Capturing Volatile Natural Products by Mass Spectrometry

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REVIEW
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Capturing volatile natural products by mass

spectrometry
Cite this: Nat. Prod. Rep., 2014, 31, 838
Jeroen S. Dickschat*
Covering: up to 2013
This review gives a modern methodic overview of how volatile natural products from various sources such
as plants, animals, bacteria and fungi can be trapped and how compound identication can be performed
even in cases of very low yields or within highly complex compound mixtures. A detailed discussion is
Received 28th August 2013
presented on how a structural proposal for an unknown analyte can be derived from GC-MS data.
Furthermore, the application of trace analytical techniques in biosynthetic studies with isotopically
DOI: 10.1039/c3np70080a
labelled compounds is presented, including a discussion of the pros and cons of dierent kinds of stable
www.rsc.org/npr
isotope labellings in GC-MS analyses.
1
2
3
3.1
3.2
3.2.1
3.2.2
3.2.3
4
5
6
7
8
9
Introduction
Sampling techniques
Interpretation of EI mass spectra
The molecular ion
Fragment ions
Fragmentations with the cleavage of one bond
Fragmentations with the cleavage of two bonds
Multistep fragmentation mechanisms
Retention time and retention index
Examples of GC-MS based structure elucidation
Feeding experiments
Conclusions and outlook
Acknowledgements
References
Introduction
Volatiles are produced by nearly every organism and may have

very important functions. A prominent example is the rst
pheromone ever reported. To identify the volatile attractant
Butenandt and coworkers extracted 500 000 pheromone glands,
yielding 280 g of crude extract from which they obtained 12 mg
of the pure pheromone that was identied by elemental analysis
and UV spectrosocopy as a hexadecadienol with conjugated
double bonds.1 Further structure elucidation by chemical
degradation2 and synthesis of the four possible stereoisomers3
resulted in the identication of bombykol as (10E,12Z)-hexadecadien-1-ol.
Technische Universit
at Braunschweig, Institut f
ur Organische Chemie, Hagenring 30,
38106 Braunschweig, Germany. E-mail: j.dickschat@tu-bs.de
838 | Nat. Prod. Rep., 2014, 31, 838861
Since the early days, analytical techniques have very much

improved with basically two revolutionary developments. The
phenomenon of nuclear magnetic resonance was discovered
independently by Bloch4 and Purcell5 in 1946, for which they
received the Noble Prize in Physics in 1952. NMR spectroscopy
was made available to chemical laboratories in the 1960s and
became a standard technology some years later. This technique
allowed for the rapid structure elucidation of organic molecules. Another milestone was the development of the rst
functional mass spectrometer by Aston in 1920 that he used to
demonstrate that most chemical elements exist in form of
isotopes.6 For his pioneering work he was awarded the Nobel
Prize in Chemistry only two years later. The rst coupled system
of a gas chromatograph with a time-of-ight (TOF) mass spectrometer was developed in the 1950s by McLaerty and Gohlke.7
Since this time, the technique has been very much improved by
the development of several ionisation techniques, such as
electron impact ionisation (EI), chemical ionisation (CI), fast
atom bombardment (FAB), and matrix assisted laser desorption
ionisation (MALDI), coupling of mass spectrometers to liquid
chromatography (especially HPLC), and various types of mass
analyzers including sector eld, TOF, quadrupole, ion trap,
Orbitrap, and Fourier transform ion cyclotron resonance
(FTICR) mass spectrometers. Details of these developments will
not be presented here, but are given in the accompanying article
by Carter in this themed issue.8
This article aims at demonstrating how the technique of GCMS, that is in our days available in almost every chemical laboratory, can be used to rapidly elucidate the structures of all kinds
of volatile natural products without isolating large quantities
as is required for chemical degradations or structure determination by NMR. The most important points brought across in
This journal is The Royal Society of Chemistry 2014
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this review are (I) how a structure for an unknown volatile can be
proposed based on GC-MS data, and (II) how GC-MS can eciently be used in biosynthetic feeding experiments that are
grounded on the usage of isotopic labellings. Rather than being
comprehensive which is nearly impossible due to the large
number of investigations on volatile natural products such as
(insect) pheromones, aroma constituents of (ouring) plants,
and volatiles from bacteria and fungi, in this article an eort has
been made to present a selection of historically interesting and
most recent work. In particular, no attempt has been made to
cover the extremely large number of more than 3000 reports on
volatile plant constituents in essential oils.9
Recent previous reviews on volatile natural products
summarise the work on bacterial volatiles,10 plant volatiles,11
halogenated volatiles from marine algae,12 biosynthetic pathways to volatiles in plants,13 and the importance of volatiles in
soil ecosystems.14
2 Sampling techniques
The principle setups of two important sampling techniques for
the collection of volatiles from various biological probes are
shown in Fig. 1. The closed-loop stripping apparatus (CLSA) was
originally developed by Grob and Z
urcher for stripping of
volatiles from aqueous solutions,15 but can easily be modied
for the investigation of volatiles released by biological
samples,16 such as bacterial or fungal agar plate cultures
(Fig. 1A).17,18 Even volatiles from plants,16 insects,16 mites,19 and
spiders20 have been collected by use of a CLSA. The method is
based on a closed system in which a continuous air stream is
circulated. The air stream is directed through a small chamber
containing the biological sample and then through a charcoal
lter for trapping of any emitted volatiles. Aer a collection time
of several hours up to one day the charcoal lter is removed and
the absorbed volatiles are eluted with an organic solvent, e.g.,
dichloromethane. The obtained headspace extract can then be
Jeroen S. Dickschat studied
chemistry at TU Braunschweig. In
2005 he obtained his PhD under
the guidance of Prof. Stefan
Schulz, working on bacterial
volatiles and terpene biosynthesis as a fellow of the Fonds der
Chemischen Industrie. Aer a
postdoctoral stay at the Universit
at des Saarlandes with Prof.
Rolf M
uller he moved to the lab of
Prof. Peter Leadlay at the
University of Cambridge (UK) as
a fellow of the Deutsche Akademie der Naturforscher Leopoldina.
Since 2008 he is a group leader and Emmy Noether fellow at TU
Braunschweig. His research interests include biosynthetic pathways
to secondary metabolites, the identication and structure elucidation of new natural products, and their total syntheses, cumulating
in his habilitation in 2013.
NPR
Schematic view of sampling techniques for volatiles. A) Closedloop stripping apparatus (CLSA), and B) solid phase micro-extraction
(SPME).
Fig. 1
analysed by GC-MS. Since the biological sample is contained in

a closed system that is well separated from the environment, the
experiments can be performed without the danger of infecting
samples, such as bacterial agar plate cultures, with microbial
contaminants. This non-invasive technique yields several
microliters of extract that can be used for repeated GC-MS
analyses, e.g., on chiral stationary phases, or for derivatisations
by microreactions for structure elucidation. A drawback of the
method is that due to the closed-loop principle a sample can be
contaminated by compounds from a previous analytical run,
and thus clean blank runs are particularly important.
Mechanically damaged charcoal lters should never be used to
avoid accumulation of carbon particles in the pump that may
cause memory eects.21
An alternative method for the analysis of volatiles from
biological samples is solid phase microextraction (SPME).
Originally developed for the analysis of trace compounds in
water,22 SPME is also very useful for headspace samplings.23 The
method makes use of a poly(dimethylsiloxane) bre as adsorbant that is presented to the headspace above a biological
sample in a closed vessel such as an agar plate (Fig. 1B). Aer
equilibration, usually within a few minutes, the bre can be
withdrawn into a needle that is directly inserted into the GC
injector where the bre is expelled for thermal desorption of the
collected volatiles. The method is solvent-free, fast, cheap, and
non-invasive. Initially, SPME was mainly used for the analysis of
environmental pollutants, food and drug analysis,24 but insect
pheromones,25 plant volatiles,26 or volatiles emitted by bacterial
liquid and agar plate cultures27,28 can also eciently be analysed. A drawback of the method is the dierential anities of
volatiles from varying compound classes for the adsorbent
bre.29
3 Interpretation of EI mass spectra

Large electronic databases containing EI mass spectra of many
known volatile natural products and small synthetic organic
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molecules are available, some of them summarising specialised

knowledge, e.g., the mass spectra of terpenes.30,31 These databases are of utmost importance for analytical work on volatile
natural products since they allow for the rapid identication of
many known compounds. One remaining challenge for the
highly ecient identication of already known compounds in
the future is the development of strong chemoinformatic tools
that interconnect all the accumulated knowledge from the
various databases and make it available to all analytical chemists. Herein, the chemical sciences can certainly learn from
biology that has been revolutionised during the last decade by
the development of strong bioinformatic tools and large databases that today make bioinformatic data available to every
scientist. However, signicant progress in the eld of natural
products chemistry can only be made when new compounds
can be identied. The GC/EI-MS technology delivers two
dierent kinds of important information, the mass spectrum
and the gas chromatographic retention time, both of which
should always be considered for the development of a structural
proposal for an unidentied analyte. EI mass spectra are characterised by a molecular ion [M+] that arises by collision of a
highly energetic electron (usually 70 eV) and the neutral analyte.
The electron impact leaves the ionised analyte in a high energy
state that causes a series of fragmentation reactions to give a
characteristic pattern of fragment ions of which the most
intensive fragment ion is referred to as the base peak ion (or
briey base peak). A major diculty, particularly in EI-MS, is
that the molecular ion may be of very low abundance due to its
very ecient fragmentation, e.g., aliphatic alcohols easily
undergo a neutral loss of water and their mass spectra are very
similar to the mass spectra of the corresponding olens. In
some cases, the analyte may undergo thermal reactions during
the chromatographic process, e.g., decarboxylations or Cope
rearrangements. These diculties of the GC-EI-MS technique
should always be kept in mind to prevent erroneous data
interpretation. In this chapter a choice of interesting examples
will be presented to show how structural information can be
extracted from a mass spectrum. In practice, the successful
development of such a structural proposal from EI-MS data will
require an experienced analytical chemist.
3.1
the strongly increasing number of possible molecular

