Development and Validation of Spectroscopic Simultaneous Equation Method for Simultaneous Estimation of Itopride Hydrochloride and Omeprazole in Synthetic Mixture

 

Hiral J. Varachhiya, Rohan K. Barse, Suresh Jain*

Department of Pharmaceutical Quality Assurance, Shree Dhanvantary Pharmay College,

Kim (Surat)-394110, India.

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

 

ABSTRACT:

A Simple, rapid, accurate, precise spectrophotometric method was developed and validated for simultaneous estimation of Itopride Hydrochloride and Omeprazole in synthetic mixture. The method is based on the simultaneous equation method. Itopride Hydrochloride has absorbance maxima at 258 nm and Omeprazole has absorbance maxima at 300 nm in methanol. Both drugs obey Beer’s law in the concentration range of 10-50 µg/ml (R2= 0.9994) for Itopride Hydrochloride and 2.5-12.5µg/ml (R2=0.9992) for Omeprazole. Limit of detection is 0.105 and 0.052 µg/ml and Limit of quantification is 0.320 and 0.160 µg/ml for Itopride Hydrochloride and Omeprazole, respectively. Percentage recovery for assay was found to be 99.68 % and 100.06 % for Itopride Hydrochloride and Omeprazole, respectively. The accuracy and reproducibility of the praposed method was statistically validated by recovery studies. The method was validated according to the ICH guidelines.

 

KEYWORDS: Itopride Hydrochloride, Omeprazole, Simultaneous Equation Method, Validation.

 

 


INTRODUCTION:

Itopride hydrochloride (ITO), N-({4-[2-(dimethylamino) ethoxy] phenyl} methyl)-3, 4-dimethoxybenzamide hydrochloride is novel prokinetic agent used in treatment of functional dyspepsia and other gastrointestinal conditions. It is white amorphous powder, Soluble in DMSO, methanol, water, sparingly soluble in ethanol, Polyethylene glycol, very slightly soluble in hexane, dichloromethane, methyl benzene.1 Omeprazole, 5-methoxy-2 [(4-methoxy-3, 5-dimethylpyridin-2-yl) methansulphinyl]-1H-benzimidazole is selective and irreversible proton pump inhibitor.

 

 

 

It is used in treatment of GERD, peptic ulcer, zollinger-syndrome. It is A white or almost white powder, Very slightly soluble in water, freely soluble in ethanol and methanol, slightly soluble in acetone, isopropanol.2

 

A combination therapy of Itopride Hydrochloride and Omeprazole produce synergistic effect and has shown more effectiveness than mono therapy of proton pump inhibitors, clinically safe, has good efficacy, lower adverse effect. Formulation containing Itopride HCL and Omeprazole which proved to be safe for use in treatment of GERD by clinical trial.3 Itopride Hydrochloride useds as prokinetic agent. It act by inhibiting dopamine D2 receptor and acetylcholinesterase, so it increases the concentration of acetylcholine, in this way it increases the GI peristalsis, promote gastric emptying. Omeprazole used as proton pump inhibitors. It inhibit H+/K+-ATPase system present at secretory surface of gastric pariatal cells. So, it decrease the gastric acid secretion. So, the combination of prokinetic agent (Itopride Hydrochloride) and Proton pump inhibitors (Omeprazole) produce synergistic effect in GERD treatment by increasing gastric motility and reducing gastric acid secretion.4

 

Literature survey reveals that many analytical methods are reported for determination of ITO5-9 and OME10-17 individually or with other drugs in combination. However, no method is reported for simulataneous determination of these two drugs in combination. So, in the present work, a successful attempt has been made to estimate both these drugs simultaneously using RP-HPLC method in synthtic mixture. This study attempts to describe a rapid and sensitive HPLC method with UV detection, useful for routine quality control of ITO and OME in pharmaceutical formulation.

 

Itopride Hydrochloride

 

Omeprazole

Figure 1 Chemical structure of Itopride Hydrochloride and Omeprazole

 

MATERIAL AND METHODS:

Chemicals and reagents:

Itopride Hydrochloride and Omeprazole raw materials were received as gift sample from Prayosha Health Care Pvt. Ltd, Ankleshwar and R. P Industries, Panoli, respectively. All chemicals were of analytical reagent grade and solutions were prepared with methanol AR grade (FINAR).

 

Instrumentation:

A double beam UV-visible spectrophotometer (Simadzu model 2450, Japan) with spectral width of 2nm, 1cm quartz cell was used to measure absorbance of solutions.

 

Selection of solvent:

On the basis of solubility study methanol was selected as the solvent for dissolving ITO and OME.

 

 

Preparation of Standard Stock Solutions of ITO and OME:

Itopride Hydrochloride Stock Solution:

An accurately weighed amount of ITO (10mg) was transferred to a 10ml volumetric flask and dissolved, diluted up to mark with methanol to obtain standard solution having concentration of ITO (1000µg/ml).

 

Omeprazole Stock Solution:

An accurately weighed amount of OME (10mg) was transferred to a 100ml volumetric flask and dissolved, diluted up to mark with methanol to obtain standard solution having concentration of OME (100µg/ml).

