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Sample preparation for HPLC-ESI-MS/MS analysis

LC-ESI-MS in parent mode and multiple reaction monitoring (MRM) mode for determination of fatty acids and phospholipids.

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Sample preparation for HPLC-ESI-MS/MS analysis

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  1. LC-ESI-MS in parent mode and multiple reaction monitoring (MRM) mode for determination of fatty acids and phospholipids • Fatty acids, particularly arachidonic acid (AA, 20:4 FA), and phospholipids, especially lysophospholipids, including LPA, LPC, LPE, LPG, LPI, and LPS were determined by LC-ESI-MS in parent mode and multiple reaction monitoring (MRM) mode. a. AA has been implicated as inflammatory mediators; it’s metabolites, prostaglandin E2,can promote tumor growth; b. Lysophospholipids (Lyso-PLs) are important cell signaling molecules, such as lysophosphatidic acid (LPA), which enhances the invasion and migration of cancer cells; c. Phospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production, and its expression is upregulated in several kinds of carcinomas, like melanoma; PLA2 is also implicated in cancer development; d. LC-ESI-MS in scan mode does NOT provide any lipids information in samples.

  2. Sample preparation for HPLC-ESI-MS/MS analysis • the MeOH method 1 for extraction of lipids: In brief, 10 µL of plasma were added into 1 mL of MeOH with 500 pmol of 12:0 LPC, 100 pmol of 14:0 LPA and 100 pmol of 17:0 Cer as the internal standard (IS). After vortex and incubation on ice for 10 min, the mixture was centrifuged (10,000g, 5min, room temperature), and 120 µL of supernatant were directly used for mass spectrometry (MS) analysis. • Accurate measurement of lysophosphatidic acid by ESI-MS/MS in plasma samples requires pre-separation of lysophosphatidylcholine. 2 1. Zhenwen Zhao, Yan Xu, Anextremely simple method for extraction of lysophospholipids and phospholipids from blood samples. J Lipid Res, 2010, 51: 652-659. PMID: 19783525. 2. Zhenwen Zhao, Yan Xu, Accurate measurement of lysophosphatidic acid by ESI-MS/MS in plasma samples requires pre-separation of lysophosphatidylcholine. J Chrom B, 2009, 877: 3739-3742. PMID: 19734112.

  3. Data analysis The experiment results showed that: • 1. 18:1 LPE was significantly different in patients with polyp or colon cancer compared with healthy controls. • 2. The contents of AA, 22:6 FA, total FA, 18:0 LPI, 20:4 LPI, and total LPI in polyp patient were the most abundant, while those in colon cancer were the lest. However, since only 9 controls, 12 patients with polyp, and 12 patients with colon cancer samples were analyzed, whether these lipids could be used for early diagnosis of colon cancer merits further studies.

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