UV Spectroscopic Method Development and Validation for Simultaneous Estimation of Rebamipide and Omeprazole Magnesium in Synthetic Mixture

Introduction to illness ^[1-3].

Gastric ulcer is a condition where the lining of the stomach is eroded by acid and other substances.  Gastric ulcers, also known as stomach ulcers, are open sores that develop on the inside lining of the stomach. These ulcers can cause significant discomfort and pain, often manifesting as a burning or gnawing sensation in the upper abdomen. Gastric ulcers are a form of peptic ulcer disease, which also encompasses duodenal ulcers found in the duodenum, the initial section of the small intestine.

Treatment:

Proton pump inhibitors (ppis): medications like omeprazole reduce stomach acid production, allowing ulcers to heal. Antiulcer: rebamipidea mucosal protective agent with anti-oxidative and anti-inflammatory properties. Patients are advised to either stop or reduce their use of nonsteroidal anti-inflammatory drugs.

Role of rebamipide and omeprazole Magnesium:

Rebamipide is a substance that helps protect the mucous membranes in the body, has anti-inflammatory effects, and also acts as an antioxidant. It improves blood flow to the mucous membranes, which can assist in the healing of ulcers. Research has indicated that rebamipide can be just as effective as omeprazole in treating H. pylori caused by Pylori bacteria after treatment.  Omeprazole magnesium is a type of omeprazole, a medication that decreases the amount of acid produced in the stomach. It is frequently employed to treat gastric ulcers by establishing an environment that promotes healing.

Role of Rebamipide and Omeprazole Magnesium in the mechanism of disease.:

Rebamipide improves the protective mucosal barrier by increasing blood flow and reducing inflammation, while omeprazole decreases acid production, creating a less acidic environment that aids in the healing process. By working together, they can successfully address the physiological alterations that occur in gastric ulcers.

Overview of medication.

Introduction to Rebamipide: ^[4]

Rebamipide is a medication that is used in combination with other similar treatments (e.g., proton pump inhibitors) to alleviate gastritis and safeguard the lining of the stomach. It was first approved by the U.S. Food and Drug Administration (FDA) in February 2013. Rebamipide was accepted by the CDSCO in July 2004.

Fig 1.1 Structure of Rebamipide ^[4]

Mechanism of action Reba (MOA)

1. Mucosal defense and recovery.

2. enhanced prostaglandin production.

3. increased mucus production.

4. enhanced circulation.

5. activation of epithelial cell proliferation.

It stimulates the growth and multiplication of epithelial cells, aiding in the repair of damaged mucosal surfaces^.

Fig 1.2 Marketed Formulation of Rebamipide

Omeprazole magnesium: ^[5]

Omeprazole magnesium is a medication that treats conditions that cause too much stomach acid like heartburn, stomach ulcers and reflux disease. Omeprazole magnesium was first approved by the FDA approved October, 2010. Omeprazole magnesium got approval from CDSCO in December, 2016.

Fig 1.3 Structure of Omeprazole Magnesium ^[5]

Mechanism of action Ome (MOA)

Omeprazole is a targeted inhibitor of the proton pump in parietal cells, which is responsible for the production of stomach acid. This enzyme plays a crucial role in the final stage of acid secretion, omeprazole inhibits acid secretion in response to all stimuli.

Fig 1.4 Marketed Formulation of Omeprazole magnesium

MATERIALS AND METHOD

Chemical and Reagent:

Table 1.1 List of Chemical and Reagents

Melting Point Determination

Solubility Study

UV spectroscopy Method:

The solutions were scanned in the range of 400-200 nm against methanol as reference, and the peaks were observed in the spectra of rebamipide(reba) at 302nm(figure1) and omeprazole magnesium(ome) at 230nm(figure 1.5).

Fig 1.5 UV spectra of Rebamipide(302nm)

Figure 1.6 UV spectra of Omeprazole magnesium(230nm)

Figure 1.7 Overlay of Rebamipide and Omeprazole magnesium

Procedure for creating and testing our method.

The choice of solvent: rebamipide (reba) and omeprazole magnesium (ome) are both soluble in methanol. As a result, methanol was selected as the solvent for the UV analysis.

Preparation of Control Solutions.

1. rebamipide (reba):

1000 µg/ml: measure 100 mg of reba, mix it with methanol in a 100 ml flask.  100 µg/ml: take 10 ml from the above and mix it with 100 ml of methanol.

2. omeprazole magnesium (ome):

A concentration of 1000 µg/ml can be achieved by weighing 20 mg of ome in methanol and sonicating it for 10 minutes in a 20 ml flask. The resulting solution can then be diluted to the desired mark. 100 µg/ml: extract 10 ml from the top, then mix it with 100 ml of methanol.

Test preparation:

Step 1: take synthetic mixture equal to 100 mg reba and 20 mg ome in 100 ml flask, add methanol, and sonicate for 10 min. (final strength: 1000 µg/ml reba and 200 µg/ml ome).

Step 2: take 1 ml from the above, dilute to 10 ml with methanol. (strength: 100 µg/ml reba and 20 µg/ml ome)

Step 3: Take 1 milliliter from the previous mixture and dilute it to 10 milliliters. (final concentration: 10 µg/ml reba and 2 µg/ml ome)

Wavelength determination procedure: take 1 ml each of 100 µg/ml reba and ome solutions, mix and dilute to 10 ml with methanol. (final: 10 µg/ml of each) scan the solution in uv spectrophotometer from 200–400 nm using methanol as blank to find the absorption maxima (λmax).