 

Preparation of working standard solutions:

From the above stock solution desired concentrations were prepared by transferring specific volume to 10ml with methanol.

 

Preparation of Test Solution:

The preparation of synthetic mixture was as per patent:4

 

Table 1 Composition of formulation (Synthetic Mixture)

Sr. No.

Drug-Excipient Name

Quantity (mg)

1

Itopride Hydrochloride

150

2

Omeprazole

40

3

Magnesium stearate

400

4

Starch

410

 

Total

1000

 

Above all ingredients were shift and blend to make uniformity of mixing. Take synthetic powder equivalent to 10mg of Itopride Hydrochloride in 100ml volumetric flask. Dissolve in 25ml of Methanol and sonicated for 15 min. Dilute up to 100ml with solvent shake vigorously. Filtered through Whatman filter paper No. 42 and further diluted.

 

Finally the solution had concentration of 100µg/ml and 25µg/ml for ITO and OME, respectively. From that pipette out 2.0ml in 10ml volumetric flask and volume was made up to mark with Methanol to make final concentration of mixture 20µg/ml and 5.0µg/ml for ITO and OME, respectively.

 

Determination of λ max of Individual Components:

By appropriate dilution of standard solutions of ITO and OME with methanol, solutions containing 10µg/ml of both drugs were scanned separately in the range of 200-400nm against methanol as blank. ITO shows λ max at 258nm and OME at 300 nm. (Figure 2)

 

Overlay Spectra of Itopride Hydrochloride and Omeprazole:

The overlain spectrum of ITO and OME was recorded (Figure 2) and two wavelengths 258nm (λ max of ITO) and 300nm (λ max of OME) were selected for further study.

 

Figure 2 Overlain spectra of Itopride Hydrochloride and Omeprazole

 

Methods:

Simultaneous Equation Method:

Standard Stock solutions of ITO and OME in the concentration range 10-50µg/ml and 2.5-12.5µg/ml were prepared in the methanol and the absorbance of these solutions was measured at 258nm and 300nm. Calibration curves were plotted to verify the Beer’s law and the absorptivity values calculated at the respective wavelengths for both the drugs. Two simultaneous equations as below were formed using these absorptivity values A (1%, 1 cm).

 

At λ1 A1= ax1bCx+ay1bCy …………………............(1)

 

At λ2A2= ax2bCx+ay2 bCy………………………….(2)

 

For measurements in 1 cm cells b=1

Rearrange eq. (2)

Cy= A2- ax2Cx/ ay2

 

Substituting for Cy in eq (1) and rearranging

Cx = A2ay1-A1 ay2/ax2 ay1-ax1 ay2……………….. (3)

Cy = A1ax2-A2 ax1/ax2 ay1-ax1 ay2………………...(4)

 

Where, Cx and Cy are the concentrations of ITO and OME measured in mg/10 ml in sample solutions, A1 and A2 are absorbance of mixture at selected wavelengths 258 nm and 300 nm respectively.

 

Validation of proposed method:

The method was validated according to ICH guidelines for validation of analytical procedures in order to determine linearity, sensitivity, accuracy and precision for each analyte.18

 

Linearity:

Appropriate dilutions of working standard solutions for ITO and OME were prepared in the concentration range of 10-50μg/ml and 2.5-12.5μg/ml, respectively and analyzed as per the developed method. Calibration curves were generated and the linearity wasevaluated by the least square regression method.


Table 2: Absorbance values of ITO and OME at two wavelength (258 nm and 300 nm)

Concentration of the solution (µg/ml)

Absorbance

ITO

OME

ITO

OME

258 nm

300 nm

258 nm

300 nm

10

2.5

0.355 ± 0.0011

0.087 ± 0.0005

0.022 ± 0.0006

0.092 ± 0.0006

20

5

0.687 ± 0.0015

0.153 ± 0.0006

0.077 ± 0.0004

0.201 ± 0.0006

30

7.5

1.089 ± 0.0012

0.225 ± 0.0006

0.141 ± 0.0009

0.327 ± 0.0012

40

10

1.454 ± 0.0021

0.303 ± 0.0008

0.208 ± 0.0006

0.456 ± 0.0011

50

12.5

1.814 ± 0.0009

0.376 ± 0.0008

0.271 ± 0.0008

0.580 ± 0.0013

 


 

 


Figure 2 Calibration curves of ITO at 258 nm and 300 nm


 

Figure 3 Calibration curves for OME at 258 nm and 300 nm

 

Accuracy (Recovery studies):

Accuracy of the method was determined by recovery study from synthetic mixture at three levels (80 %, 100 %, and 120%) of standard addition.

 

Percentage recovery for ITO and OME by this method was found in the range of 99.3% to 100.02 % and 99% to 100.8 %

 

The value of % Recovery within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

 

Table 3: Recovery studies for DOX and ERD for synthetic mixture

Recovery level %

conc

ITO % recovery

(mean ± SD)

conc

 

OME % recovery

(mean ± SD)

Placebo

20

99.3 ± 0.235

5

99.60 ± 0.432

80

36

99.83 ± 0.155

9

99.00 ± 0.089

100

40

99.41 ± 0.062

10

100.7 ± 0.205

120

44

100.02 ± 0.009

11

100.8 ± 0.141

 

Precision:

Precision was determined as intraday and interday variations. Intraday precision was determined by analysing ITO (10, 20, 30 µg/ml) and OME (2.5, 5.0, 7.5 µg/ml) for three times on the same day. Inter day precision was determined by analysing same concentration of solutions for three different days.

