Analysis of FAMEs Using Cold EI GC MS for Enhanced Molecular Ion Selectivity

A P P L I C AT I O N N O T E
Gas Chromatography/
Mass Spectrometry
Author:
Adam Patkin
PerkinElmer, Inc.
Shelton, CT
Analysis of FAMEs
Using Cold EI GC/MS
for Enhanced Molecular
Ion Selectivity
Introduction
Characterization of fatty acid methyl esters
(FAMEs) is used in several important fields
ranging from biofuel analysis to fat content in
foods and blood. They are generally derivatized
from free fatty acids and mono-, di-, and
triglycerides. FAMEs may be saturated, mono- or polyunsaturated, linear or branched, and
of variable chain lengths.
Electron Ionization Gas Chromatography/Mass Spectrometry (EI GC/MS) is often used to
characterize FAMEs, but may fail to produce a useful molecular ion (M+•) for short,
unsaturated, or branched chains, making compound identification more difficult.
Contrastingly, Cold Electron Ionization GC/MS (Cold EI GC/MS) can substantially increase
the M+• intensity of these compounds, while retaining the EI fragmentation pattern for
spectral library searching without modification to established GC methodologies.
Experimental
Cold EI GC/MS Ion Source
All work was carried out on a PerkinElmer AxION iQT® GC/MS/
MS system. Experimental conditions are shown in Table 1.
The enhanced Cold EI M+• abundance allows use of this ion
for more selective compound analysis, even for short-chained
or polyunsaturated FAME isomers which may have unstable
molecular ions with low or no intensity in EI.
Results and Discussion
A mixture of FAMEs was characterized using EI and Cold EI
GC/MS. The Cold EI chromatogram is shown in Figure 2.
Figure 1. Cold EI GC/MS Ion Source.
• Velocity is increased (kinetic energy in the range of 1-20 eV)
• Velocity is directed along the beam (jet separation)
• Vibrational Energy is decreased (supercooling “Cold EI”)
• GC column flow rate compatibility to 100 mL/min
Molecules exit the GC column and are mixed with makeup gas.
Supersonic expansion through a nozzle then ‘cools’ the analyte
molecules from about +250 ºC to about -255 ºC by adiabatic
expansion, forming a molecular beam.
Excess carrier gas is skimmed off, and cold analyte molecules
enter the ion source for ionization, molecular ion formation
and subsequent mass analysis.
When used in a novel MS/MS configuration the enhanced
molecular ion in Cold EI provides enhanced selectivity and
valuable information on FAMEs molecular weight.
The compound retention times were identified using the NIST
2014 and Wiley 10th Edition mass spectral libraries. Of the
37 FAMEs in the test mix, all except 3 (coeluting at 12.21 min)
were chromatographically resolved or could provide “clean”
spectra through background subtraction.
Specific attention is given to those compounds which have a
relative intensity percentage, RI (%), of <5% of the M+• in NIST,
as increasing their M+• intensity would be most beneficial to
identification and quantification. These are highlighted red in
Figures 2, 5 and Table 2, and include a number of the shorter
chain FAMEs such as Methyl hexanoate and Methyl decanoate,
and polyunsaturated isomers such as cis-5,8,11,14,17Eicosapentaenoic acid methyl ester and cis-4,7,10,13,16,19Docosahexaenoic acid methyl ester. Also shown are the
Chemical Abstracts Service number (CAS No.), the Cold EI RI
(%) of the M+•, the enhancement ratio of the Cold EI to EI M+•
RI (%), and the number of double bonds in the compound.
To better understand any pattern of EI M+• relative intensities,
the compounds were sorted by the number of double bonds
and then molecular weight, as shown in Figure 3.
Table 1. Cold EI GC/MS Ion Source.
