Der Pharma Chemica
Journal for Medicinal Chemistry, Pharmaceutical Chemistry, Pharmaceutical Sciences and Computational Chemistry

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Research Article - Der Pharma Chemica ( 2018) Volume 10, Issue 8

Analytical Method Development and Validation for Simultaneous Estimation of Cinitapride Hydrogen Tartrate and Pantoprazole Sodium in Pharmaceutical Dosage Form by RP-HPLC

Sravanthi Macharla1,2* and Ravindar Bairam1,2

1Department of Pharmaceutical Analysis, School of Pharmacy Anurag Group of Institutions, Venkatapur (V), Ghatkesar (M), Ranga Reddy (Dist), Telangana- 501301, India

2Department of Pharmaceutical Chemistry, Vignan Institute of Pharmaceutical Sciences, Deshmukhi, Yadadribhuvanagiri (Dist), Telangana- 508284, India

*Corresponding Author:
Sravanthi Macharla
Department of Pharmaceutical Analysis
School of Pharmacy Anurag Group of Institutions
Venkatapur (V), Ghatkesar (M), Ranga Reddy (Dist), Telangana- 501301, India

Abstract

The aim of the research is method development and validation of Reversed-Phase High-Performance Liquid chromatography (RP-HPLC) method for simultaneous determination of Cinitapride hydrogen tartrate and Pantoprazole sodium in its pharmaceutical dosage form. The method is simple, precise, economic, less time consuming and suitable for routine quality control analysis of both the drugs in formulation. The chromatographic separation was achieved on Thermoscientific BDS Hypersil C18 (250 × 4.6 mm, 5μl) column using a mixture of methanol and 0.1% v/v triethylamine (pH 6) in the ratio 85: 15 % v/v at a flow rate of 1.0 ml/min and UV detection at 264 nm. The retention times of Cinitapride and Pantoprazole were found to be 4.73 and 2.86 min respectively. The method shows linearity in the concentration range of 0.5-1.3 μg/ml for both the drugs with r2=0.9922 for Cinitapride and r2 =0.9974 for Pantoprazole. The LOD of Cinitapride and Pantoprazole were found to be 0.00164 μg/ml and 0.00042 μg/ml respectively. The LOQ of Cinitapride and Pantoprazole were found to be 0.00496 μg/ml and 0.00126 μg/ml respectively. The percentage recovery was found to be within the limits. The method for the determination of assay was below 2.0% RSD. Hence the developed HPLC method was applied for the estimation of Cinitapride and Pantoprazole in its pure form as well as in tablet dosage form and results was found to be in good agreement with the labeled claim. The developed method was found to be simple, accurate, precise, and specific and is useful in the quality control of bulk and pharmaceutical formulations.

Keywords

RP-HPLC, Cinitapride hydrogen tartrate, Pantoprazole sodium, Simultaneous estimation, Validation.

Introduction

Cinitapride hydrogen tartrate (Figure 1) is chemically 4-Amino-N-[1-(cyclohex-3-en-1yl methyl) piperidin-4-yl]-2-ethoxy-5-nitrobenzamide. It is gastroprokinetic agent and anti-ulcer agent of Benzamide class [1]. It acts as an agonist of 5HT1 and 5HT4 receptors and as an antagonist of 5HT2 receptors. Cinitapride is indicated for gastrointestinal disorders associated with motility disturbances such as gastrooesophageal reflux disease, non-ulcer dyspepsia and delayed gastric emptying.

derpharmachemica-hydrogen-tartrate

Figure 1: Chemical structure of Cinitapride hydrogen tartrate

Pantoprazole (Figure 2) is chemically 6-(diflouromethoxy)-2- {[(3,4-dimethoxypyridine-2yl)methane]sulfinyl}-1H-1,3-benzodiazole. It is a proton-pump inhibitor that inhibits gastric acid by blocking H+/K+ adenosine triphosphate enzyme system (proton pump) of gastric parietal cells. It is substituted benzimidazole indicated for stomach ulcers, intestinal ulcers, gastrooesophageal disease (GERD) by reducing amount of acid production in stomach. It is used to treat stomach ulcers caused due to medication with NSAIDs and by bacteria called H. pylori. Cinitapride and Pantoprazole are available in combined dosage form as hard gelatin capsule (CINTODAC). Each capsule contains 3 mg of Cinitapride hydrogen tartrate equivalent to Cinitapride (as extended release pellets) and 40 mg of Pantoprazole sodium equivalent to Pantoprazole (as enteric coated tablet) [2-6].