formulae with increasing molecular weight, but this is usually
not a problem when analysing volatiles that are per denitionem low molecular weight compounds. The most recent
innovations that keep pushing the boundaries of what is
possible in terms of mass resolution include the developments of Fourier transform ion cyclotron resonance mass
spectrometry (FT-ICR) and Orbitrap mass spectrometry.
These techniques are certainly of high relevance for modern
metabolimics and proteomics studies, but only of limited
interest for studying volatile natural products for which the
GC-MS coupling is still the best method. However, the very
interesting GC-TOF technology may soon become aordable
to every chemistry lab for high-resolution MS analyses of
volatiles.
If no high resolution data are available, every suggested
structure of an unidentied compound must accord with the
observed molecular ion. The detected isotope pattern of the
molecular ion gives very useful information about the elemental
formula of an unknown analyte, as will be exemplied here for
gibepyrone A (1). Its mass spectrum (Fig. 2A) shows a molecular
ion at m/z 164 with an isotope pattern of m/z 164 (100%),
165 (11.2%), and 166 (1.0%). Assuming that the analyte
contains only carbon (natural isotope distribution of 12C/13C
98.9% : 1.1%), hydrogen (1H z 100%), and oxygen (16O/18O
99.8% : 0.2%), the expected isotope patterns for dierent
elemental formulae matching the molecular ion at m/z 164
can be calculated (Table 1). The best match is obtained for
C10H12O2, which is equal to the molecular formula of 1, thus
demonstrating the robustness of the method.
The molecular ion
Several important pieces of information can be extracted from

an EI mass spectrum. The molecular ion [M+] points towards
the elemental formula of an analyte. Unambiguous information about the elemental formula can be concluded from high
resolution (HR-MS) data that are, due to recent advances in
mass spectrometer developments, of good availability. To give
an example, the molecular formulae of acetone (C3H6O) and
butane (C4H10), both resembling a molecular weight of 58 Da,
can easily be distinguished from their accurate masses
(58.041316 Da versus 58.077601 Da; note that the electron
mass, 0.000549 Da, has to be subtracted from the exact
molecular weight since the positively charged molecular ion
and not the neutral molecule is detected by HR-MS). A major
limitation in the interpretation of HR-MS data results from
840 | Nat. Prod. Rep., 2014, 31, 838861
Mass spectra of A) gibepyrone A (1) and B) 2,5-dimethylpyrazine

(2) and isotope pattern of the molecular ions.
Fig. 2
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Other frequently occurring elements in natural products

include sulfur, chlorine and bromine, which can also easily be
recognised from their isotope patterns (32S/33S/34S
95.0% : 0.8% : 4.2%; 35Cl/37Cl 75.8% : 24.2%; 79Br/81Br
50.7% : 49.3%). The characteristic isotope pattern of sulfur was
used to delineate the sulfur content of a series of polythiacycloalkanes from marine bacteria (39) and from shiitake
mushrooms (Lentinus edodes, 1018) including lenthionine 16
(Scheme 1).32,33 Selenium and tellurium compounds exhibit
even more complex isotope patterns that can be very useful in
the identication of volatiles, such as dimethyl diselenide,
dimethyl triselenide, or dimethyl ditelluride, which are released
by some bacteria during growth on selenium or tellurium
oxyanions.28,34,35
Considering nitrogen as a component of an unidentied
analyte complicates the situation, but careful analysis of the
molecular ion still delivers useful information about the
molecular formula. The presence of an odd number of nitrogen
atoms in a natural product containing only the usual elements
(C, H, O, N, S, Cl, and Br) is indicated by an odd molecular ion,
while no nitrogen or an even number of nitrogen atoms results
in an even molecular ion (nitrogen rule, this rule is also strictly
valid for molecules containing F, P, and I, but these elements
occur very rarely in volatile natural products). Furthermore, the
natural isotope pattern of nitrogen (14N/15N 99.6% : 0.4%)
can be used for delineating the nitrogen content of an unknown
volatile. This isotope pattern leads to slightly decreased [M + 1]+
and [M + 2]+ ions as compared to a hydrocarbon of the same
molecular weight, whereas oxygen results in a slightly decreased
intensity of the [M + 1]+, but increased [M + 2]+ signal (Table 2
exemplarily summarises the possible molecular formulae for a
compound with a molecular weight of 108 Da containing C, H,
N, and O).
An example is given by the mass spectrum of 2,5-dimethylpyrazine (2, Fig. 2B). From the isotope pattern of the
molecular ion the presence of sulfur, chlorine, and bromine can
be excluded. Considering only C, H, and O does not give a
satisfactory match of the calculated and measured isotope
patterns (Table 2), suggesting that an even number of nitrogen
atoms is contained in the analyte. Indeed, the calculated
isotope pattern for C6H8N2 perfectly agrees with the analytical
data.
In the case of high quality EI mass spectra, the accuracy of
such an in depth analysis of the isotope pattern of the molecular
ion is very high. The method reaches its limits in the case of
mass spectra with a molecular ion of low intensity, resulting in
comparably large measuring errors for the isotope pattern due
Table 1 Calculated isotope patterns for compounds with the

molecular ion [M]+ 164, composed of C, H, and O
Formula
m/z 164
m/z 165
m/z 166
C12H20
C11H16O
C10H12O2
C9H8O3
C8H4O4
100%
100%
100%
100%
100%
13.3%
12.2%
11.1%
10.0%
8.9%
0.8%
0.8%
0.9%
1.0%
1.1%
Scheme 1 Polythiocycloalkanes from marine bacteria (39) and from

shiitake mushrooms (1018).
to a larger signal-to-noise ratio. Furthermore, in the case of

compounds of high molecular weight, a large number of
possible molecular formulae composed of the elements C, H, N,
and O matches this particular molecular weight, and consequently, the calculated isotope patterns of two or more of these
formulae may be in accordance with the measured data.
However, in the case of volatiles that have a molecular weight
below 300 Da the isotope pattern of the molecular ion gives in
most cases valuable structural information.
3.2
Fragment ions
More detailed structural information can be obtained from the

fragment ions that must, however, always be interpreted in the
light of the molecular ion. A very detailed and didactically
perfectly structured discussion of all kinds of fragmentation
reactions with importance for EI mass spectra is summarised in
a text book by two leading analytical chemists in the eld,
McLaerty and Turecek.36 Some of the most important fragmentation reactions and their use for interpretations of mass
spectra will also be presented here.
The large body of information that can be extracted from the
fragmentation pattern is one of the major advantages of EI-MS
compared to other mass spectrometric methods. In this chapter
only analytes that have an even number of electrons prior to
ionisation will be discussed, since this is the usual case for
almost all volatile organic molecules. Ionisation proceeds with
the loss of one electron, producing an odd-electron molecular
ion [M]+c from a non-radical analyte. This initially formed
molecular ion is obtained in a high energy state due to the
ionisation process that is usually performed at an electron
energy of 70 eV. In consequence, several fragmentation reactions lead to a characteristic pattern of fragment ions via well
known mechanisms. Aer ionisation of a molecule AB to the
initial (AB)+c radical cation, fragmentation by the breaking of
one of the bonds results in the fragments Ac and B+ or A+ and Bc,
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Table 2 Calculated isotope patterns for compounds with the

molecular ion [M]+ 108, composed of C, H, N, and O
Formula
m/z 108
m/z 109
m/z 110
C8H12
C7H8O
C6H4O2
C6H8N2
C4H4N4
C5H4N2O
100%
100%
100%
100%
100%
100%
8.8%
7.7%
6.6%
7.3%
5.8%
6.2%
0.3%
0.4%
0.6%
0.2%
0.1%
0.3%
yielding an even-electron cation and an odd electron radical in

both cases. As has been recognised already in 1968 by Audier,
the fragmentation proceeds preferably with the positive charge
remaining on the fragment with the lowest ionisation
potential.37
3.2.1
Fragmentations with the cleavage of one bond
Fragmentation mechanisms by the cleavage of one bond are

summarised in Scheme 2. Ionisation of saturated hydrocarbons
results in a radical cation with subsequent bond-breaking of the
ionised s-bond (Scheme 2A). The fragment ions that are
observed with highest intensities are formed by bond breaks
next to chain branches, thus resulting in the most stable, i.e.,
tertiary or secondary carbocations. An interesting example is
given by the doubly alkyl-branched fatty acid methyl ester
(FAME) 19. A structural proposal for this compound, that was
observed in headspace extracts from the actinomycete Micromonospora aurantiaca, was derived from its EI mass spectrum
(Fig. 3A).38 The molecular ion of 19 was very weak, but the
pattern of fragmentations of the alkyl chain pointed to a
molecular ion at m/z 256. Alkyl chain fragmentations resulted
in intense fragment ions of m/z 87 and 115, but not of m/z
101, pointing to a g-methyl branch (the high intensity of the ion
m/z 87 is due to fragmentation via double hydrogen rearrangement as discussed in chapter 3.2.3, while the fragment ion
at m/z 74 by McLaerty rearrangement is in agreement with a
methyl ester, chapter 3.2.2). Furthermore, fragment ions were
observed at m/z 157 and 199, but not at m/z 171 and 185,
that seemed to suggest an h-ethyl branch (position 8) that was
further corroborated by a weak fragment ion at m/z 227 ([M
C2H5]+). However, this is a good example that shows the dangers
of a premature interpretation of mass spectra. As was described
by Ryhage and Stenhagen,39 based on extensive labelling
experiments and investigation of various alkyl branched methyl
esters, the main contribution to the fragment ion at m/z 199
is due to a loss of the C2- to C4-portion, including any attached
alkyl substituents, plus a proton, and not due to the alphafragmentation as shown in Fig. 3. Compound 19 was the only
ethyl-branched compound identied among a large number of
other FAMEs emitted by M. aurantiaca and was suggested to
arise from 2-ethylhexanoic acid, the oxidation product of the
widespread contaminant 2-ethylhexanol that originates from
plasticisers.
Another important fragmentation mechanism that
frequently results in fragment ions with high intensities is the
842 | Nat. Prod. Rep., 2014, 31, 838861
a-cleavage (Scheme 2B). In this process a CC single bond of the

