Handbook of instrumental techniques from CCiTUB

ChT.6

Gas Chromatography - Mass Spectrometry

Lourdes Berdié1, Irene Fernández2, and Pilar Teixidor1

1Unitat de Cromatografia de Gasos i Espectrometria de Masses, CCiTUB. Lluís
Solé i Sabarís 1-3. 08028 Barcelona, Spain.
2Unitat d’Espectrometria de Masses (Química), CCiTUB. Facultat de Química.
Martí i Franquès s/n. 08028 Barcelona, Spain

email: lourdesb@ccit.ub.edu, i.fernandez@ccit.ub.edu, teixidor@ccit.ub.edu

Abstract. This article briefly describes the possible configurations of the gas
chromatography- mass spectrometry (GC/MS) technology available at the
CCiTUB. Some developed examples of different applications are shown.
 Gas Chromatography - Mass Spectrometry

1. Introduction concluding paragraph:

The technology of gas chromatography-mass spectrometry (GC/MS) combines the fine separating
power of GC (complex mixtures of hundredths of compounds) with the two main competences of
MS:
• Powerful detection
• Strong capacity of unknown identification

The applied GC/MS unit of the Scientific and Technological Centers has the ability to select,
practice and develop the different appropriated methods of extraction, clean-up, concentration and
derivatization of organic compounds in a great variety of natural and synthetic samples, from
mineral to biological world in order to analyze the prepared extracts by GC/MS towards the
process of identifying and quantifying the extracted organic compounds.
Method developments and subsequent validation following the International Conference of
ChT.6 Harmonization (ICH) guidelines are also performed if they are required.

2. Methodology

Several GC/MS systems with capillary column separation and different ionization methods are
available in the Unit. These systems are summarized in Table 1.
Table 1. Summary of the different GC/MS systems available at the CCiTUB
Ionization method Analyzer
Electron ionization (EI) Quadrupol Mass Filter IonTrap Quadrupol
Positive chemical ionization (PCI) Quadrupol Mass Filter
Negative chem. ion. (NCI) Quadrupol Mass Filter

The detection in the Quadrupol mass filter MS instruments can be performed in scan or single-
ion monitoring (SIM) mode. In the scan mode, a specified mass range is scanned at regular
intervals during the chromatographic process and stored in the data system. This mode provides
reproducible mass fragmentation patterns that originate the mass spectra. With this information it is
often possible to identify the GC separated unknown compounds. There are some spectral
databases (commercial libraries) that assist the analyst in the process of identification. In spite of
this, experienced analysts are required and considerable care must be exercised in interpreting the
results of such comparisons. The identifying process can be assisted by the additional measurement
of the retention index for each peak eluted and quantification is also possible by integration of the
different ion peaks present in the chromatogram using internal and/or external standards.
With the MSn ability of the ion trap analyzer, the possibility to detect an unknown in a complex
mixture is enhanced and this tandem mode also provides additional structural information to help in
the identifying process.
According to the nature of the sample and the volatility of compounds to be analyzed, some
different sample introduction systems can be applied:

• Static Head Space (HS)

• Solid phase microextraction (SPME)
• Thermal desorption (TD)
• Direct liquid/gas injection

A summary of the different sample introduction systems used for volatile and semivolatile
compounds is given in Table 2.

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 Gas Chromatography - Mass Spectrometry

Table 2. Summary of the different sample introduction systems for volatile (VOC) and
semivolative (SVOCs) compounds
Compound Sampling
VOCs HS TD SPME Direct
SVOCs Direct SPME

Volatile compounds (VOCs): This group includes low-molecular weight compounds (ca < 250
uma) with high-vapour pressures and low-to-medium water solubilities. Some of them have natural
origin but the majority is anthropogenic, which are used and produced in the manufacture of
cleansers, paints, adhesives, plastics, pharmaceuticals, refrigerants, etc. They often are compounds
of fuels, solvents, lubricants, paint thinners and dry-cleaning agents commonly used in urban
settings. Most of them cause environmental problems and are potential carcinogens, and therefore,
their control becomes obligatory and regulated.
Semivolatile compounds (SVOCs): There are organic compounds with molecular weights up
1000 amu, which may vaporize when exposed to temperatures above room temperature (usually ChT.6
with a boiling point >100ºC). In this group, some compounds such as common pesticides (OC’s,
OP’s, triazines), persistent pollutants (phthalates, nonylphenols, PCBs, PAHs), neutral lipids, small
metabolites, aliphatic and aromatic hydrocarbons, fragrances, essential oils and relatively non-polar
drugs are analyzed in different matrices. In order to increase the volatility of compounds containing
polar functional groups (-OH, -COOH, -NH2, etc.), chemical derivatizations, e.g. silyl derivatives
for alcohols and amines, methyl esters, transterification, are often employed in order to extend the
range of suitable analytes to such compounds as steroids, polar drugs, polar lipids, organic acids,
and aminoacids.
applications of GCMS
3. Examples of applications

