Analytica Chimica Acta 465 (2002) 6379

Review

Stable isotope analysis of fatty acids by gas

chromatographyisotope ratio mass spectrometry
Wolfram Meier-Augenstein
Division of Molecular Physiology, School of Life Sciences, Old Medical School, University of Dundee, Smalls Wynd,
Dundee DD1 4HN, Scotland, UK
Received 9 November 2001; received in revised form 22 February 2002; accepted 5 March 2002

Abstract
Compound-specific isotope analysis (CSIA) of fatty acids is a relatively young analytical method. However, CSIA of fatty
acids has increasingly become the method of choice in areas where accurate and precise knowledge of isotopic composition
at natural abundance level is important. CSIA of fatty acids at natural abundance level provides information on biogenetic
and geographic origin of lipids and oils that is invaluable for research into archaeology and the environment and almost
indispensable these days for authenticity control and fraud detection in food analysis. In combination with naturally enriched
or stable isotope labelled precursors, CSIA of fatty acids has also gained increasing importance in biochemical, -medical and
-geochemical applications as it offers a reliable and risk-free alternative to the use of radioactive tracers.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Arachidonic acid; Archaeology; Authenticity; Carbon-13; Combustion; Diet; Docosahexaenoic acid; Fatty acid; Fatty acid
metabolism; Full-term; Enrichment; Infants; Isotope analysis; Isotope effect; Isotope ratio; IRMS; Gas chromatography; Incorporation;
Linoleic acid; Natural abundance; Origin; Paediatrics; Paleodiet; Pre-term; Stable isotopes; Turnover; Vegetable oil

1. Introduction

1.1. Fatty acids and IRMS

One of the aims of this review is to provide both

newcomers and experienced researchers in the field of
SIA with an overview of SIA of fatty acid both at natural abundance and enrichment level. To accommodate
a general introduction precedes this article.

IRMS is certainly a very sensitive detector, able to

yield highly precise measurements of isotope ratios
with a standard deviation in the range of fivesix significant figures. When coupled to a GC, it enables the
analyst to conduct highly precise CSIA, especially at

Abbreviations: APE, atom percentage excess; CSIA, compound-specific isotope analysis; GC, gas chromatography; GCMS, gas
chromatographymass spectrometry; GC/CIRMS, gas chromatography/combustionisotope ratio mass spectrometry; GC/PyIRMS, gas
chromatography/pyrolysisisotope ratio mass spectrometry; GC/TCIRMS, gas chromatography/thermal conversionisotope ratio mass
spectrometry; GIRMS, gas isotope ratio mass spectrometry; HPLC, high-performance (pressure) liquid chromatography; HRC, high-resolution
chromatography; HRcGC, high-resolution capillary gas chromatography; HTcGC, high-temperature capillary gas chromatography; IRMS,
isotope ratio mass spectrometry; LC, liquid chromatography; LCFA, long-chain fatty acid; MDGC, multi-dimensional gas chromatography;
MSD, mass selective detection; MS, mass spectrometry; PUFA, polyunsaturated fatty acid; SIA, stable isotope analysis; SIM, selected ion
monitoring; TMS, trimethylsilyl
Tel.: +44-1382-345514; fax: +44-1382-345514.
E-mail address: w.meieraugenstein@dundee.ac.uk (W. Meier-Augenstein).
0003-2670/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 1 9 4 - 0

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

natural isotopic abundance level. High-precision CSIA

of fatty acids at natural abundance level can provide information on biogenetic relation and origin of a given
sample. Compared with authentic reference data, subtle differences in the isotopic abundance of 2 H, 13 C
or 18 O can thus help uncover adulteration of high premium vegetable oils or reveal paleodietary habits of
our ancestors.
There is also an increasing interest in the application of high-precision CSIA of fatty acids in tracer
studies. One area of application is concerned with
quantitative studies of biochemical processes, such as
assimilation/incorporation of nutrients, turnover rates
of biologically important molecules and quantification of synthesis of unsaturated LCFAs. The other
area aims to improve detection limits of bioorganic
molecules by using labelled precursor compounds
at high enrichment levels. In both areas, the use of
non-radioactive 13 C-labelled fatty acids or presumed
precursors in conjunction with CSIA offers a safe, ethically acceptable alternative to radioactive 14 C tracers.
In either case, HRcGC is a prerequisite for
high-precision CSIA by on-line IRMS [1]. Both peak
overlap and peak distortion have a detrimental effect on accuracy as well as precision of isotope ratio