carbon bound to a heteroatom X via a CX single or C]X double
bond is cleaved (a confusing situation of nomenclature is that
by this process the a,b-carbon bond in CX systems is cleaved,
while in C]X systems the bond from the sp2 carbon to the
a-carbon is cleaved; this is not equal to cleavage of the a,bcarbon bond). The cleavage is initiated by ionisation at the
heteroatom X yielding a radical cation, followed by a cleavage
reaction that is driven by recombination of the radical electron
with an electron of an adjacent CC single bond. This results in
the loss of a radical Rc under formation of a cation that can
usually be observed with high intensity.
An example is given by the mass spectrum of 1-phenyldecan1-one (20), the major volatile compound released by the myxobacterium Stigmatella aurantiaca Sg a15.40 Fragment ions with
high intensities are observed at m/z 77 and 105 that can be
explained by two dierent a-fragmentations to either the le or
the right side of the carbonyl group (Fig. 3B). The occurrence of
intensive fragment ions at m/z 120 and m/z 133 is due to a
McLaerty rearrangement (chapter 3.2.2) and a double
hydrogen rearrangement (chapter 3.2.3).
Several other volatile compounds in which strong a-fragment ions have been observed are shown in Fig. 4. The alcohol
21 from the myxobacterium Myxococcus xanthus shows an
a-fragment ion at m/z 59 as is typical for 3-alcohols.17 Cleavage
of the other CC bond from the carbinol carbon results in m/z
143. Both fragment ions together point to the molecular ion at
[M]+c 59 + 143 30 172 (sum of masses of both a-fragment
ions mass of carbinol function that is counted twice) that is in
case of aliphatic alcohols usually hard to detect. The methyl
ketone 22, a pheromone from the desert spider Agelenopsis
aperta, shows an intensive a-fragment ion at m/z 43.20
Scheme 2
Fragmentation mechanisms with the cleavage of one
bond.
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Fig. 3
EI mass spectra of selected volatile secondary metabolites.
However, interpretation of the expected a-fragment ions of

aliphatic ketones has to be taken with care, because the same
fragment ions can arise by cleavage of a CC s-bond from
aliphatic alkyl chains (i.e., C2H3O+ and C3H7+ both yield m/z
43). HR-MS data can be used to distinguish between both
possibilities, while it is not possible in the case of fragment ions
to draw any conclusions from their isotope patterns, as discussed above in detail for the molecular ion, because a contribution of other fragment ions to the respective ions cannot be
excluded. The lactone 23 was rst identied from the blacktailed deer Odocoileus hemionus columbianus41 and is also
released as a volatile trace component by the marine bacterium
Dinoroseobacter shibae DFL-27.42 Its mass spectrum shows an
intensive base peak ion at m/z 85 that is very characteristic for
g-lactones and arises from a-cleavage of the g-lactone moiety. A
prominent a-cleavage is also realised in the isomeric lactone 24
that is known as buibuilactone, a pheromone component of the
Fig. 4 Volatile compounds showing prominent a-fragment ions in
their EI mass spectra.
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cupreous chafer beetle Anomala cuprea.43 Buibuilactone is also

released by D. shibae and the closely related bacterium Loktanella. Its a-fragmentation results in a base peak ion of m/
z 111.42
The a-cleavage next to heteroatoms is a very useful fragmentation reaction for a detailed structure elucidation of volatile alkenes. The position of an olenic double bond cannot
usually be determined from the mass spectrum of unknown
alkenes, but the iodine-catalysed addition of dimethyl disulde
(DMDS) yields a 1,2-bis(methylsulfanyl) adduct that shows
characteristic fragment ion formation via a-cleavage of the CC
bond between the two methylsulfanyl groups (Scheme 3). The
method was rst reported for the localisation of olenic double
bonds in simple alkenes44 and was subsequently also successfully applied to determine double bond positions in functionalized molecules such as unsaturated acetate esters,45
alcohols,46 aldehydes,46 ketones,47,48 fatty acids,49 methyl
esters,46,5053 lactones,54 and terpenes.55 Quantities as low as
10 ng are sucient for the procedure.46 It has been pointed out
that the derivatization proceeds as an anti-addition, thereby
resulting in the formation of threo and erythro adducts from Z
and E olens, respectively, that can be gas-chromatographically
separated.50 However, since reference DMDS adducts will be
required for stereochemical assignments by means of GC-MS,
and these will have to be obtained from the respective Z and E
olens, determination of the double bond conguration seems
to be more straightforward by direct comparison of the
geometrical isomers of the alkenes.
Only a few exemplary cases of double bond localizations by
DMDS addition will be specically discussed here, including
the identication of the female sex pheromone from the currant
stem girdler Janus integer, (Z)-octadec-9-en-4-olide (25, Scheme
3A).54 The mass spectrum of the DMDS adduct 26 showed
intensive fragment ions at m/z 201 and 173 due to a-fragmentation of the CC bond between the two methylsulfanyl
groups, which is in agreement with an olenic double bond at
the 9-position of 25. The authors did not comment on the
possibility of the alkene function in position 7 for which the
same fragment ions would be expected, but synthetic 25
matched the natural product in terms of mass spectrum and GC
retention time, and showed bioactivity in males. A similar
problem occurred in the identication of a series of volatile
unsaturated methyl ketones from marine arctic bacteria of the
cytophaga-avobacterium-bacteroides group, exemplied by
the representative compound (Z)-hexadec-10-en-2-one (27,
Scheme 3B).47 The low resolution EI mass spectrum revealed
intensive fragment ions at m/z 201 and 131, in agreement with
a C]C double bond either in the 5- or 10-position. The problem
was resolved by HR-EIMS, showing accurate masses for the
diagnostic a-fragment ions at m/z 201.129 and 131.090 that
allowed for the determination of the elemental compositions of
the fragment ions and hence in the localization of the double
bond in the 10-position.
The reaction of polyunsaturated compounds with DMDS
only yields the expected polyadducts when the double bonds
are separated by four or more methylene units, whereas
conjugated dienes result in 1,4-monoadducts, and dienes with
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from the [M 31]+ fragment ion. A recently reported example of

a sesquiterpene that was suggested to undergo an inductive
cleavage in the formation of the fragment ion m/z 109 is trichodiene (29), the main volatile emitted by the fungus Fusarium
sporotrichioides (Scheme 4B).60 A detailed discussion of the
fragmentation mechanisms for blastmycinone (30) and several
structurally related lactones from Streptomyces ambofaciens was
also recently presented, including the loss of the acyloxy side
chain by inductive cleavage (Scheme 4C).61
3.2.2
Fragmentations with the cleavage of two bonds
Some fragmentation mechanisms proceed with the breaking of

two bonds, as shown in Scheme 5. The most important reactions of this type are the retro-DielsAlder fragmentation and
the McLaerty rearrangement. The retro-DielsAlder fragmentation can proceed via two alternative mechanisms, both initiated by ionization of a double bond of a cyclohexene followed by
an a-fragmentation to a radical/allyl cation species. Subsequently, either a second a-cleavage takes place resulting in the
neutral loss of an alkene and a radical cation representing the
diene portion of the molecule, or an inductive cleavage yields
under neutral loss of a diene a radical cation that refers to the
dienophil portion of the molecule. Both charged fragments
may be observed in a mass spectrum and are easily identied
due to their even masses (if no nitrogen is contained in the
analyte), but the formation of one of the two charged fragments
is usually strongly favoured.
Scheme 3
Double bond localization by DMDS derivatization.
double bonds separated by one to three methylene groups

yield cyclic structures that likely arise via the bis-adducts
(Scheme 3C).5658 Another possibility is given by the partial
reduction of olenic double bonds using in situ-generated
diimine followed by DMDS addition which can also be performed on a ng scale.59
The last fragmentation mechanism involving bond breaking
of one bond, discussed here, is the inductive cleavage. Ionisation of a group X, either bound via CX single bond(s) or a C]X
double bond, induces heterolytic cleavage of a CX or a CC
bond with charge migration. In the case of methyl esters the
formation of [M 31]+ fragment ions can be explained by an
inductive cleavage resulting in the loss of a methoxide radical
(Scheme 4A). An example is shown for the methyl ester 19,38 in
Fig. 3A, in which the low-intensity fragment ion at m/z 225 is
due to the loss of a methoxide radical by inductive cleavage.
This fragment ion is of high diagnostic value, because the
molecular ion of 19 is of very low intensity, but can be deduced
844 | Nat. Prod. Rep., 2014, 31, 838861
Scheme 4
Volatiles showing fragment ion formations by inductive
cleavage.
View Article Online
Review
NPR
A classical textbook example for two structurally very similar

compounds, one of these showing an intensive retro-Diels
Alder fragment ion, while the related molecule is not,62 is shown
in Scheme 6A. Retro-DielsAlder fragmentation of a-ionone (31)
yields by neutral loss of isobutene a fragment ion at m/z 136
that is not observed in the mass spectrum of b-ionone (32).
Dehydrogeosmin (33) was rst identied in the ower scent of
Rebutia marsoneri and a few other Cactaceae,63 and has recently
been shown to be present in headspace extracts of several
streptomycetes.64 Its mass spectrum is dominated by a retroDielsAlder fragment ion at m/z 126. Its rst identication
aer isolation of 6 mg of impure material solely relied on the
interpretation of the mass spectrum followed by synthesis of a
reference compound. The homomonoterpene 34 is released in
trace amounts by several 2-methylisoborneol producing actinomycetes.65 The structure of this compound was also suggested from its mass spectrum exhibiting a retro-DielsAlder
ion at m/z 82, and unambiguously established by synthesis.
The mass spectrum of tricho-acorenol (35) that was rst isolated
from Trichoderma koningii presents a retro-DielsAlder ion at
m/z 84.66 This fragment ion was relevant for locating the side
of incorporation from various deuterated mevalonolactone
isotopologues in a biosynthetic study as is discussed in detail in
chapter 6.67
A second fragmentation reaction in which two bonds are
broken is the McLaerty rearrangement (Scheme 6B). This type
of reaction is initiated by ionisation of an unsaturated C]X
functional group to yield a radical cation, followed by hydrogen
transfer via a six-membered transition state that shis the
radical centre into the g-position. A subsequent a-cleavage
results in neutral loss of an olen under formation of a conjugated allylic radical cation. The rearrangement step may also be
Scheme 5
Fragmentation mechanisms with the cleavage of two
bonds.
Examples of volatile natural products showing prominent

retro-DielsAlder fragment ions in their EI mass spectra.
Scheme 6
succeeded by an inductive cleavage to yield an alternative

