Analysis of whole lipid extracts using on-line high resolution LC-MS
Catharina Crone1, Eric Genin2 and Helmut Muenster1
1Thermo Fisher Scientific, Bremen, Germany, 2Thermo Fisher Scientific, Les Ulis, France
Purpose: Analysis of a complex lipid extract with high resolution and high mass accuracy including fast polarity
switching and fragmentation.
FIGURE 2. Base peak chromatogram of the lipid
extract in positive ionization mode.
FIGURE 3. Base peak chromatogram of the lipid
extract in negative ionization mode.
FIGURE 5. Positive HCD MS/MS spectrum of the PEs
at RT 5.3 minutes
FIGURE 6. Negative HCD MS/MS spectrum of the
PEs at RT 5.3 minutes. Inset shows the fatty acid
region with assignment of the fatty acids chain
length and degree of saturation.
Methods: On-line LC-MS using a stand-alone OrbitrapTM.
9.83
100
10.05
All on-line LC-MS Experiments were performed on an ExactiveTM mass spectrometer (Thermo Fisher Scientific,
Bremen, Germany) equipped with an AccelaTM HPLC system (Thermo Fisher Scientific, San Jose, CA)
using a 5 um 150x2.1 mm Hypersil Silica Column (Thermo Fisher Scientific, USA) at a flow rate of 300 ul/min.
Solvents: A – CHCl3 / MeOH / 80:20, 5 mM NH4OAc, B – CHCl3 / MeOH / H2O 60:34:6, 5 mM NH4OAc.
Gradient: 0-2 min 20% B, 2–10 min 20% B – 100% B,10–18 min 100% B,18–19 min 100% B – 20% B,
19–27 min 20% B.
The mass spectrometer was operated with standard electrospray ionization performing alternating full scan and
CID fragment ion scans carried out in the HCD collision cell (see Figure 1). Resolution settings of 100,000 were
applied in both polarity modes. For the fast polarity switching experiments a resolution setting of 25,000 was
used to achieve a full cycle (one positive full scan and one negative full scan) in less than one second.
FIGURE 1. Schematic layout of the instrument
C-Trap
40
2.34
SM
6.25
PE
1.91
20
9.44
C22H48N
3.26
LPC
11.87
11.48
6.97
5.30
0
1
2
3
4
5
7
8
9
Time (min)
10
11
12
13
14
10.11
PI
40
4.03
PE
5.36
7.67
11.97
0
15
0
1
2
3
4
5
6
7
8
9
Time (min)
10
11
12
13
14
15
Therefore, resolution settings of up to 100,000 were used in both polarity modes to ensure the best possible
mass spectrometric separation for these complex extracts. Mass accuracies in the low to sub ppm range were
obtained without averaging data and were applied to identify and confirm the different lipids in each scan.
Due to the complexity of the extracts with many isobaric compounds amongst the different lipid classes it is
essential to obtain fragmentation information in addition to the full scan accurate mass data. This allows
confirmation of the identity of the different lipid classes and furthermore to determine the fatty acid pattern within
these lipid classes.
The high mass accuracy fragmentation data inherent to HCD offers the ability to generate pseudo parent ion
mass chromatograms which are ideally suited to confirm the specific lipid classes. In combination with high
resolution (key for separation of interferences) the applied narrow mass tolerance window (as a result of stable
accurate masses) ensures correct identification of the lipid classes even for very complex samples. Examples for
the benefits of these instrumental features are shown using complex natural lipid extracts.
Split Lens
Mass
Flatapole
Lens
RF Lens
Skimmer
Tubelens
792.5536
100
Heated Capillary
80
API Ion Source
Results
As can be seen from the total ion chromatograms generated in both ionization mode polarities (figures 2 and 3), the
chromatographic conditions employed allow good separation of the different lipid classes but not complete
separation of the different lipid species within one lipid class.
