Study On The Technology For Determination Of Free Gossypol In Rats

Objective: To establish HPLC methods for determination of free gossypol (FG) in cotton seed kernel and rat plasma; To establish HPLC/MS methods for determination of FG in rat plasma, liver and muscle. Method: Determination of FG in cotton seed kernel and rat plasma using HPLC: The HPLC system consist of ODS-C18 column (4.6 mm×200 mm, 5μm), the column temperature was 25℃, methanol and 1% phosphoric acid aqueous solution (85:15) as the mobile phase with the flow rate of 1.0 mL/min, the UV detection was 235 nm. Determination of FG in rat plasma, liver and muscle using HPLC/MS: Acetone as a extractant and precipitant of sample, after vortexing and centrifuging, the supernatant was blown dried by N2, dissolved by methanol to remove interfering substance, filtered by 0.45μm micropore film, injected to the HPLC-MS. The HPLC system consist of Agilent ZORBAX SB-C18 column (2.1 mm×150 mm, 5μm), the column temperature was 25℃, acetonitrile and 1% formic acid aqueous solution (80:20) as the mobile phase for determination of FG in rat plasma, methanol and 1% formic acid aqueous solution (85:15) as the mobile phase for determination of FG in rat liver and muscle, at a flow rate of 0.2 mL/min, the internal standard was glycyrrhetinic acid. The sample was ionized in the electrospray ionization (ESI) source of the mass spectrometer and was detected in the selected ion monitoring (SIM) mode, the detected ion of gossypol was [M-H]- m/z 517.2, the detected ion of glycyrrhetinic acid was [M-H]- m/z 469.3. Result: 1. Determination of FG in cotton seed kernel using HPLC: The standard curve is linear within the range of 1.90～35.00μg/mL, r = 0.999 6 and the relative recovery was between 98.6% and 100.4%, the content of FG in 12 cotton seed kernel was between 0.60% and 1.20%. 2. Determination of FG in rat plasma using HPLC: The standard curve is linear within the range of 0.33～3.30μg/mL, r = 0.999 4, the limit of detection was 0.04μg/mL (S/N = 3) and the relative recovery was between 94.7% and 97.3%, the content of FG in rat plasma was between 0.68μg/mL and 2.14μg/mL. 3. Determination of FG in rat plasma, liver and muscle using HPLC/MS: In rat plasma, the standard curve is linear within the range of 0.075～7.500μg/mL, r = 0.998 2, the limit of detection was 5 ng/mL and the relative recovery was between 93.3% and 97.2%, the content of FG in rat plasma was between 0.28μg/mL and 1.05μg/mL; In rat liver, the standard curve is linear within the range of 0.61～61.58μg/g, r = 0.999 0, the limit of detection was 10 ng/g and the relative recovery was between 92.8% and 98.0%, the content of FG in rat plasma was between 3.09μg/g and 6.38μg/g; In rat muscle, the standard curve is linear within the range of 0.03～4.12μg/g, r = 0.999 2, the limit of detection was 2 ng/g and the relative recovery was between 92.5% and 98.3%. Conclusions: HPLC/MS methods for determination of FG in rat plasma liver and muscle are specific, sensitive, exact and reliable with convenient sample treatment method feasible. These methods are suitable for the determination of FG in biological specimens and will be provided reference to the determination standard of FG in animal food.