Introduction
Erdosteine (ERD), a potent mucolytic agent, shows pharmacological activity by lowering the viscosity of mucus in the respiratory system and thus reducing the ability of the bacteria to adhere to the cellular membrane. ERD has anti-inflammatory properties in the bronchial airways and scavenges free radical compounds from the airways. Chemical structure of ERD consists of thiolactone and carboxyl group. Its chemical formula is 2- [2-oxo-2 - [(2-oxothiolan-3-yl) amino] ethyl] sulfanylacetic acid (Figure 1) (1-3).
Various analytical methods such as UV spectrophotometric method (4-8) and high performance liquid chromatography (9-14) have been found in the literature for the determination of ERD from pharmaceutical preparations and biological fluids. In this study, two spectrophotometric methods were proposed to determine the amount of ERD in pharmaceutical preparations using easily accessible materials and equipment. In the proposed methods, CA and tetracyanoquinodimethane (TCNQ) reagents were reacted with ERD to form charge transfer complexes (CT complexes). For quantitative analysis of pharmaceutical compounds in pharmaceutical dosage forms, the use of CA and TCNQ reagents to obtain CT complexes is preferred because they do not require a buffer, they are fast, precise and cost-effective (15-18). Therefore, in this study, we considered it appropriate to develop two methods for the analysis of ERD using these two markers, and these developed methods were successfully applied in the analysis of ERD in pharmaceutical formulations.
Method
Devices
Spectrophotometric measurements were performed using a Hitachi spectrometer Model U-2900 equipped with a xenon lamp and 1 cm quartz cells.
Reagents and Solutions
ERD was obtained from EnzyChem Lifesciences (Korea), TCNQ Fluka (Neu-Ulm, Germany) and CA Merck (Darmstadt, Germany). The pharmaceutical preparation (ERDOSTIN® 300 mg) was obtained from the pharmacy. All chemicals and reagents were used for analytical purity.
Stock Solutions
Stock solutions of ERD were prepared in methanol to make up 1 mg/mL. Solutions of 0.2% (w/v) for TCNQ and 0.2% (w/v) for CA were prepared in acetonitrile. The solutions were determined to be stable for 1 week at 4 °C.
General Analysis Method
ERD stock solution in volumes of 0.050-2.5 mL and 0.100-3.0 mL was added to 5 mL calibrated flasks for CA and TCNQ methods, respectively. The volume of the stock solutions in each flask was brought to 2.5 mL with acetonitrile for the CA method and 3.0 mL for the TCNQ method, and 0.75 mL CA and 1 mL TCNQ reagents were added to them. The reaction mixture was heated at 80 °C for 5 min for the TCNQ method and then stood at room temperature for 5 min for CA. After the cooling process, it was diluted to 5 mL with methanol and its absorbance was measured against the blank test at 454 and 843 nm for CA and TCNQ methods, respectively. Calibration charts were prepared by measuring the absorbance against the ERD concentration.
Analysis Method for Capsules
The amount equivalent to 300 mg ERD was weighed and dissolved in 125 mL of methanol. Then it was extracted in a mechanical mixer for 20 minutes and in an ultrasonic bath for 20 minutes. The volume was made up to 250 mL and then filtered through filter paper. The filtrate was diluted with methanol and studied as in the preparation of the calibration curve. The amount of substance in the capsule was measured using the calibration graph and the corresponding regression equation.
Results
The maximum absorption of the CT complexes obtained as a result of the reaction formed by ERD with CA and TCNQ reagents was observed at 454 and 843 nm, respectively (Figure 2).
Optimum conditions such as reaction time, temperature, type of solvent, amount of reagents used and reaction stoichiometry were also investigated for the reaction, which were explained in detail below.
Choosing the Most Suitable Solvent
Various solvents commonly used in analytical procedures including acetonitrile, chloroform, methanol, acetone, ethanol, 1,4-dioxane and methylene chloride were used to determine the most suitable solvent. It was observed that the most suitable solvent was obtained by using methanol.
Reagent Concentration
The optimum reagent concentration was investigated by changing the concentrations of TCNQ and CA reagents and keeping the ERD concentration constant. As shown in the figure, the optimum reagent amount was 0.75 mL CA [0.2% (w/v)] and 1.0 mL TCNQ [0.2% (w/v)] (Figure 3).
Reaction Time
The time required to complete the reaction between ERD and CA and TCNQ was studied spectrophotometrically at room temperature and 60-80 °C, respectively. A reproducible color development was achieved in 5 minutes for CA and TCNQ, respectively, at room temperature and 80 °C. The color reaction resulting from the CT complexes was observed stably for 12 hours (Figure 4).
Reaction Stoichiometry
Job’s continuous change method was used for the reaction stoichiometry (19). According to the results, the equivalent molarity of ERD and reagents was defined as the 1:1 ratio (compound/reagent).
Method Validation
The proposed analytical methods were validated according to the ICH guideline Q2 (R1) (20). Calibration curves were generated for all methods under the above conditions. Regression equation, correlation coefficients, Beer’s law limits, limit of observability (LOD) and determination limit (LOQ) data for each method are given in Table 1.
According to the results obtained, a linear correlation between 10-500 µg mL-1 and 20-600 µg mL-1 was observed for CA and TCNQ methods, respectively.
The formula of LOD/LOQ = κSDa/b was used to calculate LOD or LOQ. Here the value of is 3 for LOD and 10 for LOQ. SDa indicates the standard deviation of the scale curve intersept and b is the slope. The results are shown in Table 1.
Sensitivity values of intra-day and inter-day were examined at 50, 100 and 500 µg/mL for the TCNQ and CA method (n=5 for each) for 5 consecutive days. The % RSD values for the inter-day precision % and the inter-day precision results for all proposed methods provided good reproducibility. Results are given in Table 2. The accuracy of the developed methods was examined using the standard addition technique. Pure analyte was mixed with standard solutions at 3 different concentration levels on the sample solution and analyzed. The results obtained are presented in Table 3. It was observed that the average recovery percentages calculated were 100.31% for CA and 100.82% for TCNQ, proving the method to be of high accuracy (Table 2). The methods developed were been successfully applied in the analysis of the drug substance in pharmaceutical preparations, and according to these results, no interference from additives and excipients was observed. The results are given in Table 4. Small changes were made to the method developed to test the robustness of the method and the effect of these changes on the method was examined. For this, changes in TCNQ and CA reagent concentrations (%, w/v ±0.05) and reaction times (optimum time ±0.5 min) were made, and when the results were examined in terms of recovery and RSD values, it was observed that there was no significant difference.
Conclusion
As a result, the new spectrophotometric methods developed are very practical and applicable. Not requiring complicated sample preparation processes before hand increases the applicability of the method. The developed methods enable the analysis of ERD in pharmaceutical preparations with high accuracy and precision. This method can be used in routine analysis of the drug.
Peer-review: Externally peer reviewed.
Authorship Contributions
Concept: C.Ö., Design: C.Ö., Data Collection or Processing: C.Ö., D.D., Analysis or Interpretation: C.Ö., D.D., Literature Search: D.D., Writing: C.Ö., D.D.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.