1. INTRODUCTION:

Rabeprazole sodium is chemically, Sodium; 2-[[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulfinyl] benzimidazol-1-ide.It is a proton pump inhibitor and it is a prodrug. Rabeprazole sodium is converted to sulfenamide and decreases gastric acid secrection. It inhibits proton pumps activity and used in the therapy of gastroesophageal reflux and peptic ulcer disease.¹

Mosapride citrate is chemically, 2-hydroxypropane-1,2,3-tricarboxylic acid; 4-amino-5-chloro-2-ethoxy-N-({4-[(4 fluorophenyl) methyl] morpholin-2-yl}methyl) benzene-1-carboximidic acid. It is prokinetic drug. It is a 5-HT₃receptor antagonist. The metabolite of Mosapride produces 5-HT₃ receptor antagonism and suppresses the inhibitory transmission in myenteric plexus. These all results in increased oesophageal peristaltic activity, lower oesophageal sphincter tone.so it is used in the treatment of gastroesophageal reflux disease.^2,3Structure of Rabeprazole sodium and Mosapride citrate is given in figure 1 and 2.

Fig. 1 (A) Structure of Rabeprazolesodium (B) Structure of Mosapride citrate

Forced degradation studies are carried out to inform the degradation mechanisms such as hydrolysis, oxidation, thermolysis or photolysis of the drug substance and drug product to solve stability related problems and generate more stable formulations. Degradation of drug substance between 5% and 20% has been accepted as reasonable for validation of chromatographic assays.^4-6

Literature survey acknowledged that RP-HPLC, TLC methods had been described for the estimation of Rabeprazole sodium and Mosapride citrate.^8-11 But there is not a single UPLC method available for the estimation of both drugs. UPLC method offer very significant increase in resolution, sensitivity and efficiency with less run time and less utilization of solvents which lowers the cost and makes the technology environment friendly also. Hence the main objective of this study is to develop simple, specific, sensitive UPLC method for estimation of Rabeprazole sodium and Mosapride citrate.⁷

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Rabeprazole sodium and Mosapride citrate were kindly provided as gift sample from Gufic Bioscience Ltd-Navsari and Pure and Cure Healthcare Pvt. Ltd- Haridwar. HPLC grade Methanol, Acetonitrile and Milli Q water and AR grade Hydrochloric acid, Sodium Hydroxide, Hydrogen Peroxide and were used. Rabeflux 20mg/15mg Tablet purchased from local market.

2.2 Chromatographic Condition:

UPLC (Acquity UPLC, Waters) used, data were processed using Empower software. Chromatographic separation was performed using UPLC BEH C₁₈ (50mm × 2.1mm, 1.7µm) Column. The mobile phase consists of Ammonium acetate buffer: Acetonitrile (60:40 %v/v/v). The flow rate was set to be 0.4ml/min. The injection volume was 2µL. The detection was carried out at 270 nm.

2.3 Method development:

In optimized condition Column used BEH C₁₈ (50mm × 2.1mm× 1.7µm), Flow rate was 0.4ml/min, Detection Wavelength - 270nm, Mobile Phase composition Ammonium Acetate buffer: Acetonitrile (60:40), Injection volume was 2µl, column temperature 35ºC.

2.4 Forced degradation studies:

The study was carried out to ensure the separation of degradants peaks and standard drug peak. To perform forced degradation study samples were exposed to acidic, alkaline, oxidation, photolysis and thermal degradation. Acidic degradation and Alkaline degradation were carried out using 0.1 M HCl and 0.1 M NaOH respectively for 1 hr and then the mixture was neutralized, diluted and filter. Oxidation studies were conducted using 3% H₂O₂for 3hrs.Thermal degradation was carried out 80ºC for 48 h. For photolytic degradation the powdered drug was exposed to UV light for 4 h.

2.5 Method Validation:¹²

Analytical validation parameters for this proposed method were determined according to ICH (Q2R1) guideline. ¹

2.5.1 System suitability:

The system suitability parameters were evaluated for tailing factor, retention time and theoretical plates of standard chromatogram.

2.5.2 Linearity:

The linearity was carried out at concentration range of 60-300μg/ml for Rabeprazole sodium and for Mosapride citrate the range was 45-225μg/ml.

