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Viswanadha Institute of Pharmaceutical Sciences.
A rapid, sensitive, and accurate RP-HPLC method was developed for the identification and quantification of Ondansetron and Rabeprazole using a Waters HPLC system equipped with a PDA detector. Chromatographic separation was achieved on an Inertsil ODS-C18 column (250 × 4.6 mm, 5 µm particle size) with a flow rate of 1.2 mL/min. The mobile phase consisted of degassed and filtered Ammonium acetate buffer: water: methanol (25:15:60). Detection was performed at 275 nm, providing reliable resolution and measurement of both analytes.
Ondansetron (6-7), a competitive antagonist of serotonin type 3 receptors, is used to treat nausea and vomiting induced by cytotoxic chemotherapy drugs, including cisplatin. In addition to its antiemetic properties, ondansetron has shown anxiolytic and neuroleptic effects. Ondansetron has evolved with the introduction of orally soluble films. These films offer a more discreet and patient friendly option, especially during episodes of vomiting.
Rabeprazole (8-9) is an antiulcer medication belonging to the proton pump inhibitor class. It acts as a prodrug, converting to its active sulphonamide form in the acidic environment of parietal cells. Rabeprazole works by inhibiting the H+, K+ ATPase enzyme in the gastric lining cells, thereby reducing both basal and stimulated gastric acid secretion in a dose dependent manner.
MATERIALS AND METHODS
Preparation of Stock solution: 40 mg each of Ondansetron and Rabeprazole was accurately weighed and is transferred into neat and dry volumetric flasks of 100ml. About 60ml of methanol was added and allow to sonicate to remove the air bubbles formed in it, which is again make up to mark with methanol to give 1000µg/ml or 1mg/ml.
Preparation of working standard solution: From the stock solutions of both standards, each 4 ml was pipetted out and transferred in to 50ml volumetric flasks, then diluted up to the mark to get 32μg/ml for both the drugs.
RESULTS AND DISCUSSION
Method validation: Validation parameters include specificity, linearity, range, accuracy, precision, limit of detection, limit of quantification, robustness and assay (1-5).
Specificity: Specificity is the ability to assessing equivocally the analyte in the presence of components which may be expected to be present. Typically, these components include impurities, degradants, matrix etc. Blank solution and standard solutions of Ondansetron and Rabeprazole were injected into the HPLC system. The peak purity data of Ondansetron and Rabeprazole were compared. There should not be any interference at the retention time of the main peaks.
Linearity: Linearity for the drugs Ondansetron and Rabeprazole (10-13) was determined by preparing the standard solutions at five concentrations levels in range of 16 to 48μg/ml of Ondansetron and 16 to 48μg/ml of Rabeprazole from stock solution. The linearity charts of Ondansetron and Rabeprazole was shown in the figure no 2&3. The correlation coefficient was found to be 0.9974 and 0.9981 for Ondansetron and Rabeprazole respectively. Linearity results were tabulated in table 2.
Accuracy: Accuracy was performed by spiking known amounts of standard solution to sample solution at three different concentrations levels (50%, 100%, 150%) and there by analyzed for %RSD which should not be more than 2.0. The % recovery was calculated and the results was reported in table no. 3 & 4.
Precision: The precision (14-18) of the analytical method was studied by injecting six replicates of standard containing 32μg/ml of Ondansetron and 32μg/ml of Rabeprazole which were injected into HPLC system. The % RSD was calculated and the results were reported in the table no.5
Limit of Detection (LOD) and Limit of Quantification (LOQ): The limit of detection was defined as the concentration which yields a signal - to – noise ratio 3:1 whereas the limit of quantification was calculated to be the lowest concentration that could be measured with signal - to – noise ratio10:1. LOD and LOQ were calculated from slope and standard deviation. The results were tabulated in table no 6.
Robustness: The smallest deliberate changes in method like change in flow rate are made but there were no predictable changes in the results and are in the range as per ICH guidelines. Conditions like decrease in flow rate (1.0 ml/min), increase in flow rate (1.4 ml/min) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. % RSD was found to be within the limits and results were tabulated in table no 7.
Assay: Assay was conducted on marketed formulation and mean % assay was found. The results were tabulated in table no. 8.
