LC/MS in Lipidomics
Michal Holčapek, Eva Cífková and Miroslav Lísa
University of Pardubice, Department of Analytical Chemistry, Czech Republic
http://holcapek.upce.cz/
Lipid Classification
Nonpolar lipids
Fatty acyls
Polyketides
COOH
Fatty acyls
Glycerolipids
Sphingolipids
Prenols
Glycerolipids
Saccharolipids
Phospholipids
Sterols
Oleic acid
O
H
O
O
Fatty acyls
O
Glycerol
O
O
Sterols
Triacylglycerol
Prenols
O
OH
OH
Cholesterol
Tocopherol
1
Lipid Classification
Polar lipids
Fatty acyls
Sterols
Glycerolipids
Prenols
Saccharolipids
Phospholipids
Polyketides
Sphingolipids
Phospholipids
Glycerol
O
O
O P
O-
O H
Sphingolipids
Polar head group
OR
R = choline (PC), ethanolamine
(PE), serine (PS), glycerol
(PG), inositol (PI), etc.
O
Nonpolar fatty acyls
O
O
H OH
O P O
NH H
OO
Nonpolar fatty acyls
N+
Choline
Glycerol
Sphingomyelin
Comprehensive Lipidomic Approach
GC/FID
MS Imaging
Hexane/MeOH/H2O
CHCl3/MeOH/H2O
Nonpolar lipid extract
Total lipid extract
MALDI-Orbitrap
1D
HILIC-LC/MS
2D
RP-LC/MS
SFC/MS
NARP-LC/MS
RP-LC/MS
Ag-LC/MS
NP-LC/MS
Chiral-LC/MS
Data processing
Statistical evaluation
2
Sample Preparation – Extraction Techniques
• Folch – CHCl3: MeOH (2:1, v/v) (J.Biol.Chem. 226 (1957) 497)
• Bligh & Dyer – CHCl3: MeOH (1:1, v/v) (J.Bioch.Physiol. 37 (1959) 911)
• Shevchenko – MTBE : MeOH (5:1.5, v/v) (J.Lip.Res. 49 (2008) 1137)
• Nonpolar lipids - hexane : MeOH : water (2:1:0.1, v/v/v) (J.Lip.Res. 53 (2012)1690)
LC/MS in Lipidomic Analysis
Lipid class approach
• HILIC-LC/ESI-MS - for polar lipid classes, Si, NH2 or diol columns, mobile phases
with acetonitrile, methanol, 2-propanol, hexane and ≥ 2.5% water
• NP-LC/APCI-MS - nonpolar lipid classes, Si or NH2 columns, mobile phases with
hexane, 2-propanol, chloroform, heptane, etc.
• SFC/MS – all lipid classes, C18 or Si columns, mobile phase supercritical CO2 with
polar organic modifiers (typically methanol)
Lipid species approach
• RP-LC/ESI-MS – all lipid species, C18 and C8 columns, mobile phases aqueous
mixtures of acetonitrile, methanol or 2-propanol, often with volatile buffers
• NARP-LC/APCI-MS – nonpolar lipid species, C18 columns, mobile phases 2propanol, acetonitrile, aceton, dichlormethane, etc.
• Ag-LC/APCI-MS – nonpolar lipid species, ion-exchange column with Ag+, mobile
phases with hexane – acetonitrile (+2-propanol) or dichlormethane – acetonitrile
• Chiral-LC/APCI-MS – nonpolar lipid species, chiral columns, normal phase mobile
system with hexane and 2-propanol
• SFC/MS – all lipid species, C18 columns, supercritical CO2 with polar modifiers
3
Comprehensive Lipidomic Approach
Lipid extract
1D
•
HILIC-LC/MS
Separation of lipid classes
Optimization of HILIC-LC/ESI-MS
• Optimized parameters: column type (9 columns), mobile phase composition (mainly
organic solvent and concentration of water), gradient steepness, pH, salt content, etc.
Effect of ammonium acetate
5 mM
0 mM
Effect of pH value
pH = 7
pH = 4.5
Conditions: column Spherisorb Silica (250*4.6 mm, 5 μm, Waters), ESI-MS detection,
separation temperature 40°C, gradient of acetonitrile / aqueous ammonium acetate
4
HILIC-LC/ESI-MS Separation of Lipid Classes
Standards containing oleoyl acyls ∆9–C18:1
• Separation of 16 lipid classes + 3 regioisomeric pairs of lysophospholipids.
