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INTRODUCTION
Chromatography is a powerful method for measuring many analytes in complicated materials, making it an indispensable tool for analytical chemists.1,4 Chromatography is useful for determining the chemical structure and concentration of a mixture of compounds known as analytes.5,7 Chromatography is divided into three categories: gas, liquid, and supercritical fluid chromatography.8 Ion exchange, size separation, and gel-based electrophoretic procedures are all examples of liquid chromatography techniques. This volume provides a fundamental introduction to liquid and gas chromatography.9 A 2022 technique for APIs, a 2023 method for the fixed-dose tablet, and a 2025 QbD-based RP-HPLC method are examples of simultaneous RP-HPLC methods that have been reported in recent literature.10 Significantly, chromatographic separation, void-volume elution, placebo interference, and/or thorough stability-indicating examination are among the shortcomings of the preceding techniques.11,12 Figure 1 depicts the chromatographic categorisation. The recommended method was carefully tuned to offer sufficient chromatographic resolution in a short analysis time and validated in compliance with the applicable ICH requirements.
MATERIALS AND METHODS
Statement of Ethics
Neither experimental animals nor human subjects were used in this investigation. Consequently, ethical clearance was not needed.
Drug Profile: Bupropion
Bupropion is an aromatic ketone derived from propiophenone that has a chlorine substituent at position 3 on the phenyl ring and a tert-butylamino group at position 2. It is a secondary amino chemical, an aromatic ketone, and a type of monochlorobenzene that has antidepressant, xenobiotic, and environmental polluting properties.13
Mechanism of Action
As a Norepinephrine/Dopamine-Reuptake Inhibitor (NDRI), bupropion slightly inhibits the enzymes responsible for the absorption of dopamine and norepinephrine from the synaptic cleft, extending the duration of action of the neurotransmitters within the neural synapse and their downstream effects.14 The structure was shown in Figure 2.
Absorption: Poorly absorbed
Dextromethorphan
Dextromethorphan is a synthetic, non-opioid antitussive (cough suppressant) widely used to relieve dry, non-productive cough accompanying common cold, influenza and also additional upper respiratory tract infections.15 It is the dextrorotatory isomer of levorphanol but lacks significant analgesic or addictive effects at therapeutic doses. N-methyl-D-aspartate NMDA receptor antagonist dextromethorphan is used to treat dry cough.16
The mode of action
Dextromethorphan is an agonist of the sigma-1 receptor and an uncompetitive NMDA antagonist with modest affinity. The structure was shown in Figure 2.
Chemicals and Instruments
Chemicals and Solvents used:
The investigation employed only analytical-grade chemicals and solvents that were purchased from Rankem.
• MeCN Trimethyl Amine - A.R Grade used.
• Orthophosphoric acid - A.R Grade used.
• N HCl.
• Potassium dihydrogen ortho phosphate - AR Grade.
• Perchloric acid.
• NaOH.
Devices
1. Denver's electronic balance.
2. BVK Enterprises Ultrasonicator.
3. BVK Enterprises' pH gage.
4. HPLC Waters 2695.
5. Double beam UV spectrophotometer from Lab India.
Sample Processing
Standard Preparation of Stock Solutions
A 50 mL volumetric flask was filled with dextromethorphan and bupropion after they had been carefully weighed. The mixture was sonicated until it was totally dissolved after adding roughly 30 to 40 mL of diluent (50:50 v/v water). Subsequently, the diluent was appropriately added to the volume and well mixed. The exact amounts weighed were used to calculate the concentrations of dextromethorphan and bupropion in the standard stock solution.
Working Standard Solution
To achieve the desired higher concentrations, a suitable portion of the standard stock solution was moved into a volumetric flask and diluted to the necessary volume with the diluent of bupropion and dextromethorphan. All dilution calculations were verified to ensure consistency between the stock concentration, aliquot volume, final volume, dilution factor, and final concentration.
Preparing the Sample
The average pill weight was determined after weighing each of the five tablets independently. A carefully weighed quantity of the powdered formulation, which contained the required doses of bupropion and dextromethorphan, was finely pulverized and then added to a 50 mL volumetric flask. The mixture was sonicated to enable full analyte extraction after 25-40 mL of diluent was added. Diluent was added to get the volume up to the necessary level after letting the solution cool if necessary. After thoroughly mixing the liquid, a 0.45 µm nylon syringe filter was used to filter it. An appropriate fraction of the filtrate was further diluted with the diluent to reach the required working concentration for HPLC analysis.
