lc/ms in lipidomicsmab.uochb.cas.cz/iochb/elm2013/holcapek.pdfcholesterol tocopherol o o o o o o h...

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LC/MS in LipidomicsMichal Holčapek, Eva Cífková and Miroslav Lísa

University of Pardubice, Department of Analytical Chemistry, Czech Republic

http://holcapek.upce.cz/

Lipid Classification

Fatty acyls

Glycerolipids

Phospholipids

Sphingolipids

Sterols

Prenols

Saccharolipids

Polyketides

COOH

Nonpolar lipids

Fatty acyls

Glycerolipids

Glycerol

Fatty acyls

Sterols

OH

O

Prenols

Oleic acid

Triacylglycerol

Cholesterol Tocopherol

O

O

O

O

O

O

H

OH

2

Lipid Classification

Fatty acyls

Glycerolipids

Phospholipids

Sphingolipids

Sterols

Prenols

Saccharolipids

Polyketides

Polar lipids

Sphingolipids

Phospholipids

Nonpolar fatty acylsR = choline (PC), ethanolamine (PE), serine (PS), glycerol(PG), inositol (PI), etc.

Polar head groupGlycerol

GlycerolCholine

Sphingomyelin

O

O-N+OO P

NH H

O

OHH

Nonpolar fatty acyls

O

O-ORO

O

O P

O H

O

Comprehensive Lipidomic Approach

Total lipid extract Nonpolar lipid extract

CHCl3/MeOH/H2O

HILIC-LC/MS NP-LC/MS

Hexane/MeOH/H2O

RP-LC/MS NARP-LC/MS Chiral-LC/MS

GC/FID

2D

1D

MALDI-Orbitrap

MS Imaging

SFC/MS

Data processing

Ag-LC/MS

Statistical evaluation

RP-LC/MS

3

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 AnalysisLipid class approach

• HILIC-LC/ESI-MS - for polar lipid classes, Si, NH2 or diol columns, mobile phaseswith acetonitrile, methanol, 2-propanol, hexane and ≥ 2.5% water

• NP-LC/APCI-MS - nonpolar lipid classes, Si or NH2 columns, mobile phases withhexane, 2-propanol, chloroform, heptane, etc.

• SFC/MS – all lipid classes, C18 or Si columns, mobile phase supercritical CO2 withpolar organic modifiers (typically methanol)

Lipid species approach

• RP-LC/ESI-MS – all lipid species, C18 and C8 columns, mobile phases aqueousmixtures of acetonitrile, methanol or 2-propanol, often with volatile buffers

• NARP-LC/APCI-MS – nonpolar lipid species, C18 columns, mobile phases 2-propanol, acetonitrile, aceton, dichlormethane, etc.

• Ag-LC/APCI-MS – nonpolar lipid species, ion-exchange column with Ag+, mobilephases with hexane – acetonitrile (+2-propanol) or dichlormethane – acetonitrile

• Chiral-LC/APCI-MS – nonpolar lipid species, chiral columns, normal phase mobilesystem with hexane and 2-propanol

• SFC/MS – all lipid species, C18 columns, supercritical CO2 with polar modifiers

4


HILIC-LC/MS

Lipid extract

1D

• Separation of lipid classes

Optimization of HILIC-LC/ESI-MS

Conditions: column Spherisorb Silica (250*4.6 mm, 5 μm, Waters), ESI-MS detection,separation temperature 40°C, gradient of acetonitrile / aqueous ammonium acetate

• Optimized parameters: column type (9 columns), mobile phase composition (mainly organic solvent and concentration of water), gradient steepness, pH, salt content, etc.

Effect of pH value Effect of ammonium acetate 5 mM

0 mM

pH = 7

pH = 4.5

5

HILIC-LC/ESI-MS Separation of Lipid Classes

M. Lísa, E. Cífková, M. Holčapek, J. Chromatogr. A, 1218 (2011) 5146

• Separation of 16 lipid classes + 3 regioisomeric pairs of lysophospholipids.