radical cation. Which of the two cleavage reactions is preferred
depends on the ionisation potential of the two fragments.37
McLaerty ions are oen easily recognised, because they have
even masses (if no nitrogen is contained in the molecule).
McLaerty rearrangements occur in all types of compound
classes with C]X double bond systems including aldehydes,
ketones, esters, carboxylic acids, and alkenes, but also in
aromatic compounds such as alkylated benzenes, pyridines,
pyrazines, and many others. A few exemplary volatile
compounds with characteristic McLaerty fragment ions in
their mass spectra are shown in Scheme 7. The myxobacterium
Myxococcus xanthus releases, besides the alcohol 21 discussed
above, the structurally related ketone 9-methyldecan-3-one
(36).17 As is typical for ethyl ketones, its mass spectrum shows a
characteristic McLaerty ion at m/z 72. However, the interpretation of the mass spectra should always be performed very
carefully, because not only ethyl ketones, but also a-methyl
branched methyl ketones and a-ethyl branched aldehydes such
as compound 37 exhibit a McLaerty ion at m/z 72. The latter
compound was identied in paracloacal gland secretions of
several Caiman spp.68 As can be seen in the mass spectrum of
the methyl ester 19 (Fig. 3), mass spectra of compounds from
this class are characterized by a McLaerty ion at m/z 74. The
mass spectra of linear methyl esters and alkyl branched
compounds have been extensively studied by Ryhage and
Stenhagen in 1959 and 1960.39,69,70 This work was particularly
important, because fatty acid methyl esters have extensively
been used as taxonomic markers of bacteria especially in the
pre-genome era. Recently, a series of volatile methyl esters have
been detected in headspace samplings from the actinomycete
Micromonospora aurantiaca and the gliding bacterium Chitinophaga.38,71 The ndings of Ryhage and Stenhagen served in the
identication of branched compounds, as exemplied by the
structure of methyl 2,7-dimethyloctanoate (38) that exhibits a
McLaerty fragment ion at m/z 88, as is typical for a-methyl
branched methyl esters. The mass spectrum of the second
compound in Fig. 3, the phenyl ketone 20 from Stigmatella
aurantiaca, is dominated by a McLaerty ion at m/z 120.
Another interesting and widespread class of volatiles are
alkylated pyrazines. A series of mono- to tetra-alkylpyrazines
Nat. Prod. Rep., 2014, 31, 838861 | 845
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NPR
Review
the synthesis of two pyrazines, 3-methoxy-2-(1-methylpropyl)-5(2-methylpropyl)pyrazine and 3-methoxy-2,5-bis(1-methylpropyl)pyrazine, for comparison to volatiles emitted by
C. crocatus.73 Due to the published mass spectra these
compounds could also be identied as bacterial attractants for
the pineapple beetle Carpophilus humeralis.77
3.2.3
Scheme 7 Examples of volatile natural products showing prominent

McLaerty fragment ions in their EI mass spectra.
including 2,5-diisobutylpyrazine (39) was identied in Paenibacillus polymyxa.72 All alkylated pyrazines with side chains
longer than two carbons for which McLaerty rearrangements
via a six-membered transition state are possible exhibited the
expected McLaerty fragment ions in high intensity, usually
resulting in the base peak of the mass spectrum. Various
alkylated pyrazines have also been detected in the headspace
extracts of the myxobacteria Chondromyces crocatus and Nannocystis exedens,73,74 and the actinobacterium Corynebacterium
glutamicum.75 Structural suggestions for these compounds
were derived by interpretation of their EI mass spectra that
likewise were dominated by McLaerty ions in the cases of
alkyl substituents of a chain length of more than two carbons.
However, since the substitution pattern of di-, tri-, and tetraalkylpyrazines cannot be derived from the mass spectrum, all
structural suggestions were unambiguously proven by
synthesis of reference compounds. Dierentiating between
the various constitutional isomers of alkylated pyrazines is
also a perfect example of a problem that could be solved by
comparison of the analyte's mass spectrum to database
spectra, but for unambiguous compound identication the
mass spectra of all constitutional isomers should ideally be
available. If this is not the case, it cannot be excluded that an
isomer that is not included in the database may have a very
similar mass spectrum to a compound that is included. In
extreme cases this may require the synthesis of all possible
isomers, as was recently performed for the identication of
two chlorinated anisole derivatives from the fungus
Geniculosporium.76
If mass spectra of unidentied volatile compounds are
communicated in the original work, this may in some cases
assist in the later identication by synthesis of reference
compounds in a totally dierent context. An example is given by
846 | Nat. Prod. Rep., 2014, 31, 838861
Multistep fragmentation mechanisms
Compounds such as esters, aldehydes, and ketones usually

show the formation of intensive fragment ions via McLaerty
rearrangementation that are of high diagnostic value as these
ions point to substituents attached to the a-position. A second
intensive fragment ion in these types of compounds arises via a
double hydrogen rearrangement initiated cleavage of the
b,g-carbon bond (Scheme 8).78 In the case of unsubstituted
esters, this process results in the fragment ions m/z 87 (R
OMe, e.g., compound 19, Fig. 3A) or m/z 101 (R OEt), while
ketones yield the fragment ions m/z 71 (R Me), m/z 85
(R Et), or m/z 133 (R Ph, compound 20 in Fig. 3B), etc.
This fragmentation is of diagnostic value to locate substituents
in the a- or b-positions, and, in conjunction with analysis of the
McLaerty fragment ions, substituents can be assigned
precisely to one of these two positions.
In a few cases, fragmentation reactions via multiple steps
have been investigated. Two examples are the fragmentation
reactions that give rise to the base peak ions in the mass spectra
of geosmin (40) and 2-methylisoborneol (41), two terpenoid
bacterial volatiles that are both released by many actinomycetes, myxobacteria and cyanobacteria,10,17,74,7982 which will be
discussed here.
A multistep fragmentation pathway for the formation of the
base peak ion at m/z 112 in the mass spectrum of geosmin (40,
Fig. 5A) was suggested by Boland and coworkers (Scheme 9).83
Aer ionisation of the hydroxy function the molecule undergoes
a preferred a-cleavage under formation of the more stable
secondary radical (a primary radical is formed by an inferior
a-cleavage of the other ring). The subsequent steps reveal that
rearrangements in mass spectrometric fragmentation reactions
may not only proceed via six-membered transition states, as in
the case of the McLaerty rearrangement, but can also proceed
via other ring sizes. First, a rearrangement via an eightmembered transition state transfers a hydrogen radical from
the oxygen into the alkyl side chain, which is followed by the
combination of a hydrogen radical transfer via a six-membered
Scheme 8 Fragmentation of esters and ketones via double hydrogen

rearrangement and cleavage of the b,g-carbon bond.
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Review
transition state and subsequent a-cleavage, i.e., a McLaerty

rearrangement. The rst hydrogen radical transfer via the eightmembered transition state was proven by a deuterium labelling
experiment with labelling at the hydroxy function of 40.
The formation of the base peak ion at m/z 95 in the mass
spectrum of 2-methylisoborneol (41, Fig. 5B) was also suggested
to arise via a multistep procedure (Scheme 10).74 Ionisation of
41 at the hydroxy function is followed by a-cleavage to a tertiary
radical intermediate. A subsequent inductive cleavage under
neutral loss of acetone yields a radical cation that in a second
a-fragmentation results in an allyl cation of m/z 95.
4 Retention time and retention index

A volatile compound should only be regarded as unambiguously
identied by GC-MS, if both its mass spectrum and the retention time match those of a synthetic or commercially available
reference compound. Special problems that are frequently
observed for structurally complex volatiles (e.g., terpenes) are
the high similarities of mass spectra of closely related structural
isomers, such as diastereoisomers. In the case of two or more
coeluting terpenes the mixed mass spectrum may even be
similar to the mass spectrum of a third unrelated terpene,
further impeding the identication of compounds (particularly
from this class) solely based on the mass spectrum. For a
comparison of GC retention times authentic standards are
highly desirable. However, since these standards are not always
available, a more practicable approach is the comparison of
retention indices84 that can be calculated from the compound's
retention time in comparison to retention times of a
Fig. 5 Mass spectra of the widespread bacterial volatiles A) geosmin

(40) and B) 2-methylisoborneol (41).
NPR
Scheme 9
Fragmentation mechanism for geosmin (40).
homologous series of n-alkanes obtained under the same

experimental conditions:
I 100 n
tR tR;n
:
tR;n1 tR;n
Herein, n is the number of carbons of the n-alkane directly

eluting before the analyte for which the retention index I is to be
determined, tR is the retention time of the analyte, and tR,n and
tR,n+1 are the retention times of the n-alkane directly eluting
before and aer the analyte. The retention index of a compound
mainly depends on the type of GC column (or more specically
on its polarity), but almost never on the precise GC settings,
such as gas ow, temperature programme, or length of the GC
column. Therefore, in contrast to GC retention times, retention
indices of a particular compound can be directly compared,
even if the GC runs were performed under slightly dierent
experimental conditions. Large databases with tabulated
retention indices85,86 exist with a high value for analytical
chemists. In addition, GC retention indices are listed in many
scientic publications that can be found via publicly available
web-based compound libraries such as the NIST Chemistry
Webbook (http://webbook.nist.gov/chemistry/) or the Pherobase (http://www.pherobase.com/). How the retention index of
a compound in conjunction with a retention index increment
system can be of use for GC-MS based structure elucidations
will be discussed in chapter 5.
As pointed out earlier, the comparison of mass spectra and
retention indices is crucial for the identication of terpenes that
may be regarded as the structurally most complex class of
volatile natural products. The GC-MS analysis of terpenes in
Scheme 10 Fragmentation mechanism for 2-methylisoborneol (41).
Nat. Prod. Rep., 2014, 31, 838861 | 847
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NPR
headspace extracts together with a comparison of retention

