# Photodegradation Assessment of Calicotriol in the Presence of UV Absorbers by UHPLC/MSE
## 3 Results and Discussion
### Identification of Degradation Photoproducts  
In the present study, the identification of calcipotriol degradation products was accomplished using UHPLC/MSE experiments. The proposed structures of the degradation products are shown in Table 2. The proposed fragmentation patterns of calcipotriol and  
Figure 1: UHPLC/MSE analysis of photostability of calcipotriol in methanol solution after exposure to UVA irradiation for 1 h: (**A**) calcipotriol with oxybenzene; (**B**) calcipotriol with meradimate; (**C**) calcipotriol with sulisobenzene; (**D**) calcipotriol with avobenzene; (**E**) calcipotriol with homosulate, and (**F**) pure standard substance.  
its degradation products are shown in Table 3. The degradation process was found to affect primarily the E-4-cyclopropyl-4-hydroxy-1-methylbut-2-en-1-yl moiety, causing its isomerization to the \(Z\) isomer. Additionally, the products CP-2 to CP-5 are formed by intramolecular [4+2] photoaddition in the hexa-1,3,5-triene fragment of calcipotriol, giving diastereomers with the R or S configuration at the resulting spiro carbon atom. Regarding the activation mechanism of sulisobenzene, upon UV irradiation, it generates an active triplet state on the ketone moiety, which may subsequently activate calcipotriol in a following step. When benzophenone is exposed to ultraviolet light, its photoreactive benzophenone moiety absorbs the energy and undergoes photolysis. Benzophenone absorbs UV photons, which excite the molecule from its ground state to the singlet excited state. The molecule efficiently undergoes intersystem crossing, transitioning from the singlet excited state to the triplet excited state. The resulting triplet-state ketone is highly reactive, exhibiting pronounced biradical character. The triplet-state intermediate is significantly more reactive than the ground state, enabling benzophenone to initiate further chemical transformations [32; 33; 34]. To the best of our knowledge, this is the first report describing these photodegradation pathways and the detailed structures of calcipotriol photoproducts.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Compound** & \begin{tabular}{c} **RT** \\ **[min]** \\ \end{tabular} & **[M+H]\({}^{\star}\)** & **Fragmentation Ions** & **Structure** \\ \hline Calicipotriol & 7.93 & 395.2945 \({}^{\star}\) & \
\begin{tabular}{c} 151.1481, 243.2107, \\ 359.2733, 377.2839 \\ \end{tabular} \\ \hline CP-1 & 8.08 & 395.2945 \({}^{\star}\) &
\begin{tabular}{c} 151.1481, 243.2107, \\ 359.2733, 377.2839 \\ \end{tabular} \\ \hline CP-2 & 9.62 & & & \\ CP-3 & 9.72 & & & \\ CP-4 & 9.87 & & & \\ CP-5 & 9.95 & & & \\ \hline \hline \end{tabular}
\end{table}
Table 2: Proposed structures of degradation products of calcipotriol.  
Previous studies have confirmed that calcipotriol is susceptible to photodegradation, and several chromatographic methods have been developed to separate the parent compound from its photoproducts. [PERSON] et al. [35] demonstrated the separation of calcipotriol from its degradation products by HPLC and examined the protective effects of various solvents and UV filters; however, they did not characterize the degradation products themselves. In the experiment, a xenon lamp was employed due to its spectral distribution being similar to that of sunlight. After 3 and 6 min of irradiation, two unknown degradation products (DG1 and DG2) were obtained, as well as DG3, which appears when DG2 diminishes, suggesting a complex degradation pattern [35]. [PERSON] et al. also observed a decrease in detectable calcipotriol concentrations in the ointment after exposure to UVA radiation [14]. In research conducted by [PERSON] et al., the degradation product of calcipotriol obtained under the influence of UVA radiation was identified as DP-4, which is pre-calcipotriol, based on UV and LC-MS fragmentation patterns [15].  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Calcipotriol** & **Calcipotriol** \\ \hline \hline
**HO** & **HO** \\ \hline \hline
**CP-2** & **CP-