors in the neuronal synapses of the myenteric plexi, thereby increasing gastrointestinal motility which facilitates the movement of acid contents further down in the intestine preventing reflux esophagitis3,4 (Fig. 2). Thus, pantoprazole and cinitapride in combined dosage form use to treat hyperacidity frequently associated with gastro intestinal dysmotility.
Figure 1: Structure of Pantaprazole Sodium Sesquihydrate
Figure 2: Structure of Cinitapride hydrogen tartarate
In the literature, estimation of pantoprazole by UV-spectrophotometric5 and colorimetric6 methods in pharmaceuticals, and RP-HPLC7 method in pharmaceuticals and human plasma are reported. Various RP-HPLC methods for estimation of pantoprazole in combination with other drugs in pharmaceuticals8-11 and in biologicals12,13 have been reported.
Estimation of cinitapride by UV-spectrophotometric14 and colorimetric15 methods in pharmaceuticals, and RP-HPLC16,17 methods in pharmaceuticals have been reported. VariousUV-spectrophotometric18-20 and HPTLC21 methods for estimation of pantoprazole and cinitapride in combined pharmaceuticals formulation have been reported. However, there is no method yet reported for estimation of pantoprazole and cinitapride in combined dosage pharmaceutical formulation of by Reverse-Phase High Performance Liquid Chromatography. The aim of the present work was the development of a RP-HPLC method for simultaneous estimation of pantoprazole and cinitapride and its validation according to ICH guidelines22.
2. EXPERIMENTAL:
1) Preparation of standard stock solutions.
i) Stock Solution A: An accurately weighed quantity about 100 mg of CNT was dissolved in 20 ml of water and the volume was made up to 100 ml (conc. 1 mg/ml).
ii) Stock Solution B: An accurately weighed quantity about 100 mg of PNP was dissolved in 20 ml of water and the volume was made up to 100 ml (conc. 1 mg/ml).
2) Study of UV-Spectra and Selection of Wavelength
The stock solution A and B were diluted with distilled water to get final concentration of 10 µg/ml for CNT and PNP. The solutions were scanned in UV-Range (200-400 nm) in 1.0 cm cell against distilled water as blank. The overlain spectrum of drugs is depicted in fig. no.3.
CNT shows peak maximum at 265.5 nm while PNP shows peak maximum at 268 nm. Both the drugs showed same absorbance at 236 nm (isoabsorptive point). The two wavelengths i.e. 236 nm and 265.5 nm were selected for analysis by absorbance ration method.
Fig. No. 3 : Overlain Spectra of CNT and PNP
3) Study of Beer Lambert’s Law
The stock solutions A and B were individually diluted with water to get final concentration in the range of 5 µg to 30 µg/ml for CNT and 5-30 µg/ml for PNP. The mixtures of above two drugs were prepared in the concentration ratio of 1:13.33 (CNT: PNP) and diluted to get final concentration of mixture in the range 1-6 µg/ml of CNT and 13.33 to 79.98 µg/ml for PNP. The absorbance of the diluted solutions was measured at 236 nm and 265.5nm in 1.0 cm cell against water as blank.
The relationship between concentration and absorbance for individual drugs and in mixture was studied. A linear relationship was observed for the two drugs individually. The mixture of two drugs also obeys the Beer-Lambert’s law.
The graphs of absorbance Vs concentration were plotted for individual drug and mixture and are shown in Fig. no. 4, 5and 6.
Fig. No. 4 :Beer Lambert’s Law for CNT
4) Determination of A (1 %, 1 cm) at selected wavelength.
Aliquot portions of stock solution A and B were diluted individually with distilled water to get final concentration of 10 µg/ml of each drug. The absorbance of each of the diluted solution was measured at 236 nm and 265.5 nm in 1.0 cm cell against distilled water as blank. The A (1%, 1 cm) value were calculated by using following formula and are shown in Table No. 1.
Absorbance
A(1%, 1cm) = -----------------------------------
Concentration (g/100ml)
A (1%, 1 cm) values of CNT and PNP at selected wavelengths are as follows:
A (1%, 1 cm) for CNT at 236nm- 269.6 (SD= ± 1.1401)
A (1%, 1 cm) for CNT at 265.5 nm 599.6 (SD= ± 1.1401)
A (1%, 1 cm) for PNP at 236 nm 269.8 (SD= ± 0.8366)
A (1%, 1 cm) for PNP at 265.5 nm 340.2 (SD= ± 1.4832)
The method was first applied for the estimation of drugs in standard laboratory mixture. It gave satisfactory results and hence it was extended for estimation in marketed formulation. The results of estimation of laboratory mixture and marketed formulation are shown in Table No. 1.
