Tretinoin soft capsules: development of an HPLC method using the design of experiments (DoE) approach and identification of degradation products by HRMS

Tretinoin (ATRA) soft capsule formulation is commonly used in oncology, in the treatment of acute promyelocytic leukemia. However, there are no pharmacopoeial methods to assay this formulation. To the best of our knowledge, this study reports the first stability-indicating HPLC method for the quantification of ATRA soft capsules, using the Design of Experiments (DoE) approach. The CCRD (Central Composite Rotatable Design) was used to evaluate the effects of relevant analytical conditions on chromatographic responses; the extraction conditions from the complex capsule matrix were established using the CCD (Central Composite Design). The optimal conditions were identified using response surface methodology and applied in the method validation. The analytical method was specific, linear (2.5–30 µg/mL), precise (1.25%), accurate (mean recovery of 101.14%), robust, and specific, with no interference of degradation products and its main related substance, isotretinoin. The developed method was also applied to the uniformity of dosage units by content uniformity. Forced degradation studies indicated photolysis as the preferred degradation route, exhibiting second-order kinetics under the experimental conditions (UV-A radiation). Additionally, two degradation products obtained under thermolysis and oxidative conditions were identified by high-resolution mass spectrometry (HRMS): an epoxide derivative (DP1, 317.2114 m/z) and an ATRA isomer (DP2, 301.2169 m/z). Graphical abstract