Transcript LECITHINS: FUNCTIONAL COMPONENTS FOR FOODS

AOCS Annual Meeting - 2006
Structural analysis of phospholids in complex
mixtures using Electrospray Ionization
Tandem Mass Spectrometry
Fabiola Dionisi
Elif Büyükpamukçu
Nestlé Research Centre
Switzerland
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Outline of the presentation
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
Definition and properties of PLs

Analytical methods for PLs

LC-ESI-MS/MS method

Applications

Conclusions
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DEFINITIONS AND PROPERTIES OF PLs
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What are phospholipids (PLs)?
Large class of molecules classified
according to the polar head group
present (eg.):
sn-1
O
R
R'
C
C
O
O
CH2
C
H
O
O
sn-2
PCs: phosphatidylcholines
PEs: phosphatidylethanolamines
PIs: phosphatidylinositols
PSs: phosphatidylserines
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H2C
O
P
O
X
OH
sn-3
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Why study phospholipids?
Nutritional functions
Technological functionality
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Physiological importance
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Nutritional importance of PLs
Found naturally in many foods.
Source of;
•Essential fatty acids (e.g. LC-PUFA from egg PLs in infant formula)[1-2]
•Choline (from sphingomyelin and phosphatidylcholine)
Important in brain development of fetuses and infants[3]
[1] Matthews et al, 2002, J Nutr, 132: 3081.
[2] Amate et al, 2001, J Nutr, 131: 1250.
[3] Buchanan et al, 2001, Nutr, 17: 18.
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Nutritional importance of PLs

Lipid metabolism[4]
– Lowering elevated serum lipid levels (cholesterol & TAGs)
– Balancing effect on HDL/LDL
– Effects on cardiovascular health

Liver therapy
– Protective, curative, regenerative effects on liver cell membranes[5]

Neurological effects
– Assisting in enhancing acetylcholine production in degenerative neurological
disorders [6]

Memory/cognitive effects
– Use of bovine brain derived PS or similar enzymatic “derived” PS[7]

Others
– Reduction of fatigue by using a dietary supplement containing PLs[8]
– Physical and athletic performance[9]
[4] Knuiman et al., 1989, J Am Clin Nutr, 49: 266.
[5] Niederau et al., 1998, Hepatogastroenterol, 45: 797.
[6] Cohen and Wurtman, 1976, Science, 191: 561.
[7] Jorissen et al., 2001, Nutr Neurosci, 4: 121.
[8] Ellithorpe et al., 2003, JANA, 6 (1): 23.
[9] Fahey et al., 1998, Biol Sport, 15: 135.
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Physiological importance of PLs
PLs are major constituents of biological membranes serving as
structural components
Main Organs:
Brain
Liver
Reproductive tract
Muscles
Cellular membranes
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Main Functions:
Cellular transport of nutrients and
waste
Intercellular pressure regulation
Ion exchange
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PLs are important constituents
of commercial Lecithin
Typical Composition of Soy Lecithin
1%
Triglycerides
29%
Diglycerides
Free fatty acids
Sterols
Glycolipids
1%
57%
5%
Carbohydrates
1%
Phospholipids
1%
Water
5%
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Phospholipids are found in different
proportions in nature
80
% in total PL*
70
PC
60
PE
50
PI
40
PA
30
PS
20
SM
10
0
Soy
Corn
Sunflower
Rape
seed
Egg
Bovine
Brain
Source
PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PA: phosphatidic acid;
PS: phosphatidylserine; SM: sphingomyelin
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PLs are different in terms of their
fatty acid compositions
80
Soya
Corn
% in total fatty acids
70
Sunflower
60
Rape seed
Egg
50
Bovine brain
40
30
20
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fatty acids
Others
DHA* (22:6)
Arachidonic (20:4)
Linolenic (18:3)
Linoleic (18:2)
Oleic (18:1)
Stearic (18:0)
0
Palmitic (16:0)
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ANALYTICAL METHODS FOR PLs
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Traditional methods for PL analysis:
multi-step approaches
Extraction
–
of lipids
TAGs, PLs, glycolipids etc
Chromatographic
–
–
TLC: (followed by derivatisation, scraping
off bands, detection, quantification)
LC: normal or reversed-phase
Separation
–
–
of PL classes
HPLC: with UV/fluorescence detection
(w/wo derivatisation)
HPLC: with ELSD detection
Fraction
–
separation of lipids
There are also:

enzymatic hydrolysis to determine
fatty acid positions

determination of phosphorous content
Lack of universal HPLC detector
for direct measurement of PLs.
collection
Collection of separated PL classes
Hydrolysis
–
–
Fatty acids
Polar head group
Derivatisation
–
Derivatisation of fatty acids
Identification
of derivatised fatty acids using
GC and/or GC-MS
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These approaches give a global
overview of compositional
information regarding PLs
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What do actual methods measure?
Lipophilic tail
Hydrophilic head
O
Gas chromatography
(GC) : information on
fatty acyls
R
R'
C
C
O
O
CH2
C
H
O
O
H2C
O
P
O
X
OH
Liquid
chromatography
(LC): information
on individual PL
classes (PC, PE,PI)
Total phosphorous analysis:
information on total lecithin
content
Different methods give different information and
results are difficult to compare
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LC-ELSD separation and detection of
PLs in soya lecithin
3000
response / mV
2500
2000
PC
1500
PE
1000
500
PI
PA
0
0
5
10
15
20
time / min
An LC-ELSD chromatogram of an ILPS soya lecithin standard.
Diol, NP column. Hexane:isopropanol to water:isopropanol gradient (with TEA and acetic
acid). PL-EMD 950 Polymer Labs ELSD detector, Waters 2590 LC. Millenium software.
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LC-ESI-MS/MS method
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Mass spectrometric approaches