Fig. No. 5 : Beer Lambert’s Law for PNP
Fig. No. 6 : Beer Lambert’s Law for Mixture
Table No. 1: Summary of results of Laboratory Mixture and Marketed Formulation
Sr. No |
Sample |
StatisticalData |
Results (% Estimated* / % Label Claim**) |
|
|
CNT |
PNP |
|||
|
1 |
Laboratory* mixture |
Mean |
100.06 |
100.00 |
|
± SD |
0.5856 |
0.0286 |
||
|
CV |
0.5852 |
0.0286 |
||
|
RSD |
0.0059 |
0.0003 |
||
|
2 |
Marketed** formulation Cintodac |
Mean |
99.06 |
99.74 |
|
± SD |
0.5950 |
0.1448 |
||
|
CV |
0.6007 |
0.1451 |
||
|
RSD |
0.0060 |
0.0014 |
||
5) Analysis of Laboratory Mixture
Accurately weighed quantities, about 25 mg of CNT and 333.25 mg of PNP were transferred to 25 ml volumetric flask and dissolved in 10-15 ml of water. The volume was made up to the mark with same solvent. An accurately measured 10 ml portion of the solution was diluted to 100 ml with distilled water. A 10 ml portion was further diluted to 100 ml with distilled water.
The absorbance of the final dilution was measured at 236 nm and 265.5 nm. The contents of CNT and PNP were calculated by using following formula:
Cy may be determined in similar manner
Where, Cx and Cy is the concentration (g/100 ml) of CNT and PNP in final dilution respectively.
= 0.449 = ratio of
absorptivity of x at 236 nm and 265.5 nm.
= 0.793 = ratio of
absorptivity of y at 236 nm and 265.5 nm.
= ratio of absorbance of
mixture at 236 nm and 265.5 nm
= Absorbance of
mixture at isoabsorptive point.
= 269.7 =
absorptivity value of CNT and PNP at isoabsorptive point.
The amount of each drug estimated in laboratory mixture was calculated by using following formula.
Amount of drug estimated (mg) = C x d x v
Where, C is the concentration of drug, d is the dilution factor, v is the volume of stock solution.
6) Application of proposed method to marketed formulation
Details of marketed formulation
Trade Name: Cintodac
Mfg.: Cadila Healthcare Pvt. Ltd.
Composition: CNT (3 mg) : PNP (40 mg)
Average weight: 0.3933 g
Procedure: Twenty capsules were accurately weighed with and without shell and average weight was calculated. An accurately weighed quantity of powder equivalent to about 25 mg of PNP was transferred to 25 ml of volumetric flask and shaken with 10-15 ml of water for 5 min.
The volume was made up to the mark with the same solvent. The solution was then filtered through whatman Grade I filter paper.
Aliquot portion of the filtrate was further diluted to get final concentration of about 10 µg/ml of CNT and 133.3 µg/ml of PNP on the basis of labeled claim. The absorbance of final dilution was measured at 236 nm and 265.5 nm. The concentration of CNT and PNP was calculated by using the formula used for laboratory mixture and the percent estimation was calculated by using following formula.
7) Recovery Studies
Recovery Studies were carried out by standard addition method.
Preanalyzed powder equivalent of about 19.66, 21, 22.33, 23.66, 25 mg. of PNP was transferred to 25 ml of volumetric flask separately (Flasks A, B, C, D, E). The amount of pure drug added to each of the flask was as follows:
Flask A – about 1.525 mg CNT and about 20.34 mg PNP
Flask B – about 1.425 mg CNT and about 19 mg PNP
Flask C – about 1.325 mg CNT and about 17.67 mg PNP
Flask D – about 1.225 mg CNT and about 16.34 mg PNP
Flask E – about 1.125 mg CNT and about 15 mg PNP
The samples are shaken with 10-15 ml of water and the volume was made up to the mark with the same solvent. The solutions are mixed and filtered through whatman grade 1 filter paper. The aliquot portion of the filtrate was further diluted to get final concentration of about 10 ug/ml of CNT and 133.3 ug/ml of PNP. The absorbances of final dilutions were measured at 236 nm and 265.5 nm.
The percent recovery was calculated by using following formula.
Where,
A = Total amount of drug estimated in sample (mg)
B = Amount of drug contributed by capsule powder (mg)
C= Amount of pure drug added (mg)
Validation of the proposed method was carried out and the results for accuracy, precision, specificity and ruggedness are given in Table No. 3.
The specificity studies were carried out by exposing the samples to different stress conditions such as acidic, alkaline, oxidation, heat and UV-light. The results were comparable in case of alkali exposed samples to normal samples. In case of acid, oxide and heat treated samples the results were on lower side for CNT and PNP. Results of UV light exposed samples were on much lower side for CNT and PNP indicating degradation.
Ruggedness studies were carried out under three different parameters i.e. different times, different days and different analysts shows that the results of estimation by proposed method are much reproducible under different conditions. The study signifies reproducibility of the method under different conditions. The results of standard laboratory mixture and capsule formulation indicate that the proposed method is simple, rapid and avoids prior separation of component. Hence this method can be adopted for the routine estimation of CNT and PNP in marketed formulations.
8) Linearity and range
The study was performed by measuring absorbance of different concentration of standard solution at 236 nm and 265.5 nm. The results are summarized in following Table No. 2.
The summary of the results of validation parameters are given in Table No.3.
RESULTS AND DISCUSSION
The molar absorptivity and sandell’s sensitivity values show the sensitivity of cinitapride and pantoprozole at respective and at isoabsorptive point wavelengths, while precision is confirmed by % RSD (%relative standard deviation). The reproducibility, repealability and accuracy of these methods were found to be good, evidenced by low standard deviation.
Thus the proposed method for simultaneous estimation of CNT and PNP in capsules was found to be simple, accurate, sensitive & economical. Therefore, the method can be useful in routine equality quality control analysis.