Sil 20ac xr prominence autosampler

The molecular size distribution of the various CPs from seaweeds and abalone viscera was determined by gel permeation chromatography (GPC) using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Japan), a degasser, a column oven controller, and an evaporative light scattering detector (ELSD; Shimadzu, Japan). An Ultrahydrogel 500 (300 × 7.8 mm) column (Waters Co., Miliford, MA, USA) in combination with a guard column (Ultrahydrogel, 6 × 40 mm, Waters, Milford, MA, USA) was used to maximize the resolution. Standard pullulans including 1300, 6000, 12,000, 22,000, 50,000, 110,000, 200,000, 400,000, and 800,000 Da were used as molecular mass markers. The injection volume was 20 µL and eluted with distilled water at 45 °C with a flow rate of 0.5 mL/min. Each sample was analyzed in duplicate.

Liquid chromatographic separation and tandem mass spectrometric detection were performed using an ABI Sciex 3200 Q Trap mass spectrometer (ABI Sciex, UK) interfaced with a Shimadzu UFLC system (UFLC XR system with CBM-20A controller, SIL-20 AC XR Prominence autosampler, CTO-20 AC Prominence column oven and LC-20 AD XR pumps (Shimadzu, UK). For the analysis of lipid extracts from clinical specimens, positive and negative controls, UPLC was combined with enhanced product ion identification (LC-EPI) or multiple reaction monitoring (LC-MRM) as previously described (15).

Purified FX and SX were analyzed by reversed phase HPLC using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Kyoto, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Kyoto, Japan), a degasser, a column oven controller, and a photodiode array detector (PDA; Shimadzu, Kyoto, Japan). The FX and SX were separated on a reverse-phase Sunfire C₁₈ column (5 μm particle size, 250 × 4.6 mm ID, Waters, Milford, MA, USA) coupled to a C₁₈ guard column (5 μm particle size, 15 × 4.6 mm ID), regulated at 25 °C with 20 μL sample injections. The mobile phase for FX consisted of methanol and water with a flow rate of 1 mL min⁻¹. For solvent gradient conditions, methanol/water ratio was increased from 60:40 (v/v) to 100:0 (v/v) over 20 min, 100% methanol was held for next 15 min, and then methanol/water ratio was decreased from 100:0 (v/v) to 60:40 (v/v) over 25 min. To separate SX, the isocratic mobile phase was acetonitrile, methanol, and 0.1% ammonium acetate (75:15:10, v/v/v) at a flow rate of 1 mL min⁻¹. Chromatographic peaks were identified at a wavelength of 450 nm by comparing the retention times and spectra against the known standards (Sigma-Aldrich) (St. Louis, MO, USA). Each sample was analyzed in duplicate.