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After the sample was injected to the autosampler, the phospholipid fractions were divided into a step gradient with mobile phase A (acetonitrile/methanol/water = 2:2:1 v/v/v containing 0.1% formic acid and 0.028% ammonia): mobile phase B (isopropanol containing 0.1% formic acid and 0.028% ammonia) ratios of 100:0 (05 min), 50:50 (525 min), 50:50 (2559 min), 100:0 (5960 min), and 100:0 (6075 min) at a flow rate of 70 l/min and a column temperature of 30 C