Method development
A series of experiments conducted to purify a compound called LDT and separate it from its impurities. The methods used in these experiments include reversed phase chromatography, UV-Vis spectroscopy, solubility testing, and in different HPLC columns. In the reversed phase chromatography experiments, the stationary phase of the column was coated with a hydrophobic material and the mobile phase was a polar solvent. The compounds of interest were retained on the stationary phase based on their hydrophobicity, with more hydrophobic compounds being retained for longer periods of time. This method was used to successfully separate LDT-Impurity-A, LDT-Impurity-B, LDT-Impurity-C, LDT-Impurity-D, and LDT-Impurity-E impurities. The chemical structures of LDT, LDT-Impurity-A, LDT-Impurity-B, LDT-Impurity-C, LDT-Impurity-D, and LDT-Impurity-E shown as Scheme 1. UV-Vis spectroscopy was used to identify and quantify the compounds in the mixture. The absorbance of a compound at a specific wavelength can be used to determine its concentration in a solution. The LDT and its known impurities all showed absorbance at 260 nm (Fig. 1) when dissolved in methanol, suggesting that they contain functional groups that absorb UV light at this wavelength.
The solubility of LDT was tested in various solvents and at different pH values. The highest solubility was found in dimethyl sulfoxide at 1000 mg mL-1, followed by methanol at 33.3 mg mL-1. The solubility of LDT was also highest at pH 1.2 at 100 mg mL-1, while at other pH values the solubility was much lower. Methanol was chosen as the solvent for LDT due to its higher solubility compared to other solvents. 10:90 v/v ratio of water: methanol was used as a diluent to remove interference from the blank. The pKa of LDT was determined to be 7.99, and various buffers with different pH values tested for their ability to separate LDT from its impurities. The optimal pH for separating LDT from its impurities was found to be around 7.2, using a buffer solution of di potassium hydrogen phosphate and diluted orthophosphoric acid. Finally, various types of columns tested for their ability to separate LDT and its known impurities. The columns tested included Eclipse XDB C8, Primasil C18, Inertsil ODS 3V, and Zorbax SB-phenyl. The results showed that the Primasil C18 and Eclipse XDB C8 columns were the most effective at separating the impurities from LDT. Overall, these experiments demonstrated the effectiveness of using a combination of reversed phase chromatography, UV-Vis spectroscopy, solubility testing, and various HPLC columns for the separation and purification of LDT from its impurities.
Method Validation The method was validated according to ICH guidelines, which are a set of internationally recognized guidelines for the validation of analytical methods. As part of the method validation process, several tests were conducted to ensure that the method was specific, sensitive, precise, linear, and accurate. These tests are designed to ensure that the method is suitable for its intended use and that it can reliably measure the concentration of LDT and its impurities in samples.
The system suitability tests were performed by injecting known concentrations of LDT (1 µg mL-1) into the analytical system and calculating the relative standard deviation (% RSD) of diluted LDT. The % RSD was found to be below 10%, which is within the acceptance criteria. Table 1 and Fig. 2 summarize the results of the system suitability tests and show a typical chromatogram, respectively. Specificity was tested by spiking known concentrations of LDT impurities (1.5 µg mL-1 ) into a sample of LDT (1 mg mL-1) and injecting the sample into the system. No peak interference was observed at the retention time of the analyte or impurities in the blank solution, and the purity angle was found to be less than the purity threshold in the spiked sample. Table 2 and Fig. 3 summarize the results of the tests and show a typical chromatogram, respectively.
The limit of detection (LOD) and limit of quantitation (LOQ) were determined using the signal-to-noise ratio method. The LOD was found to be 0.14 µg mL-1 for the impurities and 0.09 µg mL-1 for LDT, while the LOQ was found to be 0.45 µg mL-1 for the impurities and 0.30 µg mL-1 for LDT. The precision of the LOQ was tested by injecting multiple preparations of the LOQ solution and calculating the % RSD, which was found to be less than 15%. Table 2 and Fig. 4 summarize the results of the tests and show a typical chromatogram, respectively. The recovery of LDT and its impurities was also tested by spiking known concentrations of the impurities into a sample of LDT and measuring the recovery. The recovery was found to be between 80% and 120%, which is within the acceptance criteria. The results shown in Table 3.
The linearity was tested by changing the concentrations of LDT and its impurities from 0.45 µg mL-1 to 2.25 µg mL-1. The correlation coefficient and percent Y-intercept at 100% response values were calculated and found to be 0.999 for LDT and its impurities. The intercept and slope values for LDT and its impurities were also calculated. Precision was tested by injecting multiple preparations of LDT (1 mg mL-1) and its impurities at different concentrations (1.5 µg mL-1) and calculating the % RSD. The % RSD was found to be less than 10% for all concentrations, indicating that the method was precise (Fig. 5). Accuracy was tested by measuring the recovery of LDT and its impurities at different concentrations. The recovery was found to be between 80% and 120% for all concentrations, indicating that the method was accurate. Table 3 summarize the results of the tests respectively.
