1260 infinity autosampler

Optical rotations were measured on a JASCO P-2000 polarimeter using a 1-cm cell. UV and electronic circular dichroism (ECD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, Surrey, UK). 1D and 2D NMR spectra were obtained with Bruker AVANCE III HD 850 spectrometers (Bruker, Billerica, MA, USA) at the National Center for Interuniversity Research Facilities at Seoul National University (NCIRF). UHPLC-Q/TOF-MS analyses were performed on a Waters Acquity UPLC system (Waters Co., Milford, MA, USA) coupled with a Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) that was equipped with an electrospray interface (ESI). The absolute configurations of the amino acids in compounds 1 and 2 were determined using an Agilent 6120 quadruple MSD consisting of a 1260 Infinity pump, a 1260 Infinity autosampler, a 1260 Infinity DAD (Agilent Technologies, Santa Clara, CA, USA), and an Agilent Zorbax SB-C₃ column (150 × 4.6 mm, 5 μm) at 50 °C. Semi-preparative HPLC separations were performed with a system consisting of a Gilson 321 Pump and a UV/Vis-151 detector (Gilson Inc., Middleton, WI, USA). Extra-pure grade solvents for extraction, fractionation, and isolation were purchased from Dae Jung Pure Chemical Engineering Co. Ltd., Siheung, Korea. Deuterated DMSO for NMR analyses was purchased from Merck (Darmstadt, Germany).

Liquid chromatographic analysis was performed on an Agilent HPLC-DAD system, which was equipped with an Agilent 1260 infinity DAD (DEAA306741), an Agilent 1260 infinity autosampler (DEAAC23211), an Agilent 1260 infinity column heater (DEACN25004), an Agilent 1260 infinity pump (DEAB709020), and an Agilent extend-C18 (4.6 mm × 250 mm, 5 μm) column. The binary gradient elution system consisted of solvent A (acetonitrile) and solvent B (0.1% acetic acid water). Optimum separation was achieved by using the gradient program described in Table 2. The column temperature was maintained at 30°C. The autosampler was conditioned at 25°C and injection volume was 10 μL. The flow rate was 1 mL/min. The raw data was detected by an HPLC-DAD (Agilent1260, USA) and the wavelengths were shown in Table 3.

Metabolites were extracted using acetonitrile/methanol (75:25; v/v) containing deuterated internal standards (25 μM thymine-d₄ [Sigma-Aldrich], 10 μM inosine-¹⁵N₄ [Cambridge Isotope Laboratories], 10 μM citrulline-d₇ [Sigma-Aldrich], 25 μM phenylalanine-d₈ [Cambridge Isotope Laboratories], and 10 μM valine-d₈ [Sigma-Aldrich]). The samples were separated using a 2.1 × 100 mm 3.5-μm Xbridge amide column (Waters). Mobile phase A was 95:5 (v/v) water/acetonitrile, with 20 mM ammonium acetate and 20 mM ammonium hydroxide (pH 9.5). Mobile phase B was acetonitrile. For amide-negative mode, the chromatography system consisted of a 1260 Infinity autosampler (Agilent) connected to a 1290 Infinity HLPC binary pump system (Agilent). The eluents were detected in negative mode on a coupled 6490 QQQ mass spectrometry equipped with an electrospray ionization source. The settings were as follows: sheath gas temperature, 400 °C; sheath gas flow, 12 L/min; drying gas temperature, 290 °C; drying gas flow, 15 L/min; capillary, 4000 V; nozzle pressure, 30 psi; nozzle voltage, 500 V; and delta EMV, 200 V. Detailed methods have been described previously⁶² .