Gas Chromatograph:
PerkinElmer Clarus® 680
Mass spectrometer:
PerkinElmer AxION® iQT™ MS/MS
Injector Type:
Injector temperature:
Injection:
Programmable Split/Splitless
250 ˚C
0.4 µL, split 200:1
GC Transfer Line Temperature:
Ion Source Temperature:
250 ˚C
200 ˚C
Oven Program:
50 ˚C, ramp to 240 ˚C at 20 ˚C/min
Acqusition Range:
Acqusition Time:
m/z 40-400
0.20 sec
Solvent Delay:
none
Analytical Column:
PerkinElmer Elite™ - 5MS
30 m x 0.25 mm ID x 0.25 μm
Carrier Gas:
1 mL/min Helium
99.999+% purity
Supelco 37 Component FAME Mix
Ion Source Type:
Ion Source Mode:
Background Noise Removal:
Cold EI makeup gas:
Ion Source Mode:
Cold EI makeup gas:
Cold EI
Cold EI
On
50 mL/min
Classical EI
2 mL/min
Filament:
5 µA
Sample:
2
6e7
Intensity
5e7
4e7
3e7
2e7
1e7
0
3
4
5
6
7
8
9
10
11
Retention Time (minutes)
12
13
14
15
16
Figure 2. Cold EI chromatogram of FAMEs mixture. FAMEs with NIST EI molecular ion relative intensities below 5% are highlighted in red.
Table 2. FAMEs with NIST EI molecular ion relative intensities below 5% are highlighted in red, and compared to their Cold EI results. FAMEs in retention time order.
Compound Name
CAS No.
Formula
MW
NIST EI
RI %
Cold EI
RI %
Cold EI
Enhancement
Double
Bonds
2.33
Methyl butyrate
623-42-7
C5H10O2
102.1
1.4
48
655
0
4.48
Methyl hexanoate
106-70-7
C7H14O2
130.1
0.5
4
7
0
6.33
Methyl octanoate
111-11-5
C9H18O2
158.1
3.8
16
4
0
7.83
Methyl decanoate
110-42-9
C11H22O2
186.2
1.5
50
44
0
8.50
Methyl undecanoate
1731-86-8
C12H24O2
200.2
2.0
85
57
0
9.14
Methyl laurate
111-82-0
C13H26O2
214.2
6.0
130
37
0
9.74
Methyl tridecanoate
1731-88-0
C14H28O2
228.2
4.0
196
84
0
10.24
Methyl myristoleate
56219-06-8
C15H28O2
240.2
4.0
96
358
1
10.31
Methyl myristate
124-10-7
C15H30O2
242.2
4.0
259
117
0
10.79
Methyl cis-10-pentadecenoate
90176-52-6
C16H30O2
254.4
4.2
102
374
1
10.84
Methyl pentadecanoate
7121-64-1
C16H32O2
256.2
6.0
405
140
0
11.26
Methyl palmitoleate
1120-25-8
C17H32O2
268.2
6.0
89
356
1
11.36
Methyl palmitate
112-39-0
C17H34O2
270.3
10.0
590
115
0
11.76
cis-10-Heptadecenoic acid methyl ester
77745-60-9
C18H34O2
282.3
3.5
77
808
1
11.85
Methyl heptadecanoate
1731-92-6
C18H36O2
284.3
7.9
698
216
0
12.11
Methyl γ-linolenate
16326-32-2
C19H32O2
292.2
23.0
269
22
3
12.18
Methyl linolelaidate
2566-97-4
C19H34O2
294.3
30.0
358
162
2
12.23
cis-9-Oleic acid methyl ester
112-62-9
C19H36O2
296.3
6.0
56
650
1
12.32
Methyl stearate
112-61-8
C19H38O2
298.3
14.0
993
172
0
12.92
cis-5,8,11,14-Eicosatetraenoic acid methyl ester
2566-89-4
C21H34O2
318.3
0.8
41
78
4
12.96
cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester
2734-47-6
C21H32O2
316.2
0.2
4
28
5
13.00
cis-8,11,14-Eicosatrienoic acid methyl ester
21061-10-9
C21H36O2
320.3
3.0
340
234
3
13.07
cis-11,14-Eicosadienoic acid methyl ester
2463-02-7
C21H38O2
322.3
10.0
194
523
2
13.09
Methyl cis-11-eicosenoate
2390-09-2
C21H40O2
324.3
2.8
31
1965
1
13.11
cis-11,14,17-Eicosatrienoic acid methyl ester
55682-88-7
C21H36O2
320.3
8.0
