Development of Analytical Methods and Estimation of the Bulk and Pharmaceutical Dosage Form of Etodolac using UV-Visible Spectrophotometer

Etodolac is a non-steroidal anti-inflammatory drug (NSAID) belonging to the pyranocarboxylic acid class, widely prescribed for the management of pain, inflammation, and stiffness associated with rheumatoid arthritis, osteoarthritis, and other musculoskeletal disorders [1]. It exerts its therapeutic effect primarily by inhibiting the cyclooxygenase (COX) enzymes, thereby reducing the synthesis of prostaglandins responsible for pain and inflammation [2]. Unlike some other NSAIDs, Etodolac exhibits relative COX-2 selectivity, which may contribute to a lower incidence of gastrointestinal side effects [3].

Chemically, Etodolac is (±)-2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetic acid, a weakly acidic compound that is practically insoluble in water but freely soluble in organic solvents such as methanol and water (70:30) and ethanol [4]. It is officially listed in major pharmacopoeias including the British Pharmacopoeia (BP) and United States Pharmacopoeia (USP), where its assay is typically carried out using titrimetric or chromatographic methods [5].

A comprehensive literature survey reveals that several analytical methods have been reported for the quantitative estimation of Etodolac in bulk drug and pharmaceutical formulations. These include spectrophotometric [6–8], high-performance liquid chromatographic (HPLC) [9,10], and liquid chromatography–mass spectrometric (LC–MS) techniques [11]. However, chromatographic methods are often time-consuming, require expensive instrumentation, and involve complex mobile phases, limiting their routine application in quality control laboratories.

In contrast, UV–Visible spectrophotometric methods offer simplicity, cost-effectiveness, sensitivity, and reliability for drug analysis, particularly when the compound exhibits a well-defined absorption maximum in the UV range [12]. Therefore, the development of a simple, accurate, and economical UV–Visible spectrophotometric method for the estimation of Etodolac in bulk and pharmaceutical dosage forms is of significant analytical and industrial interest.

The objective of the present work was to develop and validate a rapid, precise, and reproducible UV–Visible spectrophotometric method for the quantitative determination of Etodolac in bulk drug and tablet dosage form as per ICH Q2(R1) guidelines.

UV–Visible Spectrophotometric Method

UV–Visible spectrophotometry is a widely employed analytical technique based on the measurement of light absorption by a substance as a function of wavelength. It is extensively used for the qualitative and quantitative estimation of analytes that possess chromophoric groups capable of absorbing ultraviolet or visible radiation [4]. The technique is founded on the Beer–Lambert law, which states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the sample cell.

Compared to other instrumental methods such as HPLC or LC–MS, UV–Visible spectrophotometry offers several advantages — it is simple, rapid, cost-effective, and precise, making it particularly suitable for routine analysis in quality control laboratories [5]. While chromatographic methods have been previously reported for the determination of Etodolac, only a limited number of studies have focused on developing an optimized and validated UV–Visible spectrophotometric method for its estimation in bulk and pharmaceutical dosage forms [6].

The present work focuses on the development and validation of a reliable and economical UV–Visible spectrophotometric method for the estimation of Etodolac in bulk and tablet formulations. The method was designed and validated in accordance with the International Council for Harmonisation (ICH) guidelines Q2(R1) for analytical method validation parameters such as linearity, precision, accuracy, and sensitivity [7].

Distilled water was selected as the solvent after preliminary solubility and absorbance trials. The choice of solvent was based on factors such as sensitivity of the method, solubility of the drug, cost-effectiveness, and environmental safety. The λmax of Etodolac was determined by scanning the standard drug solution in the UV range (200–400 nm) against a solvent blank, and it was found to exhibit maximum absorbance at 224.6 nm. This wavelength was selected for all subsequent analytical measurements.

MATERIALS AND METHODS

Materials

All chemicals and reagents used were of analytical or HPLC grade. Etodolac was used as the API. Methanol (analytical grade) and distilled water were used as solvents for the preparation of standard and sample solutions. Pharmaceutical-grade excipients were procured from an authorized research laboratory.

