Quantitative HPLC Analysis of Metabolites

Sample reactions were analyzed to quantitate metabolites by absorbance. Analytes were separated on a Waters XBridge BEH C18 3.5 μM column (4.6 mm × 100 mm) using an Agilent 1100 series HPLC instrument equipped with a VWD detector (280 nm). The mobile phase consisted of solvent A (10% solvent B, 90% water) and solvent B (0.1% formic acid/acetonitrile). A gradient method started at 80% solvent A for the first minute, then decreased to 70% for the next 6 min. The method held 70% A for 3 min before decreasing to 15% A over 11 min, then returning to 80% over 3 min and holding for the final 2 min. The flow rate was 1.2 mL/min for a total run time of 25 min. Parent drug and metabolite responses were normalized to the internal standard acenocoumarol and quantitated using a standard curve generated with 5OH-APINACA. The absorbance response corresponds to the indazole ring, which remained unmodified in these reactions [28 (link)], so that the relative absorbance response for all analytes were approximately the same, making inference for quantitation purposes possible.