derpharmachemica-Pantoprazole-sodium

Figure 2: Chemical structure of Pantoprazole sodium

The combination of Cinitapride and Pantoprazole is used to treat gastro intestinal disorders in particular hyperacidity associated with gastro-intestinal dismotility [7]. Extensive literature survey reveals that several analytical methods have been reported for the estimation of Cinitapride and Pantoprazole in pharmaceutical dosage form.

The aim of the current research is to develop simple and accurate RP-HPLC method for simultaneous determination of Cinitapride and Pantoprazole and extend it for their determination in formulation and validate as per the ICH guidelines [8-10].

Materials and Instruments

Chemicals and solvents used [11-15]

• Acetonitrile

• Water

• Methanol

• Orthophosphoric acid

• Triethylamine

All reagents and chemicals were used of HPLC grade.

Pure drug samples (Table 1) [16]

Drugs Supplier Quantity Purity
Cinitapride hydrogen tartrate Comprime Labs 10.0 g 99.98% w/w
Pantoprazole sodium Comprime Labs 10.0 g 99.98% w/w

Table 1: Pure drug information

The drugs used for the present investigation were donated as gift samples.

Marketed formulation available (Table 2) [17]

Brand Name Mfg By Content Quantity
CINTODAC hard gelatin capsule Zydus Cadila Healthcare Ltd. Cinitapride hydrogen tartrate 3 mg
Pantoprazole sodium 40 mg

Table 2: Marketed formulation information

The marketed formulation was purchased from local market.

Instrumentation (Table 3) [18]

Name of Equipment Make Model
HPLC Shimadzu 996 PDA Detector
LC Solutions Software
pH Meter Lab India® SAB 5000
Digital Electronic Balance Shimadzu BL 220H
Column Thermoscientific (5 µm) BDS HYPERSIL C18 [4.6 x 250 mm (id)]

Table 3: Required Instruments information

Experimental

HPLC method [19]

From the various trials (Figures 3a-3e) (Table 4), chromatograms using mobile phase containing different ratios of methanol and 0.1% triethylamine such as 90: 10, 85: 15, 80: 20, 75: 25 and 70: 30 %v/v were recorded at 264 nm at a flow rate of 1.0 ml/min. The ratio of methanol and 0.1% triethylamine in 85: 15 %v/v (Figure 1b) gave good resolution with symmetric peaks.

Effect of pH of mobile phase

The mobile phase consisting methanol and 0.1 % triethylamine in ratio 85:15 %v/v was adjusted to different pH such as 3, 4, 5, and 6 using 1% Orthophosphoric acid.

From the trials (Figures 4a-4d and Table 5), at the pH of 6 (Figure 4d), both the drugs showed symmetric peaks and hence selected for the study. Chromatographic condition also determined (Table 6).

Preparation of standard solutions

Accurately weighed 10 mg of Cinitapride and 10 mg of Pantoprazole were transferred into a separate clean, dry 100 ml volumetric flasks and dissolved with sufficient volume of mobile phase. The volume was made up to 100 ml with mobile phase to get 100 μg/ml concentration of each drug.

From the stock solution, dilutions were made in the concentration range of 0.5-1.3 μg/ml for both the drugs with mobile phase and chromatograms were recorded at 264 nm. The peak areas were plotted against concentration and calibration graphs were constructed for both the drugs. Concentration range of 0.5-1.3 μg/ml was found to be linear and obeys Beer’s law.