• Individual fractions are collected for off-line 2D-LC/MS characterization
Conditions: column Spherisorb Silica (250*4.6 mm, 5 μm, Waters), ESI-MS detection,
separation temperature 40°C, gradient of acetonitrile / aqueous ammonium acetate
M. Lísa, E. Cífková, M. Holčapek, J. Chromatogr. A, 1218 (2011) 5146
Nontargeted Quantitation of Lipid Classes
1
Sphingosyl PE (d17:1/12:0)
Lipid
class
tR [min]
a
b
r2
Response
factor (RF)
PI
8.5
45.7
-23.8
0.9995
1.272
PG
4.7
183.1
14.6
0.9996
0.318
LPG
8.4
271.8
23.1
0.9993
0.214
PE
24.8
196.6
2.2
0.9991
0.296
IS
33.9
58.1
-0.6
0.9998
1.000
LPE
36.1
112.7
-1.1
0.9984
0.516
PS
37.3
43.9
25.2
0.9994
1.325
PC
39.8
550.7
9.5
0.9997
0.106
SM
45.3
857.6
3.8
1.0000
0.068
LPC
51.2
488.6
2.8
0.9998
0.119
E. Cífková, M. Holčapek, M. Lísa, et al., Anal. Chem. 84 (2012) 10064
5
Comprehensive Lipidomic Approach
Lipid extract
1D
•
NP-LC/MS
HILIC-LC/MS
Separation of lipid classes
NP-LC/APCI-MS of Nonpolar Lipid Classes
Standards containing nonadecanoyl acyls (C19:0)
• Separation of 6 nonpolar lipid classes and 2 regioisomers of diacylglycerols
Conditions: column Acquity UPLC BEH HILIC (50*2.1 mm, 1.7 μm, Waters), APCI-MS
detection, 30°C, gradient of hexane / acetonitrile / 2-propanol
6
NP-UHPLC Analysis of Plasma of CVD Patients
• NP-UHPLC/APCI-MS measurement of plasma extract of nonpolar lipids
•
Conditions: column HILIC - Acquity UPLC (2.1×50 mm, 1.7 µm, Waters), flow rate 1
mL/min, separation temperature 30°C, gradient hexane/2-propanol/acetonitrile
Comprehensive Lipidomic Approach
Lipid extract
1D
•
HILIC-LC/MS
SFC/MS
NP-LC/MS
Separation of lipid classes
7
SFC/MS of Polar Lipid Classes
Mixture of lipids standards from Avanti
1.00x108
PC
7.50x107
Intensity
SM
LPC
5.00x107
PE
PG
2.50x107
LPE
10 minutes
CER
0.00
0.00
1.20
2.40
3.60
4.80
6.00
Minutes
7.20
8.40
9.60
10.80
12.00
Conditions: column Acquity UPC2 BEH, methanol gradient, additive ammonium formate, ESIMS detection
Data courtesy: Giorgis Isaac (Waters)
Comprehensive Lipidomic Approach
Lipid extract
1D
•
•
HILIC-LC/MS
SFC/MS
RP-LC/MS
NP-LC/MS
Separation of lipid classes
Separation of lipid species
8
RP-LC/ESI-MS of Total Lipid Extract
Blood plasma
K. Sandra et al., J. Chromatogr. A 1217 (2010) 4087
LC-MS Feature Maps of Plasma
ESI+
ESI-
K. Sandra et al., J. Chromatogr. A 1217 (2010) 4087
9
RP-LC/ESI-MS of Total Lipid Extract
• both polar and nonpolar lipids are separated in one chromatographic run
using the coupling of two columns
Conditions: ACQUITY UPLC BEH C18 column (150 and 150 x 2.1 x 1.7 mm); flow rate 0.18
mL/min, separation temperature 40°C, 5 mM aqueous ammonium acetate/5 mM ammonium
acetate in acetonitrile:2-propanol (1:2) gradient.
RP-LC/ESI-MS of Total Lipid Extract
13 FA and 6 LPC
EIC 184
34 TG
27 PC andEIC
12 369
SM
15 CE
Conditions: ACQUITY UPLC BEH C18 column (150 and 150 x 2.1 x 1.7 mm); flow rate 0.18
mL/min, separation temperature 40°C, 5 mM aqueous ammonium acetate/5 mM ammonium
acetate in acetonitrile:2-propanol (1:2) gradient.
10
Comprehensive Lipidomic Approach
Lipid extract
1D
HILIC-LC/MS
2D
•
•
SFC/MS
RP-LC/MS
NP-LC/MS
RP-LC/MS
Separation of lipid classes
Separation of lipid species
Off-line 2D HILIC x RP-LC/ESI-MS of Polar Species
1D: HILIC sepation
16:0/18:1-PC
2D: RP-LC separation
PC
Conditions: column Kinetex C18
(150*2.1
mm,
2.6
μm,
Phenomenex), ESI-MS detection,
separation temperature 40°C,
acetonitrile
/
2-propanol
/
aqueous ammonium acetate
gradient.
M. Lísa, E. Cífková, M. Holčapek,
J. Chromatogr. A, 1218 (2011) 5146
11
Off-line 2D HILIC x RP-LC/ESI-MS of Polar Species
1D: HILIC separation
16:0/18:1-PE
2D: RP-LC separation
PE+pPE
Conditions: column Kinetex C18
(150*2.1
mm,
2.6
μm,
Phenomenex), ESI-MS detection,
separation temperature 40°C,
acetonitrile
/
2-propanol
/
aqueous ammonium acetate
gradient.
Off-line 2D HILIC x RP-LC/ESI-MS of Polar Species
1D: HILIC separation
d18:1/16:0-Cer
d18:1/16:0-HexCer
2D: RP-LC separation
HexCer + Cer
Conditions: column Kinetex
C18 (150*2.1 mm, 2.6 μm,
Phenomenex),
ESI-MS
detection,
separation
temperature 40°C, acetonitrile /
2-propanol
/
aqueous
ammonium acetate gradient.
