Captiva

Freeze-dried skin was weighted (0.0140 g) and extracted with 200 μL of methanol and 1.4 mL of acetone/water/TFA (70:29.95:0.05). The solution was extracted under sonication for 10 min and stirred at 40 rpm for 20 min, repeated twice. The extraction was carried out at 4 °C in the absence of light and the extract was centrifuged and thermostated at 4 °C, 10,000 rpm, for 10 min. Then, a fraction of 0.5 mL of the supernatant was dried with nitrogen under pressure. At this stage, and unless chromatographic analysis was followed immediately, the samples were stored as concentrates in the deep freezer (−80 °C). The concentrate was redissolved with a mixture of 250 μL methanol and 750 μL water 0.134% formic acid. Subsequently, the solution was led to an ultrasonic device again at 4 °C in the dark for 30 min. The extract was centrifuged and thermostated at 4 °C, for 14,000 rpm, for 15 min, and the supernatant was filtered through 0.22-μm RC syringe filters (RC) (Captiva, Agilent Technologies) prior to chromatography.

HEK293T cells were transfected with MCT7/pEGFP-C1 by using polyethylenimine and cultured for 48 h. The cells were incubated in the Na⁺-free buffer consisted of 10 mM Hepes/Tris (pH 7.4), 273 mM mannitol, 5.4 mM KCl, 4.2 mM KHCO₃, 1.3 mM CaCl₂, 0.44 mM KH₂PO₄, 0.49 mM MgCl₂, 0.4 mM MgSO₄, 0.34 mM K₂HPO₄, and 5.6 mM D-glucose for designed time. The amino acid contents of the cells were analyzed by LC-MS/MS system. The cells were scraped with ice-cold distilled deionized water, and the cell suspensions were deproteinated by adding acetonitrile. The samples were passed through a 0.45-μm PVDF filter (Captiva; Agilent Technologies), and the filtrates were used for analysis. The analysis was operated in positive ionization mode using a Waters Acquity UPLC H-Class connected with the Xevo TQD system (Milford). LC separation was performed at a 0.6 ml/min flow rate on an Intrada Amino acid analytical column (50 × 3 mm, 3 μm) (Imtakt). The separation was done by using a gradient program. The gradient program was composed of solvent A (20 mM ammonium formate buffer in water) and solvent B (acetonitrile) as follows: 10% A for 0 to 1.0 min, 10% to 100% A for 1.0 to 2.3 min, 100% A for 2.3 to 2.9 min, and 10% A for 2.9 to 3.8 min. The column temperature was set at 35 °C. Mass Lynx software, version 4.1, software was used to control the instrument and to collect data.