• Individual fractions are collected for off-line 2D-LC/MS characterization

Standards containing oleoyl acyls ∆9–C18:1

Conditions: column Spherisorb Silica (250*4.6 mm, 5 μm, Waters), ESI-MS detection,separation temperature 40°C, gradient of acetonitrile / aqueous ammonium acetate

Nontargeted Quantitation of Lipid Classes

E. Cífková, M. Holčapek, M. Lísa, et al., Anal. Chem. 84 (2012) 10064

Lipidclass

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

Sphingosyl PE (d17:1/12:0)

1

6


HILIC-LC/MS

Lipid extract

1D NP-LC/MS


NP-LC/APCI-MS of Nonpolar Lipid Classes

• Separation of 6 nonpolar lipid classes and 2 regioisomers of diacylglycerols

Standards containing nonadecanoyl acyls (C19:0)

Conditions: column Acquity UPLC BEH HILIC (50*2.1 mm, 1.7 μm, Waters), APCI-MSdetection, 30°C, gradient of hexane / acetonitrile / 2-propanol

7

NP-UHPLC Analysis of Plasma of CVD Patients

• 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

• NP-UHPLC/APCI-MS measurement of plasma extract of nonpolar lipids


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS


8

Inte

nsi

ty

0.00

2.50x107

5.00x107

7.50x107

1.00x108

Minutes0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80 12.00

SFC/MS of Polar Lipid Classes

Mixture of lipids standards from Avanti

CER

PGPE

PC

SMLPC

LPE

Data courtesy: Giorgis Isaac (Waters)

Conditions: column Acquity UPC2 BEH, methanol gradient, additive ammonium formate, ESI-MS detection

10 minutes


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS RP-LC/MS


• Separation of lipid species

9

RP-LC/ESI-MS of Total Lipid Extract

K. Sandra et al., J. Chromatogr. A 1217 (2010) 4087

Blood plasma

LC-MS Feature Maps of Plasma

K. Sandra et al., J. Chromatogr. A 1217 (2010) 4087

ESI+

ESI-

10


• 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 0.18mL/min, separation temperature 40°C, 5 mM aqueous ammonium acetate/5 mM ammoniumacetate in acetonitrile:2-propanol (1:2) gradient.

13 FA and 6 LPCEIC 184

27 PC and 12 SM34 TG

15 CEEIC 369

Conditions: ACQUITY UPLC BEH C18 column (150 and 150 x 2.1 x 1.7 mm); flow rate 0.18mL/min, separation temperature 40°C, 5 mM aqueous ammonium acetate/5 mM ammoniumacetate in acetonitrile:2-propanol (1:2) gradient.


11


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS

RP-LC/MS

RP-LC/MS

2D



Off-line 2D HILIC x RP-LC/ESI-MS of Polar Species1D: HILIC sepation

PC

2D: RP-LC separation

Conditions: column Kinetex C18

(150*2.1 mm, 2.6 μm,Phenomenex), ESI-MS detection,separation temperature 40°C,acetonitrile / 2-propanol /aqueous ammonium acetategradient.

16:0/18:1-PC

M. Lísa, E. Cífková, M. Holčapek,J. Chromatogr. A, 1218 (2011) 5146

12

16:0/18:1-PE1D: HILIC 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 acetategradient.

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

HexCer + Cer

Conditions: column KinetexC18 (150*2.1 mm, 2.6 μm,Phenomenex), ESI-MSdetection, separationtemperature 40°C, acetonitrile /2-propanol / aqueousammonium acetate gradient.


Off-line 2D HILIC x RP-LC/ESI-MS of Polar Species

13

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

P. Dugo et al., J. Chromatogr. A 1278 (2013) 46

PE in cow´s milk

14

Stop-Flow 2D HILIC x RP-LC/MS of Polar Lipids

P. Dugo et al., J. Chromatogr. A 1278 (2013) 46

PC in plasma


HILIC-LC/MS

Lipid extract

1D

• Individual lipid classes

• Individual lipid species

NP-LC/MSSFC/MS

RP-LC/MS

RP-LC/MS

2D NARP-LC/MS

15

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 from6 to 28 carbon atoms and from 0 to 6 DBs) identified in more than 200 differentmaterials (plant oils, animal fats, fish oils, body fluids) and statistically evaluatedby PCA

OCH2 COR1

CHOCH2 O COR3

R2CO Triacylglycerolsn-2

sn-3

sn-1

M. Lísa, M. Holčapek, J. Chromatogr. A 1998-1999 (2008) 115M. 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

OR2COCH2O COR1CHCH2O COR3

+ H+

[M+H]+ MW

[M+H-RiCOOH]+ - fragment ions A(identification of FA)