12
Orthogonality of Offline 2D-LC/MS of Lipids
M. Lísa, E. Cífková, M. Holčapek, J. Chromatogr. A 1218 (2011) 5146
Stop-Flow 2D HILIC x RP-LC/MS of Polar Lipids
PE in cow´s milk
P. Dugo et al., J. Chromatogr. A 1278 (2013) 46
13
Stop-Flow 2D HILIC x RP-LC/MS of Polar Lipids
PC in plasma
P. Dugo et al., J. Chromatogr. A 1278 (2013) 46
Comprehensive Lipidomic Approach
Lipid extract
1D
2D
•
•
SFC/MS
HILIC-LC/MS
RP-LC/MS
RP-LC/MS
NP-LC/MS
NARP-LC/MS
Individual lipid classes
Individual lipid species
14
NARP-LC/APCI-MS of Nonpolar Lipids
• Nonpolar lipids - TGs, DGs, wax esters, cholesterol and its esters
• Separation of TGs according to ECN and also inside ECN groups
• Equivalent carbon number (ECN) = CN – 2*DB (CN - carbon number in all acyl
chains, DB - double bond number)
• Our optimized LC/MS method >400 TGs containing 36 FAs with acyl lengths from
6 to 28 carbon atoms and from 0 to 6 DBs) identified in more than 200 different
materials (plant oils, animal fats, fish oils, body fluids) and statistically evaluated
by PCA
CH 2 O COR 1 sn-1
Triacylglycerol
sn-2 R 2CO O CH
CH 2 O COR 3 sn-3
M. Lísa, M. Holčapek, J. Chromatogr. A 1998-1999 (2008) 115
M. Holčapek, M. Lísa, P. Jandera, N. Kabátová, J. Sep. Sci., 28 (2005) 1315
M. Holčapek, P. Jandera, P. Zderadička, L. Hrubá, J. Chromatogr. A 1010 (2003) 195
Important Ions in APCI-MS Spectra of TGs
[M+H-RiCOOH]+ - fragment ions A
(identification of FA)
[M+H]+  MW
CH2 O COR1
R2CO O CH
+ H+
CH2 O COR3
-R1COOH
-R2COOH
-R3COOH
B2
CH2 OH
2
CH
O
R CO
+
CH2
B1(3)
CH2+
R2CO O CH
CH2 O COR3
CH2 O COR1
CH
CH2 O COR3
+
CH2 O COR1
R2CO O CH
CH2+
1(3)
CH2 O COR
HO CH
+
CH2
C
[RCO]+
15
Off-line 2D HILIC-NARP-LC/MS of Nonpolar Lipids
1D: HILIC separation
• TG are separated according to ECN
and also inside ECN groups
ECN = CN – 2*DB
CN – carbon number in all acyl chains
DB – double bond number
2D: NARP-LC separation
TG + Chol
Conditions: 2 columns NovaPak C18 (300+150*3.9 mm, 4 m,
Waters), APCI-MS, temperature
25°C, acetonitrile / 2-propanol
gradient
Resolution of Isomers Differing in DB Position
• Different retention times both in NARP-LC and Ag-LC modes
• Different relative abundances of fragment ions
NARP-LC/APCI-MS analysis of unusual FA in conifer seed oils
-linolenic acids (C18:39,12,15)
15
12
COO H
9
Pinolenic acid (C18:35,9,12)
12
9
5
COOH
• 18 carbon atoms and 3 double bonds in different positions
16
NARP-LC/APCI-MS Analysis of Conifer Seed Oils
LLPi
Fatty acids
CN:DB Common ∆9(6)
∆k=0.4
Pinolenic (Pi)
18:2
Linoleic (L)
Taxolic (Ta)
LLLn
∆k(Pi)=0.4
∆k(Ta)=0.6
LLL
LLTa
TaLPi
∆k=0.6
∆k=0.6
PiLPi
LnLPi
PiLnPi
PiPiPi
Linolenic (Ln)
Retention factor...k=(tR-tM)/tM
∆k=0.4
∆k=0.4
Unusual ∆5
18:3
Larch
(Larix decidua)
APCI-MS Analysis of Unusual TGs
LLL
[M+H]+
LnLnLn
[M+H]+
A / [M+H]+ = 25 / 100%
A / [M+H]+ = 42 / 100%
A
A
C
C B
LLTa
B
PiPiPi
A
A
[M+H]+
[M+H]+
100 / 87%
100 / 74%
C-H2O
C B
C-H2O
B
C
17
NARP-LC/APCI-MS Separation of IsomericTGs
cis/trans
linear / branched
RIC 579
linear / branched
•
Conditions: Nova-Pak C18 columns (300+150*3.9 mm, 4 m, Waters),1 mL/min, APCI-MS
detection, separation temperature 25°C, injection 10 L, acetonitrile / 2-propanol gradient
M. Lísa et al., J. Chromatogr. A 1218 (2011) 7499
Enzymatic
Hydrolysis of
Blackcurrant Oil
1
TG
CH2 O COR
R CO O CH
3
CH2 O COR
DG
CH2 O COR
2
R CO O CH
CH2 OH
0,75
Native oil
TG
(83 TGs
from 14 FAs)
0,55
AU 0,35
2
0,15
1
-0,05
0
10
20
30
1
MG
FA
40
50
60
70
80
90
Time [min]
CH2 O COR
HO CH
CH2 OH
0,95
Enzymatic
hydrolysis
FA + MG
0,75
i
R COOH
DG
0,55
AU
• Hydrolysis with stereoselective
immobilized enzyme Lipozyme in
supercritical fluid CO2 extractor
0,35
TG
0,15
-0,05
0
10
20
30
40
50
60
70
80
90
Time [min]
18
Enzymatic Hydrolysis of DG in Blackcurrant Oil
Regioselectivity of
enzyme is ca. 97%
DG
1,3-DG
1,2-DG
LLn
3.67
96.33
LLn
1.92
98.08
LL
2.57
97.43
M. Lísa, M. Holčapek, H. Sovová, J. Chromatogr. A 1216 (2009) 8371
UHPLC – Reduction of Analysis Time
0,72
HPLC
0,55
UHPLC
Positional DB isomers:
Ln (9,12,15-C18:3)
γLn (6,9,12-C18:3)
0,52
0,35
0,32
0,15
0,06
HPLC:
-0,04
47,5
60
65
70
75
80
50 50min
-0,08
85
90
3
95
4
5
6
0,16
-0,04
4,5
7
8
9
5 min
5
γLnγLnLn
55
10
11
12
γLnγLnγLn
50
LnLnLn
γLnLnLn
45
γLnγLnLn
40
LnLnLn
35
γLnγLnγLn
-0,05
γLnLnLn
0,12
5,5
UHPLC:
Acquity BEH C18 column (150*2.1 mm, 1.7 m,
Nova-Pak C18 columns (300+150*3.9 mm,
4 m, Waters),1 mL/min, 25°C, injection 10 L, Waters), 0.4 mL/min, 30°C, injection 1 L,
acetonitrile / 2-propanol gradient.