3.1. Authentication analysis of personnel care products

In order to detect differences between two similar commercial deodorant spray samples (original
branded and imitation), the analysis of volatile organic compounds was performed. The obtained
results revealed multiple coincident compounds in both samples but the synthetic musk galaxolide
(1,3,4,6,7,8-Hexahydro-4-6-6-7-8-8-hexamethyl cyclopenta-[g]-2-benzopyran) was only detected
in the original spray. However, in the imitation sample, high amount of ethanol was found and
floropal (2,4,6-Trimethyl-4-phenyl-1,3-dioxane) was detected instead of the polycyclic musk. The
GC/MS ability to filter specific ions permitted to detect this 1,3-dioxane odorant which coeluted
with a menthol derivative. The different chromatograms and mass spectra obtained in this example
are shown in Figs. 1-5.

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 Gas Chromatography - Mass Spectrometry

ChT.6

Figure 1. Comparison of the chromatograms corresponding to the original and defective samples
obtained by HS-GC/MS on a DB-624 column. Note: Coelution between p-menth-1-en-8-ol acetate
and floropal (only in sample 2).

Figure 2. Mass chromatograms corresponding to the original and defective samples. Trace ions
m/z 136 corresponding to p-menth-1-en-8-ol acetate and m/z 191 to floropal

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 Gas Chromatography - Mass Spectrometry

ChT.6

Figure 3. Mass spectra of p-menth-1-en-8-ol acetate (top) and floropal (bottom)

Figure 4. Comparison of the chromatographic profiles corresponding to the original and defective
samples obtained by GC/MS on a VF-5MS column.

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 Gas Chromatography - Mass Spectrometry

ChT.6

Figure 5.Mass spectrum of galaxolide with the corresponding library search result.

The original deodorant could be distinguished from the copy with the GC-MS technique. Even
in the case of a coelution, it was possible to find differences between both samples.

3.2. Odour markers in food containers

In order to detect some odour markers in defective tin containers, samples were analyzed by head-
space gas-chromatography mass spectrometry (HS-GC/MS). The VOCs chromatograms revealed
some significant quantitative differences between a blank sample (correct) and an odorant sample
(defective). In the latter sample, higher concentrations of chlorinated compounds and phenol were
detected. These compounds could be responsible for the unpleasant odour (see Fig. 6)

Figure 6. Comparison of the chromatographic profiles corresponding to the original and defective
samples obtained by HS-GC/MS on a DB-624 column

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 Gas Chromatography - Mass Spectrometry

3.3. Biomarkers in archaeological residues

Lipid biomarkers extraction and analysis of cooking wares residues (5th century AD) from Sa
Mesquida (Mallorca, Spain) were performed by GC/MS. The origin of food contained in the
archaeological ceramics could be distinguished from the fatty acids profile (Pecci) .

ChT.6

Figure 7. Chromatograms corresponding to a different total lipid extract samples. The fatty acids
profile in sample A, where the saturated acids are clearly dominant, suggests an animal fat trace. In
contrast, the fatty acids pattern showed in sample B suggests that a vegetal fat origin as
unsaturated acids is relevant.

3.4. Determination of fenthion in liver samples (GC/MSn example)

Fenthion is an organophosphorous (OPs) pesticide of regulatory concern for health and safety
problems due to its lipophilic properties and large degree of persistence in the environment. One of
the most important organophosphorous compounds reactions is water hydrolysis. Moreover, OPs
inhibit the phosphorylate esterases, particularly the enzyme acetylcholinestarase, thus causing an
accumulation of the neurotransmitter acetylcoline. Other effects are mutagenicity and
carcinogenicity as well as specific organ toxicity to heart, liver, kidney and other organs.
For this compound, extraction and clean-up procedures are long and generally involve several
steps (e.g. extraction, GPC and SPE clean-up), which can imply systematic losses and consequent
inaccuracy (Russo et al., 2002). Ion trap tandem mass spectrometry is a useful approach to reduce
clean-up steps in such a complex matrix. Using this technique, analysis of fenthion in liver samples
can be achieved with a simple extraction (Figs. 8-11)

