RP-HPLC bioanalytical Method of Itopride hydrochloride for determination and its Pharmacokinetic applications

 

Meenakshi Bhatia1, Rameshwar Dass1,2*, Goutam Rath3

1Department of Pharmaceutics, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India

2Guru Gobind Singh College of Pharmacy, Yamunanagar, Haryana, India.

3Department of Pharmaceutics, Siksha O Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India.

*Corresponding Author E-mail: rdladwa01@gmail.com

 

ABSTRACT:

Itopride hydrochloride (ITH) is currently used for the management of acute prokinetic disorders. In recent research, the determination of ITH in plasma, a new economic, prompt, and isocratic RP-HPLC bioanalytical approach, was established. This method is valid for quantifying the low concentration of drugs in plasma trials which is the demand to be improved. A HILIC column with a separation phase of MeOH: Buffer (30:70 v/v) and a flow with one millilitre per minute, as well as a UV sensor at 258nm, was used to achieve chromatographic high separation. The pharmacokinetic parameters were determined using blood from rabbits after administration of oral suspension containing ITH particles (1mg/kg). The retention time for both ITH and ornidazole, an internal standard (IS), was found to be 6.249min and 4.843 respectively. The statistical interpretation of the results was confirmed. The linearity assortment of the drug was 2-15µg/mL, and the recovered ITH and IS were found to be 100.1%  and 99.8% with accuracy, whereas the analysis of the sample was 99.87%, respectively. In addition, the oral pharmacokinetic parameters were successfully analyzed by using this method for ITH and OND. The highest plasma concentration (Cmax) of ITH was 6.805 (mcg/mL) at (Tmax) 1hr and half-life was found at 4.022 hours, correspondingly. The present analytical method has been efficiently used to study the pharmacokinetics of drug combinations.

 

KEYWORDS: Bioanalytical, Itopride hydrochloride, Pharmacokinetic, RP-HPLC, Plasma.

 

 

INTRODUCTION:

Chemically, itopride hydrochloride (ITH) (Figure 1a) is a benzamide derivative. It is used as a prokinetic agent. It is active against esophageal reflux disease1. The chemical name of ITH is N[{4-(2-(dimethylamino) ethoxy) phenyl} methyl]-3,4-dimethoxy-benzamide hydrochloride. This benzamide assembly has two types of linkage of amide and ether in itopride molecules and these are vulnerable to breakdown1. The ITH has pka values of 14.71 (Strongest acidic) and 8.77 (Strongest basic)2. ITH works via dopamine D2 and cholinesterase inhibition.

 

The ITH’s half-life is approximately 6 hrs.3 and metabolite and/or unchanged drugs excreted by kidneys4,5. On the other hand, Internal standard ornidazole (Figure 1b) is an imidazole derivative possessing antiulcer activity. Its chemical arrangement is 1-chloro-3-(2-methyl-5-nitroimidazole-1-yl)-2-propanol with a melting point of 77.5°C6. It is mentioned under pharmacopoeia as an official drug4. The oral bioavailability of OND was ≥90% against absorption through the small intestine5. The plasma half-life of OND is 11-12 hrs. and has low water solubility6. The pka value of OND is linear 13.89 (strongest acidic), 3.08 (strongest basic) and it acts as the strong base2. The stability studies on OND at acidic and basic environmental conditions show 27.18% and 22.21% degradation respectively7.

 

Figure 1. Chemical arrangement of a. Itopride HCl (drug) and b. Ornidazole (OND)

 

The RP-HPLC practice for the coexistent appraisal of both ITH and OND in formulations4 has not been reported to date. In addition, the bioanalytical process advancement and justification with its functional pharmacokinetics did not exist for both drugs. The acceptable ICH procedures Q2 (R1), that is grounded on pharmaceutical and therapeutic science8-13.

 

Experimental Procedure:

Reagents and Chemicals:

The API’s ITH and OND were received as gift samples from Abbott Healthcare India Private Limited, India. Sodiumdihydrogen phosphate and dipotassium hydrogen phosphate were used in the AR range. Methanol and water were of HPLC level. The animals (Newzealand rabbits) were used for the plasma samples3.

 

Instrumentation: (Chromatographic Conditions):

The RP-HPLC performed on (Waters Milford, USA) composed with a 515 series pump, manual porter apparatus with a 20µl decreed loop and hamilton injector of 50µl and a UV-2487 detector. The uncoupling and determination of both the drugs were made through the Atlantis HILIC column (250x4.6) mm, 5µm. The wavelength of the detector was adjusted at 258nm. The methanol: phosphate buffer, pH 5.8 of mobile separation phase, (30:70V/V) adjusted with o-phosphoric acid was verified on a microprocessor pH meter4.

 

Solutions:

Stock standard solution:

The stock solutions of ITH and OND were made by adding each 100mg/100ml of HPLC water. The various dilutions (2-15µg/ml) of both ITH and OND were prepared using a mobile solvent to produce the different working solutions4,7.