acetonitrile / 2-propanol gradient.
52,5
19
Comprehensive Lipidomic Approach
Lipid extract
1D
2D
•
•
SFC/MS
HILIC-LC/MS
RP-LC/MS
NARP-LC/MS
RP-LC/MS
NP-LC/MS
Ag-LC/MS
Separation of lipid classes
Separation of lipid species
Silver-ion LC/APCI-MS of TGs
Regioisomers
OOP
• Separation mechanism according to the double bond (DB) number
• Resolution of cis- / trans-, regio- and DB positional isomers
Geometrical isomers
2trans 3trans
1cis 2cis 3cis 4cis
POP
OPP
OPO
1trans
[OP]+
100%
[PP]+
83%
[OP]+
100%
[PP]+
32%
Conditions: 3 columns ChromSpher (250*4.6 mm, 5 m, Varian) connected in series, 1
mL/min, 25 °C, injection 1 L, hexane / 2-propanol / acetonitrile gradient
M. Lísa et al., Anal. Chem. 81 (2009) 3903 and J. Chromatogr. A 1218 (2011) 7499
20
Randomization Synthesis of TG Regioisomers
• Chemical transesterification (random) of fatty acids in TGs
• Catalyst sodium methanolate, temperature 75°C, reaction time 30 min
• Applied for the synthesis of regioisomeric TG standards
Mixture of 2 mono-acid TGs (AAA + BBB)
Regioisomers Regioisomers
(AAB:ABA=2:1) (BBA:BAB=2:1)
M. Lísa et al. Anal. Chem. 81 (2009) 3903
Randomization Mixture of PPP / OOO / LnLnLn
Products: initial PPP, OOO, LnLnLn +6 regioisomeric doublets +1 regioisomeric triplet
Double bonds
6
2
5
7
3
0
1
4
9
Conditions: 3 columns ChromSpher (250*4.6 mm, 5 m, Varian) connected in series, 1
mL/min, APCI-MS detection, separation temperature 25 °C, injection 1 l, hexane / 2-propanol /
acetonitrile gradient
21
Preference of sn-2 Occupation by Silver-ion LC
Regioisomers
Plant oil
Animal fats
Sunflower
Pork
Wild boar
Cattle
Duck
Rabbit
POP/OPP
100/0
8/92
10/90
63/37
52/48
51/49
OOP/OPO
98/2
12/88
28/82
94/6
53/47
77/33
PLP/LPP
100/0
1/99
8/92
61/39
56/44
52/48
LLP/LPL
97/3
9/91
9/91
62/38
58/42
54/46
OLP/LOP/OPL
63/36/1
3/12/85
11/14/85
49/36/15
41/36/23
47/35/18
• Plant oils – strong preference of unsaturated fatty acids in sn-2, mainly linoleic
acid (L)
• Pork and wild boar fats – preference of saturated fatty acids (P)
• Other studied animals fats – in between above mentioned two cases
M. Lísa et al. Anal. Chem. 81 (2009) 3903
M. Lísa et al., J. Chromatogr. A 1218 (2011) 7499
Silver-ion LC/MS of TGs in Beef Tallow
Double bonds
0
1 trans
2 trans 3 trans
1 cis
2 cis 3 cis 4 cis
• Clear resolution of cis- / trans- isomers, e.g., 9cis-18:1 (oleic) and
9trans-18:1 (elaidic) containing triacylglycerols
22
OEE+EOE
NARP-LC
NARP-LC conditions:
Nova-Pak C18 columns (300+150*3.9 mm,
4 m, Waters) connected in series, APCI-MS
detection, 1 mL/min, 25°C, injection 10 L,
acetonitrile / 2-propanol gradient
OOE
OEE
Ag-HPLC
Ag-LC conditions:
3 columns ChromSpher (250*4.6 mm, 5 m,
Varian) connected in series, APCI-MS
detection, 1 mL/min, 25 °C, injection 1 l,
hexane / 2-propanol / acetonitrile gradient
OOO
OEO
EOE
EEE
OOO
O – C18:19cis
E – C18:19trans
EEE
OOE+OEO
Off-line 2D LC/MS of TGs – Orthogonality
• NARP and silver-ion modes provide orthogonal separation, their off-line 2D
coupling yields superior resolution including the resolution of regioisomers
M. Holčapek et al., J. Sep. Sci. 23 (2009) 3672
Off-line 2D NARP x Ag-LC/APCI-MS of TG
98
ECN 42
LOSt
γLnOγLn
γLnOLn