PLs and MS
GC-MS
– analysis of derivatised fatty acids
FAB-MS/MS
– first applications of characterising intact PLs
– suffered from background suppression effects
API techniques
ESI-MS is most widely used
– with chromatographic separation (RP and NP phase)
– without chromatographic separation (direct infusion experiments)
Schematic of MS/MS experiments
Selected mass
Q1
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collision cell
Fragments from selected mass
Q2
Q3
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Separation and identification of PC and
PE species by LC-MS/MS
After LC separation, PL species are detected using special scanning
function in a triple quadrupole MS
Parent / precursor ion scan, used for PCs
Q1
Q2
masses
collision cell
Constant neutral loss scan, used for PEs
Q1
Q2
masses
collision cell
Q3
fragment ion
specific
detection
Q3
fragment ions
mass - fragment = loss
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RP-HPLC-ESI-MS/MS*
Mass Spectrum of POPE: 1-palmitidyl, 2-oleyl-3-sn-phosphatidylethanolamine
100
Parents of 184
LaLaPC
13.08
SOPC
21.10
LLPC
17.09
OOPC
19.34
%
SSPC
17.09
0
100
OOPE
19.60
Neutral Loss 141
Mass Spectrum of POPC: 1-palmitidyl, 2-oleyl-3-sn-phosphatidylcholine
POPE
PPPE
LaLaPE
13.14
SSPE
23.13
%
0
12


19
14
16
18
20
22
24
min
LC-ESI-MS/MS*: C18, RP column, methanol:H20 (9:1) to chloroform mobile phase, with 0.5%
formic acid. Positive ionisation, parent/precursor scanning for m/z 184.
*API4000, triple quadrupole, Agilent 1100 LC-MS/MS, Analyst 1.3.1 software.
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Identification of Fatty Acyls using
negative ESI / MS / MS

In negative ionisation mode, PLs produce
fatty acyl carboxylates as anions as the
major fragments. This enables quick
assignment of individual PLs.
100 ES1.38e5
0281.3
PO-PC
P255.2
[M+44]804.8
%







PCs in negative ionisation produces;
[M+44]- formate adduct,
[M-CH3]- loss of a methyl group from the
choline head group.
Carboxylate anions are forming from both
[M+44]- and [M-CH3]-.
PEs in negative ionisation produces;
[M-H]- ions due to the facile loss of a
proton from the ammonium ion in PEs.
the major fragments arising from [M-H]are the fatty acyl anions
[M-CH3]744.8
0
200
300
400
500
600
700
m/z
O281.3
100
3.59e3
ES-
PO-PE
P255.1
%
M-H716.6
563.7
452.5
0
100
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200
300
400
500
600
700
800 m/z
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Mass Spectrum of POPE: 1-palmitidyl, 2oleyl-3-sn-phosphatidylethanolamine
Constant neutral loss scan
O
+MS2 (718.70) CE (30): 15 MCA scansPOPE_1.wiff (Turbo Spray)
577.6
R
Max. 1.4e7 cps.
+H
+H
C O CH2
1.4e7
[DAG]+
1.3e7
R'
C
C O
1.2e7
O
O
H2C O P
1.1e7
Intensity, cps
H
NH3
OH
[DAG]+
1.0e7
O
9.0e6
8.0e6
7.0e6
6.0e6
5.0e6
4.0e6
3.0e6
[M+H]
-[141]
2.0e6
+
718.6
1.0e6
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
m/z, amu
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Mass Spectrum of POPC: 1-palmitidyl, 2oleyl-3-sn-phosphatidylcholine
Parent / precursor ion scan
Max. 3.4e7 cps.
+MS2 (760.70) CE (42): 15 MCA scans POPC_1.wiff (Turbo Spray)
3.4e7
O
R
184.1
3.2e7
R'
3.0e7
C
C O
+H
CH2
C
O
H2C O
2.6e7
[M+H]+
H
O
2.8e7
Intensity, cps
O
P
O
N(CH3 )3
OH
2.4e7
[Phosphocholine+H]+, m/z 184
2.2e7
2.0e7
1.8e7
1.6e7
1.4e7
1.2e7
1.0e7
8.0e6
6.0e6
760.7
4.0e6
2.0e6
50
22
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150
200
250
300
350
400
450
500
m/z, amu
550
600
650
700
750
800
850
900
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APPLICATIONS
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Identification of PE species in lecithins by LC-ESIMS/MS*
TIC of +NL (141.00): from Sample 7 (PE-soya, 0.1 mg/mL,NLS141)
PE
17.99
1.00e7
Max. 1.0e7 cps.
O
716.8
PLPE
9.50e6
R
740.8
LLnPE/OLnPE
9.00e6
8.50e6
17.25
R'
8.00e6
7.50e6
7.00e6
742.8
SLnPE/OLPE
6.50e6
Int
en
sit
y,
cp
s
6.00e6
5.00e6
3.50e6
+H
CH2
C
H
O
H2C
18.30
16.38
4.00e6
O
[DAG]+
718.8
POPE
4.50e6
C
O
O
744.8
OOPE/SLPE
738.7
LLnPE
5.50e6
C
O
P
O
NH3
OH
[M+H]+
19.08
3.00e6
2.50e6
19.72
736.8
LnLnPE
2.00e6
746.7
SOPE
1.50e6
1.00e6
5.00e5
0.00


