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Abstract
The choline head group containing phosphatidylcholine (PC) and sphingomyelin (SPM) are major eukaryotic lipid components playing
an important role in forming membrane microdomains and serve as precursor of signaling molecules. Both lipids can be monitored by
positive ion mode electrospray tandem mass spectrometry using a parent ion scan of m/z 184. Although PC species appear at even m/z and
SPM species at odd m/z, there may be a significant overlap of their isotopes. In order to separate PC and SPM species, an isotope correction
algorithm was established, which utilizes calculated isotope percentages to correct the measured peak intensities for their isotopic overlap.
We could demonstrate that this approach was applicable to correct the isotope overlap resulting from spiked PC and SPM species.
Quantification was achieved by addition of different PC and SPM species prior to lipid extraction. The developed assay showed a precision,
detection limit and robustness sufficient for routine analysis. Furthermore, an analysis time of only 1.3 min combined with automated data
analysis using self-programmed Excel Macros allows high-throughput analysis. In summary, this assay may be a valuable tool for detailed
lipid analysis of PC and SPM species in a variety of sample materials.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Mass spectrometry; Electrospray ionization; Phosphatidylcholine; Sphingomyelin; Phospholipid; High-throughput
1. Introduction
A major component of eukaryotic lipid membranes are
phospholipids, including the glycerol-based phosphatidylcholine (PC) and the sphingosine-based sphingomyelin
(SPM) [1]. These lipids vary in the degree of saturation
and chain length of the fatty acid moiety. In addition, there
exists a minor PC fraction containing an ether bond at sn-1
position instead of the ester bond [2], which may serve as
precursor for the generation of platelet-activating factor
(PAF), a potent lipid mediator [3]. These structural differ-
109
110
Fig. 1. Parent ion scan of m/z 184 of crude plasma lipid extract. The spectrum shows a typical parent ion scan of m/z 184 from crude plasma lipid extract
averaged from a 1.3-min measurement. The internal standards PC 28:0 and PC 44:0 were added prior to lipid extraction. The PC species appeared at even and
the SPM species at odd m/z values. The inset shows a zoom of the area m/z 805818. The combined spectra were smoothed according to Savitzky Golay.
111
Table 1
Isotope correction of overlapping isotope peaks
m/z
731
732
782
783
784
810
811
812
813
814
815
C (mM)
SPM 36:1
PC 32:1
PC 36:4
SPM 40:3
PC 36:3
PC 38:4
SPM 42:3
PC 38:3
SPM 42:2
PC 38:2
SPM 42:1
0.306
0.366
0.452
0.577
0.731
0.999
1.205
1.539
1.931
2.460
0.470
0.568
0.721
0.900
1.128
0.865
0.882
0.923
0.964
0.998
0.569
0.793
1.126
1.513
2.037
0.428
0.533
0.693
0.879
1.123
0.390
0.419
0.461
0.527
0.584
0.542
0.546
0.565
0.574
0.588
0.260
0.269
0.275
0.284
0.279
0.157
0.159
0.166
0.166
0.164
0.982
1.186
1.517
1.907
2.434
0.030
0.037
0.042
0.046
0.040
0.748
0.741
0.744
0.743
0.724
0.528
0.752
1.086
1.472
2.000
0.173
0.173
0.177
0.182
0.180
0.245
0.247
0.249
0.269
0.266
0.397
0.395
0.406
0.397
0.403
0.036
0.044
0.044
0.053
0.046
0.088
0.087
0.093
0.089
0.090
A. Uncorrected ratios
0
0.149
0.045
0.310
0.113
0.549
0.188
0.821
0.300
1.229
The displayed values are ratios counts(analyte) to counts(PC 28:0 as internal standard). SPM 36:1, PC 36:4 and PC 38:4 were added at the indicated
concentrations to plasma samples prior to lipid extraction. Table A shows the uncorrected ratio and table B the ratios after correction by calculated isotope
percentages using self-programmed Excel Macros as described in Materials and methods.