LLγLn
OLnLn
LLLn
γLnPLn γLnγLnP
ECN 44
88 DB 6
LOγLn
SLSt
LOLn
GLγLn
OLγLn
OLLn
LnLnP
LLL
78
ECN 46
GLLn
γLnLP
LnLP
DB 5
OOLn
OOγLn
LOL
OLL
SLnL
GLL
γLnOP
OOL
LnOP
DB 4
LLP
OLO
nd
68
SLL
DB 3
58
58
LOP
OLP
63
68
73
st
1 dimension (NARP-HPLC) [min]
78
nd
2 dimension (Ag-HPLC) [min]
(B)
2 dimension (Ag-HPLC) [min]
(A)
ECN 40
DB 7 LnOLnOLSt
1st dimension (NARP-HPLC) [min]
23
Schematic Layout of LC x LC Instrumentation
E.J.C. van der Klift et al., J. Chromatogr. A 1178 (2008) 43
Online 2D Ag x NARP-LC/APCI-MS of TG
ECN = CN – 2*DB
E.J.C. van der Klift et al., J. Chromatogr. A 1178 (2008) 43
24
Online 2D Ag x NARP-LC/APCI-MS of TG
ECN = CN – 2*DB
L. Mondello et al., J. Sep. Sci. 34 (2011) 688
Dual Parallel MS for Lipid Analysis
W. C. Byrdwell, J. Chromatogr. A, 1217 (2010) 3992
25
Dual Parallel MS for Lipid Analysis
HILIC-LC separation of polar lipids
Diverted flow
RP-LC separation of nonpolar lipids
W. C. Byrdwell, J. Chromatogr. A, 1217 (2010) 3992
Comprehensive Lipidomic Approach
Lipid extract
1D
HILIC-LC/MS
2D
•
•
RP-LC/MS
SFC/MS
NARP-LC/MS
RP-LC/MS
Ag-LC/MS
NP-LC/MS
SFC/MS
Separation of lipid classes
Separation of lipid species
26
SFC/MS of Cholesterol Esters
3
TAGs_08022012_025
100
1: TOF MS ES+
BPI
4.15e5
2
%
4
5
Peak
Lipid Species
1
18:3 CE
2
3
18:2 CE
17:0 CE and 18:1 CE
4
18:0 CE
5
19:0 CE
6
23:0 CE
1
6
4 minutes
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
Time
3.80
Conditions: ACQUITY UPC2instrument, column Acquity UPC2 HSS C18 SB, methanol
gradient, additive ammonium formate, ESI-MS detection
Data courtesy: Giorgis Isaac (Waters)
SFC/MS of Triacylglycerols
Peak
2
TAGs_08022012_029
100
1
Lipid Species
1
6
4
5
%
7
3
1: TOF MS ES+
BPI
2.08e5
2
15:0/15:0/15:0 TG
18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:3(∆9,12,15
Cis) TG
3
16:0/16:0/16:0 TG
4
18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,12Cis) TG
5
18:1(∆9Cis)/18:1(∆9Cis)/18:1(∆9TCis) TG
6
17:0/17:0/17:0 TG
7
18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG
8
18:0/18:0/18:0 TG
20:0/20:0/20:0 TG
9
8
9
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
4 minutes
3.80
Time
UPC2instrument,
column Acquity UPC2 HSS C18 SB, methanol
Conditions: ACQUITY
gradient, additive ammonium formate, ESI-MS detection
Data courtesy: Giorgis Isaac (Waters)
27
Comprehensive Lipidomic Approach
Lipid extract
1D
2D
•
•
SFC/MS
HILIC-LC/MS
RP-LC/MS
NARP-LC/MS
RP-LC/MS
NP-LC/MS
Chiral-LC/MS
SFC/MS
Ag-LC/MS
Separation of lipid classes
Separation of lipid species
Chiral Separation of Triacylglycerols
CH 2 O COR 1 sn-1
sn-2 R 2CO O CH
CH 2 O COR 3 sn-3
OH
O
O
A
A
O
O
H+
A
O
OH
OH
A
O
O
=
*
O
O
OH
A
O
O
A
H+
OH O
OH
A
O
O
A
A - C20:0
O - ∆9-C18:1
• Column: cellulose-tris-(3,5-dimethylphenylcarbamate) (2 x 250*4.6 mm, 3 µm, Lux
Cellulose-1, Phenomenex)
• Gradient: hexane - 2-propanol (0.1% 2-propanol/hour)
• Temperature: 35°C
M. Lísa, M. Holčapek, Anal. Chem. 85 (2013) 1852
28
Synthesis of Triacylglycerol Isomers
Randomization reaction of monoacyl-TGs
• random transesterification of fatty acyls in TGs
regioisomers
R1
R1 +
R1
R2
R2
R2
R1
R1 +
R1
MeONa
75°C, 30 min
R2
R1
R1
R1
R2
R1
R1
R1 +
R2
R2
R2
R1
R2
R1
R2
R1
R2 +
R2
R2
R2