24
11
12
13
14
21.00
15.72
13.77
15
16
17
18
19
20
Time, min
21
24.23
22
23
24
25
26
27
28
LC-ESI-MS/MS*: C18, RP column, methanol:H20 (9:1) to chloroform mobile phase, with 0.5%
formic acid. Positive ionisation, neutral loss scanning for 141.
*API4000, triple quadrupole, Agilent 1100 LC-MS/MS, Analyst 1.3.1 software.
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Identification of PC species in lecithins by LC-ESIMS/MS*
TIC of +Prec (184.00): from Sample 6 (soya lecithin std, 0.1 mg/mL)
18.08
5.9e7
O
PC
757.4
PLnPC
5.5e7
5.0e7
783.2
LLPC
4.5e7
R'
759.1
PLPC
R
C O
C O
C
O
H2C O
496.9
P-OH-PC
521.1
L-OH-PC
2.0e7
781.2
LLnPC
19.27
17.08
1.5e7
1.0e7
521.1
L-OH-PC
5.0e6
25
N(CH3)3
785.1
SLnPC
2.5e7

O
[Phosphocholine+H]+, m/z 184
3.0e7

P
OH
3.5e7
0.0
[M+H]+
H
O
18.95
4.0e7
+H
CH2
4
7.52 8.189.26
6
8
10
522.8
O-OH-PC
21.13
778.8
LnLnPC
12
16.13
14
16
787.1
SLPC
761.3
POPC
789.1
SOPC
22.79
18
20
Time, min
22
24
26
28
30
32
LC-ESI-MS/MS*: C18, RP column, methanol:H20 (9:1) to chloroform mobile phase, with 0.5%
formic acid. Positive ionisation, parent/precursor scanning for m/z 184.
*API4000, triple quadrupole, Agilent 1100 LC-MS/MS, Analyst 1.3.1 software.
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Analysis of soya and sunflower lecithins:
advanced methods give detailed results
3000
Sunflower lecithin
Soya lecithin
PLnPC
, LLPC
PLnPC,
LLPC
8.0e7
PLPC
PLPC
2500
response / mV
7.0e7
2000
6.0e7
PC
1500
PE
1000
5.0e7
PI
2.0e7
1.0e7
5
LLnPC
LLnPC
10
POPC
POPC
3.0e7
0
0
SLnPC
SLnPC
OOPC,
SLPC
OOPC,
SLPC
4.0e7
PA
500
17.71
15
time / min
20
0.0
16.19
19.54
LnLnPC
LnLnPC
18.85
20.83
15.43
14
15
SOPC
SOPC
16
17
18
19
20
21
22
Time, min
•Separation and quantification of
phospholipid classes in soya lecithin
using LC-ELSD
Separation, identification and
quantification of phospholipid classes
in soya lecithin using LC-MS/MS
(tandem mass spectrometry)
LLnPC is present substantially in
soya lecithin, trace amounts in
sunflower lecithin: potential marker
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Regioisomerism in negative
ionisation mode?
Example: SO-PC vs OS-PC
O
O
H3C
O
O
O
H3C
H
O
H3C
O
P
O
+
N
-
O
CH3
CH3
CH3
O
H
O
O
H3C
O
100
283.2
[S]-
+
N
CH3
CH3
CH3
281.2
[O]283.2
[S]-
281.2
[O]-
[M-CH3]772.5
%
[M-CH3]772.5
%
[M-44]832.6
[M-44]832.6
772.6
506.3
488.3
0
200
O
-
O
100
100
P
O
300
400
500
600
700
800
900
m/z
1000
490.3
0
100
200
300
400
832.6
508.3
500
600
700
800
900
m/z
1000
There appears to be some differences in the ratio of fatty acyl when they are on sn-1
and sn-2 positions for the same regioisomers. This should be further studied
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Conclusions
Developed
a RP-HPLC-ESI-MS/MS method for detection,
separation and identification of various phospholipids
Successfully
characterized individual phospholipids in
complex mixtures
Demonstrated
the direct applicability of our method to
lecithins
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Acknowledgements

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Elif Buyukpamukcu
NRC/QS-FDI
Nestlé Research Center