112
Table 2
Cholesterol and concentration dependence of the calibration lines
Dilution
PC 34:0
Slope
PC 36:0
Intercept
PC 34:1
PC 36:4
PC 38:4
SPM 34:1
SPM 36:1
Slope
Intercept
Slope
Intercept
Slope
Intercept
Slope
Intercept
Slope
Intercept
Slope
Intercept
5.32
5.25
5.38
5.30
5.31
1.0
0.028
0.027
0.022
0.023
0.025
11.9
4.82
4.84
4.82
4.71
4.80
1.2
1.076
1.075
1.080
1.094
1.081
0.8
5.23
5.25
5.12
5.07
5.17
1.7
0.730
0.725
0.719
0.740
0.729
1.2
6.12
6.14
6.10
5.93
6.07
1.6
0.262
0.277
0.268
0.282
0.272
3.3
3.12
3.05
2.87
2.69
2.93
6.6
0.369
0.369
0.363
0.343
0.361
3.4
2.81
2.76
2.63
2.41
2.65
6.7
0.110
0.106
0.105
0.106
0.107
1.8
5.40
5.20
5.20
5.10
5.23
2.4
0.027
0.014
0.016
0.008
0.016
46.8
4.90
4.90
4.90
4.80
4.88
1.0
1.158
1.158
1.169
1.152
1.159
0.6
5.30
5.40
5.20
5.10
5.25
2.5
1.437
1.434
1.422
1.432
1.431
0.4
6.20
6.30
6.30
6.10
6.23
1.5
0.620
0.625
0.626
0.622
0.623
0.4
3.10
3.11
3.05
2.78
3.01
5.2
0.570
0.548
0.527
0.527
0.543
3.8
2.80
2.81
2.64
2.58
2.71
4.3
0.151
0.148
0.150
0.141
0.148
3.2
Calibration lines were established by standard addition of naturally occurring PC and SPM species to plasma prior to lipid extraction. The ratio counts(analyte)
to counts(PC 28:0 as internal standard) was plotted against the spiked concentration in AM plasma and the regression line was calculated by linear least square
fit. Regression line coefficients were all above 0.99 and the displayed values show the slope and intercept of the regression line. The calibration lines consisted
of five different concentrations ranging from 0 to 300 AM. Sample dilution corresponds to the extracted plasma volume.
Table 3
Precision intra day and inter day of PC and SPM species
Phosphatidylcholine
Sphingomyelin
Saturated
IS
PC 28:0
PC 44:0
PC 28:0
PC 44:0
Mean
CV%
Mean
CV%
mean
CV%
mean
CV%
0.05
3.7
0.05
6.3
0.06
11.7
0.05
10.1
Monounsaturated
0.47
3.0
0.44
5.8
0.56
9.2
0.49
10.9
Polyunsaturated
2.01
3.3
1.93
6.3
2.17
8.8
1.95
11.5
O-alkyl
0.17
2.7
0.16
5.8
0.17
9.7
0.15
10.8
Total
2.53
3.2
2.42
6.2
2.78
8.8
2.50
11.2
Total
IS
PC 28:0
PC 44:0
Inter day
PC 28:0
PC 44:0
mean
CV%
mean
CV%
0.56
3.6
0.56
6.1
mean
CV%
mean
CV%
0.59
8.7
0.56
12.1
The intra day values are mean in mM and the coefficient of variation (CV) from 10 sample aliquots measured in series using PC 28:0 and PC 44:0 as internal
standard, respectively. The inter day values are mean in mM and CV of a plasma sample calculated from seven different runs.
113
4. Conclusion
In order to achieve a separate quantification of PC and
SPM using a parent ion scan of m/z 184, an automated
sequential isotope correction algorithm was developed. A
detailed evaluation of this approach revealed a good
precision as well as a low limit of detection. Combined
114
PC Species
Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl
PC
29:1
PC O
30:1
PC
29:0
PC O
30:0
0.7
0.3
0.03
0.8
0.5
0.03
0.9
0.3
0.04
Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl
PC
35:2
PC O
36:2
PC
35:4
PC O
36:1
PC
35:0
PC O
36:0
PC
36:5
PC
36:5
PC
36:4
PC
36:4
PC
36:3
PC
36:3
PC
36:2
PC
36:2
PC
36:1
PC
36:1
Mean
S.D.