Ruggedness is the ability of an analytical method to produce consistent results despite variations in certain conditions, such as the analyst performing the test or the day on which the test is conducted. In this case, ruggedness was tested by conducting analyst-to-analyst variability, day-to-day variability, using different HPLC systems, using different HPLC columns on different days, and with different analysts. The % RSD was calculated for each impurity in these conditions, along with the cumulative % RSD. The % RSD was found to be below 10 in all conditions, indicating that the method is rugged. Robustness is the ability of an analytical method to produce consistent results despite variations in certain parameters, such as temperature or pH. In this case, the robustness of the method was tested by changing the flow rate, column oven temperature, and pH. The flow rate was changed from 1.0 mL/min to 1.2 mL/min and 0.8 mL/min, the column oven temperature was changed from 45°C to 40°C and 50°C, and the buffer pH was changed from 7.2 to 7.0 and 7.4. The system suitability and % RSD were evaluated in these conditions, and the results showed that the system suitability complied and there was no significant change in the % RSD of LDT and its impurities. These results indicate that the method is robust. Table 4 and Table 5 summarize the results of the tests of ruggedness and robustness respectively.
Overall, these results indicate that the analytical method used to measure the concentration of LDT and its impurities in a sample is specific, sensitive, precise, linear, and accurate, and therefore it can used for the regular analysis.
Degradation effects Forced degradation is the deliberate exposure of a sample to conditions that can cause partial degradation of the sample. Forced degradation studies are performed to show that an analytical method is suitable for analyzing degraded samples, and to provide information about the conditions in which the sample is unstable. This information can be used to design formulations that can avoid potential instabilities. In this case, the LDT sample subjected to various forced degradation conditions to partially degrade the drug. The degradation conditions included treatment with 5 N HCl solution (acid degradation, Fig. 6), 1 N NaOH solution (alkali degradation, Fig. 7), 5% H2O2 (oxidation, Fig. 8), water (hydrolysis degradation), exposure to heat at 105°C (thermal degradation), exposure to UV light at 254 nm (photolytic degradation), and exposure to 75% relative humidity (hygroscopicity). The as such LDT chromatogram shown in Fig. 9. Samples were prepared at concentrations of 1000 µg mL-1 and 200 µg mL-1 for related substances and assay by HPLC, respectively. The results showed that degradation was observed up to 6% in acidic degradation, 3% in alkali, oxidation, hydrolytic, and humidity conditions, and 2% in photolytic degradation conditions.. It is important to understand the conditions under which a sample is unstable, as this information can be used to design formulations that can minimize or prevent degradation. In addition, it is important to have analytical methods that are suitable for analyzing degraded samples, as this is.
LCMS Study LC-MS analysis performed using two mobile phases: mobile phase A was 0.1% v/v formic acid in water, and mobile phase B was a mixture of acetonitrile and water (90:10 v/v). A 1000 µg mL-1 LDT sample was prepared and injected into the LC-MS system in a volume of 10 µL. The results of the LC-MS analysis are summarized in Table 8, along with the identified mass numbers. A typical LC-MS chromatogram for acid hydrolysis is shown in Fig. 10, LC-MS chromatogram for base hydrolysis is shown in Fig. 11, LC-MS chromatogram for oxidation is shown in Fig. 12. The plausible chemical structures of degradant impurities shown in Scheme 2.
Assay By Hplc
An assay is a method used to determine the concentration or amount of a substance in a sample. In this case, an HPLC method was developed and validated for the assay of LDT and its related impurities. The method used a di potassium hydrogen phosphate buffer and solvent methanol to separate and detect the analytes. The buffer pH was adjusted to 7.2 with diluted orthophosphoric acid, which was used as solvent A. A mixture of methanol and water was used as solvent B. The Inertsil ODS-3V column (250 x 4.6 mm, 5 µm) and a detection wavelength of 260 nm were used to analyze the sample. A 200 µg mL− 1 sample was prepared in a diluent of methanol and water (1:1 v/v) and injected into the chromatographic instrument. The developed method was validated according to the ICH guidelines. The % RSD was found to be 0.2 for five repeated injections, the correlation coefficient was 0.999 for linearity, and the recovery was found to be between 98% and 102%. These results summarized in Table 6, and a typical chromatogram and linearity graph is shown in Fig. 13 and Fig. 14.