2.5.3 Specificity:

Specificity was performed by injecting diluent, placebo and sample solution to check the interference of excipients.

2.5.4 Accuracy:

The accuracy was carried out by spiking three different concentration 50, 100 and 150% (100, 200 and 300 μg/ml for Rabeprazole sodium and 75, 150 and 225 μg/ml for Mosapride citrate) to placebo and recovery was calculated.

2.5.5 Precision:

Repeatability was performed under 6 replicates of Rabeprazole sodium (200μg/ml) and Mosapride citrate (150μg/ml). Intra-day and inter-day variations of Rabeprazole sodium and Mosapride citrate were performed in triplicate at three different concentration levels 50, 100, 150% (100, 200 and 300μg/ml for Rabeprazole sodium and 75, 150 and 225μg/ml for Mosapride citrate).

2.5.6 LOD and LOQ:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by formula.

2.5.7 Robustness:

The robustness of method was established by introducing small deliberate changes in experimental condition. The changes made in wavelength ± 1 nm (269 nm, 270nm, 271nm) for flow rate ±0.05ml/min (0.35ml/min, 0.4ml/min, 0.45ml/min) and for Temperature ±5ºC (33ºC, 35ºC, 37ºC).

3. RESULT AND DISCUSSION:

3.1 Optimized Condition:

Initially different concentration of Methanol, Acetonitrile and water were tried for the selection of mobile phase. The optimized mobile phase was Ammonium acetate buffer: Acetonitrile (60:40% v/v). Chromatographic separation was performed using UPLC BEH C₁₈ (50mm × 2.1mm) × 1.7µm. The flow rate was set to be 0.4ml/min. The injection volume was 2µL. The detection was carried out at 270nm.

Fig.2 Chromatogram of Optimized condition

Fig. 3 Chromatogram of (A) Acid degradation (B) Base degradation (C) Oxidative degradation (D) Photo degradation (E) Thermal degradation

Table No:1 Degradation condition

Degradation Condition

% Degradation

RAB

Purity Threshold

Purity

Angle

% Degradation

MOSA

Purity Threshold

Purity

Angle

Acid degradation

Base degradation

Oxidative degradation

Photo degradation

Thermal degradation

28.8

11.6

10.6

11.9

21.7

1.273

0.291

0.557

11.510

2.178

0.623

0.182

0.448

0.353

0.458

17.3

8.8

12.8

4.7

6.5

2.970

0.249

0.959

0.5860

2.026

0.432

0.045

0.773

0.340

0.341

3.2 Forced degradation study:

Degradation order for Rabeprazole sodium was acidic >thermal >photo>basic >oxidation and for Mosapride citrate acidic > oxidation> basic >thermal>photo. Both drugs were not stable in acidic condition. Photo Degradation is more in Rabeprazole sodium because it is light sensitive and Mosapride Citrate is more stable. Degradant peaks were generate in acidic and thermal condition. (Table-1).

3.3 Method Validation:

3.3.1 System suitability parameter:

Table No: 2 System suitability parameter

System suitability parameter

Rabeprazole sodium

Mosapride citrate

Theoretical plate

Resolution

Tailing factor

9055

1.2

2231

15.6

1.2

3.3.2 Linearity:

Linearity spectra and graph is given in Figure 4 and 5.

3.3.3 Precision and Accuracy:

Repeatability and intraday, interday precision for RP-UPLC method was measured in terms of RSD and it was found to be less than 2. Accuracy was found between the range of 98-102%. On the basis of accuracy and precision data we can conclude that method is accurate and precise. Results are shown in table 3.

Fig.4 Linearity graph of (A) Rabeprazole sodium (B) Mosapride citrate

Table-3: Intra and Inter day precision and Accuracy of method

Precision			Interday Precision	Intraday Precision	Accuracy
Drug Name	Level (%) (n = 3)	Conc. (μg/ml)	RSD	RSD	%Recovery Range
RAB	50 100 150	100 200 300	0.097627 0.186614 0.021691	0.091925 0.186614 0.085927	99.89- 100.17
MOSA	50 100 150	75 150 225	0.550894 0.753738 0.976938	0.626573 0.769506 0.917422	99.13- 99.96

3.3.4 LOD and LOQ:

LOD and LOQ were found to be 0.04374μg/ml and 0.132546μg/ml for Rabeprazole sodium and 0.03282 and 0.099454μg/ml for Mosapride citrate respectively.