measurements. It is, therefore, not surprising that scientists working on high-precision CSIA of fatty acids
by on-line IRMS invariably employ HRcGC methods.
1.2. Principle of isotope abundance analysis
by IRMS
GIRMS or simply IRMS is probably the oldest type
of MS used in analytical chemistry [2]. IRMS has been
a standard tool in areas, such as geochemistry, quaternary sciences and environmental sciences [3]. However, only since the commercial availability of IRMS
instruments coupled to a gas chromatograph via a combustion interface in 1990 (Fig. 1), has IRMS received
the attention of other areas of applied analytical chemistry; areas where GCMS and LCMS have already
been commonly used.
In contrast to organic mass spectrometers that yield
structural information by scanning a mass range over
several hundred Dalton for characteristic fragment
ions, IRMS instruments achieve highly accurate and
precise measurement of isotopic abundance at the
expense of the flexibility of scanning MS. Since
GCMS can be used to measure stable isotope enrichment, the question arises why one should embrace

Fig. 1. Set-up of an isotope ratio mass spectrometer coupled to a gas chromatograph via a combustion interface to measure
15 N/14 N isotope ratios. The schematic for the IRMS shows the cup configuration for 13 C/12 C isotope ratio measurement.

13 C/12 C

or

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

GC/CIRMS. Scanning mass spectrometers use a

single detector and therefore, cannot simultaneously
detect particular isotope pairs for isotope ratio measurement. For isotope ratio measurement, the MS is
best operated in SIM mode to optimise sensitivity to
selected masses. Even in SIM mode, limited accuracy
and precision of such isotope ratio measurements
impose a minimum working enrichment for 13 C and
15 N of at least 0.5 APE [4,5]. In other words, organic
MS cannot provide accurate and precise information
on isotopic composition at natural abundance level
or in cases where a low rate of tracer incorporation
in a minor biochemical pathway results in isotopic
enrichment of <0.5 APE.
IRMS instruments achieve highly precise measurement of isotopic abundance at the expense of the flexibility of scanning MS. For isotope ratio measurement,
the analyte must be converted into a simple gas, isotopically representative of the original sample, before
entering the ion source of an IRMS. Consequently,
continuous flow isotope ratio measurements of 2 H/1 H,
13 C/12 C and 18 O/16 O are performed on H , CO and
2
2
CO, respectively, with 13 C abundance measurements
accounting for almost 70% of all gas isotope ratio
analyses made. It is also important to note that IRMS,
in fact, determines differences in isotope ratios with
great precision and accuracy rather than the absolute
isotope ratio. IRMS measurements yield the information of isotopic abundance of the analyte gas relative
to the measured isotope ratio of a standard or reference gas.
To achieve accurate and highly precise measurement of isotope ratios, obviously great care must be
taken to ensure that no part of the analyte data is lost.
In the case of CO2 , the data comprise three ion traces
for the different isotopomers 12 C16 O2 , 13 C16 O2 and
12 C18 O16 O with their corresponding masses at m/z
44, 45 and 46, respectively. The three ion beams are
registered simultaneously by a multiple Faraday cup
(FC) arrangement with a dedicated FC for each isotopomer. The resulting ion currents are continuously
monitored, subsequently digitised and the resulting
peak data (points) transferred to the host computer.
Here, the peak area for each isotopomer is integrated
quantitatively and the corresponding ratios are calculated.
Due to simultaneous measurement of two or three
isotopomeric ions, GC/CIRMS can measure isotopic

65

composition at low enrichment and natural abundance level with a high degree of accuracy and
precision. This means that minute variations in very
small amounts of the heavier isotope are detected in
the presence of large amounts of the lighter isotope.
Since the small variations of the heavier isotope habitually measured by IRMS are of the order of 0.07
to +1.09 APE, the -notation in units of per mil ()
has been adopted to report changes in isotopic abundance as a per mil deviation compared to a designated
isotopic standard:



Rs Rstd
s =
1000 []
(1)
Rstd
where Rs is the measured isotope ratio for the sample
and Rstd the measured isotope ratio for the standard. To
give a convenient rule-of-thumb approximation, in the
-notation, a 13 C-enrichment in the range of 0.033 to
+0.0549 APE corresponds to 13 C value range of 30
to +50. In 13 C isotopic abundance terms, a change
of +1 is approximately equivalent to a change of
+0.001 APE.
The sensitivity of GC/CIRMS is such that
tracer/tracee (mol/mol) ratios down to 105 can reliably be detected [6]. In the same review, Brenna
et al. also provide an in-depth discussion of notations
and elementary calculations, such as mass balance
and pool mixing equations. Over the last decade several review articles have collated publications in the
field of GC/CIRMS [1,3,711]. Since these articles
aimed to cover a wide area of applications, CSIA of
fatty acids was always mentioned as one of many.
The following article will focus exclusively on CSIA
of fatty acids and the wealth of information that can
be obtained by this technique.