 

Plasma preparation:

Previously, frozen rabbit plasma was defrosted at laboratory temperature and treated with 2ml methanol and vortexed for 10 minutes. The supernatant was separated from the centrifuged plasma for 10 minutes at 4000rpm, and filtered via a syringe filter. Pipette mixed plasma (0.5ml) to 10ml closed tube, then add 2.0ml acetonitrile. Vortexed the above mixture and stood up for 5 min. at laboratory temperature, the composite was spin-settled at linear 4000rpm for 6-7 min. Transferred carefully the supernatant into the test tube and filtered supernatant was injected into the HPLC injector system9,10,12.

 

Preparation of resolution solution:

The resolution solution was prepared via mixing phosphate buffer Ph 5.8 and methanol by the parts of 70:30v/v. The above mixture was sonicated for 5 minutes to remove the air and to mix proper solutions.  

 

Validation procedure:

Selectivity, accuracy, precision, linearity, and robustness of the method were assessed, as well as stability under various stress situations with least instability, freezing and thawing stability, stock solution stability, and dilution fidelity and recovery11-14.

 

Selectivity:

Selectivity was estimated with blank plasma by the absenteeism of intrusive same retention time peak of analytes and internal standard.

 

Linearity:

The Linear regression for calibration curves was determined by preparation of dilutions from 2-15mcg/ml of itopride hydrochloride.

 

Recovery:

Recovery of the drug was assessed by comparing the results of 3 different samples (lower, medium and highest concentrations) of ITH solutions15.

 

Precision with accuracy:

For precision studies of six samples, replicates were prepared. Precision and accuracy were assessed via inter and intraday sample peaks.

 

Fortified Samples:

This was estimated by changing the sample concentration approx. 1600 ng/ml (approx. two times of HQC) with 1/5 and 1/10 dilutions and quantified against the calibration curve to evaluate the ability to dilute the pharmacokinetic samples.

 

Stability studies:

The stability of ITH was found in both solutions and plasma procedure validation. The drug's two concentrations were selected as low and high 2 and 15 mcg/ml respectively. The stability was evaluated by plasma samples kept at freezer-thawing 3 cycles and equated to fresh ITH samples with the same concentration4,8,13.

 

Pharmacokinetic Studies:

The GGS college of pharmacy, institutional animal ethical committee (IAEC) Yamunanagar (Approval Number: Regn. No. 873/PO/Re/05/CPCSEA) approved the rabbits. The pharmacokinetics of ITH and ONZ in rabbits were successfully validated by the RP-HPLC method (Table 5). New Zealand, either sex rabbits (2.5-3.0kg) were albedo to water and food. A pharmacokinetic evaluation of the drug on healthy male rabbits (n=3). The plasma samples (0.6mL) were accumulated from overnight fasting rabbits via ear marginal veins after oral administration of 1.8 mg of ITH. The various plasma tests were collected at 0, 1, 2, 3, 4, 6, 8, 12, and 24 h, tubes having EDTA. The tubes with blood were centrifuged at 4000rpm for 10 minutes at four-degree Celsius, to recover the plasma. Promptly succeeding samples were exposed to freezing and kept at -20°C for further use. Plasma drug samples were processed via liquid-liquid extraction. Plasma concentration-time data of ITH was analyzed by the excel solver extension. The pharmacokinetic study in rabbits was described as a method. After oral administration of a single ITH dose (2.14mg/kg/day) to rabbits, plasma levels were determined up to 24 h following administration15,19-21.

 

RESULT:

Optimization of Procedure:

The isocratic HPLC parting of active substances was accomplished with the mobile separating system. The mobile system of pH 5.8 was prepared by mixing methanol: phosphate buffer (30:70 v/v) and samples were passed via a 0.45-micron nylon membrane filter. The sample is inserted with a fixed volume of 20µl at the flow with 1ml/min at a pressure of 3160 psi. This method was selected because of its reliable resolution with this mobile phase. This method is best accepted with applied chromatographic conditions (Figure 2).

 

 

Figure 2. Chromatographic representative of Itopride HCl in Plasma.

 

Implementation of the method:

This developed process was effectively exercised to evaluate plasma for ITH and OND and develop pharmacokinetic parameters. Both drugs were found in a matrixed particulate system by the utilization of the assay as depicted below. The precision percent of RSD is upto 2, indicating that the procedure is acceptably precise8. (Figure 3).

 

Figure 3. Illustrative Chromatographic image of itopride hydrochloride and OND in Plasma.

 

Validation:

Specificity:

This RP-HPLC bioanalytical method can quantify and distinguish the bioactive substances in a combined sample. The bioanalytical technique development and validation for ITH were employed, and this method was commenced in a chromatographic state for the bioanalytical investigation20,21. (Figure 4).

 

 

Figure 4. Prototypical Chromatogram of absolute blank plasma.