Apparatus

A Shimadzu UV–Visible double-beam spectrophotometer (Model UV Mini-1900, Shimadzu Corporation, Kyoto, Japan) equipped with matched quartz cuvettes of 1 cm path length was used for all absorbance measurements. All spectral analyses were performed using the instrument’s software in both spectrum and photometric modes as appropriate.

Determination of Wavelength of Maximum Absorption (λmax)

A stock solution of Etodolac was prepared by dissolving 10 mg of the drug in 100 ml of methanol and water (70:30) to obtain a concentration of 100 µg/ml. From this stock, 1 ml was further diluted to 10 ml with methanol and water (70:30) to prepare a clear and stable 10 µg/ml working solution. The working solution was scanned in the wavelength range of 200–400 nm against a methanol and water (70:30) blank using a UV–Visible spectrophotometer. The obtained spectrum showed a distinct absorption maximum (λmax) at 224.6 nm, which was used for all subsequent analytical measurements. [8].

Assay Determination

An accurately weighed quantity of Etodolac equivalent to 100 mg was transferred to a 100 ml volumetric flask. The drug was dissolved in approximately 10 ml of methanol and water (70:30) with gentle shaking, and the volume was made up to the mark using methanol and water (70:30) to obtain a clear stock solution (1000 µg/ml). The solution was sonicated for 5 minutes to ensure complete dissolution and then filtered through Whatman No. 40 filter paper. Suitable aliquots of this stock solution were further diluted with methanol and water (70:30) to obtain a final concentration of 10 µg/ml. The absorbance of the resulting solution was measured at 224.6 nm using methanol and water (70:30) as the blank. The percentage assay of Etodolac in the sample was calculated using the standard calibration curve (Table 1) [9].

Validation Parameters

Linearity

Linearity was evaluated by preparing a series of Etodolac standard solutions in the concentration range of 5–35 µg/ml. The absorbance of each solution was measured at 224.6 nm, and a calibration curve was plotted between absorbance and concentration. The method showed a linear relationship in accordance with Beer–Lambert’s law, and regression analysis was used to determine the correlation coefficient (R²).

Repeatability

Repeatability was assessed by preparing six replicates of 15 µg/ml Etodolac solution. The absorbance of each replicate was measured, and the mean, standard deviation (SD), and percentage relative standard deviation (%RSD) were calculated to evaluate the repeatability of the method within the linearity range.

Accuracy (Recovery Studies)

Accuracy was determined by performing recovery studies at three concentration levels: 80%, 100%, and 120%. A pre-analyzed tablet sample equivalent to 15 µg/ml of Etodolac was spiked with pure drug to obtain final concentrations of 12 µg/ml, 15 µg/ml, and 18 µg/ml, respectively. Each concentration was analyzed in triplicate, and the percentage recovery was calculated to assess the accuracy of the method [12].

Precision

Precision was evaluated by conducting both intra-day and inter-day studies. For intra-day precision, three concentrations (10 µg/ml, 15 µg/ml, and 20 µg/ml) were analyzed three times within the same day (morning, afternoon, and evening). For inter-day precision, the same concentrations were analyzed over three consecutive days. The precision of the method was expressed in terms of %RSD [13].

Ruggedness

Ruggedness was determined by analyzing six replicates of 15 µg/ml Etodolac solution under different conditions — by two different analysts and using two different spectrophotometers. The %RSD values were compared to confirm the ruggedness and reproducibility of the proposed method [14].

Limit of Detection (LOD)

The Limit of Detection (LOD) was established based on the standard deviation of the response and the slope of the calibration curve. It represents the lowest concentration of Etodolac that can be detected but not necessarily quantified with accuracy.

Limit of Quantitation (LOQ)

The Limit of Quantitation (LOQ) was determined from the standard deviation of the response and the slope of the calibration curve. It represents the minimum concentration of Etodolac that can be quantitatively determined with acceptable precision and accuracy in accordance with ICH Q2(R1) guidelines.

RESULTS AND DISCUSSION

Etodolac is a non-steroidal anti-inflammatory drug (NSAID) widely prescribed for the treatment of pain and inflammation. In the present work, a simple, precise, and cost-effective UV–Visible spectrophotometric method was developed and validated for the quantitative estimation of Etodolac in bulk and pharmaceutical dosage forms.

