Optimization and Validation of a Stability-Indicating RP-HPLC Method for Dual Drug Analysis of Aspirin and Omeprazole

1. Introduction

In contemporary pharmaceutical analysis, the concurrent quantification of multiple active ingredients in fixed-dose combinations has become increasingly important because such products provide better therapeutic outcomes, greater patient adherence to therapy, and fewer daily doses [1,2,3,4,5]. A particularly relevant example is the pairing of Acetylsalicylic acid with Omeprazole, which is frequently prescribed for individuals needing extended antiplatelet treatment along with protection against gastric injury.

Acetylsalicylic acid, belonging to the non-steroidal anti-inflammatory drug (NSAID) class, is widely utilized for its pain-relieving, fever-reducing, anti-inflammatory, and blood-thinning effects. It serves as a fundamental treatment for preventing cardiovascular incidents including heart attacks and strokes [6,7,8,9]. Nevertheless, long-term intake of Acetylsalicylic acid often leads to notable gastrointestinal complications such as mucosal damage, peptic ulcers, and gastric bleeding due to its suppression of prostaglandin synthesis. To counteract these harmful effects, Omeprazole—a proton pump inhibitor (PPI)—is frequently given alongside Acetylsalicylic acid. Omeprazole works by irreversibly blocking the H⁺/K⁺-ATPase enzyme in the stomach’s acid-producing cells, thereby reducing gastric acid secretion and shielding the stomach lining from damage induced by Acetylsalicylic acid.

Given the widespread clinical application of this two-drug regimen, creating a trustworthy, precise, and responsive analytical technique for their simultaneous determination holds significant pharmaceutical and regulatory value. Such a method is necessary for product quality testing, bioavailability assessments, and pharmacokinetic investigations. High-performance liquid chromatography (HPLC) continues to be the most preferred analytical tool in pharmaceutical testing owing to its excellent selectivity, sensitivity, precision, and potential for automation. Reverse-phase HPLC (RP-HPLC) is especially common for analyzing drugs with different polarity levels within complex mixtures [10,11,12,13,14].

A stability-indicating analytical procedure is a validated method that can accurately and exclusively quantify the active pharmaceutical ingredient (API) in the presence of its decomposition products, manufacturing impurities, and formulation excipients [15,16,17]. Developing such methods is a regulatory necessity according to International Council for Harmonisation (ICH) guidelines Q1A(R2) and Q2(R1), which require that the analytical technique must prove its capacity to distinguish the unchanged drug from its breakdown products formed under forced degradation conditions, including exposure to acid, alkali, oxidizing agents, light, heat, and reducing agents.

Although several analytical methods have been reported for measuring Acetylsalicylic acid and Omeprazole separately, few validated stability-indicating RP-HPLC methods exist for their simultaneous analysis. The currently available procedures often involve prolonged run times, complex mobile phase mixtures, or inadequate validation according to modern ICH standards. Consequently, this research aimed to create, refine, and validate a straightforward, fast, accurate, and stability-indicating RP-HPLC method for the concurrent estimation of Acetylsalicylic acid and Omeprazole in pharmaceutical formulations, adhering to ICH Q2(R1) recommendations. The method was further challenged with forced degradation experiments to confirm its stability-indicating capability, making it suitable for routine quality control operations in pharmaceutical laboratories.

2. Methodology

2.1. Instrumentation and Chemicals Used in the Present Work

An RP-HPLC technique was established and validated for the simultaneous measurement of Acetylsalicylic acid and Omeprazole in drug products. The analysis employed a Waters Alliance HPLC system fitted with a PDA detector. Chromatographic separation was achieved using a Luna Phenyl Hexyl column (250 × 4.6 mm, 5 µm particle diameter). The mobile phase comprised Acetonitrile and 0.1% Perchloric acid in a 20:80 (v/v) ratio, which was filtered using a 0.45 µm membrane filter and degassed before use. The flow rate was set at 1.0 mL/min, and the detection wavelength was fixed at 249 nm, chosen as the isosbestic point for the two drugs. The injection volume was 10 µL, and the total analysis time was 5 minutes. The equipment and reagents employed are summarized in Table 1 and Table 2.