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 Gas Chromatography - Mass Spectrometry

28061114 # 2563-2701 RT: 25.76-26.87 AV: 70 SB: 148 27.46-28.63 , 24.31-25.53 NL: 1.32E4
F: + c Full ms [50.00-550.00]
278.14
100

95

90

85

80

75

70

65

Relative Abundance
60

55

50

45

40

35

30

25

20 169.14
245.17

ChT.6 15
125.13

153.15
10 79.16
231.1
5 213.19
298.49 327.23 368.51 388.32 447.34 489.28 549.38
0
50 100 150 200 250 300 350 400 450 500 550
m/z

Figure 8. Fenthion mass spectrum (EI) obtained in an ion trap analyzer from a 2 ppm standard
solution

In a liver sample, a coelution at 25.98 min is observed due to palmitic acid present in the liver.
RT: 23.62 - 28.11
NL:
13 1.69E8
12
Liver with fenthion 25.98
TIC F: + c Full
ms
11 [50.00-550.00]
a) Full scan MS 24031106
10 26.05
9
Relative Abundance

8
7
6
5 25.61
4
3
2 26.25
25.17 26.67 26.89 27.24 27.88 28.02
1 25.08
23.96 24.29 24.83
0
NL:
13 3.50E4
12 TIC F: + c SRM
2 26.25
11
b) MS m/z 278→m/z 245 ms2
278.00@cid1.1
4
10
[244.00-246.00]
9 MS 24031106
8
7
6
5
4
3
2
1 26.03
23.89 24.16 24.47 24.91 25.26 25.43 25.83 26.42 27.17 27.34 27.81 28.09
0
24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0
Time (min)

Figure 9. Liver extract zoom chromatogram. a) TIC chromatogram and b) SRM 278 to 245 amu

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 Gas Chromatography - Mass Spectrometry

24031106 #2681-2699 RT: 26.21-26.35 AV: 10 SB: 12 26.78-26.88 , 26.05-26.12 NL: 1.53E4
24031106 #2605-2670 RT: 25.62-26.11 AV: 33 SB: 12 26.78-26.88 , 26.05-26.12 NL: 3.71E5 F: + c Full ms [50.00-550.00]
F: + c Full ms [50.00-550.00] 277.91
100
87.07
100
95
95
90
90
85
85
80
80
75
75

70 70

65 65

60 60

Relative Abundance
Relative Abundance

55.10 129.04 255.99

55 55

50 157.02 185.01
50

45 45
213.01
40 40
35 35
73.10
115.03
30
30 151.05
25 244.97
25
20
227.02 20
15
15
10
10
5 230.98
280.95 104.09 184.08
304.03 341.92 370.45 428.87 460.91 502.77 532.90 5 328.88
0 62.06 212.11 354.83 386.97 462.89 496.06 523.75
137.08
50 100 150 200 250 300 350 400 450 500 550 0
m/z 50 100 150 200 250 300
m/z
350 400 450 500 550 ChT.6
Figure 10. Mass spectra of palmitic acid (left) and fenthion (right) in liver extract.

Signal to noise ratio observed for SRM from 278 to 245 amu is better than that observed for the
278 mass chromatogram.

RT: 22.26 - 30.94

26.25 NL:
100 4.93E4
Mass chromatogram m/z=
90
m/z 278 277.50-278.50
F: + c Full ms
80 [50.00-550.00]
MS 24031106
70
Relative Abundance

60
28.62
50
28.76
40

30 28.82
28.50
20 25.96
28.30 29.38
25.58
25.39 26.59 27.17 27.95 30.26 30.81
10 22.75 24.29
23.45
0
26.25 NL:
100 4.05E3
2 TIC F: + c SRM
90 MS m/z 278→m/z 245 ms2
278.00@cid1.1
80 4
[244.00-246.00]
70 MS 24031106

60

50

40

30

20

10 26.03
23.00 23.89 24.16 25.21 25.43 26.42 27.34 28.24 28.32 29.23 29.59 30.25 30.86
0
23 24 25 26 27 28 29 30
Time (min)

Figure 11. Mass chromatogram m/z 278 and SRM from 278 to 245 amu.

