HILIC UPLC/MS UPLC LC/ MS LC/MS HPLC 2-4 HPLC 4,5 HILIC / HILIC 80 ACQUITY UPLC Xevo QTof MS ACQUITY BEH HILIC HILIC TOF ESI 1.7 µm BEH HILIC UPLC HIL

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1 HILIC UPLC/MS UPLC LC/ MS LC/MS HPLC 2-4 HPLC 4,5 HILIC / HILIC 80 ACQUITY UPLC Xevo QTof MS ACQUITY BEH HILIC HILIC TOF ESI 1.7 µm BEH HILIC UPLC HILIC UPLC HPLC 1

2 100 µl 900 µl µl 500 µl 470 µl / 1/1 120 µl 10 LC LC ACQUITY UPLC : ACQUITY UPLC HILIC 2.1x100 mm 1.7 µm : 30 : 500 µl/min A 10 mm / 95:5 ph=8.0 B 10 mm / 50:50 ph= %B/10 PC SMm/z Da PC SM 1 8 HILIC UPLC/MS 16 HILIC 1 6 N- heptadecanoyl -sphing-4-enine Cer d18:1/17:0 dimyristoyl-phosphatidylglycerol PG 14:0/14:0 dimyristoyl-phosphoethanolamine PE 14:0/14:0 dimyristoyl-phosphatidylcholine PC 14:0/14:0 lauroyl sphingomyeline SM d18:1/12:0 eicosanoyl-sn-glycero-3-phosphocholine LPC 20:0 MS E 3 µl PC(14:0/14:0) 6.08 MS LPC(20:0) MS Xevo QTof MS PG(14:0/14:0) SM(d18:1/12:0) 7.78 ESI / 2800 V /1900 V Cer(d18:1/17:0) 0.59 PE(14:0/14:0) V L/Hr HILIC UPLC m/z Analysis of Intact Lipids from Biologics Matrices by UPLC/HDMS

3 HILIC UPLC 9 UPLC 2 2A PCSM LPC 9,10 HILIC Cer PG PI PE PC 2B Cer PC 2A LPC SM 2A ESI+ HILIC UPLC PC 2B PI, PE LPC PG 2B ESI- HILIC UPLC A HILIC-Based UPLC/MS Method for the Separation of Lipid Classes from Plasma 3

4 HILIC PC SM UPLC PC 32:1 m/z SM d18:1/18:0 m/z Da HILIC 3 2 SM(d18:1/18:0) PC(32:1) 3 PC 32:1 m/z SM d18:1/18:0 m/z mda 4 A HILIC-Based UPLC/MS Method for the Separation of Lipid Classes from Plasma

5 HILIC UPLC Shui G, et al. Sensitive profiling of chemically diverse bioactive lipids. J. Lipid Res. 2007, 48; Han X and Gross R W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples. Mass Spectr. Rev. 2005, 24; Sommer U, et al. LC-MS based method for the qualitative and quantitative analysis of complex lipid mixtures. J. Lipid Res. 2006, 47; Castro-Perez J M, et al. Comprehensive LC MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients. J. Proteome Res. 2010, 9; Peterson B L and Cummings B S. A review of chromatographic methods for the assessment of phospholipids in biological samples. Biomed. Chromato. BMC 2006, 20; Perona J S and Ruiz-Gutierrez V. Simultaneous determination of molecular species of monoacylglycerols, diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography. J Chromato B. 2003, 785; Henderson M A and McIndoe J S. Ionic liquids enable electrospray ionisation mass spectrometry in hexane. Chem. Commun. 2006, Shui G, et al. Characterization of substrate preference for Slc1p and Cst26p in S cerevisiae using lipidomic approaches and an LPAAT activity assay. PLoS One. 2010, 5; e Shockcor J, et al. Analysis of intact lipids from biological matrices by UPLC/high definition MS. Waters Application Note no en, February Rainville P D, et al. Novel Application of Reversed-Phase UPLC-oaTOF-MS for Lipid Analysis in Complex Biological Mixtures: A New Tool for Lipidomics. J. Proteome Res. 2007, 6; Waters UPLC ACQUITY UPLC Xevo Waters Corporation The Science of What s Possible Waters Corporation 2011 Waters Corporation. Produced in Japan JA PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F:

1 WSV SIR m/z Analyte RT (Min) SIR m/z Cone voltage (V) Ascorbic Acid (C).91 177 2 Thiamine (B1) 1.1 265 5 Nicotinic Acid (B3) 1.27 124 15 Pyridoxal (

1 WSV SIR m/z Analyte RT (Min) SIR m/z Cone voltage (V) Ascorbic Acid (C).91 177 2 Thiamine (B1) 1.1 265 5 Nicotinic Acid (B3) 1.27 124 15 Pyridoxal ( ACQUITY UPLC H-Class ACQUITY QDa Mark E. Benvenuti, Dimple Shah, and Jennifer A. Burgess Waters Corporation, Milford, MA, USA MS ACQUITY QDa UPLC HPLC UV ACQUITY QDa LC EC 1925/26 CFR 21 HPLC 1 LC-MS ACQUITY

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