2.2. Selection of Detection Wavelength and Preparation of Solutions

Standard solutions of Acetylsalicylic acid and Omeprazole were scanned across the 200–400 nm range using a PDA detector. An isosbestic point at 249 nm was observed for both drugs, indicating equal absorbance at this wavelength. Hence, 249 nm was selected for simultaneous measurement. Precisely weighed quantities of 81 mg Acetylsalicylic acid and 40 mg Omeprazole were placed into a 10 mL volumetric flask, dissolved in diluent (acetonitrile), sonicated, and diluted to the mark. Further dilution produced final concentrations of 81 µg/mL for Acetylsalicylic acid and 40 µg/mL for Omeprazole. A sample corresponding to 20 mg of each drug was accurately weighed, transferred into a 10 mL volumetric flask, sonicated for 30 minutes, and diluted to volume with diluent. Additional dilution and filtration through a 0.45 µm filter were carried out before injection. Several experimental runs were performed by modifying chromatographic parameters such as mobile phase composition, column type, and flow rate. Among these, Trial-6 proved optimal, yielding well-separated peaks with satisfactory system suitability parameters including resolution, asymmetric factor, and theoretical plate number.2.3. Method optimization and system suitability

Several trials were conducted by varying chromatographic conditions such as mobile phase composition, column type, and flow rate. Among the trials, Trial-6 was found to be optimal, providing well-resolved peaks with acceptable system suitability parameters including resolution, tailing factor, and plate count as shown in Table 3.

System suitability was evaluated by injecting standard solutions and assessing parameters such as retention time, theoretical plates, tailing factor, and resolution. The acceptance criteria were a tailing factor less than 2, plate count greater than 2000, and resolution greater than 2.

2.3. Method Validation

The developed method was validated according to ICH criteria for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ. Linearity was examined by preparing six different concentration levels of each drug. A calibration graph was constructed by plotting concentration against peak areas. Accuracy was assessed using the standard addition approach at three levels (80%, 100%, and 120%). Percentage recovery was calculated to evaluate accuracy. Precision was studied as system precision, method precision, and intermediate precision. Six replicate injections were analyzed, and %RSD was determined. Robustness was tested by introducing intentional changes in chromatographic conditions including flow rate and mobile phase composition. The limit of detection (LOD) and limit of quantification (LOQ) were computed using Equation 1 and 2.

$$LOD={\displaystyle \frac{3.3\times \sigma }{S}}$$

(1)

$$LOQ={\displaystyle \frac{10\times \sigma }{S}}$$

(2)

Where $\sigma$ is standard deviation of response and $S$ is slope of calibration curve. The percentage assay of the sample was calculated using Equation 3.

$$\%Assay={\displaystyle \frac{AT}{AS}}\times {\displaystyle \frac{WS}{DS}}\times {\displaystyle \frac{DT}{WT}}\times {\displaystyle \frac{P}{LC}}\times 100$$

(3)

Where $AT$ is area of test sample, $AS$ is area of standard, $WS$ is weight of standard, $DS$ is dilution of standard, $DT$ is dilution of sample, $WT$ is weight of sample, $P$ is purity of standard, and LC is label claim.

3. Results and Discussion

An RP-HPLC method was established for the simultaneous determination of Acetylsalicylic acid and Omeprazole by assessing various chromatographic parameters. Several trials (Trial 1 to Trial 6) were carried out by modifying mobile phase composition, column type, and detection wavelength. Among these, Trial-6 was chosen as the optimized method because of its superior chromatographic performance. The final optimized chromatogram (Figure 1) displayed well-resolved and symmetric peaks for both compounds. The retention times recorded were 2.038 min for Acetylsalicylic acid and 2.995 min for Omeprazole, indicating fast separation within a short runtime. The resolution between the two peaks was 4.63, significantly above the acceptable limit of 2, confirming effective separation.

The detailed optimized chromatographic conditions are presented in Table 4, and the corresponding results are summarized in Table 5. The system suitability parameters such as tailing factor and plate count were within acceptable limits, indicating that the developed method is efficient and reliable.

System suitability was evaluated by injecting standard solutions, and the outcomes are shown in Table 6. The plate count for Acetylsalicylic acid and Omeprazole was 11485 and 7693, respectively, both exceeding the minimum requirement of 2000. The tailing factors were 0.97 and 1.03, respectively, indicating symmetrical peak shapes. The resolution between the two peaks was 4.63, confirming adequate separation. The %RSD values for peak areas were 0.17% for Acetylsalicylic acid and 0.42% for Omeprazole, both well within the acceptable limit of 2%. These results confirm that the system is appropriate for analysis.

