HPTLC Method Development and Validation for the Estimation of Rabeprazole Sodium and Itopride Hydrochloride in Tablet Dosage form

 

B Dhandapani, N Anjaneyulu, K Vinod Kumar1 and Shaik Harun Rasheed and M Ramakotaiah2

1K.M.C.H. College of Pharmacy, Kovai Estate, Kalappatti Road, Coimbatore – 641035, Tamilnadu.

2Department of Pharmaceutical Analysis, Donbosco College of pharmacy, Pulladi Gunta, Etukuru,Guntur, A. P.

*Corresponding Author E-mail: dhandapanirx@gmail.com

 

ABSTRACT:

The simple, accurate and precise method for the quantitative determination of Rabeprazole sodium (RP) and Itopride hydrochloride (IH), from its tablet dosage form by HPTLC method, the chromatograms were developed using a mobile phase of Ethyl acetate : Methanol : Ammonia ( 8.5:1:0.5 v/v ) on precoated plate of silica gel 60 F254 and quantified by densitometric absorbance mode at 285 nm. The Rf values of IH and RP were 0.21 and 0.41 respectively. Linearity of Itopride hydrochloride (IH) and Rabeprazole sodium (RP) was in the range of 75 - 375 ng /ml and 10 - 50 ng /ml. Recovery studies of 98.88 – 102.41%, percentile relative std deviation of not more than 0.8 and correlation coefficient (linearity range) of   0.9954 – 0.9999 for IH and RP, it shows that developed methods were accurate and precise. The LOD and LOQ values were found to be 10ng/ml, 30ng/ml and 5ng/ml, 10ng/ml for IH and RP respectively. The mean percentage recovery values close to 100% it indicates there is no interferences of additives with Rabeprazole sodium (RP) and Itopride hydrochloride (IH)  present in tablet dosage forms. The method has been validated as per ICH guide lines. This method can be employed for the routine analysis of tablets containing IH and RP.

 

KEYWORDS: High performance liquid thin layer chromatography (HPTLC); Rabeprazole sodium (RP); Itopride hydrochloride (IH).

 


INTRODUCTION:

Rabeprazole sodium1 is chemically (RP) 2-[[[4-(3-methoxypropoxy)-3-methyl-2-      pyridil] - methyl] sulfinyl]-1H- benzimidazole Fig-1. It act orally by suppresses gastric acid secretion by inhibiting  the  parietal cell H+/K+ ATP pump and used in Short-term treatment in healing and symptomatic  relief of duodenal ulcers and erosive or ulcerative gastro esophageal reflux disease (GERD); long- term treatment of pathological hypersecretory conditions, including Zollinger-Ellison syndrome and in combination with amoxicillin and clarithromycin to eradicate Helicobacter pylori.

 

Itopride hydrochloride (IH) is chemically N-[[4-(2-dimethylaminoethoxy) phenyl] methyl] -3, 4- dimethoxy-benzamide hydrochloride2 Fig-2 used as a prokinetic agent. It act orally by increase the acetylcholine concentration by inhibiting dopamine D2 receptors and acetylcholine esterase higher acetyl choline concentration increases the GI peristalsis increase the lower esophageal sphincter pressure stimulate the gastric motility accelerate the gastric empting and improve the gastro duodenal co-ordination.

 

Literature survey reveals that various analytical methods 3-10 have been reported for rabeprazole and itopride hydrochloride in single dosage forms. The present paper aims to report a simple HPTLC method for estimation of rabeprazole and itopride hydrochloride in their combined dosage form.

 

MATERIALS AND METHODS:

Instrumentation:

Pre coated silica gel 60F254­ on aluminium sheets (200μm thick) of E-Merck, Germany were used as stationary phase.  Pre-washing of plate was done with methanol and then it was activated by keeping in an oven at 115° C for 10 minutes. Camag HPTLC System (with TLC Scanner), Win CATS Softwar V 4.0 and Linomat 5 as application device) used for the analysis.