Validation parameters
• System suitability parameters:* 6 set of injections was injected.
Precision
• Std working solutions:* 1 mL of stock sol was put into a 10-mL volumetric flask and diluted. (50µg/mL bupropion, 25µg/mL dextromethorphan).17
Linearity
From stock composition subsequent volumes are pipetted out in diverse flasks. Over a wide concentration range of 25-150% of the target concentration, the approach's linearity was shown. Aliquots of 0.25 mL, 0.5 mL, 0.75 mL, 1 mL, 1.25 mL, and 1.5 mL of the stock solution were added to a 50 mL volumetric flask. These aliquots were diluted with mobile phase until the concentration reached 25, 50, 75, 100, 125, and 150 ppm, respectively. The HPLC system was filled with the solutions in accordance with the test protocol. Plotting a calibration curve for concentration versus peak area was done.
Accuracy
By spiking the drug substances bupropion and dextromethorphan on a placebo in the range of approximately 50 to 150 of test concentration, a series of solutions were generated in triplicate (6 preparations for 50 and 150% level), and they were analyzed according to specification level triplicate injections for 50%, 100%, and 150%. RSD, individual recovery, mean recovery, and linearity were calculated at each level.
• LOD:* The standard deviation of the intercepts was calculated after the calibration curve was replicated three times.
• LOQ:* Standard deviation of the intercepts was computed after three repetitions of the calibration curve.
Specificity
By examining the diluent blank, placebo solution, standard solution with bupropion and dextromethorphan, and commercial formulation, the specificity of the suggested RP-HPLC method was assessed. We looked for any interfering peaks at the analyte retention periods on the chromatograms of the blank and placebo formulations.
Forced Degradation Studies
Bupropion and Dextromethorphan were subjected to independent forced degradation investigations in acidic, alkaline, oxidative, thermal, photolytic, and hydrolytic settings in order to assess the suggested RP-HPLC method's capacity to indicate stability. The controls were unstressed drug solutions. Under the same stress conditions, corresponding blank solutions were made without the medication ingredient.18
Acidic degradation
2 mL of 5% HCl and a tiny amount of diluent were added to a 20-mL volumetric flask after 5 mL of the corresponding drug stock solution had been transferred. 20 min were spent heating the mixture to 80ºC. The solution was cooled to room temperature and neutralised with 2 mL of 5% NaOH following the conclusion of the stress treatment. Next, diluent was added to bring the volume up to 20 mL. Prior to HPLC analysis, a 0.45 µm nylon syringe filter was used to filter the residual solution after the first 4 mL of the produced solution was discarded.
Alkaline degradation
A 10 mL volumetric flask was filled with 1 mL of the matching drug stock solution, and then 1 mL of NaOH was added. For 3 min, the mixture was refluxed at 60ºC. After the solution cooled to room temperature, diluent was added to bring the volume up to 10 mL after neutralising it with 2 mL of 5% HCl. The remaining solution was filtered using a 0.45 µm nylon syringe filter prior to HPLC analysis, with the first 4 mL being discarded.
Oxidative degradation
A millilitre of the corresponding medication stock solution was treated with 20 % v/v Hydrogen Peroxide (H2O₂) and kept at 60ºC for half an hour. To achieve the necessary concentration for HPLC analysis, the solution was cooled and suitably diluted with the diluent after the stress treatment was finished. A 0.45 µm nylon syringe filter was employed to filter the fluid before injection.
Thermal degradation
5 mL of the relevant medication stock solution was put into a 20 mL volumetric flask and diluted with diluent. The mixture was heated to 105ºC for an entire day. After cooling to room temperature, the solution was filtered through a 0.45 µm nylon syringe filter before being subjected to HPLC analysis.
A comparison was made between the stressed and unstressed samples. From the chromatographic response, the percentages of recovery and degradation were computed. The chromatograms were also examined for additional peaks and their separation from the Bupropion and Dextromethorphan peaks.
OUTCOMES AND DISCUSSION
Observation
Dextromethorphan and bupropion separated at 2.21 and 2.87 min with good resolution. This approach was optimised and validated by the excellent plate count and tailing factor.