Therefore, resolution settings of up to 100,000 were used in both polarity modes to ensure the best possible mass
spectrometric separation for these complex extracts. Mass accuracies in the low to sub ppm range were obtained
without averaging data and were applied to identify and confirm the different lipids in each scan.
Due to the complexity of the extracts with many isobaric compounds amongst the different lipid classes it is
essential to obtain fragmentation information in addition to the full scan accurate mass data.
This allows confirmation of the identity of the different lipid classes and furthermore to determine the fatty acid
pattern within these lipid classes.
Relative Abundance
Orbitrap Mass Analyzer
O
651.5340
C 43 H 71O 4
-1.03 ppm
792.5530
C 45H79O8 NP
-0.99 ppm
18:0
0
350
400
450
500
550
m/z
600
650
700
750
40
16:0
303.2335
C20H31O2
1.86 ppm
22:4
283.2646
C 18 H35O 2
1.34 ppm
331.2648
C22H35O 2
1.83 ppm
255.2332
C16 H31 O 2
0.93 ppm
332.2682
317.0679
303.2335
0
260
270
280
290
300
m/z
310
320
330
40
0
C40 H75 O10 N P
0.60
C40 H79 O10 N P
90
786.5289
-0.22
C42 H77 O10 N P
788.5442
-0.58
C42 H79 O10 N P
790.5599
-0.56
C42 H81 O10 N P
70
792.5758
-0.21
C42 H83 O10 N P
65
794.5920
0.40
C42 H85 O10 N P
60
806.4986
1.03
C44 H73 O10 N P
808.5142
0.94
C44 H75 O10 N P
810.5296
0.63
812.5447
0.05
C44 H79 O10 N P
35
814.5597
-0.86
C44 H81 O10 N P
30
818.5924
0.91
C44 H85 O10 N P
820.6062
-1.33
C44 H87 O10 N P
832.5124
-1.25
C46 H75 O10 N P
834.5293
0.30
C46 H77 O10 N P
836.5455
0.92
C46 H79 O10 N P
838.5608
0.55
C46 H81 O10 N P
842.5929
1.48
C46 H85 O10 N P
85
80
75
60
776.5591
40
748.5280
20
0
752.5594
702.5433 718.5383
728.5592744.5544
706.5406
690
700
710
720
730
740
750
768.5543 780.5906
796.5852
764.5229
814.5358
760 770
m/z
780
790
800
810
820
830
700.5277
702.5432
704.5600
716.5222
716.5586
718.5382
724.5262
728.5588
730.5744
732.5540
740.5220
742.5389
744.5535
746.5693
748.5275
750.5429
752.5579
754.5751
756.5898
758.6060
764.5223
766.5385
768.5533
772.5850
774.5426
774.6008
776.5586
778.5750
840 780.5898
790.5375
792.5534
796.5848
Composition
Delta(ppm)
C39 H75 O7 N1 P1
C39 H77 O7 N1 P1
C39 H79 O7 N1 P1
C39 H75 O8 N1 P1
C40 H79 O7 N1 P1
C39 H77 O8 N1 P1
C41 H75 O7 N1 P1
C41 H79 O7 N1 P1
C41 H81 O7 N1 P1
C40 H79 O8 N1 P1
C41 H75 O8 N1 P1
C41 H77 O8 N1 P1
C41 H79 O8 N1 P1
C41 H81 O8 N1 P1
C43 H75 O7 N1 P1
C43 H77 O7 N1 P1
C43 H79 O7 N1 P1
C43 H81 O7 N1 P1
C43 H83 O7 N1 P1
C43 H85 O7 N1 P1
C43 H75 O8 N1 P1
C43 H77 O8 N1 P1
C43 H79 O8 N1 P1
C43 H83 O8 N1 P1
C45 H77 O7 N1 P1
C43 H85 O8 N1 P1
C45 H79 O7 N1 P1
C45 H81 O7 N1 P1
C45 H83 O7 N1 P1
C45 H77 O8 N1 P1
C45 H79 O8 N1 P1
C45 H83 O8 N1 P1
0.20
-0.01
1.43
-0.37
-0.38
0.02
-1.91
-0.12
-0.16
0.24
-0.60
1.09
-0.42
-0.21
-0.05
-0.49
-1.25
0.74
-0.51
0.18
-0.19
0.50
-0.65
-0.16
-0.79
0.13
-0.35
0.56
-0.41
-0.75
-0.47
-0.30
835.5328
50
789.5488
45
281.2489
800
Composition
1.18
331.2648
200
250
300
40
774.5459
766.5410
792.5467
621.4468
255.2332
253.2175
25
431.5618
20
700.5304
350
400
450
500 550
m/z
600
650
700
750
800
850
Detailed structural analysis of the phosphatidylethanolamines (PE)
Within the different lipid classes it is difficult to separate by LC the individual species. The lack of
chromatographic separation is compensated for by high resolution in the mass spectrometer with the full scan
and HCD fragmentation scans. We used a resolution setting of 100,000 for the alternating full scan / HCD scan
experiment and performed separate runs in each polarity mode.