The specificity of the method was assessed by analyzing blank, placebo, and standard solutions. No interfering peaks appeared at the retention times of Acetylsalicylic acid and Omeprazole. This confirms that the method is specific and can accurately measure the analytes in the presence of excipients.

Figure 9. Representative chromatograms showing (a) blank, (b) placebo, and (c) standard solution of Aspirin and Omeprazole, demonstrating the specificity of the RP-HPLC method with no interference observed at the retention times of the analytes.

Figure 9. Representative chromatograms showing (a) blank, (b) placebo, and (c) standard solution of Aspirin and Omeprazole, demonstrating the specificity of the RP-HPLC method with no interference observed at the retention times of the analytes.

System precision was evaluated by six replicate injections of standard solution, and the results are shown in Table 7 and Figure 10. The %RSD values were found to be 0.17% for Aspirin and 0.42% for Omeprazole, indicating excellent repeatability of the instrument.

The system precision of the developed RP-HPLC method was evaluated by performing six replicate injections of the standard solution. The chromatograms (Figure 11) showed consistent retention times of approximately 2.03 min for Aspirin and 2.99 min for Omeprazole with no significant variation in peak shape or area.

The method precision results are presented in Table 8. The %RSD values were 0.55% for Acetylsalicylic acid and 0.75% for Omeprazole, which are within acceptable limits (<2%). Intermediate precision results are shown in Table 9, with %RSD values of 0.38% for Acetylsalicylic acid and 0.69% for Omeprazole. These results confirm that the method is precise under different conditions.

Figure 12. Intermediate precision (inter-day) results showing peak area consistency for (a) Aspirin and (b) Omeprazole over six replicate injections, with %RSD values of 0.38% and 0.69%, respectively, indicating good reproducibility of the RP-HPLC method.

Figure 12. Intermediate precision (inter-day) results showing peak area consistency for (a) Aspirin and (b) Omeprazole over six replicate injections, with %RSD values of 0.38% and 0.69%, respectively, indicating good reproducibility of the RP-HPLC method.

Linearity was evaluated over a concentration range of 20.25–121.50 µg/mL for Aspirin and 10–60 µg/mL for Omeprazole. The calibration curves are shown in Figure 13, and the results are summarized in Table 10. The correlation coefficients (R²) were found to be 0.99983 for Aspirin and 0.99981 for Omeprazole, indicating excellent linearity.

Accuracy was evaluated using the standard addition method at three levels (80%, 100%, and 120%), and the results are presented in Table 11 (Acetylsalicylic acid) and Table 12 (Omeprazole) and Figure 14. The mean percentage recovery was 99.9% for Acetylsalicylic acid and 100.0% for Omeprazole. These values fall within the acceptable range of 98–102%, indicating that the method is accurate.

The assay results for Aspirin and Omeprazole are presented in Figure 15. The percentage assay values were found to be 99.7% for Aspirin and 100.2% for Omeprazole. These results indicate that the method is suitable for quantitative analysis of both drugs in pharmaceutical formulations.

Robustness was evaluated by intentionally varying chromatographic parameters such as flow rate and mobile phase composition. The results are presented in Table 13 (Acetylsalicylic acid) and Table 14 (Omeprazole). The variations did not significantly affect retention time, peak area, or system suitability parameters. The %RSD values remained within acceptable limits, confirming that the method is robust. Representative chromatograms are shown in Figure 16, Figure 17, Figure 18 and Figure 19.

Forced degradation studies were carried out under acid, alkali, peroxide, reduction, thermal, photolytic, and hydrolytic stress conditions to assess the stability-indicating ability of the developed RP-HPLC method. The degradation results for Acetylsalicylic acid and Omeprazole are presented in Table 15 and Figure 20. Under acidic conditions, Acetylsalicylic acid showed 11.9% degradation, whereas Omeprazole showed 1.9% degradation. Under alkaline conditions, degradation was 11.2% for Acetylsalicylic acid and 12.3% for Omeprazole. Oxidative degradation using peroxide resulted in 14.1% degradation for Acetylsalicylic acid and 13.3% for Omeprazole, indicating that both drugs were highly susceptible to oxidative stress. In contrast, reduction, thermal, and photolytic conditions produced relatively low degradation, while hydrolytic stress caused moderate degradation in both analytes.