R2
enantiomers
Chiral HPLC/MS of TGs
3
2
4
5
6
Randomization mixture of
AAA / OOO / LnLnLn
(C20:0 / C18:1 / C18:3)
- enantiomers
1
7
- regioisomers
0
9
A (C20:0), O (∆9-C18:1), Ln (∆9,12,15-C18:3 )
29
APCI Mass Spectra of TG Isomers
OAO
15%
AOO
OOA
43%
40%
A (C20:0), O (∆9-C18:1)
Stereospecific Synthesis of mixed-acyl TGs
2,3-isopropylidene-sn-glycerol
OH
O
O
R1
DMAP, DCC
22°C, 90 min
R1
O
O
TFA
-20°C, 30 min
R1
OH
OH
R2
DMAP, DCC
22°C, 90 min
R2 + R3
1,2-isopropylidene-sn-glycerol
O
O
OH
R1
DMAP, DCC
22°C, 90 min
O
O
R1
TFA
-20°C, 30 min
OH
OH
R1
R2
DMAP, DCC
22°C, 90 min
R2 + R3
R1
R2
R2
R1
R2 +
R2
R1
R2
R3
R1
R3 +
R2
R1
R3
R3
R2
R2
R1
R2
R2 +
R1
R2
R3
R1
R3
R2 +
R1
R3
R3
R1
DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid
M. Lísa, M. Holčapek, Anal.Chem. 85 (2013) 1852
30
Chiral HPLC/MS of TG Enantiomers
O
O
OH
OH
O
O
Ri =
O
O
O
O
O
H
+
OH Ri
OH
O
Ri
Ri
O
O
O
O
H+
O
Ri
OH
OH
O
Ri
Ri
R iR iO
ORiRi
S (C18:0)
O (∆9-C18:1)
L (∆9,12-C18:2)
Ln (∆9,12,15-C18:3)
Chiral HPLC/MS of Hazelnut oil
•
•
SSO / SOS / OSS
0 / 100 / 0
PPO / POP / OPP
0 / 100 / 0
SOO / OSO / OOS
39 / 0 / 61
POO / OPO / OOP
54 / 0 / 46
PLO / OLP / POL+LOP / OPL
27 / 29 / 44 / 0
OLO / LOO / OOL
46 / 39 / 15
LPL / PLL+LLP
0 / 100
LLO / OLL / LOL
55 / 23 / 22
unsaturated FAs in sn-2 position
more DBs in sn-1 position
P (C16:0), S (C18:0), O (∆9-C18:1), L (∆9,12-C18:2 )
31
Chiral HPLC/MS of Breast Cancer
Tumor tissue
•
PPO / POP / OPP
38 / 62 / 0
SOO / OSO / OOS
100 / 0 / 0
POO / OPO / OOP
94 / 0 / 6
PLP / LPP+PPL
56 / 44
PLO / OLP / POL+LOP / OPL
55 / 0 / 45 / 0
OLO / LOO / OOL
42 / 28 / 30
LPL / LLP+PLL
0 / 100
LLO / OLL / LOL
36 / 41 / 23
more DBs in sn-3 position
P (C16:0), S (C18:0), O (∆9-C18:1), L (∆9,12-C18:2 )
Chiral HPLC/MS of DGs and MGs
• Column: Lux Cellulose-1 (250*4.6 mm, 3 µm, Phenomenex)
• Gradient: hexane - 2-propanol (3% 2-propanol/hour)
• Temperature: 35°C
Randomization mixture of
OOO and glycerol
DGs
R1
R1 +
R1
OH
OH
OH
R1
MeONa
75°C, 30 min
R1 +
R1
OH
R1
R1
R1
OH
R1
MGs
R1
R1 +
OH
OH
OH
R1
OH
R1
OH
R1
OH
OH
O (∆9-C18:1)
32
Stereospecific Synthesis of DGs
1-benzylglycerol (2,3-DG)
O
R1
R1
OH
OH
R
O
DMAP, DCC
22°C, 90 min
1
R1
CAN
0°C, 60 min
HO
R1
3-benzylglycerol (1,2-DG)
R1
OH
HO
O
DMAP, DCC
22°C, 90 min
1
R
R1
O
CAN
0°C, 60 min
1
R1
R
OH
DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid,
CAN - ammonium cerium(IV) nitrate
Chiral HPLC/MS of DGs
randomization reaction
of OOO and glycerol
enantiomers
3-benzylglycerol
1,2-OO
OH
HO
O
33
Stereospecific Synthesis of MGs
2,3-isopropylidene-sn-glycerol (1-MG)
TFA
-20°C, 30 min
R1
OH
OH
1,2-isopropylidene-sn-glycerol (3-MG)
O
O
R1
O
O
DMAP, DCC
TFA
R1 -20°C, 30 min
OH 22°C, 90 min
OH
OH
R1
OH
O
O
R1
DMAP, DCC
22°C, 90 min
R1
O
O
DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid
Chiral HPLC/MS of MGs
randomization reaction
of OOO and glycerol
1,2-isopropylidene
-sn-glycerol
enantiomers
3-O
O
O
OH
34
Comprehensive Lipidomic Approach
GC/FID
MS Imaging
Hexane/MeOH/H2O
CHCl3/MeOH/H2O
Nonpolar lipid extract
Total lipid extract
MALDI-Orbitrap
1D
HILIC-LC/MS
2D
RP-LC/MS
SFC/MS
NARP-LC/MS
RP-LC/MS
Ag-LC/MS
NP-LC/MS
Chiral-LC/MS
Data processing
Statistical evaluation