Issues encountered during diverse trials
In order to successfully separate bupropion and dextromethorphan during the original method development, a number of chromatographic studies were carried out. Various ratios of organic solvent and aqueous phase were assessed. While increasing the organic-phase proportion led to decreased retention and insufficient analyte separation, greater aqueous-phase proportions were associated with longer analysis times and longer retention times. Poor peak shape and peak broadening were noted in certain experiments, suggesting that additional chromatographic condition optimisation was necessary. Retention duration, peak resolution, and peak symmetry were also impacted by variations in flow rate. In order to achieve an appropriate balance between resolution and analysis duration, the flow rate and mobile-phase composition were methodically changed. The Figure 3 displayed the optimised chromatogram for both drugs and linearity data for bupropion.
Validation parameters
System Suitability
Table 1 displayed data about Optimized chromatographic conditions and system appropriateness. Tables 2 validation parameters of the proposed method were shown. In Table 3 accuracy and assay results were displayed.
Linearity
Observation: Y=52904x + 8801.4 for linearity of Bupropion and Y=48512x+3943.5 for linearity of Dextromethorphan 0.999 for correlation of bupropion and 0.9998 for linearity of dextromethorphan. The linearity data for dextromethorphan and LOD chromatogram for both drugs was shown in Figure 4.
Precision
Observation: The System precision area for bupropion and dextromethorphan were found to be 0.6 and 0.4 correspondingly. Tables 4 and 5 contained data for precision.
Intermediate Precision
For bupropion and dextromethorphan, the percentage Relative Standard Deviation (% RSD) determined as 0.8% and 0.5%, respectively. The suggested approach has adequate intermediate precision and is repeatable for the simultaneous estimation of bupropion and dextromethorphan, since both values were well below the permissible limit of not more than 2.0%.
Repeatability
The reproducibility of the proposed RP-HPLC method was evaluated by looking at duplicate samples of dextromethorphan and bupropion under identical experimental conditions. For bupropion and dextromethorphan, the percentage relative standard deviation determined as 0.5% and 0.4%, respectively. The suggested analytical approach was deemed repeatable and accurate for the simultaneous quantification of bupropion and dextromethorphan since the % RSD values were determined to be less than 2.0%.
Accuracy
Recovery trials for bupropion and dextromethorphan were utilised to evaluate the accuracy of the proposed RP-HPLC technology. The mean percentage recovery was found to be 99.60% for dextromethorphan and 99.56% for bupropion. The recovery results falling within acceptable boundaries demonstrate the accuracy and suitability of the recommended method for the contemporaneous estimate of bupropion and dextromethorphan in medicinal formulations.
Sensitivity
The limits of detection for dextromethorphan and bupropion discovered to be respectively, 0.60 µg/mL and 0.80 µg/mL. It was discovered that the limits of quantification for bupropion and dextromethorphan were 2.42 µg/mL and 1.81 µg/mL, respectively. These findings show that both analytes can be detected and quantified using the suggested RP-HPLC method with adequate sensitivity. Chromatogram of working standard and LOQ Chromatogram of both drugs were indicated in Figure 5. Chromatogram of working sample was shown in Figure 6.
Specificity
In the blank or placebo chromatograms, neither the bupropion nor the dextromethorphan retention time showed any notable interference peaks. Bupropion and dextromethorphan were represented by well-resolved peaks in both the standard and sample chromatograms. The two analytes' chromatographic separation was adequate. Blank and placebo samples showed no chromatographic interference. Figure 7 displayed the specificity data of blank and Figure 8 displayed the specificity data of placebo.
Robustness
Observation
It was discovered that the robustness of dextromethorphan and bupropion was within the recommended bounds.
Assay
The mean percentage assay for bupropion was determined to be 99.37 %, with a 0.48 standard deviation and a 0.48% Relative Standard Deviation (% RSD). The mean percentage assay for dextromethorphan was determined to be 99.43 %, with a 0.35 standard deviation and a 0.35 percentage Relative Standard Deviation (% RSD). The suggested method's low % RSD number shows good accuracy and consistency. The approach is appropriate for the precise and trustworthy estimation of the analyte in the pharmaceutical formulation, as evidenced by the assay result falling within the allowed bounds.19
Forced degradation studies were accomplished to assess the stability-indicating potential of the recommended RP-HPLC technique for the concurrent detection of bupropion and dextromethorphan. The therapeutic compounds were subjected to various stress situations, such as oxidative degradation, thermal degradation, acidic and alkaline hydrolysis, and photolytic degradation. The homogeneity of the analyte peaks under all stress situations was validated by the peak purity data. As a result, the suggested RP-HPLC approach may be regarded as specific and stability-indicating. It is appropriate for routine quality-control analysis and tracking the degradation behavior of bupropion and dextromethorphan in pharmaceutical formulations. Data for degradation was indicated in Table 4.
