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Analytical processes were performed in random order, including extraction, derivatization, reconstitution, and MS analysis. Metabolite was extracted by mixing 50 µL of mid-trimester maternal serum and tertiary solvent mixture (750 µL, methanol:isopropanol:water, 3:3:2, v/v/v) followed by 10-min sonication and centrifugation (13,200 rpm for 5 minutes at 4°C). Aliquot (750 µL) was transferred into a new 1.5-mL tube and concentrated to complete dryness in a speed vacuum concentrator (SCANVAC, Korea). The dried extracts were stored at -80°C.

LC separations were carried out on an Acquity UPLC BEH C18 column (100 x 2.1 mm, particle size 1.7 µm, Waters, USA). Column temperature and flow rate were set to 40 °C and 0.3 ml/min, respectively. The mobile phases used were 10 mM ammonium formate in an acetonitrile:water mixture (90:10, v/v) (A) and acetonitrile:water mixture (10:90, v/v) (B). The linear gradients were as follows: 40–65% B for 5 min, 65-70% B for 7 min, 70-99% B for 3 min, 99% B for 2 min, 40% B for 3 min. The injection volume of the sample was 2 μl using partial loop mode for both positive and negative ionization polarity modes. Quality control (QC) samples, pooling identical aliquots of the samples, were measured regularly throughout the run for data reproducibility.

MS analysis was performed using Q-Exactive Plus Orbitrap (ionization mode: negative, Scan parameter: full scan/ddMS2, scan range: 70-1,000 m/z, resolution: 70,000, automatic gain control: 1e6, maximum injection time: 100 ms, HESI-II voltage: 3500v, S-lens radio frequency level: 50 v, capillary temperature: 300°C). MS/MS analysis was conducted in data-dependent manner (HCD, 30 eV). Raw data (.raw format) was transformed to Analysis Base File (ABF) format by Reifycs Abf Converter (http://www.reifycs.com/AbfConverter/index.html). Data process (compound identification and semi-quantification) was done by the MS-DIAL software and Lipid Blast library (MS1 tolerance : 0.005Da, MS2 tolerance : 0.01Da, similarity score: 75%).