Data processing
• Manual interpretation and processing is almost impossible due to the enormous
amounts of data, individual peaks, number of samples from clinical studies
• Dedicated lipidomic softwares from main manufacturers are available:
- LipidView (AB SCIEX)
- TransOmics – Metabolomics and Lipidomics (Waters)
- Lipid Search (Thermo)
• Freewares and open-source tools for all platforms:
- LipidXplorer (A. Shevchenko) - https://wiki.mpi-cbg.de/lipidx/
- mMass (M. Strohalm) - http://www.mmass.org/
- Mzmine 2 (T. Pluskal, M. Orešič) - http://mzmine.sourceforge.net/
•
Lipid databases and comprehensive internet resources:
- http://www.lipidmaps.org/
- http://lipidlibrary.aocs.org/
- http://www.cyberlipid.org/
- http://www.lipidbank.jp/
35
Principal Component Analysis (PCA) of TG
355 TGs
93 plant oils
PCA Loading Plots for Individual TG
0.6
LLL
0.4
OLL
LLP
p[2]
0.2
0
SLL
LLLn
OLLn
LnLP
LnLLn
PLPSLO
LLyLn
SLP
ALL
BLL
GLL
LnLnLn
LnOLn
LLPi
SLS
ALO
yLnLLn
LLM
LLMa
C20:2LL
yLnLyLn
OLyLn
yLnLP
SLLn
OLPi
LgLL
LnLnP
BLO
C24:1LL
GLP
ALP
SOLn
GLO
ALLn
OOyLn
yLnOP
SLPi
yLnLnLn
yLnLSt
LnOyLn
OLSt
StLP
C20:2LO
SLyLn
LnLSt
LLSt
PiLPi
yLnyLnP
PLPi
LLMo
C23:0LL
ALS
BLS
PLnP
OOPi
LLTa
SLnLn
BLP
LgLP
LgLO
SLM
SOyLn
ALyLn
GyLnP
BLnLn
SOPi
SOLa
PPM
SPLa
SMM
BPiPi
SyLnP
MoOMo
C19:0LL
MaLP
C23:0LnLn
OOC15:0
MoOP
C21:0LLn
C20:2OO
C24:1LyLn
C22:1OLn
C20:2OP
GOM
C22:1yLnP
AOLn
BLPi
GyLnS
BLyLn
AOPi
ALnP
BLLa
AOLa
SSLa
SyLnS
SOM
C25:0LnLn
C23:0LLn
C21:0LL
SPM
APLa
MaOP
SLMa
C23:0LPi
GLG
C20:2OS
GLS
C22:1LP
C24:1OyLn
LgLLn
C24:1yLnP
BOLn
LgLyLn
LgLPi
BLnP
PPoP
C22:1LL
GOPo
BLLn
SLnS
C22:1LO
POM
SLnP
OLMa
SOPo
AOPo
PPP
OOMa
GOLa
OOTa
OLM
GLPi
OOM
PLPo
GOLn
GOyLn
C20:3OO
SLLa
PLTa
PLM
PyLnP
SOSt
PMM
LnOMa
C19:0LPi
OLC15:0
MoLP
ALnLn
LaLaCo
LaCCy
LaLaC
LaLCy
StLnSt
MLaCo
LaOCy
yLnLnSt
LnLnSt
MMCo
PLaCo
MLaC
PLaLa
LnLnMa
LnLMo
PPiPi
SMCy
SyLnSt
SLaC
SLnSt
C20:2LLn
C20:3LL
PoLPo
LnLMa
ALnS
SSM
ByLnP
BOLa
AOM
PLaCy
MMCy
StStP
PiPiPi
PiLnPi
yLnyLnyLn
OLCy
LaLLa
LaOC
MOCy
PLCy
LnLPi
yLnOSt
PLaC
MMC
LnLnC15:0
LnLnMo
StyLnP
SLaCy
PMCy
StLnP
LLLa
C20:2LnLn
OLC
OOCy
C20:3LPi
MOC
POCy
TaLPi
C20:2LPi
LnOPo
GLnLn
OOC
MLM
GyLnyLn
PLnPo
OOSt
PLLa
SOCy
StOP
SLSt
TaOPi
SLnyLn
POC
SPiPi
MoLnMo
C22:1yLnC2
C22:1LG
SOMa
C23:0OLa
C21:0LP
C19:0OO
C23:0OLn
C21:0LO
C24:1LP
C22:1OP
C24:1yLnS
LgOLn
LgLM
BLnS
LgLnP
LgOLa
APP
BPoP
C25:0LL
C22:1OG
C22:1LC22:
C19:0OS
C23:0LP
C24:1LS
C22:1OS
C24:1OP
AOG
LgOPo
LgPoP
GLLn
C20:3LnTa
C25:0LO
C26:0LO
C24:1OS
C24:1OG
C22:1OC22:
C25:0LP
C26:0LP
ASS
LgOG
C23:0OS
C22:1LyLn
C20:2LP
POLa
MOM
POPi
SMLa
OLMo
OLTa
PPoPo
LaLaCy
MLaCy
LaLaLa
PiOPi
MMLa
PoPoPo
OLLa
LLC15:0
SPP
yLnLnyLn
MLaLa
LnOSt
MLLa
yLnOyLn
yLnLnP
SLaLa
PMLa
SyLnyLn
C22:1OO
GOG
C24:1LO
BOPo
SSP
C21:0OO
C23:0LO
C24:1OO
C26:0LL
C20:3LO
GLyLn
LgLS
AOA
BOS
C25:0OO
C26:0OO
LLPo
OOLa
MOLa
GOS
LaOLa
SSS
C23:0OO
BOP
LgOP
OOMo
GOP
PoOPo
AOS
AOP
LgOO
LnOP
OLPo
POPo
BOO
SOS
GOO
OOLn
AOO
SOP
OOPo
POP
OLP
OLO
SOO
-0.2
OOP
-0.4
OOO
-0.6
0
0.1
0.2
0.3
0.4
0.5
0.6
• 355 determined TGs (variables) in 93 plant oils (objects)
• TGs with the highest effect on PCA scores: OOO, LLL, OLL, OLO, OOP, LLP, OLP
and SOO
M. Lísa, M. Holčapek, M. Boháč, J. Agric. Food Chem. 57 (2009) 6888