| Parameter | Optimised condition | ||
|---|---|---|---|
| Mobile phase | 0.01 N Na₂HPO₄ : Acetonitrile (70:30, v/v) | ||
| Column | Intesil C18 (4.6 × 150 mm, 5 µm) | ||
| Flow rate | 1.0 mL/min | ||
| Detection wavelength | 230 nm | ||
| Run time | 10 min | ||
| Injection volume | 10 mL* | ||
| Column temperature | 24ºC | ||
| Retention time (min) | 2.210-2.229 | 2.864-2.878 | - |
| Theoretical plates | 7616-658 | 9500-9587 | - |
| Tailing factor | 1.25-1.26 | 1.19-1.20 | - |
| Resolution (Rs) | - | - | 5.9-6.0 |
| Validation parameter | Bupropion | Dextromethorphan |
|---|---|---|
| Regression equation | Y=52904x + 8801.4 | Y=48512x + 3943.5 |
| Slope | 52,904 | 48,512 |
| Intercept | 8,801.4 | 3,943.5 |
| Correlation coefficient (r) | 0.9990 | 0.9998 |
| Repeatability (%RSD) | 0.5 | 0.4 |
| Intermediate precision (%RSD) | 0.8 | 0.5 |
| LOD | 0.80 | 0.60 |
| LOQ | 2.42 | 1.81 |
| Robustness: F (−) | 0.2 | 0.2 |
| Robustness: F (+) | 0.7 | 0.5 |
| Robustness: M (−) | 0.1 | 0.1 |
| Robustness: M (+) | 0.3 | 0.3 |
| Robustness: T (−) | 0.3 | 0.3 |
| Robustness: T (+) | 1.1 | 1.2 |
| Drug | Spiking level (%) | Amount spiked | Amount recovered | Recovery (%) |
|---|---|---|---|---|
| Bupropion | 50 | 21 | 21.053 | 100.25 |
| Bupropion | 50 | 21 | 20.825 | 99.17 |
| Bupropion | 50 | 21 | 20.957 | 99.80 |
| Bupropion | 100 | 42 | 41.657 | 99.18 |
| Bupropion | 100 | 42 | 41.751 | 99.41 |
| Bupropion | 100 | 42 | 41.954 | 99.89 |
| Bupropion | 150 | 63 | 62.694 | 99.51 |
| Bupropion | 150 | 63 | 62.721 | 99.56 |
| Bupropion | 150 | 63 | 62.549 | 99.28 |
| Dextromethorphan | 50 | 9 | 8.947 | 99.41 |
| Dextromethorphan | 50 | 9 | 8.925 | 99.17 |
| Dextromethorphan | 50 | 9 | 8.943 | 99.37 |
| Dextromethorphan | 100 | 18 | 18.045 | 100.25 |
| Dextromethorphan | 100 | 18 | 17.838 | 99.10 |
| Dextromethorphan | 100 | 18 | 17.962 | 99.79 |
| Dextromethorphan | 150 | 27 | 26.927 | 99.73 |
| Dextromethorphan | 150 | 27 | 26.859 | 99.48 |
| Dextromethorphan | 150 | 27 | 27.021 | 100.08 |
| Degradation condition | Bupropion area | Recovered (%) | Degraded (%) | Dextromethorphan area | Recovered (%) | Degraded (%) |
|---|---|---|---|---|---|---|
| Acid | 2,139,445 | 95.28 | 4.72 | 841,267 | 94.57 | 5.43 |
| Base | 2,141,514 | 95.37 | 4.63 | 843,054 | 94.77 | 5.23 |
| Peroxide | 2,130,109 | 94.86 | 5.14 | 832,870 | 93.63 | 6.37 |
| Thermal | 2,178,882 | 97.04 | 2.96 | 863,392 | 97.06 | 2.94 |
| UV | 2,192,287 | 97.63 | 2.37 | 872,283 | 98.06 | 1.94 |
| Water | 2,226,600 | 99.16 | 0.84 | 882,983 | 99.26 | 0.74 |
CONCLUSION
The findings of the RP HPLC estimation of bupropion and dextromethorphan were within the acceptable range. The suggested method was shown to be easy, quick, accurate, precise, and dependable for the concurrent estimate of bupropion besides dextromethorphan in a form of combination dosage. The approach can be successfully employed for routine analysis and control of quality for bupropion and dextromethorphan in dosage forms for medications, provided that the assay results and validation parameters are suitable.
