The cottonseed samples were dried at 30 °C to constant weight for 2–3 days, and then ground with a grinder to a powder. Standard gossypol solutions were prepared by dissolving 0.01 g HPLC-grade gossypol in 10 mL of acetone. Standard gossypol solution of 0.010, 0.050, 0.100, 0.200, 0.500, 0.800, 1.000 and 2.000 mL were added into 10 mL volumetric flasks and adjusted to the calibration using acetone to prepare the different levels of gossypol solutions. Similarly, 0.1 g of sample was suspended into 2 mL acetone. In addition, the sample solutions were maintained in an ultrasonic bath for 45 min. Subsequently, the suspensions were filtered through quantitative filter paper followed by filtration with a 0.45 μm syringe filter (Agela, Newark, USA). The sediment was washed three times with acetone. Then, the extraction was adjusted to 10 mL with acetone.
The HPLC analysis was performed using an Agilent 1100 (Agilent, Santa Clara, USA), equipped with an auto-sampler and UV detection. A C18 column (Dikma, Richmond Hill, USA) (250 mm × 4.6 mm, 5 μm) was deployed as the stationary phase. The mobile phase consisted of Methanol/0.2% H3PO4 (80/20, v/v). The injection volume was 10 μL, and the flow rate was 1.0 mL/min. The UV detector was set at 238 nm, and the temperature was set at 25 °C. The samples were measured in triplicates.
The HPLC analysis was performed using an Agilent 1100 (Agilent, Santa Clara, USA), equipped with an auto-sampler and UV detection. A C18 column (Dikma, Richmond Hill, USA) (250 mm × 4.6 mm, 5 μm) was deployed as the stationary phase. The mobile phase consisted of Methanol/0.2% H3PO4 (80/20, v/v). The injection volume was 10 μL, and the flow rate was 1.0 mL/min. The UV detector was set at 238 nm, and the temperature was set at 25 °C. The samples were measured in triplicates.