indices or usage of authentic reference compounds has recently
been performed on many bacteria and fungi. The number of
dierent terpenes identied in these studies is too large for a
detailed discussion in this review, but the main compounds are
shown in Scheme 11. Geosmin (40) and 2-methylisoborneol (41)
are widespread in bacteria and have been identied in many
myxobacteria,17,40,74 actinomycetes,64,65,82,87,88 and cyanobacteria.89 The sesquiterpene alcohol (1(10)E,5E)-germacradien-11ol (42) is a biosynthetic intermediate towards 40 and was
detected as the main compound in the headspace extracts from
Stigmatella aurantiaca.40 Aciphyllene (43) is less widespread and
was found among the volatiles emitted by two Nocardia
strains,90 while epi-isozizaene (44) and its antibiotic oxidation
product albaavenone (45) are produced by many streptomycetes.64,82,91,92 In contrast, the antibiotic pentalenolactone (47)
and its parent hydrocarbon pentalenene (46) are found in only a
few streptomycetes.64,93 Trichodiene (29) is the parent hydrocarbon of the T2 toxin (48)94 and is released by Fusarium verticillioides,60 while aristolochene (49) is the precursor to the PR
toxin (50) in Penicillium roqueforti.95,96 The diterpene ent-kaurene (51) is the main volatile emitted by Fusarium fujikuroi97 and
is in this species converted into the phytohormone gibberellic
acid GA3 (52).98 Bioactivity in terpenoid compounds is in many
cases only reached once the terpene backbone is heavily
modied by oxidative transformations. Oxidised terpenoids like
47, 48, 50 and 52 exhibit, in contrast to their stem hydrocarbons, a much lower volatility, but compounds such as 45 can be
observed in CLSA headspace extracts from many
streptomycetes.64
The terpene hydrocarbons are made by terpene cyclases,
many of which are encoded in bacterial genomes, especially in
actinomycetes. The products of terpene cyclases can be analysed by GC-MS, if their mass spectra and retention indices are
known or a reference compound is available. The rst bacterial
terpene cyclase that was identied by purication of the protein
and subsequent incubation experiments with the native
substrate was the pentalenene synthase from Streptomyces
exfoliatus,99 followed by the characterisation of various other
bacterial terpene cyclases whose volatile products are shown in
Scheme 12.100113 Several of these products have been identied
from the enzymatic reactions by a laborious and timeconsuming procedure of isolation and NMR spectroscopic
structure elucidation, while others were readily identied by
GC-MS. Since modern genome sequencing has turned into a
very fast and fairly cheap technique, more and more sequencing
data are made available, showing that many more uncharacterised terpene cyclases are encoded in bacteria. A recently
developed interesting approach to gain rapid insight into the
function of these terpene cyclases is based on heterologous
expression in Escherichia coli followed by direct CLSA sampling
of volatiles from the expression strain and compound identication by GC-MS.114 This procedure resulted in the characterisation of six terpene cyclases as germacrene A synthase (54,
from Chitinophaga pinensis, this enzyme is unrelated to the
germacrene A synthase from Nostoc), g-cadinene synthase (65,
Scheme 13, from C. pinensis), a-amorphene synthase (66, from
848 | Nat. Prod. Rep., 2014, 31, 838861
Review
Scheme 11 Volatile terpenes from bacteria and fungi and their

bioactive non-volatile oxidation products.
S. viridochromogenes), 7-epi-a-eudesmol synthase (67, from

S. viridochromogenes), selina-4(15),7(11)-diene synthase (68,
from S. pristinaespiralis), and T-muurolol synthase (60, from
Roseiexus castenholzii, this enzyme is also unrelated to the
T-muurolol synthase from S. clavuligerus). Comparison not only
of mass spectra, but also of retention indices to tabulated data
was of utmost importance for compound identication in
this study.
5 Examples of GC-MS based

structure elucidation
In the case of volatiles for which neither a reference mass
spectrum nor a retention index is available instantaneous
compound identication is prevented. However, these analytes
are in fact the most interesting compounds, because they oer
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Review
Scheme 12 Volatile terpenes from characterised bacterial terpene
cyclases.
the chance of nding new natural products. In some cases a

chemical structure can be rationally suggested from the mass
spectrum by interpretation of the molecular ion including its
isotope pattern and relevant fragment ions that are formed via
specic reactions, as explained in chapter 3. Positive compound
identication will then require synthesis of a reference
compound. This is, however, only a suitable approach, if a
structural proposal for a particular compound can be derived
from its mass spectrum with some certainty, i.e., without the
Volatile terpenes identied by GC-MS in headspace

extracts under heterologous expression of bacterial terpene cyclases
in E. coli.
Scheme 13
NPR
risk of investing a lot of rather undirected synthetic work.

Knowledge of the mass spectra of structurally closely related
compounds is helpful, but also not always available.
Recent examples for the identication of volatiles by
synthesis of reference compounds include a series of degraded
oxygenated sesquiterpenes (6971) from the myxobacterium
Chondromyces crocatus115 and a structurally related dimethyloctalin derivative (72),116 an intermediate in the biosynthesis of
geosmin117 that occurs in many myxobacteria and streptomycetes64 (mentioned in some publications that appeared before
the synthesis of the compound by its molecular ion, m/z 164,
and base peak, m/z 149, Scheme 14).40,86 The structures of the
trans-fused iridoids (4R,4aR,7R,7aS)-dihydronepetalactone (73),
(4S,4aR,7S,7aR)-iridomyrmecin (74), and (4S,4aS,7R,7aS)-iridomyrmecin (75) from the parasitoid wasp Alloxysta victrix were
rigorously established by synthesis of the compounds and all
their trans-fused stereoisomers,118,119 thereby overcoming the
problem that the stereochemistry of a volatile compound is in
terms of its absolute conguration in principle not, and in
terms of its relative conguration practically never deducible
from the mass spectrum. The absolute conguration of trichoacorenol (76) from the fungus Trichoderma harzianum was also
identied by an enantioselective synthesis.120 A series of
homomonoterpenes (77 and 78) and homomonoterpene alcohols (7981) was recently reported from 2-methylisoborneol
producing bacteria.65 Their structures were deduced by
comparison of their mass spectra to the mass spectra of corresponding regular monoterpenes and monoterpene alcohols,
showing diagnostic shis of 14 mass units. The structures of
these new natural products were conrmed by synthesis of
reference compounds.
The mass spectra of compounds that exist in the form of
dierent constitutional isomers can also be very similar, e.g., for
substituted aromatic and heterocyclic compounds. A structural
proposal for the female sex pheromone from the wasp Zaspilothynnus nigripes was derived from its mass spectrum,121 but the
six constitutional isomers of the suggested structure of a
hydroxymethyl-(3-methylbutyl)-methylpyrazine (Scheme 15)
could not be distinguished from the mass spectrum. Since no
tabulated retention index data for these compounds were
available and the isolated material was insucient for NMR
spectroscopic analysis, unambiguous structure elucidation
required the synthesis of all six isomers. Synthetic 82 was
identical to the pheromone and was also shown to be emitted by
the orchid Drakaea livida to attract male wasps for pollination.
Similarly, for unambiguous identication of a dimethyl furandicarboxylate in headspace extracts of Streptomyces griseoavus and S. parvulus all four constitutional isomers were
obtained by synthesis or from commercial sources, resulting in
the identication of 83 as the natural product.122 The compound
was previously, without comparison to a synthetic standard,
tentatively identied as a volatile from another streptomycete.88
The marine bacterium Ruegeria pomeroyi from the Roseobacter
clade was shown to emit the lactone 84 as a major compound,
besides a few structurally related minor volatiles.123 The structure of 84, including its absolute conguration, was fully supported by an enantioselective synthesis and GC analysis on a
Nat. Prod. Rep., 2014, 31, 838861 | 849
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NPR
Scheme 14 Volatile terpenes that were identied by the development

of structural proposals from their EI mass spectra followed by synthesis
of reference compounds.
chiral stationary phase in comparison to the natural material.

Similarly, the structure of a volatile released by the triatomine
bug Triatoma brasiliensis was suggested from its mass spectrum
and identied as (4S,5S)-2,2,4-triethyl-5-methyl-1,3-dioxolane
(85) by synthesis of all stereoisomers.124 An important hint for
the development of the structural proposal was the presence of
large amounts of pentan-3-one in the headspace extracts that is
the precursor and/or degradation product of 85, demonstrating
Scheme 15 Volatiles that were identied by the development of

structural proposals from their EI mass spectra or by isolation and
structure elucidation via NMR spectroscopy.
850 | Nat. Prod. Rep., 2014, 31, 838861
Review
that careful analysis and integrated evaluation of all volatile

constituents from one organism may be of tremendous help in
structure elucidation.
However, this approach of delineating a structural proposal
from the mass spectrum has its limitations. This is particularly
true for terpenes as an important class of volatile natural
products, because their polycyclic structures are usually too
complex. In such cases, GC-MS analysis of headspace extracts
may point to interesting new natural products for further
investigation by compound isolation and structure elucidation
via NMR spectroscopic methods. One of the most interesting
recent examples is the structurally unique volatile sodorifen (86)
from Serratia odorifera.125 The compound was trapped by
absorption on SuperQ in quantities that were sucient for
structure elucidation by NMR analyses. This approach is of
course only suitable if the organism under investigation only
emits one compound (or at least mainly one compound), and
not a complex mixture of volatiles. Headspace extracts of the
fungus Fusarium fujikuroi contained three groups of terpenoid
compounds, and each of these groups was composed of one
main compound and various trace compounds that were
regarded as side products of the respective terpene cyclase.126
One of the three main compounds was the diterpene ent-kaurene, the biosynthetic precursor of the plant hormone gibberellic
acid GA3, whereas the structure of one sesquiterpene alcohol
was unknown and only the relative conguration of the other
sesquiterpene alcohol, a-acorenol, was known. The construction of double knockout mutants resulted in simplied headspace extracts for compound isolation and identication of the
volatiles as ()-a-acorenol (87) and koraiol (88).
For the disulde 89, released by a marine bacterium from the
Roseobacter clade, a structural proposal was derived from its
mass spectrum (Fig. 6).127 The molecular ion together with its
isotope pattern (166: 100%, 167: 8.0%, 168: 8.4%) pointed to the
presence of two sulfur atoms and no (or an even number of)
nitrogen atoms. The best agreements were calculated for
C5H10O2S2 (166: 100%, 167: 7.2%, 168: 9.5%) or C6H14OS2 (166:
100%, 167: 8.3%, 168: 9.3%). The fragment ions at m/z 135
(loss of OMe by inductive cleavage) and m/z 59 (a-cleavage)
suggested the structure of a methyl ester C5H10O2S2. The fragment ion at m/z 118 (neutral loss of MeSH) pointed to a
methylsulfanyl group that was further corroborated by m/z 45
(HCS+), a secondary fragment ion that arises from MeS+ by loss
of H2, as is frequently observed in methylsulfan derivatives.
Further fragment ions were in agreement with a linear structure
of a disulde (m/z 79: MeSS+, m/z 93: MeSSCH2+, m/z 107:
MeSSCH2CH2+). The missing McLaerty ion at m/z 74 usually
observed for methyl esters is easily explained, because the
McLaerty rearrangement requires a g-hydrogen atom, but the
g-position in 89 is occupied by a sulfur atom. However, a fragment ion for cleavage of the CbS bond at m/z 87 is observed.
A synthesis of 89 and comparison of GC-MS data in terms of
mass spectrum and retention index proved the structure of this
new natural product.
From an analytical chemist's point of view it is of utmost
importance that in cases where the synthesis of a reference
compound or a compound isolation is required for the
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Review
Fig. 6 Mass spectrum of the headspace constituent methyl

