Uplc ms

The assay was performed with mouse liver microsomes from male CD-1. Incubations were performed in duplicates as follows: microsomes in 0.1 M potassium phosphate buffer pH 7.4 (0.8 mg/mL microsomal protein), tested compound (final concentration 20 μM, cosolvent methanol, final concentration 0.38%) were preincubated at 37 °C for 15 min. Metabolic reactions were initiated by the addition of the NADPH regenerating system, containing NADP, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase. Mixtures were incubated at 37 °C for various time periods (15, 30, 60 min). Enzymatic reactions were quenched by perchloric acid followed by addition of internal standard (pentoxifylline). Negative controls were performed without NADPH-regenerating system. After centrifugation, samples were analyzed by LC/MS (UPLC/MS, Waters Corporation, Milford, MA, USA). In vitro half time (t_1/2) and intrinsic clearance (Cl_int) of test compound in liver microsomes were determined according to literature procedures [26 (link),63 (link)].

Mouse liver microsomes (MLMs) were used to investigate the phase I metabolism of selected compounds (2c, 2d, 3b, 4g, 5a) and the reference substance BP-2. Samples composed of test compound (20 µM), MLMs (0.8 mg/mL) and potassium phosphate buffer (100 mM, pH 7.4) were preincubated prior to addition of NADPH-regenerating system. NADPH-regenerating system contained NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and potassium phosphate buffer (100 mM, pH 7.4). Control incubations were performed in the absence of the NADPH-regenerating system [73 (link)] and in case of esters (2c, 2d, 3b and 4g) without the mouse liver microsomes solution to determine spontaneous hydrolysis [59 (link)]. Incubations were conducted at 37 °C for at least 15 min up to 60 min. The reactions were stopped by the addition of 250 μL ice-cold methanol containing internal standard PTX (20 μM). The mixture was then centrifuged. Supernatant analysis was performed using UPLC/MS (Waters Corporation). The assays were repeated two times. The in vitro half times (t_1/2) for test compounds were determined from the slope of the linear regression of ln % parent compound remaining versus incubation time. The calculated t_1/2 was incorporated into the following equation to obtain intrinsic clearance: (Cl_int) = (volume of incubation [µL]/ protein in the incubation [mg]) × 0.693 / t_1/2 [74 (link)].

An ultra-performance liquid chromatography instrument coupled with a quatropole mass spectrometry (UPLC-MS, Waters Co., MA, USA) was used to detect hepatic bile acids. Livers were homogenized in acetonitrile (100 mg tissue/500 μl acetonitrile) followed by centrifugation at 14,300 rpm for 10 min. The supernatant was blown to dryness under nitrogen stream, then re-dissolved in methanol-water solution (methanol:water:formic acid = 50:50:0.01) followed by centrifugation at 14,300 rpm for 10 min. Samples of 5 μl of the supernatants were injected into the UPLC-MS instrument. The instrument parameters were applied based on previous study47 (link).

Extraction method of anthocyanins from grape berry skin was referred to He et al. [7 (link)] and Sun et al. [61 (link), 62 (link)]. The UPLC-MS (Waters, Milford, MA, USA) was used to determine the composition and contents of anthocyanins, and the method was referred to Sun et al. [61 (link)].