36
PCA Scatter Plots for Data From 93 Plant Oils
40
30
A
16
B
84
20
3
t[2]
10
0
1
69 43
18
20
92 17 81
91
89 82
77 70
93
90
5
83
6
30
79
37
32
7
85
13
15
86
19
23
12
36
26 4
11 29
25
40
68
-10
34
39
38
87
74
14
21
76 78
75
88
71
C
72
24
33
31
42
9
8
80
-20
D
73
41
2
10
22
35
52
20
10
0
30
t[1]
t[2]
0
28
Evening primrose (84)
40
30
Wheat Germ (86)
20
10
27
-30
B
Walnut (79)
Soya (37)
Dog rose (7)
Mandarin orange (19)
Raspberry (32)
Hemp (3)
Coffee butter (30)
Blackcurrant (18)
Kukui nut (85)
Blueberry (20)
Blackcurrant (17)
Redcurrant (81)
Norway spruce (92) Fig (89)
Borage (82)
Kiwi (1)
European larch (91)
European silver fir (93)
Linseed (77) Coconut palm (70)
Date (90)
Cocoa Butter (83)
Grapefruit (15)
Lemon (13)
Buckwheat (23)
Mango (5)
Mango (6)
Palm (68)
-10
Macadamia nut (2)
4
8
12
16
20
Identification of Adulteration of Olive Oils by PCA
40
49
45 5048
46 47
51 44
8 sunflower oils
30
olive oil “adulterated”
with sunflower oil
t[2]
20
10
10%
5%
-10
10
15 olive oils
2% 1%
0
58 61 56
55 66 65 6263
67
53 6064 54 57
59
20
30 t[1]
40
50
• Identification of falsification of expensive plant oil (e.g., virgin olive) by cheaper plant
oils (e.g., sunflower)
• Clear identification already from 1% of adulterant
M. Lísa, M. Holčapek, M. Boháč, J. Agric. Food Chem. 57 (2009) 6888
37
Ion Mobility Spectrometry in Lipidomic Analysis
Data courtesy: Fadi Abdi (AB SCIEX)
Ion Mobility Spectrometry in Lipidomic Analysis
Data courtesy: Fadi Abdi (AB SCIEX)
38
MALDI-Orbitrap MS Imaging of Rat Brain
Optical image – before sectioning
EIC of m/z 740.5 ± 0.5
EIC of m/z 740.566 ± 0.003
EIC of m/z 740.522 ± 0.003
PE 36:3
GlcA-Cer(d18:1/18:0)
Concluding Remarks
• Combination of multiple LC/MS, MALDI-MS, MSI and GC/MS methods provides the
most comprehensive information on the lipidome (all types of isomers)
• Advantage is also a bottleneck of our approach – enormous requirements on the
data processing, automation, instrumental time, statistics and bioinformatics
• Suitable methods can be selected according to the biological problem to be solved
Acknowledgements
• Grant projects: 206/11/0022 and 203/09/0139 (Czech Science Foundation)
• University of Pardubice: M. Ovčačíková, B. Červená, R. Jirásko, V. Chagovets, H.
Dvořáková, P. Česla
• Clinical samples: J. Galuszka, J. Vostálová, B. Melichar, D. Vávra (FN Olomouc)
• SFC-MS: G. Isaac (Waters, USA)
• Statistical evaluation: M. Hill (Prague)
• Rat samples: I. Vokřál (Hradec Králové)
39
Our Key References - http://holcapek.upce.cz/
• Chiral HPLC of TGs: Anal. Chem. 85 (2013) 1852
• Nontargeted quantitation of polar lipid classes: Anal. Chem. 84 (2012) 10064
• Silver-ion LC and NARP of TGs in animal fats: J. Chromatogr. A 1218 (2011) 7499
• Off-line 2D-LC/MS of PLs: J. Chromatogr. A 1218 (2011) 5146
• Off-line 2D-LC/MS of TGs: J. Sep. Sci. 32 (2009) 3672
• PCA of TGs: J. Agric. Food Chem. 57 (2009) 6888
• Silver-ion LC of regioisomers: Anal. Chem. 81 (2009) 3903
• NARP of TGs: J. Chromatogr. A 1998-1999 (2008) 115
• Unusual TGs: J. Chromatogr. A 1146 (2007) 67
• CAD quantitation: J. Chromatogr. A 1176 (2007) 135
• APCI-MS quantitation: J. Sep. Sci. 28 (2005) 1315
40