-R2COOH CH2O COR1+CHCH2O COR3

OR2COCH2O COR1CHCH2

+

OR2COCH2

+

CHCH2O COR3-R1COOH

-R3COOH

CH2

CH

CH2+

OH

OR2COCH2

CH

CH2+

O

HO

COR1(3)

[RCO]+

B2 B1(3) C

16

Off-line 2D HILIC-NARP-LC/MS of Nonpolar Lipids1D: HILIC separation

Conditions: 2 columns Nova-Pak C18 (300+150*3.9 mm, 4 m,Waters), APCI-MS, temperature 25°C, acetonitrile / 2-propanolgradient

• TG are separated according to ECN and also inside ECN groups

ECN = CN – 2*DB

CN – carbon number in all acyl chainsDB – double bond number

2D: NARP-LC separation

TG + Chol

Resolution of Isomers Differing in DB Position

• Different retention times both in NARP-LC and Ag-LC modes• Different relative abundances of fragment ions

• 18 carbon atoms and 3 double bonds in different positions

COOH

-linolenic acids (C18:39,12,15)

15 12 9

COOH

Pinolenic acid (C18:35,9,12)

12 95

NARP-LC/APCI-MS analysis of unusual FA in conifer seed oils

17

NARP-LC/APCI-MS Analysis of Conifer Seed Oils

Fatty acids

CN:DB Common ∆9(6) Unusual ∆5

18:3 Linolenic (Ln) Pinolenic (Pi)

18:2 Linoleic (L) Taxolic (Ta)

Retention factor...k=(tR-tM)/tM

∆k(Pi)=0.4

∆k(Ta)=0.6

∆k=0.4

∆k=0.4

∆k=0.4

∆k=0.6∆k=0.6

LL

Pi

LL

Ln

PiL

nP

iP

iPiP

i

Ln

LP

iP

iLP

i

TaL

Pi

LL

LL

LTa

Larch(Larix decidua)

APCI-MS Analysis of Unusual TGsLLL LnLnLn

LLTa PiPiPi

C B

A

[M+H]+

C B

A

[M+H]+

C B

A

[M+H]+

C-H2O CB

A[M+H]+

C-H2O

A / [M+H]+ = 42 / 100%

100 / 74%

A / [M+H]+ = 25 / 100%

100 / 87%

18

NARP-LC/APCI-MS Separation of IsomericTGs

linear / branchedcis/trans

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

EnzymaticHydrolysis of

Blackcurrant Oil

-0,05

0,15

0,35

0,55

0,75

0 10 20 30 40 50 60 70 80 90

Time [min]

AU

-0,05

0,15

0,35

0,55

0,75

0,95

0 10 20 30 40 50 60 70 80 90

Time [min]

AU

TG

TG

FA + MG

DG

TG

DG

MG

CH2

CH

CH2O

O

OR2CO

COR1

COR3

CH2

CH

CH2OH

O

OR2CO

COR1

CH2

CH

CH2OH

O

HO

COR1

RiCOOH

Native oil

Enzymatic hydrolysis

• Hydrolysis with stereoselective immobilized enzyme Lipozyme in supercritical fluid CO2 extractor

FA

(83 TGsfrom 14 FAs)

19

Enzymatic Hydrolysis of DG in Blackcurrant Oil

DG 1,3-DG 1,2-DG

LLn 3.67 96.33

LLn 1.92 98.08

LL 2.57 97.43

Regioselectivity of enzyme is ca. 97%

M. Lísa, M. Holčapek, H. Sovová, J. Chromatogr. A 1216 (2009) 8371

UHPLC – Reduction of Analysis Time

Acquity BEH C18 column (150*2.1 mm, 1.7 m, Waters), 0.4 mL/min, 30°C, injection 1 L, acetonitrile / 2-propanol gradient.

Nova-Pak C18 columns (300+150*3.9 mm,4 m, Waters),1 mL/min, 25°C, injection 10 L, acetonitrile / 2-propanol gradient.