% of total
13.4
3.1
0.54
8.5
2.2
0.35
1.2
0.3
0.05
20.0
7.6
0.81
235.8
60.0
9.57
175.2
51.3
7.11
314.8
74.6
12.77
54.2
16.7
2.20
Assignment
Diacyl
Assignment
Diacyl;
Alkyl/Acyl
PC
39:6
PC O
40:6
PC
39:5
PC O
40:5
PC
39:4
PC O
40:4
PC
40:7
PC
40:7
PC
40:6
PC
40:6
PC
40:5
PC
40:5
PC
40:4
PC
40:4
PC
40:3
PC
40:3
4.1
1.1
0.17
3.3
0.9
0.14
2.6
0.6
0.11
Mean
S.D.
% of total
Mean
S.D.
% of total
PC
26:0
PC
26:0
SPM Species
Sum of
sphingoid base
and FA
SPM
30:1
SPM
32:1
PC
30:1
PC
30:1
4.5
2.9
0.18
5.7
1.7
0.23
SPM
33:1
PC
30:0
PC
30:0
6.6
3.5
0.27
24.8
6.3
1.00
SPM
34:2
PC
31:1
PC O
32:1
3.4
1.1
0.14
11.1
3.3
0.45
SPM
34:1
PC
31:0
PC O
32:0
4.1
1.2
0.17
4.7
1.4
0.19
SPM
34:0
PC
32:2
PC
32:2
6.2
2.7
0.25
3.2
1.1
0.13
PC
32:1
PC
32:1
PC
32:0
PC
32:0
PC
33:3
PC O
34:3
PC
33:2
PC O
34:2
PC
33:1
PC O
34:1
PC
33:0
PC O
34:0
PC
34:4
PC
34:4
PC
34:3
PC
34:3
PC
34:2
PC
34:2
PC
34:1
PC
34:1
29.4
17.1
1.19
20.0
5.7
0.81
9.2
2.3
0.37
13.1
3.5
0.53
11.2
2.5
0.45
1.9
0.6
0.08
2.2
1.1
0.09
19.5
7.1
0.79
645.8
140.1
26.20
334.9
93.4
13.59
PC
36:0
PC
36:0
PC
37:5
PC O
38:5
PC
37:4
PC O
38:4
PC
37:3
PC O
38:3
PC
37:2
PC O
38:2
PC
37:1
PC O
38:1
PC
38:7
PC
38:7
PC
38:6
PC
38:6
PC
38:5
PC
38:5
PC
38:4
PC
38:4
16.9
3.9
0.69
13.1
2.3
0.53
4.1
1.2
0.17
2.6
0.8
0.10
1.3
0.9
0.05
2.3
0.8
0.09
87.9
23.9
3.57
PC
40:1
PC
40:1
PC
40:0
PC
40:0
PC
41:6
PC O
42:6
PC
41:5
PC O
42:5
PC
41:4
PC O
42:4
PC
42:5
PC
42:5
PC
42:4
PC
42:4
1.2
0.3
0.05
2.0
0.5
0.08
1.3
0.4
0.05
1.2
0.3
0.05
2.5
1.5
0.10
PC
40:2
PC
40:2
2.5
0.9
0.10
SPM
35:2
3.7
1.6
0.15
SPM
35:1
1.3
0.5
0.05
SPM
35:0
SPM
36:3
SPM
36:2
SPM
36:1
SPM
36:0
60.1
14.8
2.44
PC
42:0
PC
42:0
0.7
0.3
0.03
PC
35:5
PC O
36:5
PC
35:4
PC O
36:4
PC
35:3
PC O
36:3
11.4
4.2
0.46
18.1
6.0
0.74
8.2
2.1
0.33
PC
38:3
PC
38:3
PC
38:2
PC
38:2
PC
38:1
PC
38:1
PC
38:0
PC
38:0
129.0
32.5
5.23
54.8
17.2
2.22
10.5
3.7
0.43
11.0
3.6
0.45
7.1
1.9
0.29
PC
43:6
PC O
44:6
PC
43:5
PC O
44:5
PC
43:4
PC O
44:4
PC
43:3
PC O
44:3
2.5
0.7
0.10
0.7
0.2
0.03
3.0
1.8
0.12
0.8
0.3
0.03
SPM
37:1
1.5
0.4
0.06
SPM
38:3
PC
34:0
PC
34:0
4.6
3.1
0.19
SPM
38:2
SPM
38:1
SPM
38:0
SPM
39:2
Table 4
PC and SPM (panel A) species in human plasma; SPM species (panel B) concentration after saponification of PC
FA based on
