HPTLC 2017, Berlin High Performance Thin Layer Chromatography coupled to Electrospray (ESI) Tandem MS for identifying neutral lipids, sphingolipids and phospholipids in complex samples V.L. Cebolla1, M.P. Lapieza1, C.Jarne1, M. Savirón2, J.Orduna3, L. Membrado1, G.E. Morlock4, C. Jungas5, R. Garriga6, J. Vela6, J. Galbán6 1 Instituto de Carboquímica, CSIC; 2 CEQMA-CSIC; 3 ICMA-CSIC, Zaragoza, Spain; 4 Justus Liebig University Giessen, Germany; 5 CEA-CNRS-Université Aix-Marseille, France; 6 Universidad de Zaragoza, Zaragoza,Spain

With or without primuline Coupling with MS has been opening a new era in HPTLC technology MS/MS approach was hampered by the formation of sodium adducts from complex lipids when ESI+ spectra were obtained Sodium adducts from a variety of lipids can be fragmented in positive ion mode, and sodium remains as the charge of stable fragment ions, thus being useful for their unequivocal structural identification in complex samples through MSn and HR-MS HPLC pump delivering solvent Ion-trap / μ-QToF Good quality ESI(+ and -) MS, MSn, HRMS Neutral lipids in FAME-biodiesel Sphingolipids in human plasma Phospholipids in photosynthetic bacterial membranes With or without primuline impregnation

Neutral lipids (NL) in FAME-derived biodiesel IMPORTANCE: As impurities, their composition and percentages determine in part biodiesel performance ANALYSIS: Separation, profiling and individual identification of monoacylglycerides (MG), diacylglycerides (DG), fatty acids (FA), as impurities Sample preparation: pure biodiesel (2500 μg, see Fuel 2016, 177, 244–250), or 25 v/v% in MeOH AMD conditions Diesel FAME ? 15.4 24.2 41.4 As impurities t-butyl methyl ether DCM n-heptane Diesel Primuline-fluorescence 0.02 wt% in MeOH, Λexc=365/em>400 nm

(M+Na+-CH3-H2O)–CH3-(CH2)2 (M+Na+-CH3-H2O)–CH3-(CH2)3–H2O Peak 1 (15.4 mm): Mono-acylglycerides M+Na+ C18:1 M+Na+ oxidized C18:2 377.3 393.3 C20:0 C18:0 381.3 +Na+ C21H40O4Na ESI ESI-MS (+) (M+Na)+- (CH3)-H2O MS/MS 379.3Mw MS/MS/MS 333.2Mw (M+Na)+ -H2O (M+Na+-CH3-H2O) –CH3-CH2 (M+Na+-CH3-H2O)–H2O (M+Na+-CH3-H2O)–CH3-(CH2)2 (M+Na+-CH3-H2O)–CH3-(CH2)3–H2O (+Na+ - H2O) (+Na+ - 2 H2O) (+Na+ - H2O) (+Na+ - 2 H2O) m/z

Peak 2 (24.2 mm): Fatty Acids ESI HR-MS ESI-MS (-) [C18H33O2]- M-H- C18:1 ESI-MS (-) [C18H33O2]- Oleic acid M-H- C16:0 M-H- C16:2 M-H- C18:2 279.0 M-H- C20:0 M-H- C22:0 ESI HR-MS High Resolution (HR)-ESI-MS spectrum of oleic acid in FAME-biodiesel from the plate. m/z theoretical = 281.2486, err (mDa) = 1.2 and err (ppm) = 4.3 5 5

Peak 2 (24.2 mm): Fatty Acids MS/MS 281 6 C18H33O2 M+Na+ C18:1 MS/MS 281 ESI-MS/MS spectrum of m/z 281 ion from oleic acid standard 6 6