-0,08

0,12

0,32

0,52

0,72

3 4 5 6 7 8 9 10 11 12-0,05

0,15

0,35

0,55

35 40 45 50 55 60 65 70 75 80 85 90 95

HPLC UHPLC

-0,04

0,16

4,5 5 5,5-0,04

0,06

47,5 50 52,5

Positional DB isomers:Ln (9,12,15-C18:3)γLn (6,9,12-C18:3)

Ln

Ln

Ln γL

nL

nL

n

γLnγL

nγL

n

γLnγL

nL

n

Ln

Ln

Ln

γLn

Ln

Ln

γLnγL

nγL

n

γLnγL

nL

n

50 min 5 minHPLC: UHPLC:

20


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS

RP-LC/MS

RP-LC/MS

2D NARP-LC/MS Ag-LC/MS



1cis 2cis 3cis 4cis1trans 2trans 3trans

Geometrical isomers

Silver-ion LC/APCI-MS of TGs• Separation mechanism according to the double bond (DB) number• Resolution of cis- / trans-, regio- and DB positional isomers

OO

P

PO

PO

PP

OP

O

Regioisomers

Conditions: 3 columns ChromSpher (250*4.6 mm, 5 m, Varian) connected in series, 1mL/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

100%100%

32%

83%

[PP]+

[OP]+

[PP]+

[OP]+

21

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(AAB:ABA=2:1)

Regioisomers(BBA:BAB=2:1)

M. Lísa et al. Anal. Chem. 81 (2009) 3903

Randomization Mixture of PPP / OOO / LnLnLn

Double bonds

Conditions: 3 columns ChromSpher (250*4.6 mm, 5 m, Varian) connected in series, 1mL/min, APCI-MS detection, separation temperature 25 °C, injection 1 l, hexane / 2-propanol /acetonitrile gradient

Products: initial PPP, OOO, LnLnLn +6 regioisomeric doublets +1 regioisomeric triplet

0

1

2

3

4

5

6

7

9

22

Preference of sn-2 Occupation by Silver-ion LC

RegioisomersPlant 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) 3903M. Lísa et al., J. Chromatogr. A 1218 (2011) 7499

Silver-ion LC/MS of TGs in Beef Tallow

1 cis 2 cis 3 cis 4 cis1 trans 2 trans 3 trans

0Double bonds

• Clear resolution of cis- / trans- isomers, e.g., 9cis-18:1 (oleic) and 9trans-18:1 (elaidic) containing triacylglycerols

23

Off-line 2D LC/MS of TGs – OrthogonalityNARP-LC

Ag-HPLC

OO

O

OO

O

EE

E

EE

E

EO

EO

EE

OO

EO

EO

OO

E+

OE

O

OE

E+

EO

E

• NARP and silver-ion modes provide orthogonal separation, their off-line 2Dcoupling yields superior resolution including the resolution of regioisomers

NARP-LC conditions:

Ag-LC conditions:

3 columns ChromSpher (250*4.6 mm, 5 m,Varian) connected in series, APCI-MSdetection, 1 mL/min, 25 °C, injection 1 l,hexane / 2-propanol / acetonitrile gradient

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

M. Holčapek et al., J. Sep. Sci. 23 (2009) 3672

O – C18:19cisE – C18:19trans

Off-line 2D NARP x Ag-LC/APCI-MS of TG

58 63 68 73 781 dimension (NARP-HPLC) [min]st

58

68

78

88

98

2 d

imen

sion

(A

g-H

PL

C)

[min

]n

d

OLP

LOP

SLL OLO

OOL

LLPLnOP

SLnLOLL

LOL

OOLnOOγLn

GLLn

GLγLn

LnLPγLnLP

SLSt

LLLOLLn

LOLnLOγLn

OLγLnLnLnP

γLnPLnLLLn

LLγLnLnOLn

OLStLOSt

γLnOLnOLnLn

γLnOγLn

γLnγLnP

GLL

γLnOP

DB 3

ECN 46

DB 7

DB 6

DB 5

DB 4

ECN 40 ECN 42 ECN 44

(A)

(B)

1 dimension (NARP-HPLC) [min]st

2 d

imen

sion

(A

g-H

PL

C)

