The compound was eluted with 85%acetonitrile/0.1%% formic acid from Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 μm, Agilent, USA) at a flow rate of 0.4 ml/min.
Anolyte was continuously recycled using an adjustable peristaltic pump (Pump II, Model 3385 Thomas Inc, Swedesboro, New J. USA) at a flow rate of 0.27 mL Min−1, corresponding to a hydraulic retention time (HRT) of 12 h.
The mice were chemically restrained with 1.5% isoflurane (Abbott Laboratories, North Chicago, IL, USA) delivered in O2, using a model 100 vaporizer (SurgiVet, Waukesha, WI, USA) at a flow rate of approximately 1.0 L/min.
The nucleotides were separated and quantified on an RP-C-18 column that was combined with a guard column (Supelcosil LC-18-T; 15 cm × 4.6 mm, 3 μm packing and Supelguard LC-18-T replacement cartridges, 2 cm; Supelco, Bellefonte, USA) at a flow rate of 1 ml/min.
After that we administer, through a peripheral venous access of at least 18 G, 120 150 ml iodine-containing contrast medium (Iomeprolo, Iomeron350; Bracco, Milano, Italy) using a power injector (Spectris; Medrad, Pittsburgh, Pennsylvania, USA) at a flow rate of 4.0 ml/s, followed by a saline flush of 50 ml at a flow rate of 4.0 ml/s.
The samples were then pre-concentrated on the loading column (BetaBasic 20 × 2.1 mm, 5 µm particle size in DASH, Thermo Fisher Scientific, USA) with 60%% of solvent A (0.1 % NH4OH, H2O) and 40%% of solvent B (0.1 % NH4OH, MeOH) using the load pump (low-pressure quartenary pump Accela 600, from Thermo Fisher Scientific, USA) at a flow rate of 1000 μL min−1.
After centrifugation at 5,000×g for 5 min at 4°C, the gemcitabine content in the supernatant was determined by high-performance liquid chromatography (HPLC), with a Diamond C18 chromatographic column (5 μm, ID 4.6 × 300 mm, Anoka, MN, USA) and at a flow rate of 1 mL/min.
Separation was achieved on a self-packed 0.5 mm×110 mm Luna SCX (Phenomenex, Torrance, CA, USA) column at a flow rate of 18 µL/min with a steep salt gradient from 0 400 mM NH4Cl in 25% acetonitrile, 20 mM KH2PO4, pH 3.0.
Animals were anesthetized with 0.8 3% halothane delivered from a calibrated Fluotec 5 (Fluotec-Ohmeda, Tewksbury, MA, USA) vaporizer at a flow rate of 1 4 l/min oxygen.
Glucose, xylose, galactose, arabinose and mannose were separated on an Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) run at a flow rate of 0.5 ml/min at 85°C, with water as the eluent.
Supernatants were neutralised with 500 μl trioctylamine : 1,1,2-trichloro-trifluoroethane (1 : 4), centrifuged for 1 min at 10 000 g and 50 μl were injected onto a Simmetry Shield C18 5 μm, 300 × 4.6 mm column (Waters, Milford, MA, USA) eluted at a flow rate of 1 ml min−1 with 9% methanol, 6% acetonitrile and heptane sulphonic acid 5 m M, pH 3.1.
