A validated high-performance liquid chromatography (HPLC) method has been developed to analyze the (±)-gossypol in the selection of strains of Candida tropicalis culture. Since gossypol was easily degraded and oxidized, the addition of antioxidant NADPH-Na 4 and acetone extraction was chosen to prevent gossypol degradation and gradient elution assay was applied to obtain gossypol resolution. Concentrations of gossypol in C. tropicalis ZD-3 culture 20 μg/mL were determined, and

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... profiles were observed. Linearity of the gossypol standard curve by HPLC area method was ranged from 0.1 to 20 μg/mL with Y = 26.954 × X − 29.547, R 2 = 0.9991, and n = 3, with limit of detection (LOD) of 50 ng/mL and lower limit of quantification (LLOQ) of 500 ng/mL. The recovery rate is dose-dependent and ranged from 85.3% to 103.5%. It is a rapid and reliable HPLC method for gossypol quantization in microorganism culture which could be applied in solid fermentation in the feed industry.