Lc 2010a ht liquid chromatograph system

The LC-2010A HT Liquid Chromatograph system (Shimadzu Corporation, Kyoto, Japan) was used to determine the concentration of psoralen in samples. The HPLC system consisted of a double-plunger pump (Shimadzu Corporation), an autosampler (Shimadzu Corporation), an online degasser (Shimadzu Corporation), a Diamonsil C18 reverse phase column (5 μm, 4.6 mm inner diameter × 25 cm; Dikma Technologies, Inc, Lake Forest, CA, USA), a column oven (Shimadzu Corporation), an ultraviolet detector (Hamamatsu Photonics, Hamamatsu, Japan), and a recording integrator (Shimadzu Corporation). The mobile phase was methanol:water (55:45, volume/volume [v/v]) with a flow rate of 1 mL/min. The column temperature was constant at 35°C, and the detection wavelength was 246 nm. Percentage recoveries ranged from 97.4% to 102.8%. The psoralen intra- and interday relative standard deviation values were 0.87% and 2.15%, respectively. Samples from in vitro experiments were filtered through a disposable nylon syringe filter (pore size, 0.45 μm) with a 13-mm diameter (Shanghai Anpel Scientific Instrument Co, Ltd, Shanghai, People’s Republic of China) before automatic injection into the HPLC system. Samples from in vivo microdialysis were directly assayed in a timely manner without any handling.

PF in samples from in vitro studies was analyzed using an LC-2010A HT Liquid Chromatograph system (Shimadzu Corporation, Kyoto, Japan) with a Diamonsil™ C18 reverse-phase column (250×4.6 mm, 5 μm; Dikma Technologies, Beijing, People’s Republic of China). The mobile phase was a mixture of acetonitrile–water–phosphate (16:84:0.1, v/v/v) with a flow rate of 1 mL/min. The column temperature was maintained at 30°C, and the detection wavelength was 230 nm.
The in vivo synovium homogenate samples were analyzed using a TSQ-Quantum UHPLC system (Thermo Finnigan, San Jose, CA, USA). Chromatographic separations were accomplished on a Syncronis C18 reverse-phase column (50×2.1 mm, 1.7 μm; Thermo Finnigan) maintained at 30°C. The mobile phase was methanol and 5 mmol/L ammonium acetate at a ratio of 30:70 (v/v) with a flow rate of 0.2 mL/min. The mass spectrometer was operated as follows: spray voltage, 3,500 V; capillary temperature, 351°C; sheath gas (N₂) pressure, 30 psi; and auxiliary gas (N2) pressure, 10 psi. The analytes were monitored using positive selected reaction monitoring mode at m/z 498.2→179.1 for PF and m/z 318.2→121.1 for internal standard (IS). Xcalibur 1.2 data analysis system was used for data handling.