As can be seen from the mass spectrum of the PE section of the lipid extract, a number of different PEs co-elute
(figure 4). Using the accurate masses for the determination of the elemental composition and subsequently for
the identification of the different PE species, we did find a number of PEs with one ether function in the molecule
(figure 4, blue masses). In any case, as can be seen from the list of PEs in Figure 4, all compounds could be
identified with a mass error of less than 2 ppm. Similar results are obtained from the full scan data taken in
negative ionization mode (showing [M-H]-, table not shown) and ensures that all detected species within the lipid
class.
All HCD spectra show the same good mass accuracy as the full scan spectra (figure 5 and 6). Mass deviations
of less than 2 ppm are routinely obtained using external calibration. The fragmentation pathway can be easily
confirmed with the use of the accurate masses of the fragments allowing the determination of the elemental
composition of the fragments in both polarity modes.
786.5301
15
FIGURE 7. Example of alternating positive and negative scan during fast polarity switching at the
retention time of PE species.
OSPE/SOPE
5
760.5144
760
774.5306785.4658
770
780
DOPE
790
positive scan
# 348
40
DOPE
60
1.26 ppm
742.5399
C 41 H 77 O 8 N P
negative scan
# 349
DOPE: R’ = R’’ = C17H33
OSPE: R’ = C17H35, R’’ = C18H33
SOPE: R’ = C18H33, R’’ = C17H33
20
742
744
746
m/z
748
830
840
850
860
Fatty acid area
747.4981
C43 H72 O8 P
1.36 ppm
419.2576
C21 H40 O6 P
1.79 ppm
80
0
40
0
820
16:0
-C3H5O2N
(serine)
750
In order to obtain all the accurate mass information of lipids ionizing in the positive as well as in the negative ion
mode, the instrument was operated in the fast polarity switching mode. Two consecutive scans show the [M+H]+
and [M-H]- ion respectively for the different fatty acid containing phosphatidylethanolamines (figure 7). One full
cycle (one positive and one negative scan) was acquired in less than one second at 25,000 resolution. Mass
accuracy was well below 3 ppm for all ions.
834.5299
C46 H77 O10 NP
0.96 ppm
18:0
283.2647
C18 H35 O2
1.48 ppm
437.2680
C21 H42 O7 P
1.38 ppm
480.3100
327.2336
C23 H47 O7 NP
22 H31 O2
18:1 C1.89
0.97 ppm
ppm 417.2421
20:4
375.2064
557.0342
P
1.31 ppm
744.5555
C 41 H 79 O 8 N P
80
810
m/z
255.2332
C16 H31 O2
0.93 ppm
20
OSPE/SOPE
100
800
FIGURE 9. Negative HCD MS/MS spectrum of the lipid extract recorded during the elution of the PSs.
20
0
806.4984
As an example for a different lipid class the negative full scan spectrum (figure 8) and the HCD spectrum (figure
9) are shown for the phosphatityl-serines (PS).
