RP-HPLC Method Development and Validation for the Estimation of Cinitapride and Pantoprazole in Solid Oral Dosage Form

 

P.V. Hemalatha, A. Jerad Suresh and Niraimathi V.*

Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai-600003, Tamil Nadu, India.

*Corresponding Author E-mail: vnm_anr@yahoo.co.in

ABSTRACT:

 

 

INTRODUCTION:

Cinitapride^1,2 (CNP) is a substituted benzamide gastroenteric prokinetic agent acting via complex, but synergistic effects on serotonergic 5-HT2 (inhibition) and 5-HT4 (stimulation) receptor and dopaminergic D2 (inhibition) receptors in the neuronal synapses of the myenteric plexi; it is used as an anti-ulcerative drug. Pantoprazole³  is a gastric proton pump inhibitor that suppress the final step in gastric acid production by forming a covalent bond to two sites of the (H⁺,K⁺) ATPase enzyme system at the secretary surface of the gastric parietal cell. Cinitapride is chemically 4-amino –N-[1-(3-cyclohexen-1-ylmethyl)-4-piperidinyl]-2-ethoxy-5-nitrobenzamide. Pantoprazole is chemically 6-(difluoromethoxy-2-[(3, 4-dimethoxy pyridine-2-yl) methyl sulfynyl] -1H-benzimidazole. Only first derivative⁴ and HPTLC⁵ method has been reported so far for the combination. Extensive literature survey reveals that no HPLC determination of the drugs in combination has been reported so far. So an effort has been made to develop a reverse phase high performance liquid chromatography for the estimation of the drugs and also to validate the developed method.  Thus the aim of the study is to develop a simple, specific, accurate, rapid and economic RP-HPLC method for the estimation of the drugs in pharmaceutical dosage forms

 

Structures⁶:

Cinitapride

Mol wt: 402.49

 

Pantoprazole sodium sesquihydrate

Mol wt: 432.4

 

INSTRUMENTATION:

A Shimadzu Prominence LC-20AT with SPD-20A detector was used for HPLC analysis. High performance liquid chromatographic method was developed using Phenomenex C₁₈ Gemini (5 μ) 250 x 4.60 mm i.d (Internal dia) column. The instrument uses UV detector, and wavelength of 262 and 290 nm was selected to detect CNP and PNP respectively. The chromatograph makes use of rheodyne injector and an injecting volume of 20 µl was employed.

MATERIALS:

All the reagents used for preparation of mobile phase were of HPLC grade. HPLC grade methanol, water, acetonitrile were acquired from Merck India ltd. The mobile phase after preparation was filtered through 0.2µ membrane filter.

 

Preparation of mobile phase:

The developed method employs a mobile phase consisting of acetonitrile, methanol and phosphate buffer (pH7) in the ratio of 40:20:40. Phosphate buffer was made from 0.2M potassium dihydrogen orthophosphate adjusted to pH7 with triethylamine. The mobile phase was filtered through 0.2µ membrane filter.

 

EXPERIMENTS:

Preparation of cinitapride standard solutions:

Accurately weighed 30mg of standard CNP  in 25 ml volumetric flask, dissolved in methanol and made up to volume in methanol to get a concentration of 0.6mg/ml of cinitapride. The solution was filtered through 0.2µ membrane filter. Aliquots of the standard solutions were diluted with the mobile phase to get a concentration range of 84µg/ml to132µg/ml. The dilutions were injected one by one in the 20µl loop and the eluate was detected at 262nm and the chromatogram was recorded.

 

Preparation of pantoprazole standard solutions:

Accurately weighed 100mg of standard PNP in 50 ml volumetric flask, dissolved in mobile phase and made up to volume with the mobile phase to get a concentration of 2mg/ml of PNP. The solution was filtered through 0.2µ membrane filter. Aliquots of the standard solutions were diluted with the mobile phase to get a concentration range of 140µg/ml to 220µg/ml. The dilutions were injected in the 20µl loop separately; the eluate was detected at 290nm and the chromatogram was recorded.

 

Assay of formulation:

The capsule dosage form contains cinitapride and pantoprazole as tablet dosage forms.

 

Cinitapride:

Twenty tablets of CNP from the formulation were weighed accurately. Tablet powder equivalent to 6mg of cinitapride was then accurately weighed, dissolved in methanol with the aid of ultrasonication for 15minutes and made up to volume (10ml) with the methanol. The solution was then filtered through 0.2µ membrane filter and injected into the column. The eluate was detected at 262nm and the chromatogram was recorded.

 

Pantoprazole:

Twenty tablets of PNP from the formulation were weighed accurately. Tablet powder equivalent to 100mg of PNP was accurately weighed and dissolved in mobile phase with the aid of ultrasonication for 15minutes and made up to volume (50ml) with mobile phase. The solution was then filtered through 0.2µ membrane filter and injected into the column. The eluate was detected at 290nm and the chromatogram was recorded.