Analysis of marketed formulation [20-23]

20 capsules of Cintodac (Label claim: 40 mg Pantoprazole and 3 mg Cinitapride) were weighed, emptied in a glass mortar and powdered. Average weight of capsule was calculated and amount of powder equivalent to 20 mg of Pantoprazole and 18.5 mg of Cinitapride was accurately weighed and added to 50 ml volumetric flask so that sample contains 20 mg equivalent of each drug. It is dissolved in mobile phase, sonicated and made up to the mark and filtered through 0.45 μm membrane filter. Appropriate aliquot of this standard stock solution of formulation (400 μg/ml) was taken into a 10 ml volumetric flask and volume was made up to mark with mobile phase to obtain desired concentration. A 20 μl of sample was injected into injector of liquid chromatographic system and chromatogram was recorded (Figure 5 and Table 7).

Method validation

The method was developed and validated according to ICH guidelines.

Linearity

The calibration curves for Cinitapride and Pantoprazole were constructed by plotting peak area against concentration and regression equations were calculated. Both the drugs showed linearity in the concentration range of 0.5-1.3 μg/ml. Aliquots 20 μl of each solution injected under the operating chromatographic condition (Figures 6a, 6b and Table 8).

Accuracy

Accuracy studies were expressed as recovery (%), which is determined by the standard addition method at 50%, 100%, 120% of the label claim according to ICH guidelines. The %Recovery and %RSD were calculated and reported (Figures 7a-7c and Table 9).

Precision

The intraday and interday precision of the proposed method was evaluated by analyzing samples of two different concentration of Cinitapride (0.8, 0.9 μg/ml) and Pantoprazole (0.8, 0.9 μg/ml) in triplicates on same and different days (Tables 10 and 11).

Repeatability

Repeatability was determined by analyzing standard solutions of 0.8 μg/ml concentration of Cinitapride and Pantoprazole by injecting six times. The precision and % RSD were calculated and reported (Table 12).

Limit of detection and limit of quantitation (LOD and LOQ)

The LOD and LOQ of Cinitapride and Pantoprazole were calculated based on standard deviation of the response and slope values of two drugs according to ICH guidelines.

System suitability studies

System suitability is a pharmacopoeial requirement and used to verify, whether the resolution and reproducibility of chromatographic system are adequate for analysis to be done. The parameters like theoretical plate count, retention time, tailing factor and resolution of both the drugs were reported (Table 13).

Results and Discussion

Mobile phase

Effect of ratio of mobile phase

derpharmachemica-triethylamine-buffer

Figure 3a: Methanol: 0.1% triethylamine buffer (90:10 %v/v)

derpharmachemica-triethylamine

Figure 3b: Methanol: 0.1% triethylamine buffer (85:15 %v/v)

derpharmachemica-Methanol

Figure 3c: Methanol: 0.1% triethylamine buffer (80:20 %v/v)

derpharmachemica-buffer

Figure 3d: Methanol: 0.1% triethylamine buffer (75:25 %v/v)

derpharmachemica-triethylamine

Figure 3e: Methanol: 0.1% triethylamine buffer (70:30 %v/v)

From the above five trials the observations are follows:

S. No. Methanol: 0.1%triethyl amine (% v/v) Retention time
CIN PAN
1 90:10:00 4.29 2.88
2 85:15:00 4.73 2.86
3 80:20:00 5.82 3.1
4 75:25:00 4.27 3.29
5 70:30:00 4.12 3.62

Table 4: Observations for trial of effect of ratio of mobile phase

Effect of pH of mobile phase

derpharmachemica-triethylamine

Figure 4a: Methanol: 0.1% triethylamine buffer (85:15 %v/v) (pH 3)

derpharmachemica-buffer

Figure 4b: methanol: 0.1% triethylamine buffer (85:15 %v/v) (pH 4)

derpharmachemica-triethylamine

Figure 4c: Methanol: 0.1% triethylamine buffer (85:15 %v/v) (pH 5)

derpharmachemica-Methanol

Figure 4d: Methanol: 0.1% triethylamine buffer (85:15 %v/v) (pH 6)