sphingoid
base d18: 1
SPM
12:0
SPM
14:0
SPM
15:0
SPM
16:1
SPM
16:0
SPM dih
16:0
Conc. Mean
Conc. S.D.
% of total
Conc.
Saponified
% of
unsaponified
0.6
0.2
0.1
0.6
13.1
3.6
2.3
13.1
7.2
1.8
1.2
8.7
20.5
4.6
3.5
24.2
140.6
28.9
24.3
200.3
7.0
1.9
1.2
14.7
Conc. Mean
Conc. S.D.
% of total
Conc.
Saponified
% of
unsaponified
83
99
107
95
SPM
39:1
SPM
40:4
SPM
40:3
SPM
40:2
SPM
21:0
SPM
22:3
SPM
22:2
3.9
1.4
0.7
3.9
1.0
0.7
0.2
10.4
5.4
1.8
3.9
85
26
SPM
17:0
SPM
dih
17:0
SPM
18:2
SPM
18:1
SPM
18:0
SPM
dih
18:0
SPM
19:0
SPM
20:2
SPM
20:1
SPM
20:0
SPM
dih
20:0
SPM
21:1
0.6
0.2
0.1
0.9
4.2
1.3
0.7
6.9
1.1
0.4
0.2
2.5
0.9
0.4
0.1
2.2
13.0
3.3
2.3
17.4
27.1
6.6
4.7
41.9
2.6
1.4
0.5
6.7
3.1
1.4
0.5
4.5
0.6
0.4
0.1
0.7
7.9
2.7
1.4
10.1
31.8
13.8
5.5
25.0
10.1
6.9
1.8
2.9
0.7
0.3
0.1
99
132
140
118
103
99
95
SPM
40:1
SPM
40:0
SPM
41:3
SPM
41:2
SPM
41:1
SPM
41:0
SPM
42:4
SPM
22:1
SPM
22:0
SPM dih
22:0
SPM
23:2
SPM
23:1
SPM
23:0
SPM
dih
23:0
34.1
9.2
5.9
34.4
43.3
12.3
7.5
43.5
1.3
0.5
0.2
13.7
3.3
2.4
16.1
16.5
4.0
2.9
16.0
0.9
0.5
0.1
0.5
102
94
4.1
2.6
0.7
2.0
64
111
100
72
92
108
95
112
114
56
SPM
42:3
SPM
42:2
SPM
42:1
SPM
42:0
SPM
43:2
SPM
43:1
SPM
44:3
SPM
44:2
SPM
24:3
SPM
24:2
SPM
24:1
SPM
24:0
SPM
dih
24:0
SPM
25:1
SPM
25:0
SPM
26:2
SPM
26:1
4.4
2.3
0.8
5.3
36.5
8.9
6.3
39.2
80.5
19.4
13.9
108.4
27.4
6.6
4.7
33.1
0.9
0.8
0.2
0.6
0.4
0.1
4.4
2.1
0.8
3.2
0.7
0.4
0.1
0.8
0.5
0.1
2.2
96
98
97
109
80
131
PC and SPM (panel A) species were quantified in 20 healthy blood donors (IS PC 28:0). PC species were assigned according to the sum of both fatty acids containing either two acyl bonds or one alkyl and one
acyl bond (for species with an odd number of C-atoms). SPM species were assigned either as sum of sphingoid base and N-linked fatty acid or based on a dihydroxy sphingosine base with 18 carbon atoms
containing one double bond (d18:1) indicating the C-atoms and double bonds of the N-linked fatty acid. Species assignment based on a dihydroxy sphingosine base with 18 carbon atoms without double bond
(d18:0) was termed Dihydrosphingomyelin (SPM dih). The displayed values are mean and S.D. in AM and the proportion of total in percent. Only species above a detection limit of 0.6 AM were shown.