3.3.5 Robustness:

Making a purposeful changes in wavelength, flow rate, temperature were take place and RSD of peak area found to be less than 2, specify that the method is robust and results remained unaffected by small variations of these parameters.

3.3.6 Assay of tablet dosage form:

Table-4: Assay of Rabeprazole sodium and Mosapride citrate

Drug

CONC (μg/ml) (n=3)

CONC Mean±SD

% Assay

(n=3)

Rabeprazole sodium

Mosapride citrate

200

150

201.114019±0.717

150.9962529±0.301

100.55701

100.6642

4. CONCLUSION:

The developed stability indicating method for the simultaneous determination of Rabeprazole sodium and Mosapride citrate is simple, rapid, sensitive, reproducible, specific, robust, with good accuracy and precision. Stress degradation was carried out in acidic, basic, oxidation, thermal, photo condition. Developed method was capable of quantifying both drugs in presence of degradant peaks without interference. Degradation is more in acidic condition for both drugs. And Rabeprazole sodium is more light sensitive. As there is no interference of excipients at the working wavelength, it is very fast, with good reproducibility and good response. It allows reliably the analysis of Rabeprazole sodium and Mosapride citrate in tablet dosage form with its degradants.

5. ACKNOWLEDGMENTS:

The authors gratefully acknowledge Piramal pharma solutions for providing all facilities to complete this research work.

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

7. REFERENCES:

1. Desai CA, Samant BD.A Review of Rabeprazole. Journal of Postgraduate Medicine. 2002; 48-50.

2. Drug Profile and Information of Mosapride citrate, September 2019,https://pubchem.ncbi.nlm.nih.gov/compound/Mosapride-citrate

3. Drug Combination, “Rabeprazole sodium and Mosapride citrate”, September 2019, https://www.1mg.com/drugs/rabeflux-20mg-15mg-tablet-305046

4. Blessy M, Patel RD, Prajapati PN and Agrawal YK. Development of forced degradation and stability indicating studies of drugs – A review. Journal of Pharmaceutical Analysis. 2013; 4:159- 165.

5. ICH – Harmonized Tripartite Guideline, “Stability testing of New drug substance and products Q1A (R2)”, International Conference on Harmonization, IFPMA, Geneva, Switzerland, 2003.

6. Choudary SP and Chandrudu J. Simultaneous estimation of Rabeprazole and Mosapride by RP-HPLC method in tablet dosage form. International Journal of Pharmacy and Analytical Research. 2018; 7: 116-128.

7. Chawla G and Chanda R. Principle, instrumentation, and applications of UPLC: a novel technique of liquid chromatography. Open Chemistry Journal.2006; 3: 1-16.

8. Patel BH, Suhagia BN, Patel MM and Patel JR. High-Performance Liquid Chromatography and Thin-Layer Chromatography for the Simultaneous Quantitation of Rabeprazole and Mosapride in Pharmaceutical Products. Journal of Chromatographic Science. 2008; 46: 10-14.

9. Kumar SA, Debnath M and Padala V. An Isocratic Method Development and Validation for simultaneous estimation of Rabeprazole and Mosapride in Tablet Dosage Forms by using RP-HPLC. Pharma Tutor. 2016; 4: 41-51

10. Mohammed AB, Gunasekar M, Alhazmi HA, Hafiz AM and Daghriri O. Development and validation of RP-HPLC method for the estimation of Rabeprazole and Mosapride in raw and capsule formulation. Der Pharm Chemica. 2016; 8: 140-146.

11. Saravanan G, Padmaja M, Geethanjali J and Visagaperumal D. Stability Indicating RP-HPLC Method for Estimation of Rabeprazole Sodium and Mosapride Citrate in Bulk and Formulation. International Journal of pharmacy and Pharmaceutical Science. 2014; 6: 265-269.

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Received on 01.05.2020 Modified on 15.07.2020

Research J. Pharm. and Tech. 2021; 14(9):4535-4539.

DOI: 10.52711/0974-360X.2021.00789