242
51
3
13.20
Methyl arachidate
1120-28-1
C21H42O2
326.3
10.0
880
338
0
13.61
Methyl heneicosanoate
6064-90-0
C22H44O2
340.3
20.0
861
203
0
13.74
cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester
301-01-9
C23H34O2
342.3
0.0
2
na
6
13.94
cis-13,16-Docosadienoic acid methyl ester
61012-47-3
C23H42O2
350.3
12.0
139
301
2
13.95
Methyl erucate
1120-34-9
C23H44O2
352.3
6.0
22
816
1
14.06
Methyl behenate
929-77-1
C23H46O2
354.3
16.0
746
260
0
14.57
Methyl tricosanoate
2433-97-8
C24H48O2
368.4
16.0
999
281
0
15.04
Methyl nervonate
2733-88-2
C25H48O2
380.4
3.4
20
985
1
15.18
Methyl lignocerate
2442-49-1
C25H50O2
382.4
14.0
110
390
0
Ret. Time
3
EI vs. Cold EI Molecular Ion Relative Intensity
NIST EI Molecular Ion Relative Intensity
0
Methyl butyrate
Methyl hexanoate
Relative Intensity %
10
15
20
25
5
1.5
Methyl undecanoate
2.0
1200
Methyl octanoate 16
Methyl undecanoate
Methyl laurate
Methyl laurate
6.0
4.0
Methyl myristate
4.0
6.0
Methyl palmitate
10
Methyl heptadecanoate
7.9
Methyl stearate
Methyl heneicosanoate
Methyl behenate
16
Methyl tricosanoate
16
Methyl cis-10-pentadecenoate
4.2
Methyl palmitoleate
Methyl lignocerate
Methyl myristoleate
Methyl cis-10-pentadecenoate
1 Double Bonds
3.5
cis-9-Oleic acid methyl ester
6.0
2.8
Methyl erucate
6.0
Methyl nervonate
3.4
2 Double
Bonds
10
cis-13,16-Docosadienoic acid methyl ester
12
Methyl γ-linolenate
3 Double
Bonds
3.0
8.0
0.8
861
746
999
110
96
102
Methyl palmitoleate
89
cis-10-Heptadecenoic acid methyl ester
77
cis-9-Oleic acid methyl ester
56
Methyl cis-11-eicosenoate 31
Methyl erucate 22
358
cis-11,14-Eicosadienoic acid methyl ester
cis-13,16-Docosadienoic acid methyl ester
194
139
Methyl γ-linolenate
cis-8,11,14-Eicosatrienoic acid methyl ester
23
cis-11,14,17-Eicosatrienoic acid methyl ester
993
880
Methyl linolelaidate
30
cis-8,11,14-Eicosatrienoic acid methyl ester
698
Methyl nervonate 20
Methyl linolelaidate
cis-11,14-Eicosadienoic acid methyl ester
590
Methyl heptadecanoate
Methyl tricosanoate
6.0
cis-10-Heptadecenoic acid methyl ester
405
Methyl palmitate
Methyl behenate
14
4.0
259
Methyl pentadecanoate
Methyl heneicosanoate
20
Methyl lignocerate
196
Methyl myristate
Methyl arachidate
10
Methyl myristoleate
130
Methyl stearate
14
Methyl arachidate
85
Methyl tridecanoate
0 Double Bond
Methyl tridecanoate
Methyl pentadecanoate
4 Double Bonds
5 Double Bonds
6 Double Bonds
cis-11,14,17-Eicosatrienoic acid methyl ester
269
340
242
cis-5,8,11,14-Eicosatetraenoic acid methyl ester 41
cis-5,8,11,14,17-Eicosapentaenoic acid methyl…3.6
cis-4,7,10,13,16,19-Docosahexaenoic acid…2.0
NIST EI Relaˆve Intensity
Cold EI Relaˆve Intensity
Figure 3. NIST EI molecular ion relative intensity % sorted by number of double bonds
and molecular weight. Compounds are color coded by number of double bonds.
Figure 4. Much larger Cold EI (hashed pattern) molecular ion relative intensity
% compared to NIST EI (solid pattern). Values >100 have the next largest peak
defined as 100%.
