C 18 reversed phase hplc column

Spinosads were extracted from the fermentation cultures (1 mL) by mixing with 2 mL of acetonitrile, vortexed for 20 min, incubated for 30 min at room temperature, and centrifuged at 3500×g for 10 min to remove cell debris. The supernatant was filtered using a 0.22 μm syringe filter and injected into a C18-reversed phase HPLC column (5 μm, 250 × 4.6 mm, Waters, Milford, USA) at 25 °C using isocratic elution with acetonitrile: methanol: 0.05% ammonium acetate buffer (4.5:4.5:1, v/v/v) at a flow rate of 1 mL/min and detection at 250 nm (HPLC with a UV detector, SPD-20A, Shimadzu, Kyoto, Japan), or a C18-reversed phase HPLC column (5 μm, 250 × 4.6 mm, Agilent, California, USA) at same separation condition and detection at 250 nm (HPLC, 1260DAD, Thermo scientific, Waltham, USA coupled with a DAD detector, DAD3000, Dionex, Sunnyvale, USA). Spinosad titer from Streptomyces and its derivatives was determined by comparison with standard spinosyn A and D.