Simultaneous Equation^[7]:

Cx = (A2·ay1 - A1·ay2) / (ax2·ay1 - ax1·ay2)

Cy = (A1·ax2 - A2·ax1) / (ay1·ax2 - ay2·ax1)

Given,

 A2 = 1.0742, ay1 = 0.0531, A1 = 1.9750, ay2 = 0.0036, ax1 = 0.0104, ax2 = 0.0103

Cx = 0.04991902 / 0.00051049 ≈ 97.80

Cx ≈ 97.80

Cy = 0.00916482 / 0.00051049 ≈ 17.96

Cy ≈ 17.96

Validation Parameter Study ^[6,8]

Linearity: Take 1.25,2.5,3.75,5,6.25 ml in 25 ml volumetric flasks of Standard working solutions of REBA (500µg/ml REBA and 100µg/ml OME) to get a series of concentrations 25–125 ppm for REBA and 5–25 ppm for OME. The solutions were filtered using 0.45 µm membrane filters. A calibration curve was plotted using mean absorbance vs. concentration, and the correlation coefficient (R²) and regression equation were calculated for both drugs.

2. Accuracy: Accuracy was evaluated by recovery studies at three levels: 50%, 100%, and 150% by spiking known amounts of REBA and OME to a fixed concentration (50 µg/ml REBA and 10 µg/ml OME). % Recovery was calculated for each level to check method accuracy.

Spiking Details:

Table 1.4 Linearity Detected Level

3. Precision: Repeatability (intra-assay): A solution of 100 µg/ml REBA and 20 µg/ml OME was analyzed 6 times. % RSD was calculated.

Intraday Precision: Three concentrations (25, 50, 100 µg/ml for REBA and 5, 10, 20 µg/ml for OME) were analyzed at different times (0, 3, and 6 hrs) on the same day. % RSD was calculated.

Interday Precision: To evaluate interday variation, the same concentrations were analyzed over three consecutive days. The % RSD was calculated.

Wavelengths used: REBA: 302 nm, OME: 230 nm

4. LOD and LOQ: LOD and LOQ were calculated using the standard deviation (SD) of Y-intercepts and slope of the calibration curves: LOD = 3.3 × (SD / Slope), LOQ = 10 × (SD / Slope) Assay of Synthetic Mixture: A synthetic mixture of REBA (100 mg) and OME (20 mg) was prepared with common excipients. An accurately weighed amount equivalent to 10 mg REBA and 2 mg OME was transferred into a 100 ml volumetric flask. 70% methanol was added, sonicated, and diluted to volume. The solution was filtered through a 0.45 µm membrane filter, and 1 ml was further diluted to 10 ml with methanol. The assay was carried out using the developed UV method.

RESULT AND DISCUSSION:

1. Linearity:

Table 1.5 Linearity Data of Omeprazole Magnesium

Figure 1.8: Calibration curve of Rebamipide

Table 1.6 Linearity data of Omeprazole magnesium (OME) at 302nm

Figure 1.9 Calibration curve of Omeprazole magnesium

Table 1.7 Linearity data of Omeprazole magnesium (OME) at 230nm

Figure 1.10: Calibration curve of Omeprazole magnesium

Table 1.8: Linearity Data of Rebamipide at 230 nm

Figure 1.11: Calibration curve of Rebamipide

2. Accuracy: The accuracy of the method was confirmed by a recovery study from a synthetic mixture at three levels: 50%, 100%, and 150% of standard addition. The data shown in Table 7.5 indicate that the developed method is accurate. The % recovery of REBA and OME was found to be in the range of 99.46-100.31% and 99.60%-101.02%, respectively.

Table 1.9 Accuracy data of Rebamipide and Omeprazole magnesium (n=3)

3. Precision

a) Repeatability

Repeatability has been carried out by measure the absorbance of solution. Repeatability was carried out by analysing the sample solution of REBA (100µg/ml) and OME (20µg/ml) six times and absorbance was measured and % RSD which is shown in Table 7.6.

Table 1.10 Repeatability of Rebamipide and Omeprazole magnesium

b) Intraday:

Rebamipide (REBA) and Omeprazole magnesium (OME) solutions containing 25,50 and 100 µg/ml of REBA and 5,10 and 20 µg/ml of OME was examined three times on the same day and %RSD was calculated. REBA had a %RSD of 0.098-0.93%, while OME had a %RSD of 0.11-0.35%.

c) Interday:

Rebamipide (REBA) and Omeprazole magnesium (OME) solutions containing 25,50 and 100 µg/ml of REBA and 5,10 and 20 µg/ml of OME was examined three times on the three alternative days, and %RSD was calculated. REBA had a %RSD of 0.43-1.40%, while OME had a %RSD of 0.18-0.59%.

Table 1.11 Intraday and Interday Precision of REBA and OME(n=3)

4. Limit of Detection and Limit of Quantitation.

Calibration curves were repeated six times, and the standard deviation of the intercept was calculated. Then, LOD and LOQ for Rebamipide and Omeprazole magnesium were calculated and shown in Table 1.14 below.