Uplc tuv system

Fat tissues were isolated from 8-week-old male C57/Bl6J mice, which were housed for 7 days either at 23 or 4 °C. Next, microdialysis membranes (CMA 30 Linear MD Probe, catalogue no. 8010460) were implanted. Tissues were placed in oxygen saturated buffer and perfused with perfusion fluid (M Dialysis AB, catalogue no. P000034) at a flow of 1 µl min⁻¹ using a syringe pump (CMA). After 2 h the flow through of 30 min was collected. Inosine concentrations of the dialysate were measured with a ultra-high-performance liquid chromatogtaphy with tunable UV (UPLC-TUV) system (Waters).

ALR and ALR^A131K activities were determined by measuring the initial velocity of d-alanine formation from l-alanine using ultra-performance liquid chromatography (UPLC) (Waters, Tokyo, Japan). The standard reaction mixture (0.1 mL) containing 100 mM MES buffer (pH 7.0), 20 mM l-alanine, 0.05 mM pyridoxal-5′-phosphate (PLP) and 10 % (v/v) purified enzyme solution. The enzyme reaction was run for 5 min at 30 °C and was then stopped by adding 0.1 mL of 20 % trichloroacetate. The UPLC analysis was performed with an ACQUITY UPLC TUV system consisting of a Waters Binary Solvent Manager, Sample Manager, FLR Detector and AccQ-Tag Ultra 2.1 × 100-mm column (Waters). The eluent flow rate was 0.20 mL/min, the column temperature was 45 °C, and the fluorescent wavelengths of the FLR Detector were 350 and 450 nm. The eluent was linearly graduated using 80 % sodium acetate buffer (50 mM, pH 5.9) and 20 % methanol. All enzyme assays were performed more than three times under the same conditions. Mean values and standard deviations were calculated from each assay.

St6OMT2 activity was detected by quantifying the liberation of (S)-coclaurine at 37°C for 30 min using a reaction mixture in 0.5 ml PBS, 100 μg purified St6OMT2, 1 mM (S)-norcoclaurine, and 1 mM SAM. An equivalent of heat-inactivated St6OMT2 was set as the negative control. The reaction was stopped by adding of 0.25 ml methanol. A rapid analysis method with a UPLC-TUV system (Waters, Milford, United States) was established to detect the reaction products. Equipped with the same BEH C18 column. The mobile phase A and B were the 0.03% trifluoroacetic acid and methanol, respectively. The elution program was set as follow: 18% B at 0–6 min and 18–95% B at 6–15 min. The column temperature and flow rate were 30°C and 0.25 ml·min⁻¹, respectively. The reaction products were confirmed by UPLC-MS/MS (Waters, Milford, United States) based on the retention time and m/z of the external standard.