The method was optimized using distilled water as the solvent, and the absorbance maximum (λmax) of Etodolac was found to be 224.6 nm. The proposed method was found to be rapid, sensitive, accurate, and reliable, making it suitable for routine quality control analysis of Etodolac formulations. All validation parameters were evaluated as per ICH Q2(R1) guidelines, and the results are summarized in Table 1.

Table 1: Summary of validation

Selection of Wavelength

The standard solution of Etodolac prepared in methanol and water (70:30) was scanned in the UV range of 200–400 nm using methanol and water (70:30) as the blank. The UV spectrum of Etodolac showed a distinct and sharp absorption peak at 224 nm, corresponding to its maximum absorbance (λmax). This wavelength was selected for all subsequent analytical measurements, as it provides the highest sensitivity with minimal background interference. The obtained spectrum is presented in Figure 1.

Figure 1: Lambda max of Etodolac

Linearity and range

Linearity was evaluated by analyzing Etodolac standard solutions in the concentration range of 5–25 µg/ml, using five calibration levels. The calibration curve showed a strong linear relationship between absorbance and concentration. The correlation coefficient (R² = 0.9955) confirmed excellent linearity within the selected range. The data used for constructing the calibration curve are presented in Table 2, and the corresponding plot is shown in Figure 2.

Table 2: Preparation of Standard Calibration Curve

Figure 2: Calibration Curve of Etodolac

Repeatability

Repeatability was evaluated at a concentration of 15 µg/ml using five replicate measurements. After removal of one clear outlier, the remaining data showed excellent precision. The %RSD of absorbance was 1.11%, demonstrating high repeatability of the method under identical conditions. The detailed data are presented in Table 3.

Table 3: Repeatability of Etodolac

Accuracy

Accuracy was evaluated by the standard addition method at three concentration levels: 80%, 100%, and 120% of the target concentration. Known quantities of pure Etodolac were spiked into pre-analyzed samples, and the percentage recovery was calculated.

The % recovery values obtained at all levels were within acceptable limits, indicating that the method can accurately quantify small variations in drug concentration. The mean recoveries were 99.68% (80%), 91.03% (100%), and 100.40% (120%), with %RSD values below 2%, demonstrating that the analytical method is accurate and reliable. These results confirm that the proposed UV spectrophotometric method provides accurate quantification of Etodolac in bulk and formulations. The complete accuracy data are presented in Table 4, and the graphical representation is shown in Figure 3.

Figure 3: Accuracy Readings of Etodolac

Table 4: Accuracy Readings of Etodolac

The precision of the analytical method specifies, under recommended conditions, the degree of dispersion between a set of measurements obtained from multiple samples of the same homogeneous sample. Intraday precision refers to short use of the analytical technique within a laboratory using the same operator and the same equipment, while Interday precision involves the estimation of analysis differences when different analysts use a method inside a laboratory on different days. The developed method's intra-day and the inter-day precision study confirmed adequate sample stability and method reliability where all the % RSDs were less than 2% (Table 5, 6) (Figure 4, 5).

Table 5: Precision (Intraday) readings of Etodolac

Figure 4: Precision (Intraday) readings of Etodolac

Table 6: Precision (Interday) analyses of Etodolac

Figure 5: Precision (Interday) analyses of Etodolac

Ruggedness

Ruggedness refers to the degree of reproducibility of test results obtained under a variety of normal test conditions, such as different analysts, laboratories, instruments, or environmental conditions. A rugged analytical method demonstrates minimal influence from such variations. To assess method ruggedness, Etodolac standard solution (15 µg/ml) was analysed independently by two analysts following the same UV spectrophotometric procedure. The %RSD values for Analyst-1 and Analyst-2 were found to be 1.7081% and 1.9489%, respectively. Both values being less than 2% indicate that the proposed method is rugged, producing consistent results despite variations in analysts (Table 7, Figure 6).

Table 7: Results Showing Ruggedness of Method for Etodolac

Table 6: Ruggedness of Method for Etodolac