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Objective: To establish cottonseed Renhe rat plasma free gossypol (FG) content by HPLC methods, and rat plasma, liver and muscle tissue of free gossypol content of HPLC-MS detection method. Method: HPLC method for the determination of cottonseed Renhe rat plasma free gossypol content: The column ODS-C18 (4.6 mm × 200 mm, 5μm), column temperature was 25 ℃, mobile phase of methanol-1% phosphoric acid (85: 15, V / V), flow rate of 1.0 mL / min, detection wavelength 235 nm; HPLC-MS assay in rat plasma, liver and muscle tissue of free gossypol content: the extraction of the sample with acetone as precipitant agent and, after vortex spin, centrifuged, and the supernatant dried, dissolved in methanol again vortex, centrifugation, further remove interfering substances, 0.45μm filter membrane, the sample measured, using column Agilent ZORBAX SB-C18 (2.1 mm × 150 mm , 5μm), column temperature was 25 ℃, the plasma was measured with the mobile phase of acetonitrile -1% formic acid (80:20, V / V), liver and muscle tissue as measured with the mobile phase was methanol-1% formic acid (85:15 , V / V), flow rate of 0.2 mL / min, with glycyrrhetinic acid as an internal standard, the sample through electrospray ionization (ESI) source ionized using selective ion monitoring (SIM) mode measurement, detection of gossypol ions [ MH] - m / z 517.2, the detection of glycyrrhetinic acid ions [MH] - m / z 469.3. Results: 1. HPLC assay of free gossypol cottonseed kernels: free gossypol concentration range of 1.90 ~ 35.00μg/mL good linear relationship, r = 0.999 6, high, medium and low concentrations of recoveries 98.6% - 100.4%, 12 Cottonseeds free gossypol content of the sample was 0.60% to 1.20%. 2. HPLC assay of free gossypol in rat plasma: plasma gossypol in the range of 0.33 ~ 3.30μg/mL good linear relationship, r = 0.999 4, the detection limit was 0.04μg/mL (S / N = 3), high, medium and low concentrations of average recovery was 94.7% ~ 97.3%, administered rats plasma free gossypol content of 0.68 ~ 2.14μg/mL. 3. HPLC-MS method for the determination of plasma, liver and muscle tissue free gossypol content: plasma gossypol in the range of 0.075 ~ 7.500μg/mL good linearity, r = 0.998 2, a detection limit of 5 ng / mL, high, medium and low density, the average recovery was 93.3% ~ 97.2%, administered rat plasma gossypol content of 0.28 ~ 1.05μg/mL; liver gossypol in 0.61 ~ 61.58μg / g range was linear, r = 0.999 0, a detection limit of 10 ng / g, high, medium and low concentrations of average recovery was 92.8% ~ 98.0% in treatment group content of gossypol in rat liver is 3.09 ~ 6.38μg/mL; muscle tissue gossypol in 0.03 ~ 4.12μg / g was linear within the range, r = 0.999 2, a detection limit of 2 ng / g, high, medium and low concentrations of average sample recoveries were 92.5% ~ 98.3%. Conclusion: This topic was created free gossypol content in rats in vivo detection method has good sensitivity, accuracy, precision and specificity, sample handling method is simple and fast, free gossypol in biological samples for testing, The establishment of this detection method, for food of animal origin free gossypol content testing standards provide a reference.
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