The current method validation was ensured as per Q2 provisions of the ICH.

System suitability test

Six replicates of the standard solution of 20 µg/mL and 10 µg/mL of OLP and SMD were injected consecutively into the UPLC. The various system suitability parameters like percentage relative standard deviation (%RSD), USP plates (N), and USP tailing (T) were evaluated.

Linearity

The linearity represents direct proportionality between the method's peak areas and input concentrations. In the current method, linear graphs were generated for both OLP and SMD between concentrations and peak areas using concentrations ranging from 5 µg/mL to 30 µg/mL of OLP and 2.5 µg/mL to 15 µg/mL. The regression coefficient (r2) values were assessed.

Precision

Usually, it will be measured in terms of system and method precision. The system precision was assessed in the same way as system suitability by analyzing six replicates of the standard solution of 20 µg/mL and 10 µg/mL of OLP and SMD. The method precision was confirmed by determining the %RSD value of assay of six replicate injections of sample solution.

Accuracy

The standard addition technique was employed to confirm the accuracy of the stated method. In this procedure, different level of standard solution (50,100 and 150%) was added to a known amount of sample solution (20 µg/mL and 10 µg/mL of OLP and SMD) individually. The amount of standard solution recovered from spiked solutions was computed in terms of mean % recovery. Each spiked solution was analyzed in triplicate.

Specificity

The specificity of the analytical technique is defined as the capacity of the method to identify the analyte under investigation in the presence of other compounds, such as degradants, impurities, and placebo, with no interference. The specificity of the current approach was ensured by injecting blank, placebo, standard, sample, and forced degradation solutions of OLP and SMD in a successive manner. Any interference among the RT of analytes(OLP and SMD), degradants, and placebo was observed.

Sensitivity

The sensitivity of the current procedure is assessed in terms of limit of detection (LOD) and limit of quantification (LOQ). The standard deviation procedure was adopted to assess the LOD and LOQ.

LOD = 3σ/S

LOQ = 10 σ/S

Where σ is the standard deviation of the intercept (n = 3).

S is the average slope value of the linearity curve (n = 3).

Robustness

The robustness of the method was examined by slightly varying the optimal method parameters such as mobile phase ratio (± 1 mL), temperature (± 5°C), and flow rate (± 0.1 mL/min). The % RSD value of obtained peak areas by the altered method conditions was computed to confirm the robustness of the stated approach.

Forced degradation studies

In the forced degradation approach, drug material is purposefully subjected to more intense stress conditions than those for accelerated stability. These investigations were helpful in determining the drug substance's stability, which is a fundamental factor in creating a stable dosage form. The forced deterioration studies were carried out in accordance with ICH Q1A, QIB, and Q2B guidelines [21].

Acid and base hydrolysis

Equal portions of stock solution (200 µg/mL of OLP and 100 µg/mL of SMD) and 2N HCl were mixed uniformly and refluxed at 60° C for 30 min, further cooled to room temperature neutralized with 2N NaOH. The resultant solution was further diluted to obtain a concentration of 20 µg/mL and 10 µg/mL of OLP and SMD, correspondingly. The above solution was considered as acid degradation solution. Similarly, alkali or base degradation solution was prepared by replacing 2N HCl with 2N NaOH in the acid hydrolysis procedure. After 24 h, the prepared solutions were introduced into the UPLC system.

Oxidative degradation

Equal portions of stock solution (200 µg/mL of OLP and 100 µg/mL of SMD) and 10% H2O2 were mixed uniformly and refluxed at 60 °C for 30 min and further cooled to room temperature. The resultant solution was further diluted to obtain a concentration of 20 µg/mL and 10 µg/mL of OLP and SMD, correspondingly. After 24 h, the prepared solutions were introduced into UPLC system.

Thermal degradation

The standard stock solution (200 µg/mL of OLP and 100 µg/mL of SMD) was kept at 80 °C/75% RH for 24 h in the heating chamber. Further dilution of the resultant solution was done to make a solution consisting of 20 µg/mL of OLP and 10 µg/mL of SMD.

Photodegradation

The standard stock solution (200 µg/mL of OLP and 100 µg/mL of SMD) was placed in a UV chamber at 254 nm wavelength with dark control for 24 h. Further dilution of the resultant solution was done to make a solution consisting of 20 µg/mL of OLP and10µg/mL of SMD.

Neutral degradation

The standard stock solution (200 µg/mL of OLP and 100 µg/mL of SMD) was mixed with water (pH-7) and refluxed for 15 min at 60°C. Further dilution of the resultant solution was done to make a solution consisting of 20 µg/mL of OLP and10µg/mL of SMD. After 24 h, the prepared solution was used for analysis.

Assay of marketed tablets by the current method

The assay of the commercial tablets (LYBALVI) was performed by injecting both standard and sample solutions consecutively. The %purity of OLP and SMD was determined by computing the peak areas OLP and SMD of the standard and sample solution chromatograms [16].