 

Chemicals and Reagents:

Max Labs Ltd., India, generously gifted pure IH and RP. Commercial tablets (Rabium Plus, Intas Pharma) containng RP (20 mg) and IH (150 mg) were used for the study. Water, ethyl acetate, methanol, used was of Analytical grade (E. Merck, Mumbai, India). All the other chemicals used were of analytical grade (E. Merck, India).

 

Chromatographic Conditions:

The drugs were resolved using a mobile phase of Ethyl acetate : Methanol : Ammonia (8.5:1:0.5v/v),10 mins time saturation with filter paper was selected because it gave compact spots and good resolution between analytes and good separation from solvent front and sample application positions. Development chamber (20 X 10cm), migration distance ( 80mm), band length ( 8mm ), slit dimension ( 6 X0.30mm ), temperature 26.4oC, humidity 61% and UV detection was  carried out at 285 nm.

 

Fig: 1 Structure of Rabeprazole

 

Fig: 2 Structure of Itopride Hydrochloride

 

Preparation of Stock Solutions:

A stock solution was prepared by dissolving 75 mg and 10 mg of IH and RP in  10 ml of mobile phase. The stock solution were further diluted with methanol to obtain various concentration of 75 - 375 ng /ml and 10 - 50 ng /ml for IH and RP respectively.

 

Preparation of calibration curve:

Aliquots of respective standard solutions of IH and RP were spotted on precoated TLC plates, using a semiautomatic spotter under a nitrogen stream. The plate was dried in air and developed for up to 45 mm at constant temperature using a mixture of Ethyl acetate : Methanol : Ammonia (8.5:1:0.5v/v), as the mobile phase in a Camag twin-trough chamber previously saturated with the Filter paper for 10 mins. The plate was removed from the chamber and dried in air. Photometric measurements were performed at 285 nm in the absorbance/reflectance mode with the Camag TLC Scanner 3 using CATS 4 software incorporating the track optimization option. The calibration curve was prepared by plotting the peak area versus the concentration (ng/spot) corresponding to each spot.

 

Procedure for pharmaceutical formulations:

Twenty tablets were accurately weighed and finely powdered. The powder equivalent to RP (75mg) and IH (10mg) was accurately weighed, mixed with methanol (5 ml) and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41. The residue was thoroughly washed with methanol. The filtrate and washings were combined in a 10 ml volumetric flask and diluted to the mark with methanol. The filtrate was further diluted to get the concentrations of 225 ng/ml and 30 ng/ml of IH and RP. The sample solution was applied on a TLC plate under a nitrogen stream using a semiautomatic spotter. The TLC plate was developed and photometrically analyzed as described under the Chromatographic conditions. The amounts of IH and RP present in the sample solution were determined by fitting the area values of peaks corresponding to IH and RP into the equation of the line representing the calibration curve of IH and RP.

 

RESULTS AND DISCUSSION:

IH and RP are soluble in methanol; therefore, methanol was selected as the solvent. The formulation was dissolved in methanol with sonication for 20 min to assure complete release of the drug from the formulation matrix. The mixture of Ethyl acetate: Methanol: Ammonia (8.5:1:0.5v/v), could resolve IH and RP spots with a better peak shape. The combination of Ethyl acetate and methanol offered the optimum migration (Rf values of 0.21 0.02 for IH and 0.41 0.02 for RP) and resolution of RP from other components of the formulation matrix (Fig. 3) and the peak purity overlay spectra of RB and IH shown in Fig 4 & 5 it indicates no interferences of other substances present in the formulation. On the other hand, an ammonia solution helped to sharpen the peak. Even saturation of the TLC chamber mobile phase with filter paper for 10 min assured better reproducibility and better resolution. Scanning of the same spot (75 ng/spot for IH and 10 ng/spot for RP) of both drugs seven times without changing the position of the plate; the %CV for measuring the peak area was found to be 0.68% for IH and 0.82% for RP. The repeatability of the method was checked by spotting 5 ng/ml of a combined standard solution seven times on the TLC plate (n = 7); the % CV for the peak area was found to be 1.23% for IH and 0.98% for RP. Both the %CV, for measuring the peak area and sample applications (less than 1% and 3%, respectively), ensuring proper functioning of the HPTLC system. The accuracy of the method was evaluated by calculating the recovery of IH and RP by the standard addition method at different levels of the calibration curve (n = 6). Results are shown in Table-1.