 


Table 3: Intraday and Interday Precision for ITO and OME

wavelength

concentration

Intraday (n=3)

%RSD

Interday (n=3)

%RSD

258 nm

10: 2.5 µg/ml

0.393 ± 0.0012

0.317

0.392 ± 0.0020

0.523

20: 5 µg/ml

0.800 ± 0.0008

0.102

0.798 ± 0.0024

0.312

30: 7.5 µg/ml

1.201 ± 0.0012

0.103

1.201 ± 0.0016

0.135

300 nm

10: 2.5 µg/ml

0.184 ± 0.001

0.543

0.183 ± 0.0012

0.680

20: 5 µg/ml

0.367 ± 0.0012

0.339

0.367 ± 0.0016

0.444

30: 7.5 µg/ml

0.553 ± 0.0008

0.147

0.552 ± 0.0020

0.307

 


Limit of Detection (LOD) and Limit of Quantification (LOQ):

The Limit of Detection and Limit of Quantification of the developed method was assessed by analyzing ten replicates of standard solutions containing concentrations 10µg/ml for ITO and 2.5µg/ml for OME.

 

The LOD may be calculated as

                        LOD = 3.3 × SD/ Slope

 

The LOQ may be calculated as

                        LOQ = 10 × SD/ Slope

 

Where, SD = ten replicates of absorbance

      Slope = the mean slope of the 6 calibration curves

Table: 4 LOD and LOQ data for ITO and OME

Drugs

LOD (µg/ml)

LOQ (µg/ml)

ITO + OME

(10:2.5)

258 nm

0.105

0.320

300 nm

0.052

0.160

 

Robustness and Ruggedness study:

Robustness and Ruggedness study was performed by determined by subjecting the method to slight change in the method condition, such as:

·       Change in Wavelength (258 ± 0.2 nm and 300 ± 0.2 nm)

·       Change in Analyst

·       Change in Instrument


 

 

 

Table:5 Robustness and Ruggedness data of ITO and OME

Ruggedness data of Itopride Hydrochloride (10 µg/ml) and Omeprazole (2.5 µg/ml) (n=3)*

No

Factor

Level

ITO Abs.*±SD

% RSD

OME Abs.*±SD

% RSD

1.

Change in Analyst

Analyst- 1

0.391 ± 0.0009

0.240

0.181 ± 0.0012

0.686

Analyst-2

0.392 ± 0.0008

0.208

0.182 ± 0.0004

0.258

2

Change in instrument

UV-2450

0.392 ±0.0004

0.120

0.182 ± 0.0004

0.258

 

 

UV-1800

0.393 ± 0.0008

0.207

0.183 ± 0.0008

0.446

Robustness data of Itopride Hydrochloride and Omeprazole (n=3)*

3

Change in wavelenth

(± 2 nm)

λmax

(258±2nm)

ITO Abs.* ± SD

% RSD

λmax

(300±2nm)

OME Abs.* ± SD

% RSD

256

0.390 ± 0.0004

0.120

298

0.183 ± 0.0008

0.446

260

0.391 ± 0.0008

0.208

302

0.184 ± 0.0004

0.255

 


Application of praposed method for the estimation of drugs in synthetix mixture:

Take synthetic powder equivalent to 10mg of Itopride Hydrochloride in 100ml volumetric flask. Dissolve in 25ml methanol and sonicated for 15min. Dilute up to 100ml with solvent shake vigorously. Filtered through whatman filter paper No. 42 and further diluted.

 

Finally the solution had concentration of 100µg/ml ITO and 25µg/ml OME, respectively. From that solution pipette out 2.0ml in 10ml volumetric flask and volume was made up to mark with methanol to make final concentration of mixture 20µg/ml for ITO and 5µg/ml for OME, respectively.

 

A zero order spectrum of the resulting solution was recorded and measure the absorbance at 258.00 nm and 300.00 nm were noted for estimation of ITO and OME, respectively. The concentration of ITO and OME in formulation were determined using simultaneous equation.

 

Table 6: Assay data for analysis of synthetic mixture.

Drugs

% Assay ± SD

% RSD

ITO + OME

(20:5 µg/ml)

258 nm

99.68 ± 0.154

0.155

300 nm

100.06 ± 0.339

0.339

 

ACKNOWLEDGEMENT:

The authors thankful to DR. M. N. Noolvi, principal of Shree Dhanvantary Pharmacy College for providing all facilities for my research work and Prayosha Health Care Pvt. Ltd, Ankleshwar and R.P Industries, Panoli for providing Itopride Hydrochloride and Omeprazole to carry out thiswork.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 12.07.2019         Accepted on 14.08.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(4):238-242.

DOI: 10.5958/2231-5691.2019.00038.8