Transdermal Permeation Behavior Study Of Calcipotriol Ointment

AIM: The quantitation method of calcipotriol was established using highperformance liquid chromatography tandem mass spectrometry (LC-MS/MS). Thestability of calcipotriol in three kinds of matrix was evaluated to ensure the accuracyof results. Transdermal permeation behavior in skins from various animals (pig, ratand mouse) and anatomical sites (pig back, pig abdomen and pig ear) were evaluatedwith calcipotriol ointment as model drug.METHOD: After sample preparation by liquid-liquid extraction, chromatographywas performed on an Extend-C18column with isocratic elution. The mobile phasewas methanol:0.1%ammonia (80:20, v/v) at a flow rate of1mL/min.30μL ofsample was injected to LC-MS/MS system. Detection was by electrospray ionizationin the negative ion mode using multiple-reaction monitoring of the precursor toproduct ion transitions of calcipotriol at m/z411.1â†'m/z393.5, and of lovastatin(internal standard) at m/z403.2â†'m/z101.2. According to guidelines in China Foodand Drug Administration (CFDA), the method was fully validated in terms ofspecificity, linearity, LLOQ, accuracy, precision, matrix effect, extraction recoveryand stability studies.The stability of calcipotriol in three kinds of matrix (excised skin homogenate,receptor medium and ointment) was evaluated in the condition of exposure todaylight, maintaining in dark at room temperature and maintaining in dark at37oC.Skin permeation of calcipotriol was evaluated using a RYJ-6B Franz diffusionassembly with mixture of PBS and ethanol (7:3, v/v) as receptor medium. Excised pigback skin, pig abdomen skin, pig ear skin, rat dorsal skin and mouse dorsal skin wereused as model skin, and calcipotriol ointment as model drug. The permeation andretention of calcipotriol in excised skin samples were evaluated. RESULTS: A rapid and sensitive LC-MS/MS method for analysis of calcipotriol wasestablished. The assay was linear in all matrixes (1-300ng/mL for skin homogenate,0.5-100ng/mL for receptor medium and40-1200ng/mL for ointment samples) withcorrelation coefficients larger than0.996. LLOQ values in the three matrixes were:1ng/mL in skin homogenate,0.5ng/mL in receptor medium and40ng/mL inointment. Accuracies, intra-and inter-day precisions were within±15%. Extractionrecoveries were high and constant at different concentrations. No significant matrixeffects were observed in the method. Calcipotriol was stable in dark under followingconditions: at room temperature for4h, in processed samples for4h at roomtemperature, after3freeze/thaw cycles,-80°C for15days and in the stock solution at4°C for one week. The method was rapid, high sensitive, and suitable for thetransdermal permeation behavior of calcipotriol in excised animal skins.Calcipotriol was suffered from significant degradation on exposure to daylight over7h in all matrixes. When stored in dark, no decomposition of calcipotriol occurredover24h at room temperature or37°С. The results showed that dark conditionsshould be maintained during the transdermal permeation study.The drug content in receptor medium at24h increased in the order: pig back <ratback <pig abdomen <pig ear <mouse back. Drug contents in pig skins from threeanatomical sites were similar at different time points (p>0.05), drug contents in ratand mouse back skin were higher than that of pig skins at all time points before20h.Residual drug in ointment in rat and mouse back skins were lower than that in pigskins at all time points. The total recovery of calcipotriol in permeation experiments(the sum of calcipotriol in receptor medium, skin and ointment) declined rapidly over3h and then leveled off at about80%of the applied dose over the period up to24h.The results suggest metabolism of calcipotriol accompanies its penetration into skinbut the metabolic activity of skin is limited.The results show that with increasing thickness of skin, the drug in receptormedium declines indicating that the thickness of skin is a pivotal factor influencingpermeation rate which is independent of biochemical and histological differences. The residual contents in skins are dependent on biochemical and histologicaldifferences.