[min

]n

d

24

E.J.C. van der Klift et al., J. Chromatogr. A 1178 (2008) 43

Schematic Layout of LC x LC Instrumentation

Online 2D Ag x NARP-LC/APCI-MS of TG

E.J.C. van der Klift et al., J. Chromatogr. A 1178 (2008) 43

ECN = CN – 2*DB

25

Online 2D Ag x NARP-LC/APCI-MS of TG

L. Mondello et al., J. Sep. Sci. 34 (2011) 688

ECN = CN – 2*DB

Dual Parallel MS for Lipid Analysis

W. C. Byrdwell, J. Chromatogr. A, 1217 (2010) 3992

26

Dual Parallel MS for Lipid Analysis

W. C. Byrdwell, J. Chromatogr. A, 1217 (2010) 3992

HILIC-LC separation of polar lipids

RP-LC separation of nonpolar lipids

Diverted flow


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS

RP-LC/MS

RP-LC/MS

2D NARP-LC/MS Ag-LC/MS SFC/MS



27

SFC/MS of Cholesterol Esters

Time0.20 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 3.80

%

0

100

TAGs_08022012_025 1: TOF MS ES+ BPI

4.15e5

1

2

3

4

5

6

Peak Lipid Species

1 18:3 CE

2 18:2 CE

3 17:0 CE and 18:1 CE

4 18:0 CE

5 19:0 CE

6 23:0 CE

Conditions: ACQUITY UPC2instrument, column Acquity UPC2 HSS C18 SB, methanol gradient, additive ammonium formate, ESI-MS detection


4 minutes

SFC/MS of Triacylglycerols

Time0.20 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 3.80

%

0

100

TAGs_08022012_029 1: TOF MS ES+ BPI

2.08e5

Peak Lipid Species

1 15:0/15:0/15:0 TG

2 18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis) 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

9 20:0/20:0/20:0 TG

12

3

4

5

6

7

8

9


Conditions: ACQUITY UPC2instrument, column Acquity UPC2 HSS C18 SB, methanol gradient, additive ammonium formate, ESI-MS detection

4 minutes

28


HILIC-LC/MS

Lipid extract

1D NP-LC/MSSFC/MS

RP-LC/MS

RP-LC/MS

2D NARP-LC/MS Chiral-LC/MSAg-LC/MS SFC/MS



Chiral Separation of Triacylglycerols

A - C20:0O - ∆9-C18:1

H+

O

AO

OA

O

OHO

OH

AOH

O

AO

H+

A

OO

OA

A

OHOHO

O

OHOO

A

OCH2 COR1

CHOCH2 O COR3

R2COsn-2

sn-3

sn-1=

*

M. Lísa, M. Holčapek, Anal. Chem. 85 (2013) 1852

• 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

29

R1

R1

R1

R2

R2

R2

R2

R2

R2

R1

R1

R1MeONa75°C, 30 min

R2

R2

R1

R2

R1

R2

R2

R1

R1

R1

R2

R1

R1

R2

R2

R1

R1

R2+ + ++

Synthesis of Triacylglycerol Isomers

Randomization reaction of monoacyl-TGs• random transesterification of fatty acyls in TGs

regioisomers

enantiomers

Chiral HPLC/MS of TGs

Randomization mixture ofAAA / OOO / LnLnLn

(C20:0 / C18:1 / C18:3)

0

1

2

34

56

7

9

- enantiomers

- regioisomers

A (C20:0), O (∆9-C18:1), Ln (∆9,12,15-C18:3 )

30

APCI Mass Spectra of TG Isomers

AOO OOA

OAO

43% 40%

15%

A (C20:0), O (∆9-C18:1)

DMAP, DCC22°C, 90 min

TFA-20°C, 30 min

R1

R2


R1

OHOH

R1

R2

R2

OO

OHOO

R1


TFA-20°C, 30 min

R1DMAP, DCC22°C, 90 min

R2

O

OHO

OH

R1

OHR2

R1

R2

O

R1

O

R2

R2

R1

R3

R3

R1

R2

R3

R1

R3

R2

R1+ +R2 + R3

R2 + R3 ++

R1

R3

R2

R1

R2

R3

R1

R3

R3

R1

R2

R2

Stereospecific Synthesis of mixed-acyl TGs

2,3-isopropylidene-sn-glycerol


DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid

M. Lísa, M. Holčapek, Anal.Chem. 85 (2013) 1852

31

OO

OOO

OH O

OHOH

O Ri

O

Ri

RiH+

O

OO

OH

OOH

O

OHO

O Ri

Ri

ORiH+

Chiral HPLC/MS of TG EnantiomersRiRiO

ORiRi

Ri = 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 )