Table 1: Optimised Chromatographic Conditions
| Parameter | Method |
|---|---|
| Stationary Phase (column) | Inertsil -ODS C18 (250 x 4.6 mm, 5 µ) |
| Mobile Phase | Ammonium acetate buffer: water: methanol (25:15:60) |
| Flow rate (ml/min) | 1.2 ml/min |
| Run time (minutes) | 15 min |
| Temperature in the column (°C) | Ambient |
| Volume of injection loop (μl) | 20 |
| Wavelength of detection (nm) | 275nm |
| Drug RT (min) | 4.918 for Ondansetron and 6.561 for Rabeprazole |
Figure 1: Optimized Chromatogram
Table 2: Linearity Data of Ondansetron and Rabeprazole
| Ondansetron | Rabeprazole | ||
|---|---|---|---|
| Conc (µg/ml) | Peak area | Conc (µg/ml) | Peak area |
| 16 | 499407 | 16 | 450945 |
| 24 | 779751 | 24 | 710474 |
| 32 | 1025303 | 32 | 965603 |
| 40 | 1195365 | 40 | 1133888 |
| 48 | 1487048 | 48 | 1396831 |
[Figure - see the formatted PDF]
Figure 2: Calibration Curve of Ondansetron
[Figure - see the formatted PDF]
Figure 3: Calibration Curve of Rabeprazole
Table 3: Accuracy Data of Ondansetron
| Concentration | Area | RT | Statistical Analysis of % Recovery | |
|---|---|---|---|---|
| % of spiked level | ||||
| 50% - 1 | 498173 | 4.954 | MEAN | 98.84 |
| 50% - 2 | 498919 | 4.957 | %RSD | 0.48 |
| 50% - 3 | 485240 | 4.96 | ||
| 100 % - 1 | 1058995 | 4.956 | MEAN | 99.81 |
| 100 % - 2 | 1075045 | 4.958 | %RSD | 0.5 |
| 100% - 3 | 1072698 | 4.961 | ||
| 150% - 1 | 1486752 | 4.964 | MEAN | 98.87 |
| 150% - 2 | 1484370 | 4.965 | %RSD | 0.2 |
| 150% - 3 | 1480898 | 4.968 | ||
Table 4: Accuracy Data of Rabeprazole
| Concentration | Area | RT | Statistical Analysis of % Recovery | |
|---|---|---|---|---|
| % of spiked level | ||||
| 50% - 1 | 454998 | 6.623 | MEAN | 98.81 |
| 50% - 2 | 454813 | 6.629 | %RSD | 0.32 |
| 50% - 3 | 452545 | 6.634 | ||
| 100 % - 1 | 986499 | 6.637 | MEAN | 99.47 |
| 100 % - 2 | 978944 | 6.642 | %RSD | 0.31 |
| 100% - 3 | 977737 | 6.646 | ||
| 150% - 1 | 1416722 | 6.656 | MEAN | 98.51 |
| 150% - 2 | 1413967 | 6.658 | %RSD | 0.15 |
| 150% - 3 | 1409179 | 6.663 | ||
Table 5: Precision Data for Ondansetron and Rabeprazole
| S. No | Peak areas of Ondansetron | Peak areas of Rabeprazole |
|---|---|---|
| 1 | 1058995 | 986499 |
| 2 | 1075045 | 978944 |
| 3 | 1072698 | 977737 |
| 4 | 1068268 | 973847 |
| 5 | 1071754 | 976937 |
| 6 | 1046255 | 972390 |
| Mean | 99.8 | 99.2 |
| SD | 0.42 | 0.35 |
| % RSD | 0.4 | 0.4 |
Table 6: LOD and LOQ Data of Ondansetron and Rabeprazole
| Drug Name | LOD (µg/ml) | LOQ (µg/ml) |
|---|---|---|
| Ondansetron | 2.628 | 7.963 |
| Rabeprazole | 2.252 | 6.826 |
Table 7: Robustness Data of Ondansetron and Rabeprazole
| S No | Drug Name | Condition | Peak area | % RSD | |
|---|---|---|---|---|---|
| 1 | Ondansetron | Decreased Flow rate of 1.0 ml/min |
1058342 | 1.234 | |
| 2 | Increased Flow rate of 1.4 ml/min |
1045647 | 0.624 | ||
| 3 | Rabeprazole | Decreased Flow rate of 1.0 ml/min |
979365.9 | 0.785 | |
| 4 | Increased Flow rate of 1.4 ml/min |
970738.8 | 0.942 |
Table 8: Assay Data of Ondansetron and Rabeprazole
| S. No | Peak area of Ondansetron | % Assay | Peak area of Rabeprazole | % Assay |
|---|---|---|---|---|
| Sample- 1 | 1030610 | 98.49 | 969447 | 99.37 |
| 1058342 | 979366 | |||
| Sample- 2 | 1049227 | 965439 | ||
| 1058342 | 979366 |
CONCLUSION
The developed RP-HPLC method was validated as per ICH guidelines. All the system suitability parameters were within the range as stated by ICH guidelines. Interference peaks were not observed in blank, standard and sample chromatogram. Hence simple, precise and accurate, sensitive, specific and robust method was developed and validated. This can be used in quality control department with respect to routine analysis.
Acknowledgements:
Authors are thankful to the management of Viswanadha Institute of Pharmaceutical Sciences (VNIPS) for providing facilities and support to carry out this work.
REFERENCES
K. Mounica, Dr. P. V. Madhavi Latha, K. Suvarna, Dr. P. Uma Devi, Method Development and Validation for the Simultaneous Estimation of Ondansetron and Rabeprazole in Bulk and Pharmaceutical Dosage Form by RP-HPLC Method, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 10, 3666-3671. https://doi.org/10.5281/zenodo.23640004
10.5281/zenodo.23640004