Lc 20adxr system

Optical rotations were measured with a P-2000 polarimeter (JASCO, Tokyo, Japan) using a 10.0 cm tube and water as the solvent. ECD experiments were conducted on a J-1500 spectrophotometer (JASCO, Tokyo, Japan). For the purification of compounds, a Reveleris^® X2 iES flash chromatography system (Büchi, Flawil, Switzerland) and a semi-preparative UltiMate 3000 HPLC from Dionex (Thermo, Waltham, MA, USA), comprising a P580 pump, an ASI 100 automated sample injector, an UVD 170 U detector and a fraction collector, were used. Sephadex LH-20 material was purchased from Sigma-Aldrich (St. Louis, MI, USA). Analytical HPLC experiments were performed on an LC-20AD XR System (Shimadzu, Tokyo, Japan). UV spectra and molar absorption coefficients were measured with a Shimadzu UV 1800 instrument after the dissolution in water, the blank (water) measurement, the measurement of the absorbance at λ_max and the conversion to ε according to the Beer–Lambert law.

Chromatographic analysis was performed on the LC-20ADXR System (Shimadzu, Japan). The Phenomenex Kinete EVO C18 100 Å (50 mm × 2.1 mm, 5 μm) column was used as the analytical column maintained at 40°C with (A) 0.1% formic acid aqueous solution and (B) acetonitrile as the mobile phase. The gradient elution program was operated as follows: 2% B (0–0.5 min), 2–25% B (0.5–0.51 min), 25-55% B (0.51–3.0 min), 55-95% B (3.0–3.01 min), 95% B (3.01–5.0 min), 95–2% B (5.0–5.01 min), and 2% B (5.01–6.0 min). The flow rate was 0.8 mL/min, and the injection volume was 5 μL.
Mass spectrometric detection was performed on an AB-SCIEX API 4000 mass spectrometer (AB SCIEX, Singapore) with ESI in negative ion multiple reaction monitoring (MRM) mode. The optimized instrument parameters were as follows: nebulizer gas: 50 psi, heated by N₂ gas: 55 psi, ion spray voltage: −4500 V and temperature: 550°C; nebulizer, blowback gas, and collision gas were nitrogen; declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) of the four analytes and chloramphenicol (IS) are shown in Table 1.

Arginine (ARG), the amino acid required for NO biosynthesis [14 (link)], and asymmetric dimethylarginine (ADMA), an endogenously produced competitive inhibitor of NO synthase (NOS), were simultaneously measured by HPLC as described [10 (link)], with minor modifications. In brief, ARG and ADMA were extracted from plasma (100 μL) by solid-phase extraction on a polymeric cation-exchange column (HyperSep Retain-CX SPE column; 30 mg, 1 mL; Fisher Scientific) using ammonia : water : methanol (10 : 40 : 50, v : v : v). HPLC separation was performed isocratically at 1.3 mL/min on a Shimadzu LC-20ADXR system equipped with a RF-20AXL fluorescence detector programmed to 340/455 nm (excitation/emission) and a Kinetex XB-C18 column (50 × 3.0 mm, 2.6 μm; Phenomenex). o-Phthalaldehyde-derivatives of ARG and ADMA were eluted using 50 mmol/L potassium phosphate buffer (pH 6.5) and 6.5% (v : v) acetonitrile as the mobile phase. After the peak of ARG eluted from the column, the gain setting was increased at 3 min to enable the detection of ADMA. After the last peak of interest eluted, the column was washed with 50% acetonitrile for 2 min and the system was equilibrated for 2 min before the next injection. Analytes were quantified on the basis of peak area relative to internal standard (methylmonoarginine).