In panel B, SPM species concentrations of a saponified plasma sample are shown, which were compared to a non-saponified control. For saponification the plasma sample was treated with 1 M sodium hydroxide
in 90% ethanol for 1 h at 60 8C and IS were added after saponification.
Sum of
sphingoid
base and FA
FA based on
sphingoid
base d18: 1
84
SPM
17:1
115
116
[37]
[38]
[39]
[40]
117
Erratum
Table 3
Precision intra day and inter day of PC and SPM species
Phosphatidylcholine
IS
Intraday
PC 28:0
PC 44:0
Interday
PC 28:0
PC 44:0
Sphingomyelin
Saturated
Monounsaturated
Polyunsaturated
O-alkyl
Total
Total
Mean
CV%
Mean
CV%
0.05
3.7
0.05
6.3
0.47
3.0
0.44
5.8
2.01
3.3
1.93
6.3
0.17
2.7
0.16
5.8
2.53
3.2
2.42
6.2
0.56
3.6
0.56
6.1
mean
CV%
mean
CV%
0.06
11.7
0.05
10.1
0.56
9.2
0.49
10.9
2.17
8.8
1.95
11.5
0.17
9.7
0.15
10.8
2.78
8.8
2.50
11.2
0.59
8.7
0.56
12.1
The intraday values are mean in mM and the coefficient of variation (CV) from 10 sample aliquots measured in series using PC 28:0 and PC 44:0 as internal
standard, respectively. The inter day values are mean in mM and CV of a plasma sample calculated from seven different runs.
Erratum
87
[M+H]+
Monoisotopic
1 Isotope
2 Isotope
3 Isotope
4 Isotope
PC 34:0
PC 34:1
PC 34:2
PC 34:3
PC 34:4
SPM 38:0
SPM 38:1
SPM 38:2
SPM 38:3
762.6
760.6
758.6
756.6
754.5
761.6
759.6
757.6
755.6
0.6507255
0.6509208
0.6511161
0.6513115
0.6515069
0.6440499
0.6442431
0.6444365
0.6446298
0.2758635
0.275751
0.2756384
0.2755257
0.2754129
0.2822972
0.2821886
0.2820799
0.2819712
0.062164
0.062102
0.0620399
0.0619778
0.0619156
0.0628892
0.0628255
0.0627618
0.062698
0.0098257
0.009811
0.0097963
0.0097816
0.0097668
0.009494
0.0094789
0.0094639
0.0094489
0.0011971
0.0011947
0.0011924
0.00119
0.0011876
0.0010815
0.0010793
0.001077
0.0010748
PC 34:4
SPM 38:3
PC 34:3
SPM 38:2
PC 34:2
SPM 38:1
PC 34:1
SPM 38:0
Target Mass
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
Sample 11
Sample 12
Sample 13
Sample 14
Sample 15
Sample 16
Sample 17
Sample 18
Sample 19
Sample 20
Sample 21
Sample 22
Sample 23
Sample 24
Sample 25
754.5
25580
21120
50380
79400
43870
50340
82970
79690
54650
58330
65980
75910
49970
67880
60880
67490
76610
56350
66360
94010
83370
75580
77730
88880
75830
755.6
17020
23490
29320
38710
32560
37490
52150
66740
51350
49570
35880
46050
51970
50000
65190
36650
44750
40780
38950
84020
61030
65470
51300
62610
48260
756.6
264500
231100
352600
436000
356500
608200
445300
774100
450200
782300
422900
484400
529900
497200
450600
738900
659800
597200
542000
642200
771900
553300
706400
787200
500000
757.6
263800
284100
321700
409800
442100
539600
533200
571100
535700
579500
505800
467600
608800
574100
492200
548700
502000
503600
491800
671900
718600
627100
576400
812700