3-(methyldisulfanyl)propionate (89) from a marine Roseobacter clade
bacterium.
identication of a volatile, the GC retention indices together

with the type of GC column should always be included in a
scientic publication, so that future analytical work in which
these compounds may be detected again will not require a
repeated synthesis.
Retention indices are not only useful, because they can, as
standardized data in contrast to retention times, be used for
direct comparison, even if they were obtained under dierent
experimental parameters, but they are also the basis for increment systems. For the detailed analysis of the structures of
methyl branched long chain lipids like methyl esters, alcohols,
and ketones such a retention index based increment system was
developed.128 The basic idea was the theoretical separation of a
lipid molecule into its longest alkyl chain, a functional group,
and attached methyl branches that all contribute more or less
independently to the analyte's retention index I:
X
I N FG
Mei
Herein, N is an increment for the longest alkyl chain with N
100$n where n represents its number of carbons, FG is an
increment for the functional group, and Mei are increments for
the methyl branches that depend on their positioning i along
the longest alkyl chain. Several functional group increments for
HP-5 or similar GC capillaries have been determined from
unbranched representatives of the respective compound classes
including methyl esters (FG 232),128 alkyl cyanides (FG
458),128 2-alcohols (FG 210),47 3-alcohols (FG 205),17
2-ketones (FG 200),47 and 3-ketones (FG 195).17 The increments (Mei) for the methyl branches as determined in the
original work are summarized in Table 3. A recent renement of
the system for retention index increments of fatty acid methyl
esters showed a slight dependency of the functional group and
methyl branch increments on the chain length, giving better
matches between calculated and measured retention indices.38
The fundamental idea of the retention index increment
system, together with careful interpretation of mass spectra and
biosynthetic considerations, was also successfully applied to the
NPR
development of structural proposals for a series of structurally

complex lactones from Streptomyces ambofaciens. One of these
lactones was known as a secondary metabolite of Streptomyces
spp.129 and its mass spectrum was included in mass spectral
databases, thus allowing for instantaneous compound identication as bastmycinone (30). Its mass spectrum (Fig. 7A)
revealed a fragment ion at m/z 241 due to the loss of a methyl
group, and is likely to have arisen from a-cleavage from the
lactone core, while the molecular ion could not be detected.
Two even fragment ions at m/z 214 and 200 could be assigned
to McLaerty rearrangements, either initiated by ionisation of
the lactone carbonyl oxygen or the carbonyl group of the ester
side chain. Further intensive fragment ions were observed at
m/z 57 and 85, accounting for two alternative a-cleavages
within the ester side chain. These two a-fragment ions together
with the two McLaerty fragments were of high diagnostic
value, because they directly pointed to the nature of the 2-alkyl
portion and the acyl portion of the ester in other blastmycinone
derivatives. In the mass spectra of compounds 90 and 91 the
same set of a-fragment ions at m/z 57 and 85 were observed,
suggesting that the same or an isomeric acyl moiety, as in 30,
should be present in these molecules. This was further
corroborated by the fact that in all three molecules the same
McLaerty fragment ion at m/z 200 was found. The second
McLaerty ion in 90 and 91 was detected at m/z 228 and 186,
respectively. This loss of C3H6 in both cases, as in 30, ruled out a
2-methylbutyryl or 2,2-dimethylpropionyl group, but was in
accordance with a 3-methylbutyryl or pentanoyl function. By
following the further logic of the arguments, the McLaerty
ions at m/z 200 supported a C5H11 2-alkyl chain for 90 and a
2-ethyl side chain for 91. For compound 92 the a-fragment ions
were observed at m/z 43 and 71 (butyrate or isobutyrate ester),
but only one McLaerty fragment was found at m/z 186, thus
ruling out the possibility of a butyrate ester that should result in
a second McLaerty ion at m/z 214 (neutral loss of ethene).
The m/z 186 ion furthermore pointed to a C4H9 2-alkyl group.
Further structural insights into the precise nature of the
attached substituents were concluded from the retention
indices of the compounds, using the logic of the retention index
increment system as presented above. The moderate dierence
in the retention indices of 30 and 90 (55 units) is only in
agreement with two methyl branched side chains, and therefore
a 2-isopentyl group was assumed to be present in 90. Retention
index calculations in the case of 91 were less clear, because the
shorter 2-ethyl chain in 91 in comparison to a 2-butyl group
should theoretically result in a retention index dierence of
200 units, but only a dierence of 170 units was determined.
However, the retention indices of short chain methyl esters are
also dominated by a stronger inuence of the functional group
(in case of blastmycinones the lactone core can be regarded as
the functional group),38 and for biosynthetic reasons a leucine
derived acyl group in 91 seemed more likely. Finally, the
dierence between the retention indices of 30 and 92 of ca.
100 units strongly supported an unbranched 2-alkyl moiety. All
structural proposals as discussed here and of several further
blastmycinone derivatives from S. ambofaciens and a few other
streptomycetes were veried by total synthesis. This example
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Table 3
Increments (Mei) for methyl branches.128
Position i
Mei
Position i
Mei
2 or u1
3 or u2
4 or u3
5 or u4
6 or u5
60
73
56
46
40
7 or u6
8 or u7
9 or u8
10 or u9
36
33
31
28
demonstrates the power of an integrative discussion of mass

spectra, retention indices, and biosynthetic considerations for a
successful delineation of structural proposals for previously
unidentied volatiles.
The above described in-depth analysis of GC-MS data, i.e., a
detailed analysis of gas chromatographic retention indices and
of the molecular ion and fragmentation pattern in the mass
spectra of unknown compounds, is a dicult task and requires
a lot of experience to develop a successful structural proposal.
Most importantly, the proposal has to be veried by comparison
to a synthetic reference. Most analytical studies refer to the
known chemical space and compounds in such studies are
frequently identied by comparison to retention index data
published in previous reports. The danger of this approach is
that an erroneous compound identication, e.g., in case of
(stereo)isomers with highly similar mass spectra, is easily
multiplied.
Feeding experiments
A feeding experiment is an experiment in which a biosynthetic

precursor, a small molecule of the primary metabolism such as
an amino acid, a sugar, a citric cycle intermediate, etc., or an
advanced biosynthetic intermediate, is fed to an organism,
followed by investigation whether or not the fed compound is
incorporated into the target natural product. A feeding experiment may be performed for elucidating the biosynthetic
pathway to a volatile or for isotopic enrichment for structure
elucidation. Today, in most cases, the isotopic labelling is
realised by use of a stable isotope, usually of deuterium or
13
C. Many classical biosynthetic studies have used radioactive
labels (e.g., 3H or 14C), but these techniques seem to be out of
fashion, mainly due to the rapid technological developments in
mass spectrometry that allow for the very sensitive detection of
stable isotope incorporations into natural products. In earlier
days, the incorporation of radioactive isotopes into natural
products could be best detected, with very high sensitivity,
using autoradiographe, but a major drawback is that the
handling of radioactive compounds requires special safety
equipment and trained personnel.
Both stable isotopes 2H and 13C have frequently been used
for the biosynthetic investigations of volatile natural products,
but their behaviour in GC-MS analyses is very dierent.
Deuterated compounds are characterised by particularly strong
isotope eects, much stronger than in 13C-labelled compounds.
The shorter CD bond length, as compared to a CH bond,130
852 | Nat. Prod. Rep., 2014, 31, 838861
leads to a stronger electric eld strength and, consequently, to a