Peak purity evaluation further confirmed the specificity of the method shown in Figure 21. For all stressed samples, the purity angle was lower than the purity threshold for both Aspirin and Omeprazole, demonstrating that the analyte peaks remained spectrally pure and that degradation products did not co-elute with the principal peaks. For example, in the peroxide degradation study, the purity angle/purity threshold values were 1.605/3.017 for Aspirin and 2.169/3.719 for Omeprazole. Similar results were observed for acid, alkali, reduction, thermal, photolytic, and hydrolytic degradation studies. These findings establish that the developed RP-HPLC method is stability-indicating and suitable for analysis of Aspirin and Omeprazole in the presence of their degradation products.

The sensitivity of the developed RP-HPLC method was evaluated in terms of limit of detection (LOD) and limit of quantification (LOQ), and the results are depicted in Figure 22. The LOD and LOQ values for Aspirin were found to be 0.49 µg/mL and 1.62 µg/mL, respectively, while for Omeprazole, the LOD and LOQ values were 0.24 µg/mL and 0.80 µg/mL, respectively. The chromatograms corresponding to LOD and LOQ are presented in Figure 53 and Figure 54. These results indicate that the developed method is highly sensitive and capable of detecting and quantifying low concentrations of the analytes.

4. Conclusion

In this investigation, a straightforward, fast, reproducible, accurate, and stability-indicating RP-HPLC technique was effectively established, refined, and confirmed for the concurrent quantification of Acetylsalicylic acid and Omeprazole in combined drug products. The separation process was carried out using a Luna Phenyl Hexyl column (250 × 4.6 mm, 5 µm) with a mobile phase composed of Acetonitrile and 0.1% Perchloric acid mixed at 20:80 (v/v), delivered at 1.0 mL/min, and monitored at 249 nm. The overall analysis time of 5 minutes, featuring distinct peaks at 2.038 minutes for Acetylsalicylic acid and 2.995 minutes for Omeprazole, highlights the effectiveness and speed of the proposed procedure.

The technique underwent thorough validation in line with ICH Q2(R1) requirements and produced acceptable outcomes across all validation parameters. Key system suitability indicators—such as resolution (4.63), asymmetry factor, and theoretical plate number—fell well within the prescribed limits, affirming the dependability and consistency of the chromatographic setup. A strong linear relationship between analyte concentration and detector response was observed over a broad concentration range, supported by excellent correlation coefficients for both compounds. Recovery experiments conducted at 80%, 100%, and 120% levels confirmed the method’s accuracy, with mean recovery values lying between 98% and 102%. Precision assessments, covering system repeatability, method repeatability, and intermediate precision, produced %RSD values significantly below 2%, demonstrating outstanding consistency and reproducibility.

Robustness testing through intentional minor adjustments in chromatographic parameters—including flow rate and mobile phase ratio—showed that the method remained unaffected by small variations, confirming its suitability for routine laboratory use. The method’s high sensitivity was evidenced by low LOD and LOQ figures: 0.49 µg/mL and 1.62 µg/mL for Acetylsalicylic acid, and 0.24 µg/mL and 0.80 µg/mL for Omeprazole, respectively, enabling the detection of trace amounts of both substances.

Forced degradation experiments performed under acidic, alkaline, oxidative, reductive, thermal, photolytic, and hydrolytic conditions revealed considerable breakdown of both medications under severe environments, especially during oxidative and alkaline stress. Crucially, for every stress condition examined, the purity angle of both Acetylsalicylic acid and Omeprazole remained consistently lower than their corresponding purity threshold values, confirming the spectral homogeneity and chromatographic peak purity of the analytes. This firmly establishes the stability-indicating nature of the developed approach, proving its capacity to accurately measure the unchanged active ingredients even in the presence of their decomposition products without any chromatographic interference.

Finally, the analysis of a commercially available tablet formulation yielded percentage assay values within pharmacopoeial acceptance limits for both Acetylsalicylic acid and Omeprazole, further confirming the practical utility of this method for everyday quality control testing