S. No. pH Observation
1 pH 3 Asymmetric peaks
2 pH 4 Asymmetric peaks
3 pH 5 Slight tailing
4 pH 6 Symmetric peaks

Table 5: Observations for trial of effect of pH of mobile phase

Chromatographic conditions

Column Thermoscientific (250 x 4.6 mm, 5 μ)
Particle size packing 10 µm
Stationaray phase BDS HYPERSIL C18 (5 µm)
Mobile phase Methanol: 0.1% triethylamine (pH 6)
Detection wavelength 264 nm
Flow rate 1 ml/min
Run time 07 min
Temperature Ambient
Sample size 20 µl
Diluent Methanol

Table 6: Chromatographic condition

derpharmachemica-Chromatogram

Figure 5: Chromatogram of marketed formulation

Drugs Labeled amount, mg tablet-1 Amount found, mg tablet-1 % Label claim
Cinitapride hydrogen tartrate 3 3.06 102
Pantoprazole sodium 40 41.05 102.87

Table 7: Analysis of marketed formulation

Method validation

Linearity

derpharmachemica-curve-Cinitapride

Figure 6a: Calibration curve of Cinitapride

derpharmachemica-curve-Pantoprazole

Figure 6b: Calibration curve of Pantoprazole

Parameters Cinitapride Pantoprazole
λmax, nm 264 264
Beer’s Law limit (µg/ml) 0.5-1.3 0.5-1.3
Regression equation (Y*) Y = 67806x+6650 Y = 31738x-893.8
Correlation coefficient (r2) 0.992 0.997
Slope (b) 67806 31738
Intercept (a) 6650 893.8

Table 8: Linearity observation

Accuracy studies

derpharmachemica-recovery-studies

Figure 7a: Chromatogram of 50% recovery studies

derpharmachemica-Chromatogram

Figure 7b: Chromatogram of 100% recovery studies

derpharmachemica-studies

Figure 7c: Chromatogram of 120% recovery studies

Drugs % Level % Recovery % RSD
Cinitapride 50 103.18 0.72
100 102.1 1.18
120 102 1.49
Pantoprazole 50 102.59 0.94
100 101.09 1.42
120 102.41 1.09

Table 9: Accuracy studies

Precision

Concentration ( (µg/ml) Injection Peak area %RSD
CIN PAN CIN PAN CIN PAN
0.8 0.8 1 74340 23988 0.22 0.72
2 74822 23668
3 74549 23992
4 74654 23882
5 74734 23789
6 74594 23567

Table 10: Intraday precision

S. No. Concentration (µg/ml) Peak area %RSD
CIN PAN CIN PAN CIN PAN
1 0.8 0.8 73450 22452 0.13 1.08
73650 22942
73522 22752
2 0.9 0.9 80290 26941 0.22 0.38
80652 26851
80428 26734

Table 11: Interday precision

Repeatability

Concentration (µg/ml) Peak area %RSD
S. No. CIN PAN CIN PAN CIN PAN
1 0.8 0.8 74340 23988 0.32 0.77
74822 23668
74549 23992
2 0.9 0.9 81590 27941 0.18 0.12
81312 27984
81341 28009

Table 12: Observation for Repeatability

System suitability studies

Drug Theoretical plate count Retention time (min) Tailing factor Resolution (Rs)
Cinitapride 6806.03 2.86 1.03 17.14
Pantoprazole 5682.03 4.73 1.21

Table 13: Observation for System suitability studies

Conclusion

For the simultaneous estimation of Cinitapride hydrogen tartrate and Pantoprazole sodium, RP-HPLC method was developed and validated according to ICH guidelines. The proposed method was found to be simple, precise, economic, less time consuming and proved to be superior to most of the reported methods. The mobile phase was simple to prepare and economical. The sample recovery in the formulation was in good agreement with their respective label claims and suitable for routine quality control analysis of both the drugs in formulation.

Acknowledgement

The authors are grateful to Comprime Labs Pvt Ltd, Kukatpally, Hyderabad for providing gift samples of Cinitapride and Pantoprazole. The authors are also thankful to Principal Lalitha College of Pharmacy, Anurag Group of Institutions for providing all the facilities to support the research work.

References

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