lower polarisability of deuterated compounds in comparison to
their protonated analogues.131 Since weak dispersion forces play
a major role in the gas chromatographic retention of non-polar
analytes,132 deuterated compounds usually have signicantly
shorter retention times.17,34,133,134 This has important consequences for feeding experiments on the biosynthetic pathways
to volatile natural products, if using deuterated precursors. The
incorporation rates of the fed deuterated compound into the
volatile natural product are usually not 100%, therefore resulting in the production of a mixture of the labelled and the nonlabelled compounds by the respective organism. In the case of
low-resolution MS analysis, only the ecient gas chromatographic separation of the deuterated from the protonated
volatile allows for a precise determination of the incorporation
rates by simple peak integration (baseline separation of the
deuterated compound from the natural isotopomer requires the
incorporation of ca. 35 deuterium atoms). Furthermore, a
detailed interpretation of the clean mass spectrum of the
deuterated compound is possible, if mass spectral fragmentation mechanisms are known, that may even allow for a localisation of the site of deuterium incorporation in the natural
product. This is exemplied by the incorporation of deuterium
labelling from several synthetic deuterated mevalonolactone
isotopomers129 into tricho-acorenol (35) that undergoes a retroDielsAlder fragmentation in MS (Scheme 6).67 The corresponding fragment ion at m/z 84 for the unlabelled
compound (Fig. 8A) was observed at m/z 88 in the feeding
experiment with [4,4,6,6,6-2H5]mevalonolactone (Fig. 8B), corresponding to an uptake of four of the twelve incorporated
deuterium atoms into the retro-DielsAlder fragment forming
portion of 35. Similar localisations of deuterium atoms by
interpretation of the major fragment ions at m/z 138 and 151,
together with interpretations of mass spectra with several other
mevalonolactone isotopomers, allowed to precisely follow a
sequence of a 1,4- and a 1,2-hydride shi during terpene cyclisation. The interpretation of mass spectra in such detail was
only possible due to the gas chromatographic separation of the
deuterated from the non-labelled material.
Early feeding experiments with deuterated compounds in
combination with GC-MS analysis have been performed to
investigate the biosynthetic pathways to fatty acid derived insect
pheromones. The reactions on fatty acid derivatives discussed
in the next section likely proceed with coenzyme A (CoA)
derivatives or with enzyme bound substrates, but since the true
nature of the intermediates has not been investigated the
biosynthetic pathways will be discussed on the level of free acid
substrates as in most of the original articles. In a pioneering
experiment the biosynthesis of bombykol (96, Scheme 16), the
rst identied sex pheromone of the silkworm moth Bombyx
mori,135 was investigated by feeding of [11,12-2H2]-(11Z)-hexadecenoic acid (94, deuterated positions are marked by asterisks) whose incorporation into 96 was readily detected by
GC-MS.136 The deduced pathway starts from hexadecanoic acid
(93) that is converted into (11Z)-hexadecenoic acid (94) by
activity of a D11-desaturase, followed by introduction of a
second double bond under abstraction of one hydrogen from
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NPR
C-10 and one hydrogen from C-13 to yield (10E,12Z)-hexadecadienoic acid (95) and reduction to bombykol.
This successful labelling study promoted a series of similar
GC-MS based feeding experiments on biosynthetic pathways to
insect pheromones. It was shown by feeding of [16,16,16-2H3]palmitic acid (93, deuterated positions marked by asterisks)
that the biosynthesis of the sex pheromones from Agrotis
segetum, (7Z)-dodecenyl acetate (104), (9Z)-tetradecenyl acetate
(103), and (11Z)-hexadecenyl acetate (102), proceeds by introduction of the (Z)-double bond to yield 94 (Scheme 17).137 Its
reduction to the alcohol and acetylation results in 102, while
the shorter derivatives 103 and 104 require a chain degradation by b-oxidations of 94 prior to reduction and acetylation.
Similar results were obtained for the biosynthesis of 102 by
feeding [13,13,14,14,15,15,16,16,16-2H9]-93 to Mamestra
brassicae.138
The biosyntheses of all four compounds 96 and 102104
starts with a D11-(Z)-desaturation that is a key step in the
formation of a large number of lepidopteran pheromones.
Boland and coworkers have shown in a series of elegant
experiments that introduction of the (Z)-double bond by the
D11-desaturases from three investigated lepidopterae, M. brassicae, B. mori, and Manduca sexta, proceeds via a syn elimination
of C(11)-HR and C(12)-HR (Scheme 18).139 Feeding of (11S,12R)[2H14]-93 to M. brassicae resulted in the incorporation of twelve
deuterium atoms into the insect pheromone 102, as determined
by GC-MS, while a complementary feeding experiment with
(11R,12S)-[2H14]-93 resulted in the retention of all fourteen
deuterium atoms in labelled 102.139 The same stereochemical
course was found for the reaction of 93 to 94 in B. mori and
Fig. 7 Mass spectra of A) blastmycinone (30) and BD) blastmycinone

derivatives (9092) detected in headspace extracts of Streptomyces
ambofaciens.
Fig. 8 Mass spectra of A) unlabelled tricho-acorenol (35) and B) of
[2H12]-35 obtained after feeding of [4,4,6,6,6-2H5]mevalonolactone.

Asterisks indicate completely deuterated carbons.
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Scheme 16 Biosynthetic pathway to bombykol (96).
M. sexta. The incorporation of deuterium labelling into 94 was

investigated by GC-MS aer conversion into the respective
methyl ester with diazomethane and the dimethyl disulde
adduct by treatment with dimethyl disulde/iodine.140 The
stereochemical course of the lepidopteran D11-desaturases is
the same as previously determined for the D9-desaturases from
algae,141 bacteria,142 and animals,143 suggesting a common
enzyme mechanism.
The biosynthesis of the sex pheromones (5Z)-dodecenol and
(5Z,7E)-dodecadienol (110) of the pine caterpillar moth, Dendrolimus punctatus, was established by feeding experiments
with deuterated fatty acids including [16,16,16-2H3]-93,
[18,18,18-2H3]octadecanoic acid (105) and [15,15,16,16-2H4](11Z)-octadecenoic acid (106), followed by analysis of the
pheromone gland content by GC-MS (Scheme 19).144 The
pathway starts from 93, which is elongated to 105, followed by
introduction of a (Z)-double bond by a D11-desaturase to 106
Scheme 17 Biosynthesis of lepidopteran pheromones by activity of a

D11-(Z)-desaturase. The deuterated positions as relevant for the
biosynthetic investigations on 102104 in A. segetum are indicated by
asterisks, whereas additionally marked carbons (asterisks in brackets)
were only relevant for the feeding study on the biosynthesis of 102 in
M. brassicae.
854 | Nat. Prod. Rep., 2014, 31, 838861
Review
and chain shortening by b-oxidation to (9Z)-hexadecenoic acid

(107). A second (E)-double bond is introduced by a D11-desaturase, possibly the same enzyme as that acting in the reaction
of 106, to yield (9Z,11E)-hexadecadienoic acid (108). Two
subsequent b-oxidations to (5Z,7E)-dodecadienoic acid (109)
and a reduction establish the pheromone 110. The second
compound (5Z)-dodecenol is produced via the same pathway,
only with the omission of the second D11-desaturation. The
biosynthesis of the pheromones of the lappet moth Gastropacha
quercifolia, (5Z)-dodecenol and (5Z)-dodecenal, was investigated
by the feeding of [7,7,8,8,10,10,11,11-2H8]-(5Z)-dodecenoic
acid.145 Analysis of the pheromone bouquet by GC-MS
conrmed that it is the direct precursor to the pheromone
components,
while
the
saturated
analogue
[7,7,8,8,10,10,11,11-2H8]dodecanoic acid was not incorporated,
suggesting that the unsaturation is introduced at an earlier
stage of the pathway.
Such feeding experiments with insects can be dicult due to
the complex behaviour of these macroscopic eukaryotic organisms. In contrast, bacteria are much easier to handle and a
feeding experiment can simply be carried out by adding an
isotopically labelled precursor to the culture medium. One of
the rst studies on the biosynthesis of volatile secondary
metabolites was performed with the myxobacterium Myxococcus
xanthus.17 As shown by the feeding of deuterated precursors, the
main volatiles released by this bacterium, 9-methyldecan-3-one
(36) and (S)-9-methyldecan-3-ol (21), are derived from L-leucine
(113). The feeding of completely deuterated [2H10]leucine
resulted in the incorporation of nine deuterium atoms into both
metabolites; one deuterium is lost during transamination and
oxidative decarboxylation to the branched starter unit isovaleryl-CoA (112, Scheme 20). Aer chain elongation with two
malonyl-CoA units to the acyl carrier protein (ACP) bound
intermediate 114 a nal methylmalonyl-CoA building block is
incorporated, as was demonstrated by the feeding of [2H5]
Stereochemical course of D11-(Z)-desaturases involved in

the biosynthesis of lepidopteran pheromones.
Scheme 18
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Scheme 19 Biosynthetic pathway to (5Z,7E)-dodecadienol in Dendrolimus punctatus.
propionic acid. Release of the b-keto-thioester intermediate 115

and decarboxylation yields 36, which is converted to 21 by
reduction.
The major advantage of using deuterated precursors in this
study was the chromatographic separability of the deuterated
compound from the material with a natural isotope pattern.
This allowed for a location of the deuterium incorporation by
careful interpretation of fragment ions (Fig. 9). Important
fragment ions of 36 were observed at m/z 43 (cleavage of the
terminal isopropyl group), m/z 57 (a-cleavage next to the
carbonyl group), and m/z 72 (arising by McLaerty rearrangement). Feeding of [2H10]leucine resulted in a specic
increase of the fragment ion at m/z 43 to 50, while the fragment ions at m/z 57 and 72 remained unchanged, giving
evidence for deuterium uptake into the isopropyl moiety
(Fig. 9B). Feeding of [2H5]propionic acid resulted in the incorporation of three deuterium atoms into 36 and 21; one deuterium atom in the a-position was lost due to carboxylation to
methylmalonyl-CoA and a second one due to the increased C,Hacidity of methylmalonyl-CoA or of the intermediate 115. The
incorporation of labelling into the ethyl groups of 36 and 21
could be located by the specic shis of the fragment ions at
m/z 57 and 72 to m/z 60 and 75 (Fig. 9C). Since a complete
washout of deuterium from the a-carbon was observed in the
feeding experiment with [2H5]propionic acid another strategy
using [13C2]acetic acid was required to prove the biosynthetic
origin of the acetate-derived carbons in 36 and 21. As discussed
above, the isotope eect of 13C is much weaker than for
deuterium, and consequently a 13C-labelled compound is not
chromatographically separated from the 12C analog, resulting
NPR
in the superimposition of the mass spectra from the labelled

compound and the compound with a natural isotope pattern
(Fig. 9D). Nevertheless, the incorporation of up to two units of
[13C2]acetic acid was observable from the molecular ion. The
location of the incorporation was not discussed in the original
work, but a slight increase in the fragment ions at m/z 58 and
m/z 74 can be observed, which is in agreement with an
incorporation of labelling into C-2 and C-3 (compare Fig. 9A and
9D). Feeding of deuterated dimethylacrylic acid (111), that is
also linked to terpene metabolism in myxobacteria,117,146 gave
consistent results.
An interesting study was recently performed by Rui and
Boland, in which the stereochemical course of the biosynthetic
pathway from arachidonic acid to dictyoene in Ectocarpus siliculosus was followed by stereospecic deuterium labellings at
C-16 (Scheme 21).147 For this purpose the synthetic deuterated
isotopologues of arachidonic acid 117 and 118 were fed to
E. siliculosus. Along the known biosynthetic pathway148 these
isotopologues of arachidonic acid are oxidatively converted to
hydroperoxyeicosatetraenoic acids 119 and 120 by lipoxygenase
activity. The deprotonation induced fragmentation results in the
divinylcyclopropane 121 and (5Z,7E)-9-oxononadienoic acid
(122). A nal Cope rearrangement of 122 yields dictyoene (123)
that was captured by SPME and analysed by GC-MS for its
deuterium content. Deuterium labelling from the pro-S hydrogen
in position 16 of arachidonic acid was retained, whereas
labelling of the pro-R hydrogen was lost, showing that
Scheme 20 Biosynthetic pathway to 9-methyldecan-3-one and

