# Photodegradation Assessment of Calicotriol in the Presence of UV Absorbers by UHPLC/MSE
## 1 Introduction
Previous research has shown an interaction between calcipotriol and ultraviolet radiation. [PERSON] et al. [14] conducted a study in which calcipotriol ointment was applied before and after phototherapy; the ointment was collected and analyzed using reverse-phase high-performance liquid chromatography. UVA exposure led to a decrease in detectable calcipotriol concentrations, indicating that when calcipotriol is used in combination with PUFA, it should be applied after UVA exposure [14]. In research conducted by [PERSON] et al. [15], montasone furoate, calcipotriol, and their combinations were exposed to various stress conditions. The calcipotriol degradation product, identified as DP4, was formed under conditions of heat exposure, UVA radiation, and oxidation with H\({}_{2}\)O\({}_{2}\). Basedon UV and LC-MS fragmentation patterns, DP4 was identified as pre-calcipotriol [15], [PERSON] et al. [16] developed an RP-HPLC method for the separation of known (Calci Impurity-C, Calci Impurity-B, and Calci Impurity-D) and unknown isomeric impurities of calcipotriol from calcipotriol, pre-calcipotriol, and betamethasone dipropionate. The pre-calcipotriol solution was obtained in a vial by dissolving 2 mg of calcipotriol in 200 \(\upmu\)L of a solution prepared by combining 1 mL of triethylamine and 9 mL of chloroform, which was closed and kept in a water bath at 60 \({}^{\circ}\)C for 2 h. Pre-calcipotriol is an isomer of calcipotriol, formed as a result of a reversible transformation that begins when calcipotriol is prepared in solution and continues until equilibrium is reached. This process also occurs in medicinal products and is affected by temperature. Pre-calcipotriol is not considered an impurity, and the effect of calcipotriol results from both substances. Studies using LC-MS, LC-MS/MS, FT-IR, and NMR confirm the isomeric relationship and structural formula of pre-calcipotriol. It is predicted that calcipotriol undergoes Cis/Trans isomerism at carbon 7, forming Imp-B. This is followed by a rearrangement of double bonds, resulting in pre-calcipotriol [16]. In addition to pre-calcipotriol, other identified isomers of calcipotriol include trans-calcipotriol (5E, 7E), 24R-calcipotriol, and the beta-calcipotriol isomer. These compounds are primarily formed during the synthesis of calcipotriol [16; 17; 18]. Vitamin D isomers with (5E, 7E) triene geometry have higher biological activity compared to natural (SZ, 7E) vitamin D compounds [17; 19]. The beta-calcipotriol isomer can be utilized as a reference standard for analytical method development or method validation [20]. Photop protection is recommended by dermatological societies as a key strategy in the prevention of skin cancer. Therefore, a comprehensive understanding of the potential interactions between substances applied simultaneously to the skin, such as UV filters and active pharmaceutical ingredients, is essential to ensure the safe and effective use of topical drug products [12]. Previous studies on tazarotene, bexarotene, terbinafine, and bifonazole have shown that UV filters can significantly impact the stability of drug substances when exposed to UV radiation [21; 22; 23; 24]. Building on these findings, the present study aimed to evaluate how commonly used UV filters affect the stability of calcipotriol. To achieve this, a range of filters capable of absorbing UV-A and UV-B radiation was selected for the experiments. The sunscreen agents tested included salicylic acid derivatives such as homosalate (homomenthyl salicate), which exhibits maximum absorption at 306 nm; dibenzoylmethane derivatives like avobenzene (butyl methoxydibenzoylmethane), serving as UVA I filters with a peak at 357 nm; and benzophenone derivatives including oxypenzone (benzophenone-3, 2-hydroxy-4-methoxybenzophenone) which absorbs in both the UVB (280-315 nm) and short-wavelength UVA (315-355 nm) ranges. Additionally, dioxybenzene (benzophenone-8) and sulisobenzene (benzophenone-4) were included, both characterized by two absorption peaks at 286 nm and 440 nm.