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Plasma and tumor resveratrol concentrations were determined using liquid chromatography–electrospray ionization–mass spectrometry following the protocol described below.
The liquid chromatography–electrospray ionization–mass spectrometry system used was a Shimadzu LCMS-2010 EV liquid chromatograph mass spectrometer system (Kyoto, Japan) connected to the LC portion consisting of two LC-10AD pumps, a SIL-10AD VP auto injector, a SPD-10A VP ultraviolet detector and a SCL-10A VP system controller.
Data qualitation and quantitation were accomplished using Shimadzu LCMS Solutions Version 3 software (Kyoto, Japan). The analytical column used was a Phenomenex Luna C18 (150 × 4.6 mm intradermally, 5 μm particle size). The mobile phase consisted of
(i) acetonitrile and
(ii) 0.5% aqueous acetic acid (vol/vol), filtered and degassed under reduced pressure prior to use. Separation was achieved using gradient elutions of 18–31% A at 0–10 min and 31–48% A at 10–20 min at a flow rate of 1.0 ml/min at ambient temperature (25 ± 1°C). This was followed by a 5 min equilibration period with initial conditions prior to injection of the next sample. Ultraviolet detection was set at 320 nm.
The mass spectrometer conditions consisted of a curved desolvation line temperature of 200°C and a block temperature of 200°C. The curved desolvation line, interface and detector voltages were −20.0 V, 4.5 kV and 1.2 kV, respectively. Vacuum was maintained by an Edwards® E2M30 rotary vacuum pump (Edwards, UK).
Liquid nitrogen (Washington State University Central Stores, Pullman, WA) was used as a source of nebulizer gas (1.5 l/min). Resveratrol and chlorogenic acid internal standard (IS) were qualitated in selected ion monitoring negative mode. The monitored single plot transitions were resveratrol at m/z 227 and chlorogenic acid at m/z 353.
Methanolic stock solutions of resveratrol (100 μg/ml) and the IS, chlorogenic acid (100 μg/ml), were prepared. These solutions were protected from light and stored at −20°C between uses, for no longer than 3 months. Calibration standards in serum were prepared daily from the stock solution of resveratrol by sequential dilution with blank rat serum, yielding a series of concentrations, namely, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 g/ml.
Plasma samples (0.1 ml) were aliquoted and 25 μl of chlorogenic acid (IS) was added to each sample. The proteins present were precipitated using 1 ml of ice-cold high-performance liquid chromatography (HPLC)-grade acetonitrile, vortexed for 1 min (Vortex Genie-2, VWR Scientific, West Chester, PA) and centrifuged at 5 000 r.p.m. for 5 min (Beckman Microfuge centrifuge, Beckman Coulter, Fullerton, CA).
The supernatants were evaporated to dryness under compressed nitrogen gas. The residue was reconstituted with 100 μl of mobile phase, vortexed for 1 min and centrifuged at 5000 r.p.m. for 5 min; the supernatant was transferred to HPLC vials, and 25 μl of it was injected into the LC/MS system. Weighed tumors were rapidly frozen in liquid nitrogen and pulverized to a fine powder with a mortar and pestle under liquid nitrogen.
To each sample, 25 μl of chlorogenic acid (IS) and 1 ml ice-cold HPLC-grade acetonitrile were added. Samples were vortexed for 1 min and centrifuged at 5000 r.p.m. for 5 min. The supernatants were evaporated to dryness under compressed nitrogen gas. The residue was reconstituted with 100 μl of mobile phase, vortexed for 1 min and centrifuged at 5000 r.p.m. for 5 min; the supernatant was transferred to HPLC vials, and 25 μl of it was injected into the LC/MS system.
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