(S)-9-methyldecan-3-ol in Myxococcus xanthus.
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beetle Stenus bimaculatus.150 Incorporation of deuterated isotopomers of L-lysine (124), L-leucine, and sodium acetate suggested the biosynthetic pathway shown in Scheme 22.
Decarboxylation and transamination of 124 results in the amino
aldehyde 125, which is cyclised to the imine 126. The corresponding enamine 127 undergoes nucleophilic attack to the
isoleucine-derived aldehyde 128, forming the condensation
product 129, which is subsequently reduced to 130. Acetylation,
most likely with acetyl-CoA, yields the acetamide 131 that is
reduced to stenusine. The study also contained a detailed
analysis of mass spectral fragmentations to locate the sites of
incorporation.
As discussed for several examples in this article so far, the
strongest advantage of using deuterated precursors is the
possibility of gas chromatographic separation of deuterated
volatiles from their natural analogs, allowing the localisation of
deuterium incorporation, if fragmentation reactions for the
analyte are known or are at least very plausible following the
reactions as discussed in chapter 3. This method has its limits,
where the fragmentation reactions are unclear, or two or more
alternative fragmentations resulting in the same fragment ion
are possible. An example is given by the fragmentation mechanism of the base peak ion at m/z 95 for the musty odorant
2-methylisoborneol (41), for which two alternative terminal
a-cleavages with loss of stereochemical distinct methyl groups
are possible (Scheme 10). One of these methyl groups originates from the methyl group in the terpene precursor mevalonolactone (133) (in case of the myxobacterium Nannocystis
exedens that uses the mevalonate pathway to terpenes) or
from C-1 in deoxyxylulose phosphate (134) (in case of most
streptomycetes using the deoxyxylulose phosphate pathway,
Fig. 9 Mass spectra and important fragment ions of A) 9-methyldecan-3-one (36), B) [2H9]-36 after feeding of [2H10]leucine, C) [2H3]36 after feeding of [2H5]propionic acid, and D) superimposed mass
spectra of 36, [13C2]-36, and [13C4]-36 after feeding of [13C2]acetic
acid. Asterisks indicate labelled positions.
the deprotonation induced fragmentation of hydroperoxyeicosatetraenoic acid proceeds with stereospecic loss of
the pro-R hydrogen at C-16. This nding was in agreement with
previous investigations on the stereochemical course of the
reaction in Gomphonema parvulum.149
A recent example for a biosynthetic study on an alkaloid by
feeding of isotopically labelled precursors followed by GC-MS
analysis of incorporation is given by stenusine (132) from the
856 | Nat. Prod. Rep., 2014, 31, 838861
Scheme 21 Stereochemical course of the biosynthesis of dictyoene

(123) in Ectocarpus siliculosus.
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NPR
Scheme 23). However, analysis of the stereochemical course of

the terpene cyclisation of (E)-b-methylgeranyl diphosphate
(138) by feeding of an appropriately deuterated terpene
precursor cannot be conclusive due to the uncertainties in the
fragment ion formation. Feeding of a 13C-labelled precursor
followed by compound isolation and 13C-NMR spectroscopy
would give an unambiguous answer, but the isolation of
compounds such as 41 from culture extracts is signicantly
hampered by the high volatility. Recently, a highly sensitive
method for microscale biosynthetic investigations using the
techniques of headspace sampling was described.65 Feeding of
[1-13C]deoxyxylulose to an agar plate culture, collection of
volatiles on charcoal lters by CLSA, followed by GC-MS and
13
C-NMR analysis of the sample established the stereochemical
course of the cyclisation to 41. In this analytical procedure the
advantages of several techniques were combined: Headspace
sampling (CLSA) only yielded the volatile material, and unlike
culture extractions, all extractable secondary metablites, and
the loss of volatiles was prevented. GC-MS analysis of the
headspace extract showed the content of terpenes that were
identied from their mass spectra and retention indices (only
terpenes were important since a terpene precursor was fed; all
other volatiles did not incorporate the isotopic labelling and
were in consequence not visible in the 13C-NMR spectrum).
Finally, 13C-NMR spectroscopy showed into which carbon
atoms of 41 the labelling was incorporated. Due to the high
incorporation rates, even micrograms of material were sucient to detect the 13C-enriched carbons.
The same method was recently used to investigate the volatiles emitted by the PR toxin (50) producing fungus Penicillium
roqueforti.95 The parent hydrocarbon of 50 is aristolochene,
which is made by aristolochene synthase.151,152 Several side
products of this enzyme have been identied in a series of
Scheme 22
Biosynthetic pathway to stenusine (132) in Stenus
bimaculatus.
studies including GC-MS analyses of SPME headspace extracts

from PR toxin producing strains of P. roqueforti,153 and of
volatile products obtained from enzyme incubations with the
wildtype enzyme or site-specic mutants.154157 This includes the
compounds germacrene A (139), a-selinene (140), b-selinene
(141), selina-4,11-diene (142), valencene (143), and (E)-b-farnesene (144, Scheme 24). Interestingly, a single critical amino acid
switch (Y92A) turned the aristolochene synthase into an enzyme
with (E)-b-farnesene synthase activity.157
In a recent study on the volatiles released by P. roqueforti an
additional previously unrecognised sesquiterpene alcohol was
detected.95 Its mass spectrum (Fig. 10) revealed high similarities to the mass spectrum of eudesma-11-en-4a-ol (148), but
some of its stereoisomers (Scheme 25) were reported to have
similar retention indices and could not be excluded.158 Since
also the full 13C-NMR data of all stereoisomers were mentioned
in the same report, the structure of eudesma-11-en-4a-ol could
be proven by feeding of [methyl-13C]mevalonolactone,
capturing of the volatiles and direct 13C-NMR analysis of the
headspace extract, resulting in the detection of the three
expected 13C signals for 148. Finally, a third study making use
of the technique was performed to investigate the mechanistic
details of biosynthetic pathways to terpenes, including the
volatiles a-acorenol (87) and koraiol (88) in Fusarium
fujikuroi.159
In contrast to the widespread usage of hydrogen and carbon
isotope labellings, oxygen labellings are only applied in few
biosynthetic studies, largely because 17O and 18O labelled
compounds are comparably expensive and only few such
compounds are commercially available. An interesting recent
example is given by a series of studies on the biosynthesis of
spiroacetals in fruit ies of the genus Bactrocera.160,161 The
species-specic incorporation of labelling from [18O2]dioxygen
into the spiroacetals, such as 154, was followed by the
capturing of these volatiles by SPME and subsequent GC-MS
analysis (Scheme 26). In B. tryoni both oxygens in 154 were
Scheme 23 Biosynthetic pathway to 2-methylisoborneol and

stereochemical course of terpene cyclisation. Asterisks indicate
13
C-labelled carbons.
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Scheme 26 Biosynthesis of spiroacetals in Bactrocera. Asterisks

indicate uptake of labelling into both oxygens of 154 after administration of [18O2]dioxygen to B. tryoni, whereas for B. curcumis only one
oxygen of 154 was labelled.
Scheme 24 Side products of aristolochene synthase from P. roque-
forti identied by GC-MS.
Fig. 10 Mass spectrum of an eudesman type sesquiterpene alcohol

from P. roqueforti.
Scheme 25 Structures of eudesma-11-en-4a-ol (148) and its

stereoisomers. Published retention indices on a DB-1 column157 are
given in brackets.
found to be labelled aer administration of [18O2]dioxygen,160

whereas in B. curcumis the labelling was only introduced into
one oxygen.161 These results were explained by the introduction
of both hydroxy functions in the linear precursor 153 by
involvement of a cytochrome P450 in B. tryoni, while only one of
the two hydroxy functions in 153 may be introduced by a
cytochrome P450 in B. curcumis and the other hydroxy function
is derived from water during b-oxidation of a fatty acid
precursor.
858 | Nat. Prod. Rep., 2014, 31, 838861
Conclusions and outlook
As demonstrated in this article, GC-MS is an exquisite tool for

the identication of new volatile natural products. Careful
interpretation of mass spectra with respect to the molecular ion
and its isotope pattern and, most importantly, in light of
known fragmentation pathways may give the experienced
analytical chemist the possibility of delineating a structural
proposal. Retention index increment systems can provide
further valuable information. The (future) general availability
of high-resolution mass spectrometry as a standard laboratory
technique will certainly have a strong impact on the identication of new volatile natural products as the elemental
compositions of molecular and fragment ions become easily
accessible with this technique. This will provide an even
stronger fundament for structural suggestions, as it can be
obtained from low-resolution data. However, it will not be
possible, neither based on low-resolution nor on high-resolution data, to delineate the structure of a new compound from
GC-MS data alone. For verication of structural proposals the
synthesis or isolation of a reference compound is always
crucial. In the case of compounds with a known mass spectrum, the use of (web-based) mass spectral libraries is a very
powerful tool for compound identication. In this sense,
chemistry should clearly learn from the recent achievements in
biological sciences that have been revolutionised by the
development of strong bioinformatic tools and the assembly of
large databases.
The GC-MS technique is also very useful for biosynthetic
investigations by application of isotopically labelled
compounds in feeding experiments. As discussed here in
detail, deuterated compounds are of particularly high value,
because a deuterated volatile can be separated from its nondeuterated analog by gas chromatography, thus allowing for
the interpretation of the clean mass spectrum of the
deuterated compound, which is especially useful when
encountering low incorporation rates. The most recent developments in the analysis of volatiles includes the combination
of the highly sensitive GC-MS technique with feeding experiments of 13C-labelled precursors that allows for an ecient
13
C-NMR analysis of headspace samples containing only
microgram amounts of a particular analyte. The described
techniques in this article together with future developments
may allow for the ecient identication of new volatiles as an
important class of natural products and detailed insights into
their biosynthetic pathways.
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Review
8 Acknowledgements
Funding by the Deutsche Forschungsgemeinscha (DFG) with

an Emmy Noether fellowship (DI1536/1-3) and a Heisenberg
fellowship (DI1536/4-1) is gratefully acknowledged.
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