Sphingolipids (SL) in human plasma ANALYSIS: Separation, profiling and individual identification of molecular species of sphingomyelins (SMs) and Gb3 (globotriaosyl- ceramides) IMPORTANCE: Biomarkers of Lysosomal Storage diseases (Niemann-Pick, Fabry) Sample preparation: plasma extraction, centrifugation and hydrolysis (see Chromatography 2015, 2, 167-187) AMD conditions 16.7 28.4 Fabry’s patient plasma (25 μL) MeOH DCM Detection: primuline post impregnation (200 ppm); λexc=406nm; (4mm-bands, scanning 3 x 0.2 mm)

Peak 1 (16.7 mm): Sphingomyelins Human plasma,30 μL 725.6 m/z: SM+Na+,(d18:1;C16:0), [C39H79N2O6PNa]+ 835.7 m/z: SM+Na+,(d18:1;C24:1), [C47H95N2O6PNa]+ ESI-MS (+) (SM peak) SM+Na+ (d18:1;C16:0) (d18:1;C24:1) (d18:1;C22:0) (d18:1;C18:0) (d18:1;C20:0) + Na + MS C16:0 m/z= 725.6 MS C24:1 m/z= 835.7 MS2 (fragmentation of 725 m/z) d18:1;C16:0 SM-PC+Na+ (d18:1;C16:0) SM-N(CH3)3+Na+ + Na + m/z= 666.5 M-N(CH3)3+ Na + Na + m/z= 542.6 M-PC+ Na + MS2 (fragmentation of 835 m/z) d18:1;C24:1 SM-PC+Na+ (d18:1;C24:1) SM-N(CH3)3+Na+ + Na + m/z= 776.6 M-N(CH3)3+ Na m/z= 652.7 + Na + M-PC+ Na +

Peak 2 (28.4 mm): Gb3 (globotriaosylceramides) [Gb3+Na]+ (d18:1;C16:0) ESI-MS (+) Fabry’s patient plasma [C52H97NO18Na]+ [Gb3+Na]+ (d18:1;C24:0) [Gb3+Na]+ (d18:1;C22:0) [Gb3+Na]+ (d18:1;C18:0) [Gb3+Na]+ (d18:1;C20:1) + ESI-MS/MS of m/z 1046 + Na [M-hexose+Na]+ + Na +

Phospholipids (PL) in photosynthetic bacterial membranes IMPORTANCE: PL bound to MP have influence on protein activity, and in protein crystallization for isolation ANALYSIS: Separation, profiling and individual identification of molecular species of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidyglycerols (PG) bound to membrane proteins (MP) in photosynthetic purple bacteria Sample preparation: Rhodobacter blasticus and Rhodospirillum rubrum membrane extraction using DDM. Purification of Protein-Detergent-Lipid complex (see Phil. Trans. R. Soc. B 2012, 367, 3412-3419) Water PC (11-13 mm) PE (29-34 mm) PG (45-49 mm) MeOH AcOEt UV366 (AcH) PC (tracks 1,2); PE (3,4); CL (5,6); PG (7,8); blank (9); Rh. blasticus (10,11); blank (12); Rh. rubrum (13,14); blank (15); other membrane extracts and intercalated blank tracks (16-23).

Rhodobacter blasticus ESI+ of band at md 12 mm (PC) R R´ [PC(36:2)+Na]+(CH3COONa) [PC(36:2)+Na]+(HCOONa) [PC(34:1)+Na]+(CH3COONa) R R´ ESI- of band at md 48 mm (PG) ESI- of PG standard at md 48 mm (* m/z 714.6: background AcONa cluster coming from plate conditioning with AcH)

Rhodospirillum rubrum ESI--MS of band at md 30 mm (PE) R R’ The ion at 742 m/z was fragmented to obtain its HPTLC-ESI--MS/MS Loss of –OCH2-CH2(NH3) (* Ions at m/z 714.3, 796.5 and 878.6 correspond to AcONa clusters)

Thank you for your attention The obtention of MSn and HRMS spectra from the plate provides reliable structural identification and a deep characterization of complex lipid profiles Thank you for your attention