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 Gas Chromatography - Mass Spectrometry

3.5. GC-MS Structural Characterization of polyesters produced from Linseed Oil by P. aeruginosa
42A2
Poly[3-hydroxyalcanoates] (PHAs) are optically active polyesters. The PHA synthesized by
numerous bacteria are high-molecular weight polymers which form intracellular inclusion bodies
such as carbon and energy reservoir. These polymers are environmentally friendly, biodegradable
and may become an alternative for biocompatible materials. The native polymer has a random
composition of monomers ranging from C6 to C14 with 20% of the alkyl side chains exhibiting
unsaturation at C12 and C14 alkyl side chains. A strategy for the study of the nature of these PHA
by GC/MS has been developed in our Unit. Biodegradable polymer was extracted from lyophily
zed cells with CHCl3 and after purification (MeOH precipitation) was hydrolyzed (CHCl3, H2SO4,
MeOH, 100C, 3h) in order to obtain the monomers (3-hydroxy-methylesters). The monomers were
silylated and characterized by GC-MS (see Figs. 12-14).

RT: 22.00 - 27.00

ChT.6 100
C14:3 25.71 NL:
3.64E7
95

90
C12:0 C14:1 25.92
TIC MS
GCEIMonic
a14
85
RT: 12.00 - 30.00
17.74 80 NL:
100

95
75

70
C12:2 23.44
1.73E8
TIC MS

90
C8:0 65
GCEIMonic
a14
Relative Abundance

60 23.27
85
55
80 50

75 20.71 45
C14:2
70
C10:0
40

35
C12:1 25.63
65
30
Relative Abundance

60 23.19
25

55 20

15
50 23.12
10 25.97
45 22.68
22.97
5 22.09 23.89 24.31 24.60 25.09 25.37 26.50 26.64 26.93
40
22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0
35 Time (min)

30

25
25.71
25.92
20

15
C6:0 23.27
23.44

10 14.57
25.63 28.04
23.19
5 28.39
19.22 25.97
13.10 15.31 16.15 21.46 22.97 24.31
0
12 14 16 18 20 22 24 26 28 30
Time (min)

Figure 11. Ion Chromatogram (TIC) of the trimethylsilyl derivatives of (3-hydroxy-methyl

esters) monomers.

TMSiOCHCH2COOCH3
TMSi OTMSi
73 175 TMSiO O
100 89
OCH3
80
M-CH3 C12:0
60 287
133
40 131 M-OCH3
75 159 M-TMSi
20 59
105 117 176 229 288
143 255
173 181 199 215 245 271 290 303 325 343
0
73
33
TMSiO O
89
25 136
OCH3
Abundance

20
80
C12:1
Relative

15 M-CH3
55 67 95 133 150
10 96 178 285
159 210
105
5 179 201
167 211 227 253 286
257 284 300 319 328 349
0 73
58
79
50
89
40
105 134
30 93 133
67 M-CH3 C12:2
20 59 119 283
135 147
95 131 159
10 175
167 208
177 209 227 255 267 284 298 320 341
0
50 100 150 200 250 300 350
m/z

. EI mass spectra correspondig to the C12 family

Figure 12. EI mass spectra corresponding to the C12 family

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 Gas Chromatography - Mass Spectrometry

100 M-OTMSi 213

M-CH3 287

80

M+H
60

40
303 C12:0
211 229 301
181
20 175 214
271 M+C2H5
304
73 123 137 151 163 197 230 245 285 331 343
89 97 119 215 255 306
0
285
27

20 M+H
301
15
C12:1
Abundance

179 211
Relative

10

137 157 209

227
286 M+C2H5
5 161 269 299 302
95 135 155 180 329
73 83 111 207 228 253 283 341
213 305
0
283
25

177
20 135
M+H
15 209
299
10 149 C12:2
175 243

5
109
133 207 241
284 M+C2H5
73 95 159 267 300
93 121 136 178 225 285
97 205 244 327
302 339 340
0
100 150 200 250 300
m/z

CI (CH4) mass spectra correspondig to the C12:0 monomers.

ChT.6
Figure 13. CI (CH4) mass spectra corresponding to C120 monomers

Acknowledgements

We thank the Unitat de Toxicologia Experimental y Ecotoxicologia (UTOX)-Parc Científic de

Barcelona for providing the samples of section 3.4.

References

I. Fernández, M. Bassas and A. Manresa. “GC-MS Structural characterization of polyesters

produced from linseedoil by Paeruginosa 42A2”.11as Jornadas de Análisis Instrumental
“JAI”. Barcelona 2005.
A. Pecci (ERAA-UB), IEF-FP7 Profolant Marie Curie Project
Russo et al., 2002 Journal of Chromatography B, 780:431-434

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 Gas Chromatography - Mass Spectrometry

ChT.6

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