# Photodegradation Assessment of Calicotriol in the Presence of UV Absorbers by UHPLC/MSE
## 3 Results and Discussion  
### Method Validation  
The developed method was validated in accordance with the guidelines of ICH Q2 (R2), and it is suitable for the determination of calcipotriol and its photodegradation products in the tested methanolic solutions. The validated UPLC-MS/MS method was specific to the tested substance and guaranteed good peak separation both before and after UV irradiation. The proposed method was validated for the determination of calcipotriol in the presence of degradation products and chosen organic UV filters. In order to assess the specificity of the new UHPLC method, the solutions of calcipotriol with chosen UV filters after 1h of UVA irradiation were investigated. Adequate separation was achieved for both the primary compound and its degradation products. Additionally, the UV filters employed did not produce any extraneous signals in the chromatogram that might overlap with the peaks corresponding to the main substance or its degradation products. Table 1 presents the summarized results of the method validation. The linearity of the method was evaluated by examining six methanolic calcipotriol solutions within a concentration range of 15-150 \(\upmu\)g mL\({}^{-1}\). The linear regression equation was y = 83451x \(-\) 86683. The linear regression analysis showed good linearity of the method across the entire concentration range, with correlation coefficients and determination coefficients (R2) of 0.9990 and 0.9979, respectively. The y-intercept of the regression analysis for calcipotriol did not achieve statistical significance. The Shapiro-Wilk test confirmed that the residuals of the dependent variable are normally distributed, with \(p\) = 0.15490, which is higher than the significance level of \(\alpha\) = 0.05. The Durbin-Watson statistic was utilized to test for the presence of autocorrelation of the random component. The test statistic value, d = 1.77, exceeds the upper critical value, dU = 1.39, at a significance level of \(\alpha\) = 0.05 (k = 1, n = 18), indicating no autocorrelation. Furthermore, [PERSON]'s test was employed to verify the homogeneity of variance. The probability \(p\) = 0.942656 is greater than the significance \(\alpha\) = 0.05, indicating that the variance of response is homogeneous. The LOD and LOQ values are 7.39 \(\upmu\)g mL\({}^{-1}\) and 50 \(\upmu\)g mL\({}^{-1}\), respectively. The measurement range of the method is from 50 to 150 \(\upmu\)g mL\({}^{-1}\). The precision was assessed through repeatability testing, resulting in a coefficient of variation of 2.41%. The accuracy of the method was evaluated using recovery analysis at three concentration levels: 75%, 100%, and 150%. The average percent recovery ranged from 99.17% to 101.55%.  
\begin{table}
\begin{tabular}{c c} \hline
**Parameter** & **Calcipotriol** \\ \hline Retention time (min) \({}^{\text{a}}\) & 8.04 \\ \hline Limit of detection (\(\upmu\)g mL\({}^{-1}\)) & 7.39 \\ \hline Limit of quantitation (\(\upmu\)g mL\({}^{-1}\)) & 50 \\ \hline Linear range (\(\upmu\)g mL\({}^{-1}\)) & 15–150 \\ \hline \end{tabular}
\end{table}
Table 1: Method Validation Parameters.