1. 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

2. 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

3. 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

4. (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

5. 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 = , err (mDa) = 1.2 and err (ppm) = 4.3 5 5

6. 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

7. 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, )
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)

8. 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 +

9. 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 +

10. 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, )
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).

11. 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)

12. 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, and correspond to AcONa clusters)

13. 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