Model 717 plus autosampler

The analysis of MK-7 was carried out according to a previous reported procedure [8] , with slight modifications. 2-Propanol (2.4 ml) and n-hexane (4.8 ml) were mixed with 2 ml of the fermented medium. The mixture was vigorously shaken and kept standing for 1 h. After centrifugation at 3,000 rpm for 10 min (Union 32R; Hail, Korea), the organic phase was collected and evaporated under nitrogen gas. The oil residue was dissolved in ethanol. After filtering with 0.45-μm syringe filters (Millipore, USA), the sample solution was injected into a high-performance liquid chromatography system with a model 600 Controller gradient pump equipped with a degasser, and model 717 Plus autosampler (Waters, USA). The analytical column was a Nova-Pak C 18 column (WAT086344, 3.9 × 150 mm; Waters, USA), and the temperature was fixed at 40 o C. After reduction by platinum black (reduction column, 4.6 × 10 mm; JIScience, Korea), the peaks were detected with a model 474 scanning fluorescence detector (Waters, USA) using an excitation and emission wavelength of 320 nm and 430 nm, respectively. The mobile phase was 100% methanol at a flow rate of 1.0 ml/min, and the injection volume of the sample was 20 μl. The concentration of MK in the medium was expressed as μg/ml of culture medium.