 

Linearity:

The range of concentration for calibration curves from 2-12µg/mL for ITH and OND displayed a linear relationship with r2 value of 0.998, presented in figure 5. The above results revealed an excellent correlation between the ratio of peak area against the equivalent concentration of ITH and OND shown in Table 1.

 

 

Figure 5. RP-HPLC Calibration curve for itopride hydrochloride

Table 1. Result of calibration curve linearity.

Parameter

ITH

Linearity range

2-12µg/ml

Regression equation

y = 39435x + 1316.4

Correlation coefficient (r2)

0.998

Slope

39435

Y-Intercept

1316.4

 

 

Accuracy:

The proposed method's accuracy was found by calculating percentage value recovery from plasma. When medicines in plasma supernatant were injected, percentage recovery for ITH and OND was measured at concentration levels: 8, 10, 12g/ml, and 5g/mL. We determined the percentage value of recovery and RSD. shown in Table 2.

Table 2. Result of Accuracy study.

Recovery Samples Fortified Samples

S. No

Conc. (µg/mL)

Peak area

Mean Peak Area

Conc. (µg/mL)

Peak Area

Mean Peak Area

% Recovery

1

8

465270

466912

80+100

1044816

1046458

100.72

 

468968

1048514

 

466498

1046044

 

2

10

579546

575398

100+100

1159092

1154928

100.81

 

 

573728

 

1153227

 

 

572920

 

1152466

 

3

12

699196

699088

120+100

1278742

1278634

100

 

 

698239

 

1277785

 

 

699829

 

1279375

 

 

Precision:

Three samples were injected into the HPLC column for repeatability and inter-day precision testing. In all concentrations examined, the relative standard deviation was less than 2.0 percent. shown in Table 3.

 

Table 3. Itopride exactitude of Result for Precision.

Validation Parameter

% Mean

S.D.

% R.S.D

System

577865

9729.127875

1.683633353

Day to Day

565774.1667

5322.025795

0.929953965

Analyst to Analyst

572289.1667

7643.110896

1.350911962

 

Method Robustness:

The robustness of the method was determined by making deliberate adjustments to the chromatographic settings under ICH guidelines. The procedure was tested in the presence of pH fluctuations in the mobile phase (pH 4 0.1) 20,21. shown in Table 4.

 

Table 4. Consequences of robustness.

S. No.

Conc (µg/mL)

Flow (1.00 mL/min)

pH (5.8)

Wavelength

0.9

1.1

5.75

5.85

256nm

300nm

1

10

574833

542399

573931

558393

572933

589303

2

10

583833

549398

578393

568930

579292

572698

3

10

578989

559233

573909

558398

579839

573830

Mean

579218.3333

550343.333

575411

561907

577355

578610.3333

STD

4504.380683

8456.72102

2582.51118

6082.096925

3839.03

9277.402456

%RSD

0.007776654

1.53662641

0.44881158

1.082402769

0.66493

1.603393842

 

Pharmacokinetic Studies:

The mean blood concentration relationship time curve beside an oral injection of ITH (1.8 mg/ml) and the kinetic of blood parameters were condensed in Table 6. The Cmax of ITH was obtained from the curves, which was 6.805mcg/mL respectively, and the Tmax was 1.0 hrs. shown in Figure 6.

 

Figure 6. Mean rabbit plasma concentration-time Profiles of Itopride HCl.

Table 5. Assay of particles dosage system analysis.

Parameters

Mean (%)

S.D.

R.S.D. (%)

99.87

0.09255

0.09263

 

Table 6. Pharmacokinetic Studies.

Parameters

ITH

Cmax (mcg/mL)

6.805

Tmax (hrs)

1.00

AUC0-∞ (mcg/ml.h)

31.360

AUC0-t (mcg/ml.h)

30.934

t1/2 (hrs)

4.022

 

DISCUSSION:

The developed technique uses methanol with phosphate buffer (pH 5.8) as the mobile phase. The devised method was unique in that it showed no interference from the blank matrix during measurement and had a quick elution time (less than 10 minutes for itopride HCl). The described method was successfully used in rabbits for ITH pharmacokinetic research. The biological half-life (t1/2), Tmax value, and Cmax were estimated for the oral ITH, and applied in bioavailability, drug interaction and bioequivalence studies.

 

CONCLUSION:

A simple, accurate and exact RP-HPLC method was devised in this study. The described approach was effectively used for pharmacokinetic research. The biological half-life (t1/2), Tmax value, and Cmax were estimated for the oral ITH.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENT:

The authors desire to express appreciation to the management of GGS College of Pharmacy and Abbott Pharmaceutical LTD, HP, India for providing standard ITH reference standards and their liaison in the research work.

 

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Received on 10.01.2022            Modified on 06.04.2022

Accepted on 26.05.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(9):4012-4016.

DOI: 10.52711/0974-360X.2022.00672