Rpc Phospholipids Case Study

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S1. On the gradient-elution choice in MMC separations of phospholipids As explained in the main text, three different solvents or solvent mixtures, namely methanol, MeOH (solvent A) and acetonitrile, ACN (solvent B), both containing 10 % (v/v) of water and 1 mmol/L of ammonium acetate, and pure ACN (solvent C), were used to develop the MMC gradient elution program. The program was progressively modified and tested against a standard mixture of phospholipids containing PE 18:0/22:6, PE 18:1/18:1, LPE 18:0, LPE 22:6, PC 16:0/20:4, PC 12:0/12:0, LPC 18:1 and a SM mix from chicken egg yolk, whose species d34:1 and d36:1 were taken as references. The eXtracted Ion Current (XIC) chromatograms retrieved for fingerprint ions of the PL classes under consideration (m/z ratios: 196.0380 for PE and LPE; 224.0693 for PC and LPC; 168.0431 for PC, LPC, SM) from LC-MS…show more content…
Indeed, due to its high polarity, LPC 18:1 (compound # 7) was eluted quite slowly from the C18 column and was finally surpassed both by one of the PCs, the (16:0/20:4), and by all the SMs, whereas LPCs were always eluted after PCs and SMs in the previously developed HILIC separation [11]. Among other results of the MMC separation based on gradient I, the peak splitting for PE 18:1/18:1 and the peak tailing for SMs deserve to be mentioned (see traces A and C in Figure S1). Once the PLs had been all eluted and before the return to the initial composition along with the 20-min conditioning, the mobile phase was drastically changed by decreasing solvent A to 0% and solvent B to 10% and by increasing the percentage of solvent C, pure ACN, to 90%. This step was introduced as a precautionary washing procedure for the C18 column, in the perspective of future application to much more complex lipid mixture, i.e. to remove apolar lipids not eluted from that column when using the A/B mobile phase ratio 50/50

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