Purification of drug degradation products supported by analytical and preparative supercritical fluid chromatography

A stressed degradation (oxidation) was employed to produce metabolites from an active pharmaceutical ingredient (API) with large molecular weight (about 900 g/mol). An analytical chromatographic method was desired to compare the products generated by different degradation methods while a multi-gram-scale preparative chromatographic method was necessary to purify the produced metabolites. Supercritical fluid chromatography (SFC) was selected for both tasks as no other chromatographic method had achieved the resolution of the API and metabolites (two isomeric mono-oxide species and one di-oxide). First, an analytical-scale method was developed with ultra-high performance supercritical fluid chromatography (UHPSFC). Achiral stationary phases containing sub-2 μm fully porous particles or sub-3 μm superficially porous particles, and chiral phases containing 3 and 5 μm fully porous particles were selected for a first screening with gradient elution (carbon dioxide - methanol containing additives). The stationary phase providing the most promising results was ACQUITY Torus 2-PIC (100 x 3 mm, 1.7 μm, Waters). A central composite design (CCD) was conducted to optimize the gradient program and oven temperature. Final gradient conditions were as follows: 50-70% methanol in 3.8 min with oven temperature set at 36 °C, back-pressure set at 11 MPa and flow-rate at 0.8 mL/min. The optimized method was employed to analyze samples obtained with different degradation conditions. Then the method was adapted and transferred to preparative-scale SFC on a 5 μm-particles Torus 2-PIC stationary phase (150 x 30 mm). The method was modified to comprise an isocratic step followed by a gradient, favoring peak shape of the last eluting compound and minimal volume of collected fractions. Batch injections in gradient mode were carried out to purify six grams of crude product.