533900
758.6
5475000
9264000
12230000
13580000
11650000
15640000
13690000
20240000
17820000
20870000
16130000
16140000
17720000
15420000
14380000
17930000
19410000
17030000
16920000
17250000
21320000
17270000
24800000
19150000
16840000
759.6
2676000
4490000
5726000
6600000
5612000
7664000
7137000
9482000
8304000
9821000
7894000
7652000
8566000
7915000
7322000
8421000
9164000
8291000
8093000
8714000
10170000
8783000
11470000
9179000
8010000
760.6
4910000
4639000
5370000
7880000
8725000
12140000
9569000
12360000
10960000
13850000
11220000
9751000
8869000
11340000
10810000
14170000
15660000
14470000
12090000
12430000
15840000
11330000
11120000
13370000
12330000
761.6
2090000
1907000
1986000
3123000
3402000
5046000
3878000
4889000
4187000
5669000
4433000
3812000
3336000
4382000
4473000
5678000
6374000
5699000
4847000
4915000
6288000
4446000
4165000
5288000
5027000
PC 34:4
SPM 38:3
PC 34:3
SPM 38:2
PC 34:2
SPM 38:1
PC 34:1
SPM 38:0
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
21927
19750
48420
72354
41060
44771
78131
76314
50659
55322
62920
70673
6939
14837
8416
6558
14578
17326
18046
33729
29048
25515
8602
15010
259259
222687
344252
426020
346127
596180
429819
751981
432546
765781
413055
470943
153108
188152
174519
227831
293633
285019
348428
248550
349119
252228
329273
265837
5383170
9160199
12120640
13439508
11488247
15458176
13496180
20059015
17625507
20686251
15946326
15978476
378315
590500
572762
882027
714836
1083316
1383214
954818
802011
1027737
1105122
854809
4228649
3504376
3961046
6208986
7312318
10187323
7671280
10025480
8923389
11423696
9210945
7849348
180468
226716
69467
204077
61221
391628
289673
246561
62788
417677
182709
162557
1
2
3
4
5
6
7
8
9
10
11
12
88
Erratum
Appendix A (continued)
File Name
PC 34:4
SPM 38:3
PC 34:3
SPM 38:2
PC 34:2
SPM 38:1
PC 34:1
SPM 38:0
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
46207
63541
58064
67490
76610
55025
64668
86666
73552
31601
22175
40019
8120
12364
17225
11237
45751
27755
511560
481316
427483
728934
647111
584392
530883
613706
752441
388613
367362
306588
238536
225900
253878
265151
406506
396454
17500671
15212958
14204431
17755983
19249221
16862911
16753139
17012838
21074324
1111820
1431823
1272462
870129
983446
1118857
967043
1463035
1198603
6707858
9257038
8893926
12092202
13390624
12368389
10065277
10161052
13299784
121959
91293
366935
202956
315407
96110
236220
211134
219164
13
14
15
16
17
18
19
20
21
Erratum
Worksheets(bCorr_SheetQ).Cells(Corr_DataLine+Sample, Corr_DataCol+Species)
_/Isotopepercentage(0)* Isotopepercentage(IsotopeNumber)
(REM: Correction formula)
End If
Next Sample
Column_Cor=Column_Cor+1
End If
Next IsotopeNumber
End If
Next Species
End Sub
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89