Q-Absorbance ratio and Chromatographic Method for the Analysis of Aspirin and Salicylic acid

Two simple, specific, precise and accurate methods were developed for the analysis of Aspirin (ASA) in bulk and formulation forms in presence of its degradation product, salicylic acid (SA). The first method was based on determining Q-Absorbance ratios for aspirin and salicylic acid at two wavelengths; 251.4 nm (iso–absorptive point, 1) and 275.6 nm (max of ASA, 2) and obtaining their absorptivity values which form the basis for their concentrations calculations. Beer’s law was obeyed in the concentration range 50-450μg/ml and 10-90μg/ml for ASA and SA, respectively, with correlation coefficient not less than 0.999. The second method was based on development of stabilityindicating chromatographic method (HPLC), reliable for the assay of aspirin in presence of salicylic acid. The stability-indicating property of the developed method was achieved on a C18 column (250*4.6mm) using a mixture of methanol and 1% (v/v) acetic acid aqueous solution (60:40 v/v) as mobile phase at 1ml/min flow rate and a UV detector set at 298nm. The constructed calibration curves were linear in the concentration range 1.05.0mg/ml and 30-270μg/ml for ASA and SA, respectively. The developed methods were optimized and validated in accordance to ICH guidelines. The validated methods were applied for the assay of aspirin in five brands low dose formulations as well as to identify and quantify any salicylic acid present using standard reference materials. © 2020 Elixir All rights reserved. Elixir Appl. Chem. 144 (2020) 54583-54588 Applied Chemistry Available online at www.elixirpublishers. com (Elixir International Journal) Nafisa A. Osman et al./ Elixir Appl. Chem. 144 (2020) 54583-54588 54584 Materials and Methods Reagents Aspirin CDH (Central drug house (P) ltd., 99%w/w), India; Salicylic acid CDH (Central drug house (P) ltd., 99%w/w), India; Aspirin tablets (five brands) were collected from the local market; Methanol PRE, CARLO ERBA, S.A. S. ; Methanol RS HPLC, CARLO ERBA, S.A. S. ; Glacial acetic acid CDH (Central drug house ltd.), made in India. Instrumentation and Analytical conditions UV–VIS spectrophotometer instrument (UV–1800 240V, Shimadzu, Japan). HPLC chromatograph, LC-20AD Solvent delivery unit, DGU-20A3 Degasser, Rheodyne 7725i Sample injector, VP-ODS Column C18 250*4.6mm, SPD-20A UV/VIS Detector, Shimadzu, Japan). Preparation of Sample and Standard solutions Q-Absorbance ratio method Standard stock solutions Accurate quantities of ASA and SA standards were weighed, dissolved separately in methanol to obtain (solution A; 500μg/ml ASA, solution B; 2500μg/ml ASA, solution C; 500μg/ml SA). Sample solution Twenty tablets from each brand were accurately weighed and then powdered. To a quantity of the powder containing 0.3g of aspirin, 60 ml of methanol were added. The solution was shaken for 10 minutes, completed to 100 ml, mixed and filtered. 5 ml of the filtrate were diluted to 50 ml with methanol (solution D; 300μg/ml). HPLC method Standard stock solutions Accurate quantities of ASA and SA standards were weighed, dissolved separately in a mixture of 60 volumes of methanol and 40 volumes of 1% (v/v) acetic acid aqueous solution to obtain (solution E; 25000μg/ml ASA, solution F; 1500μg/ml SA). Sample solution Twenty tablets were accurately weighed and then powdered. To a quantity of the powder containing 0.3 g of aspirin, 60 ml of methanol were added. The solution was shaken for 10 minutes, completed with 1% acetic acid aqueous solution to 100 ml, mixed and filtered through 0. 45μm membrane filter (solution G; 3000μg/ml). Procedures Methods development Q–Absorbance ratio method Determination of iso–absorptive point and wavelength of maximum absorbance In order to specify the iso–absorptive point and wavelength of maximum absorbance (λmax) of ASA, six solutions of ASA and SA standards were prepared from standard stock solutions (A&C) as follows: ASA 100 g/ml, (ASA 80g/ml + SA 20g/ml), (ASA 60g/ml + SA 40g/ml), (ASA 40g/ml + SA 60g/ml), (ASA 20g/ml + SA 80g/ml) and SA 100 g/ml. All spectra were then recorded and their overlay graph was examined to determine the iso–absorptive points. HPLC method System suitability In order to obtain a column/mobile phase system that would be suitable for the quantification of ASA, different mobile phase systems with different compositions and polarities were examined. Mobile phase system of 60 volumes of methanol to 40 volumes of 1% acetic acid aqueous solution was selected to get the best chromatographic resolution of ASA and SA in an isocratic elution. The detector was set at wavelength of 298 nm after examining the overlay spectra of ASA and SA, as it gives suitable ASA response and maximizes SA response in mixture of different concentration of both. Flow rate was of 1 ml/min and the injection volume was 20L. System suitability tests as tailing factor, retention factor, separation factor, number of theoretical plates and resolution were then studied. Methods validation Calibration curves and linearity data Q–Absorbance ratio method Series of concentrations of standard ASA (50–450g/ml) and SA (10–90g/ml) were prepared from standard stock solutions (B&C) and spectra were recorded between 400 to 200 nm. Calibration curves of ASA and SA were then constructed by plotting the absorbance at selected wavelengths (251.4nm &275.6nm) versus concentration in order to determine the absorptivity values and the validation parameters. HPLC method Serial concentrations of ASA (1.0–5.0mg/ml) and SA (30–270g/ml) were prepared from standard stock solutions (E&F). Solutions were injected into the column at room temperature and their peak areas were measured. Calibration curves were then constructed, by plotting the peak areas versus corresponding concentrations, and validation parameters were determined. Precision and Accuracy The precision of the developed methods was evaluated by determining the within-day and between-day data were for three concentrations within the linearity range. The relative standard deviation (RSD) values were then calculated. The accuracy of the methods was assessed by comparing the results obtained with the official method and calculating the tvalue at 95% confidence level [36]. Assay of ASA tablet formulations Q–Absorbance ratio method The absorbance values of solution D were measured at 275.6 nm (max of ASA) and 251.4 nm (iso–absorptive point) where absorptivity values were previously determined under calibration curve. The concentrations of ASA and SA in sample solution were then calculated using the Q–Absorbance ratio method formula [37]: Where the absorptivities of X at  &  are & , the absorptivities of Y at  &  are & , the absorbance of a sample at  &  are & and the concentration of X & Y are & , respectively. HPLC method Twenty μl volumes from solutions G were injected into the column at room temperature and their peak areas were measured. Nafisa A. Osman et al./ Elixir Appl. Chem. 144 (2020) 54583-54588 54585 Results Q–Absorbance ratio method Determination of maximum wavelength and the iso-