32

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

OH

R1

OHOH

OHR1

R1

OHOH

OH

R1

R1

R1

R1

OHR1

OHR1

R1

R1

R1

OH

OHOH

R1

R1

R1 + ++MeONa75°C, 30 min

• Column: Lux Cellulose-1 (250*4.6 mm, 3 µm, Phenomenex)• Gradient: hexane - 2-propanol (3% 2-propanol/hour)• Temperature: 35°C

Randomization mixture ofOOO and glycerol

O (∆9-C18:1)

DGs MGs

33

R1

CAN0°C, 60 min


O OHOH

O R1R1

HO R1R1

HO OOH

R1 OR1

R1R1

OHDMAP, DCC22°C, 90 min

CAN0°C, 60 min

R1

DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid,CAN - ammonium cerium(IV) nitrate

3-benzylglycerol (1,2-DG)

1-benzylglycerol (2,3-DG)

Stereospecific Synthesis of DGs

Chiral HPLC/MS of DGs

HO OOH

1,2-OO

randomization reactionof OOO and glycerol

3-benzylglycerol

enantiomers

34

Stereospecific Synthesis of MGs


TFA-20°C, 30 min

R1

O

OHO

OH

R1

OHO

R1

O


TFA-20°C, 30 min

R1

R1

OHOHO

O

OHOO

R1

2,3-isopropylidene-sn-glycerol (1-MG)

1,2-isopropylidene-sn-glycerol (3-MG)

DMAP - 4-dimethylaminopyridine, DCC - dicyclohexylcarbodiimide, TFA - trifluoroacetic acid

Chiral HPLC/MS of MGs

OO

OH

3-O

enantiomers


randomization reactionof OOO and glycerol

35


Total lipid extract Nonpolar lipid extract

CHCl3/MeOH/H2O

HILIC-LC/MS NP-LC/MS

Hexane/MeOH/H2O

RP-LC/MS NARP-LC/MS Chiral-LC/MS

GC/FID

2D

1D

MALDI-Orbitrap

MS Imaging

SFC/MS

Data processing

Ag-LC/MS

Statistical evaluation

RP-LC/MS

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/

36

Principal Component Analysis (PCA) of TG93 plant oils

355

TG

s

PCA Loading Plots for Individual TG

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0 0.1 0.2 0.3 0.4 0.5 0.6

LaCCyLaLaCoLaLaCyMLaCoStLnStLaLCyLaLaCMLaCyPLaCoMMCoLnLnStyLnLnStLaOCyLnLnLnLaLaLaMLaCyLnLnLnLnLStMMCyPLaCyyLnLnyLnyLnLStyLnyLnyLnPiLnPiPiPiPiStStPLnLLn

OLCyyLnLLnLaLLaLLStLaOCyLnLyLnLnLPiPLCyMOCyLnOStMLaLaMMCPLaCyLnOStPiLPiStLnPPMCySLaCyStyLnPLnLnMoLnLnC15:0

LLLn

C20:2LnLnLLLaLLyLnOLCLnOLnLLPiOOCyLnOyLnC20:3LPiMLLaOLStLaOLaC20:3LnTaLnLnPyLnOyLnMOCyLnLnPPOCyStLPTaLPiyLnyLnPPiOPiPLaLaMMLaSLnStSLaCSyLnStSMCyPPiPiLnLMoLnLnMa

LLL

C20:2LLnLLPoPoLPoC20:3LLPoPoPoOLLn

LnOPoC20:2LPiLLMGLnLnOLyLnLLTaOLLaOLPiOOCLnLP

PLnPoGyLnyLnMLMSLnLnyLnLPPLLaOOStMOLaPLPiPOCSLnyLnTaOPiSLStStOPSOCySyLnyLnPMLaSLaLaMoLnMoSPiPiLLMoLLC15:0LnLMaC20:2LL

OLL

GLLnOLPoC20:3LO OOLnPoOPoGLyLn

LLP

OOyLnGLPiOLMPLPoOLTaSLLnOOLaOOPiLnOPPPoPoALnLnSLyLnyLnOPPLMPLTaSLLaSLPiMOMPOLaPLnPPOPiSOStPyLnPSMLaPMMOLMoLLMaMoLPOLC15:0C19:0LPiLnOMaC20:2LOGLLOLO