Sorted in this manner and extrapolating with this limited data
set, it appears that very short-chained and unsaturated FAMEs
with 4 or more double bonds are more likely to produce small
molecular ions, while longer chain saturated FAMEs and those
with 2 or 3 double bonds tend to produce larger ones.
This enhanced M+• in Cold EI provides improved selectivity
and less ambiguous molecular weight determination in complex
mixtures. It also provides a more intense and unique ion which
can enhance MS/MS selectivity and detection limits.
Cold EI was found to enhance the molecular ions in all FAMEs.
Large M+• intensity enhancements are noted for mid-length
unsaturated linear chains. Nearly all the compounds with EI M+•
RI (%) below 5% now are above 5%. This is seen in Figure 4.
The most unsaturated compound (cis-4,7,10,13,16,19Docosahexaenoic acid methyl ester) exhibited no M+• at all
in EI, but shows a discernable one in Cold EI. The average
increase of the M+• RI (%) was a factor of 33.
The large observed enhancements make the M+• clearly more
unique and thus selective for Cold EI than for EI. Cold EI M+•
mass chromatograms can be used for quantification with
better selectivity than a lower mass fragment ion which might
otherwise have to be used for EI.
4
1000
Methyl decanoate 50
Methyl decanoate
cis-4,7,10,13,16,19-Docosahexaenoic acid… 0.0
Relative Intensity %
400
600
800
Methyl hexanoate 4
3.8
cis-5,8,11,14,17-Eicosapentaenoic acid methyl… 0.2
200
Methyl butyrate 48
0.5
Methyl cis-11-eicosenoate
0
35
1.4
Methyl octanoate
cis-5,8,11,14-Eicosatetraenoic acid methyl ester
30
Cold EI spectra of some FAMEs with smaller EI M+• relative
intensities are shown in Figure 5 with their NIST library search
results, and red highlighting of the molecular ion. Some of the
names are synonyms.
Cold EI cooling is shown to increase the RI (%) of the M+•
and reduce fragmentation. This lower internal energy may
also change the preferred fragmentation energies and kinetics,
leading to the enhancement of mid-mass ions more stable
at these lower energies.
74
100
87
100
87
143
102
50
71
43 45
0
40
50
55
49
53
44
42
41
55
57
59 61
60
64
72
69
69
72
75 77 79
75
86 88 90 92
88
96
0
101
87
59
75
43 49 55 60 65
83
69
43
97
50
100
60
70
80
90
Head to Tail MF=620 RMF=627
79
170 177 185
185
200
170
200
169
157
143
100
40
119 133
150
161
119
55
175
161 175 187
87
133
203
80
90
100
110
120
130
140
150
160
Head to Tail MF=577 RMF=584
180
190
Undecanoic acid, methyl ester
348
203 217
233 247 259
318
286
233 247 262
303
3
275 287
318
0
79
80
41 55 67
111
50
91
43
100
100
120
RT12.9269
83 97 111 125
150
67
60
70
50
217
105
100
60
RT8.5085
105
87
41 49 57 67
50
100
50
40
149
149
129
74
Butanoic acid, methyl ester
91
100
41
101
87
74
43
RT2.3314
50
126
115
50
40
0
129
115
109
55
71
100
200
157
101
50
140
160
180
200
Head to Tail MF=760 RMF=771
220
240
260
280
300
320
5,8,11,14-Eicosatetraenoic acid, meth
69
83
305
152 166 180 194 208 222 236 250 264
167 181 195
125
221 236 250 264
380
330
362
2
362
2 380
334
291 306
348
97
55
40
60
80
100
RT15.0414
120
140
160
180
200
220
240
Head to Tail MF=627 RMF=641
260
280
300
320
340
360
Figure 5. Comparison of Cold EI spectra (top, red) to NIST library-searched EI reference spectra (bottom, blue) showing the enhanced Cold EI molecular ion (boxes).
Conclusion
Cold EI GC/MS enhances FAMEs molecular ion intensity,
improving analysis specificity and ease of molecular ion
identification for compounds with low EI molecular ion
relative intensity.
Not for use in diagnostic procedures.
*
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15-Tetracosenoic acid, methyl ester, (Z
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