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To extract plasma metabolites, 200 ul plasma were transferred to each 1.5 ml microcentrifuge tube. 200 µl of 50% aqueous methanol (v/v) containing 0.1% formic acid and 0.05% trifluoroacetic acid were added and vigorously mixed for 30 s. 250 µl of chloroform was added to each sample tube and mixed for 30 s. After this, the sample solution was incubated at 4°C for 10 min and centrifuged at 4°C and 13,000g for 10 min. The solution under pellet was transferred to a 1.5-ml Eppendorf tube for metabolite analysis of heart. Lipid extract of heart tissues was evaporated under a stream of nitrogen, diluted with an isopropanol/acetonitrile/water mixture (2/1/1, v/v/v), and transferred into autosampler vials after centrifugation for 10 min at 13,000g and 4°C.

LC separations were carried out on an Acquity UPLC BEH Amide column (100 x 2.1 mm, particle size 1.7 µm, Waters, USA). Colum temperature and flow rate were set to 40 °C and 0.2 ml/min, respectively. The mobile phases used were 10 mM ammonium formate in 100% DW (A) and acetonitrile:water mixture (90:10, v/v) (B). The linear gradients were as follows: 90% B for 1 min, 35-90% B for 11 min, 25-35% B for 2 min, 25-90% B for 2 min, 40% B for 6 min. The injection volume of the sample was 2 μl for positive ionization polarity modes. Quality control (QC) samples, pooling identical aliquots of the samples, were measured regularly throughout the run for data reproducibility.

To obtain MS spectral data, LC-QTOF analyses of metabolite extracts were performed on a QTOF 6546 MS/MS System (Agilent) combined with a UPLC system (Agilent).