GOLnC22:1LyLnOOPo

C20:2LPC20:3OOGOyLn

SLLOLP

GOLaALLnOOMOOTaPOPoSOLnBLnLnGyLnPALyLnSOyLnPLPSLMPPoPPOMSOLaSOPiSLnPMoOMoSyLnPBPiPiSMMSPLaPPMOOMoC19:0LLOLMaC21:0LLnMoOPOOC15:0C23:0LnLnMaLPC22:1LLC20:2OOGLOC22:1OLnC24:1LyLnGOPo

OOO

C20:2OPGLPALLGOMBLLn SLO

OOP

AOLnC22:1yLnPSOPoBLyLnGyLnSBLPiSLPBLLaALnPAOPiSLnSAOLaPOP

SOMSyLnSSSLaPPPAPLaSPMC21:0LLC23:0LLnC25:0LnLnOOMaC23:0LPiSLMaMaOPC24:1LLGLGC22:1LOGOOC24:1OyLnC22:1LPGLSC20:2OSBLLLgLLnALOGOPBOLnC24:1yLnP

SOO

LgLyLnAOPoLgLPiALPSLSBLnPALnS SOPAOMBOLaByLnPSSMSPPC23:0LLC21:0LOC23:0OLnC19:0OOC21:0LPC23:0OLaSOMaC22:1LGC22:1yLnC2C24:1LOGOGC22:1OOC24:1LPLgLLC22:1OPBLOC24:1yLnSGOSLgOLnAOOBOPoLgLMBLPALSLgOLaLgLnPBLnSAOPSOSBPoPAPPSSPC25:0LLC23:0LOC21:0OOC23:0LPC19:0OSC22:1LC22:C22:1OGC24:1OOC24:1LSC26:0LLC24:1OPC22:1OSLgLOAOGBOOLgOPoLgLPBLSBOPLgPoPAOSSSSC25:0LOC23:0OOC25:0LPC22:1OC22:C24:1OGC24:1OSC26:0LOLgOOC26:0LPLgLSLgOPBOSAOAASSC25:0OOC23:0OSLgOGC26:0OOp[2

]

• 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

37

PCA Scatter Plots for Data From 93 Plant Oils

-30

-20

-10

0

10

20

30

40

0 10 20 30 40

6

1

17

7

79

9

78

11

90

89

8

88

87

10

12

73

7675

5

814

13

14

15

16

1819

71

3

80

20

21

72

70

43

37

77

22

23

40

24

93

9291

26

74

82

84

27

69

25

6883

29

52

30

86

85

31

28

32

33

34

2

35

3638

39

4142

30

t[2]

t[1]

A

B

C

D

-10

0

10

20

4 8 12 16 20

Mango (6)

Kiwi (1)

Blackcurrant (17)

Dog rose (7)

Walnut (79)

Date (90)

Fig (89)

Mango (5)

Redcurrant (81) Lemon (13)

Grapefruit (15)Blackcurrant (18)

Mandarin orange (19)Hemp (3)

Blueberry (20)

Co (70)conut palm

Soya (37)

Linseed (77)

Buckwheat (23)

European silver fir (93)

Norway spruce (92)European larch (91)

Borage (82)

Evening primrose (84)

Palm (68)

Cocoa Butter (83)

Coffee butter (30)

Wheat Germ (86)

Kukui nut (85)

Raspberry (32)

Macadamia nut (2)

B

t[2]

Identification of Adulteration of Olive Oils by PCA

-10

0

10

20

30

40

10 20 30 40 50

44

4546 47

4849

50

51

53 5455

56

57

58

5960

6162

6364

6566 67

10%5%

2% 1%

t[2]

t[1]

15 olive oils

8 sunflower oils

olive oil “adulterated” with sunflower oil

• 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

38

Ion Mobility Spectrometry in Lipidomic Analysis

Data courtesy: Fadi Abdi (AB SCIEX)

Data courtesy: Fadi Abdi (AB SCIEX)

Ion Mobility Spectrometry in Lipidomic Analysis

39

EIC of m/z 740.5 ± 0.5

EIC of m/z 740.566 ± 0.003 EIC of m/z 740.522 ± 0.003

Optical image – before sectioning

MALDI-Orbitrap MS Imaging of Rat Brain

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)

40

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

lc/ms in lipidomicsmab.uochb.cas.cz/iochb/elm2013/holcapek.pdfcholesterol tocopherol o o o o o o h...

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