Luna 5 μm silica column

Experimental parameters of LC-MS/MS assay have been reported previously^{17 (link),20 (link),21 (link),37} . LC-MS/MS analysis was performed on an AB 4000 QTRAP LC-MS/MS System (AB Sciex, Foster City, CA, USA) equipped with a TurboIonSpray (electrospray ionization; ESI), and Agilent 1260 Infinity HPLC pump and autosampler (Agilent Technologies, Santa Clara, CA, USA). An Atlantis dC18 3 μm column (3.0 × 50 mm; Waters Corporation, Milford, MA, USA) was used for DS and HS analysis, and a Luna 5 μm Silica column (50 × 2.0 mm; Phenomenex Inc., CA, USA) was used for KS analysis. Data were acquired and processed using Analyst 1.5.2^TM software (AB Sciex). The intra- and inter-assay precision values of this MS/MS-based method were estimated and determined using three different known concentrations (low, medium, and high levels) of samples in triplicate over a period of 6 days regularly^{17 (link)}. Calibrations of DS, HS, and KS standards, internal standards, and pooled sample controls with known concentrations were performed with every batch analysis. The experimental parameters of mass spectrometer were checked routinely to ensure the quality of the MS measurement.

Dried and ground algal material (233.4 g) was sequentially extracted with dichloromethane (DCM) and ethyl acetate (EtOAc) at room temperature. The filtered organic extracts were combined and evaporated to afford 1.94 g of crude extract. Fractionation by gel filtration chromatography (Sephadex LH-20 column, n-hexane/DCM/methanol (7:2:1)) gave fractions F1–F6. Further chromatographic steps by gel filtration in Sephadex LH-20 (n-hexane/DCM/methanol (3:1:1)) of fraction F6, followed by medium pressure chromatography in a Lobar LiChroprep Si 60 (40–63 μm) column eluted with n-hexane/EtOAc (1:1) and silica gel column (step gradient from CHCl3/EtOAc (4:1) to 100% EtOAc), allowed isolation of cystomexicone B (6, 1.39 mg). Pure compounds gongolarone C (5, 0.17 mg, rt: 30.2 min), gongolarone B (1, 1.75 mg, 34.1 min), 6Z-1′-methoxyamentadione (2, 3.10 mg, 37.1 min), gongolarone A (4, 0.48 mg, 39.1 min), and 1′-methoxyamentadione (3, 6.22 mg, 42.1 min) were obtained by HPLC of fraction F6.4.3-5 (Phenomenex, Luna 5 μm Silica column, 100 Å, 250 × 10 mm; isocratic n-hexane/EtOAc (3:2), 10 min at 1 mL/min; gradient up to 100% EtOAc, 50 min at 2 mL/min; 100% EtOAc, 5 min, 2 mL/min). Physical properties and NMR data of compounds 1–6 were confirmed with those previously reported [16 (link),31 (link)].