Similar to the other lipid classes, the full scan data for the PSs are used to determine the accurate molecular
weights and subsequently their elemental compositions (figure8). In the HCD scan significant fragments are
seen for the loss of the serine group and for the fatty acid residues. This allows the determination of the lipid
group and the distribution of the fatty acids in the PS molecules (figure 9).
40
-0.13 ppm
744.5536
C 41 H 79 O 8 N P
60
791.5548
C44 H77 O10 N P
60
-0.15 ppm
746.5693
C 41 H 81 O 8 N P
80
810.5292
836.5346
816.5769
838.5600
839.5630
817.5802 832.5139
856.5104
846.6247
790.5519
10
100
100
834.5295
55
790.5404
20
300
60
20
60
60
40
80
NH2
18:1 18:0
Delta
(ppm)
764.5452
788.5454
100
0
Lens
Curved
Lens
System
P
OH
14.16
FIGURE 4. Positive full scan mass spectrum of the PE section taken at RT 5.3 min and list of identified
PEs obtained from the positive full scan experiment.
Octapole
O
20
8.42
14.16
6
60
20
O
22:6
80
2.90
80
m/z
760.5143
95
22:6
20:4
Relative Abundance
Methods
60
100
100
Relative Abundance
The analysis of lipids is challenging to mass spectrometry due to its complexity and variety of lipid classes.
Electrospray ionization both in positive and in negative ionization mode has been shown to be a useful method
for structural studies of lipids and especially for phospholipids because of their zwitterionic structure. Information
can be obtained regarding the molecular weight, the acid moieties and the residue attached to the phosphoric
acid.
Some lipid classes like the phosphatidylinositols (PI) are best analyzed in negative ionization mode where most
other lipid classes are best ionized in positive mode of operation. Nevertheless, both ionization modes deliver
complementary information and therefore, to save time, it is beneficial to run the mass spectrometer in the
alternated mode of operation. The non hybrid benchtop orbitrap mass spectrometer is capable of providing a full
cycle (one positive and one negative high resolution full scan) in less than one second while maintaining high
mass accuracy. In addition the instrument can perform CID experiments in the HCD collision cell delivering
accurate fragment mass information which is of high value for structural analysis.
Relative Abundance
Introduction
Relative Abundance
80
PS
1.92
80
385.2735
C 25 H 37O 3
-0.67 ppm
Relative Abundance
PC
SM:7G
327.2335
Relative Abundance
RT:0.00 - 15.72
Relative Abundance
Results: Accurate mass data of different lipid species within the different lipid classes ionizing in different
polarity modes could be obtained in one single LC-MS run.
HCD Collision Cell
327.2335
C22H31O 2
1.84 ppm
100
RT:0.00 - 15.25 SM: 7G
2.32
100
FIGURE 8. Negative full scan spectrum of the lipid extract recorded during the elution of the PSs
together with an accurate mass list of all detected PS species.
Relative Abundance
Overview
250
300
621.4589
C44 H62 P 701.5142
-0.91 ppm C46 H69 O5
-1.22 ppm
22:6
350
400
450
500
550
m/z
792.5774
C42 H83 O10 NP
1.72 ppm 836.5359
629.4539
600
650
700
750
800
850
Conclusions
The ExactiveTM is an ideal instrument for the analysis of complex lipid extracts. The ability to perform full scans
and MS/MS scans in both polarity modes at very high resolution ensuring accurate masse measurements
independent of the polarity allows fast, precise and unambiguous identification of the lipid classes as well as the
individual lipid species. Operation of the instrument in the alternated mode of operation saves time without
sacrificing analytical performance.
Acknowledgements
We would like to thank Anne ATHIAS, IFR 100 Santé-STIC/Plateau Technique Lipidomique, Dijon, France
and Arnaud BERNARD, LBMN - INSERM U866, Dijon, France for supplying the samples.
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IMSC09_W391_CCrone