 

 Fig 1.  Chromatogram of CNP

 

Fig 2.  Chromatogram of PNP

 

Validation of the proposed method⁷:

Selectivity of the method was assessed on the basis of elution of CNP and PNP using above mentioned chromatographic conditions and mobile phase. The developed method was validated according to ICH Guidelines. The specificity, linearity, accuracy, limit of detection, limit of quantitation, and system suitability parameters for the proposed method have been validated for the determination of CNP and PNP.

 

Table 1: System suitability parameters for CNP and PNP

 

S. no	Parameter	Cinita-pride	Pantopra-zole
1.	Theoretical Plates	6497	5091
2.	Tailing factor	0.93	1.32
3.	Capacity factor	0	0
4.	Temperature of the column	ambient	ambient
5.	Linearity (μg/ml)	84-132	140-220
6.	Correlation coefficient (r2)	1	1
7.	Limit of Detection (LOD) (μg/ml)	1.5	1.9
8.	Limit of Quantitation (LOQ) (μg/ml)	5.3	5.8

 

Linearity studies⁸:

The chromatogram of standard drugs reveals the retention time (RT) of CNP and PNP as 6.5 and 3.5 minutes (fig 1&2) under the above said chromatographic conditions and mobile phase. The peak area obtained in the chromatogram of the standard dilutions at the said RT were plotted against concentration for both CNP and PNP. A five point linearity chart was plotted for both the drugs. It was analyzed by regression analysis. CNP showed good linearity at 84-132 µg/ml and PNP showed good linearity at 140-220µg/ml (fig 3&4). The correlation coefficient was calculated to be 1 which is within the limit (Table 1).

 

Fig 3: Calibration chart of CNP

 

Fig 4: Calibration chart of PNP

 

Limit of detection (LOD) and limit of quantification (LOQ):

The LOD and LOQ for CNP and PNP were predicted based on the parameters of standard error of estimate and slope, calculated from linearity of the response data of CNP and PNP.

 

Accuracy⁹:

The accuracy of the developed method was determined by performing recovery studies. A known amount of the standard drugs were added to the respective samples and the chromatogram was recorded for the same. The recovery studies were performed on spiked samples and injected in duplicate.

 

Table 2: Assay of CNP and PNP

 

Drug	Assay*		%Recovery ±SD*
	% Amount present ±SD	%RSD
CNP	99.72 ± 0.7856	0.7878	99.44±0.4545
PNP	100.33 ±  0.4714	0.4698	99.50±0.6937

*mean of three determinations

 

Specificity¹⁰:
The specificity test of the proposed method demonstrated that the excipients from tablets do not interfere in the drug peak. Furthermore, well shaped peaks indicate the specificity of the method. Better resolution was found for the drug peak with no interference proved that the method was found to be selective to the drug.

 

RESULTS AND DISCUSSION:

RP-HPLC¹¹ method was developed for the estimation of CNP and PNP in bulk and oral dosage form. The chromatogram for CNP and PNP obtained by the proposed method showed the retention time to be 6.5 and 3.5 minutes respectively. The drugs presented a good linearity at the range of 84-132 µg/ml and 140-220µg/ml for CNP and PNP respectively which assures that the proposed chromatographic conditions and the mobile phase were suitable for the estimation. The chromatographic conditions were optimized using mobile phase of varied polarity. Finally a mobile phase consisting of acetonitrile, methanol and phosphate buffer (pH7) in the ratio of 40:20:40 gave good linearity of response and the shape of chromatogram was also perfect. The regression equation of the drugs’ concentration over peak area was calculated. The system suitability parameters are presented in table 1. The number of theoretical plates for CNP and PNP was found to be 6497 and 5091 respectively which confirms the good efficiency of the column for the drugs and nature of mobile phase. The low value of LOD and LOQ indicates the sensitivity of the method. The percentage purity of the CNP and PNP by the proposed method was appreciable and presented in table 2. The good, acceptable percentage recovery of the drugs (table 2) ascertains the accuracy of the developed method. This confirms that the results of estimation by the proposed method are not affected by the excipients present in the dosage forms. The developed method was validated with respect to linearity, specificity, accuracy and system suitability and was inferred that the proposed method is perfect.

 

CONCLUSION:

A new analytical method has been developed. The results indicate that the proposed

RP-HPLC method is specific, accurate, simple, cheap and less time consuming which could be  applied for the routine analysis of CNP and PNP in formulation.

 

ACKNOWLEDGEMENTS:

The authors are thankful to M/S Zydus Cadila, Ankleshwar, Gujarat (India) and Knis Laboratories, Chennai, TN (India) for providing Cinitapride and pantoprazole as gift samples respectively. They are also thankful to Mr. Thirugnanamoorthy, Pharma Analytical Lab, Pondicherry for his support in study. The authors are also thankful to the Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College for rendering full support to carry out the study.

 

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Received on 22.12.2011         Modified on 08.01.2012

Accepted on 15.01.2012         © AJRC All right reserved