 

Fig. 3: Chromatogram of and Rabeprazole from a tablet formulation Chromatogram of the sample showing resolution of  Itopride peak (225 ng/spot, Rf = 0.21 0.02) and  Rabeprazole (30 ng/spot, Rf = 0.41 0.02) peak from components of the formulation matrix.


Table – 1 Recovery Studies

Drug

Amount of drug added (mcg/ml)

%  Recovery

% RSD*

50% level

100% level

50% level

100% level

50% level

100% level

RP

10.0

20.0

101.11

99.58

0.2683

0.3116

IH

75.0

150.0

100.41

101.55

0.1791

0.1469

RP- Rabeprazole Sodium IH – Itopride Hydrochloride, *Each value is a mean of six observations.

 

 


Fig. 4: Peak purity Overlay Spectra of Rabeprazole sodium at 285 nm

 

Fig, 5: Peak purity Overlay Spectra of Itopride  at 285 nm

 

Different validation parameters for the proposed HPTLC method for determining the IH and RP content are summarized in Table 2 This method was applied to determine the content of IH and RP in two different combined market samples of IH and RP tablets.The content and percentage of IH and RP in market samples are presented in Table 3.The results indicate that the proposed HPTLC method is simple, specific, rapid, precise and accurate for the simultaneously estimation of IH and RP in its combined formulations.

 

Table – 2 Validation Parameters of the Proposed HPTLC Method

Parameters

Itopride

Rabeprazole

Linearity range (ng/spot)

75 - 375

10 - 50

Correlation coefficient

0.9954

0.9999

Precision (%CV)

-         Intra day (n = 3)

-         Interday (n=5)

Repeatability of Sample application (n=7)

Repeatability of Peak area (n=7)

 

1.24 – 2.58

0.37 – 3.62

1.23

0.68

 

0.68 – 2.55

0.72 – 2.99

0.98

0.82

% Recovery

100.41 – 101.55

99.58 – 101.11

Limit of Detection (ng/spot)

10

5

Limit of Quantification (ng/spot)

30

10

 

Table – 3:  Analysis of formulation

Drug

Amount(mg / Tablet)

% Label

claim

% RSD*

Labelled

Estimated

RP

20

20.125

100.62

0.1144

IH

150

149.95

99.96

0.1234

*Each value is a mean of six observations.

 

REFERENCES:

1.       Budavari S, editors. The Merck index. 13th ed. Whitehouse Station, NJ: Merck and Co, Inc; 2001. p. 1450.

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8.       Jain M, Shah L, Gupta S. Quantitation of itopride hydrochloride in human serum by HPLC with fluorescence detection and its application to bioequivalence study. J Chromatogr B 2004; 813: 247.

9.       Jinchang H, Yuxu SG, Lei L. Development of a liquid chromatography/tandem Mass spectrometry assay for the quantification of rabeprazole in human plasma. J Chromatogr B 2005; 19: 2321.

10.     Mondal U, Ganesan M, Pal TK, Jayakumar M, Chattaraj TK, Roy K, et al . Bioequivalence study of rabeprazole sodium on healthy human volunteers. J Indian Med Assoc 2004; 102: 26, 28,30.

 

 

 

Received on 27.10.2009                             Modified on 24.12.2009

Accepted on 20.01.2010                            © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 475-477