2. Sample preparation
Due to the high sensitivity of IRMS analysers, the
quality of results of CSIA cannot be better than the
quality of the sample or the quality of the chromatographic analysis, i.e. GC peak resolution. In other
words, sample preparation is a crucial part of the whole
analysis because it constitutes more often than not
the performance-limiting step [12]. For high-precision
CSIA by GC/CIRMS close attention must be paid to
the following points.

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

Every step of the sample preparation protocol (collection, purification, isolation and derivatisation)
must be scrutinised for potential mass discriminatory effects to avoid isotopic fractionation of the
target compounds.
If the potential of isotopic fractionation cannot be
ruled out conclusively, an internal standard, of a
similar chemical nature (but not requiring derivatisation) and of known isotopic composition, should
be added to the sample prior to the sample preparation procedure.
Signal size and isotopic composition of the standard(s) must match those of the analyte(s) [13].
The potential of all GC parameters (polarity of
stationary phase, carrier gas management and temperature programme) and techniques should be
exploited to their fullest.
The isotopic signature of the derivatisation agents
used should be homogenous throughout the duration of a project involving GC/CIRMS. This is
conveniently achieved by acquiring a large stock
from the same batch and by appropriate storage.
The latter may include storage over drying agents,
at low temperatures, under an inert gas and not
exposed to light.
2.1. Mass discrimination
Mass discrimination or isotopic fractionation is a
source of error that is unique to CSIA. In principle,
two different types of isotope effects can cause isotopic fractionation, kinetic isotope effects and thermodynamic isotope effects. In general, isotope effects
are caused by differences in vibration energy levels
of bonds involving heavier isotopes as compared to
bonds involving lighter isotopes. This difference in
bond strength can lead to different reaction rates for
a bond when different isotopes of the same element
are involved [14]. The most significant kinetic isotope
effect is the primary isotope effect, whereby a bond
containing the atom or its isotope in consideration is
broken or formed in the rate-determining step of the
reaction. Rieley presented an excellent in-depth discussion of kinetic isotope effects and associated theoretical considerations in 1994 [15].
The second kind of isotope effect is associated with
differences in physicochemical properties, such as
infrared absorption, molar volume, vapour pressure,

boiling point and melting point. Of course, these

properties are all linked to the same parameters as
those mentioned for the kinetic isotope effect, i.e.
bond strength, reduced mass and hence, vibration energy levels. However, to set it apart from the kinetic
isotope effect, this effect is referred to as the thermodynamic isotope effect [16] because it manifests itself
in processes where chemical bonds are neither broken
nor formed. Typical examples for such processes in
which the results of thermodynamic isotope effects
can be observed are infrared spectroscopy, distillation
and any kind of two-phase partitioning. This thermodynamic isotope effect or physicochemical isotope
effect, is the reason for the higher infrared absorption
of 13 CO2 as compared to 12 CO2 , the enrichment of
ocean surface water with H2 18 O and the isotopic fractionation observed during chromatographic separations.
2.1.1. Gas chromatographic isotope effect
In continuous flow-IRMS (CF-IRMS) systems used
for gas isotope analysis of gases or bulk isotope analysis in combination with an elemental analyser, on-line
gas purification steps and overall interface length lead
to Gaussian-shaped signals. This is evidently even
more pronounced in GC/CIRMS systems, where analyte peaks eluting from the GC column are fed into
an on-line microchemical reactor to produce, e.g. CO2
peaks. However, due to the chromatographic isotope
effect [17,18] the m/z 45 signal (13 CO2 ) precedes the
m/z 44 signal (12 CO2 ) by 150 ms on average [19], an
effect not observed in ordinary CF-IRMS systems.
This time displacement depends on the nature of the
compound and on chromatographic parameters, such
as polarity of the stationary phase, column temperature and carrier gas flow [20]. Therefore, loss of peak
information due to unsuitably set time windows for
peak detection and hence, partial peak integration will
severely compromise the quality of the isotope ratio
measurement by GC/CIRMS. Accurate and precise
isotope ratio measurement is also compromised even
if traces of peak data from another sample compound overlap with the sample peak to be analysed
(Fig. 2). Due to the fact that isotope ratios cannot
be determined accurately from the partial integration
of a combusted GC peak, HRcGC resulting in baseline separation for adjacent peaks is of paramount
importance for high-precision CSIA.

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

67

Fig. 2. Ion chromatogram of mass trace m/z 44 of fatty acids (as FAMEs) from rapeseed oil showing excessive peak tailing and resulting
peak overlap for fatty acids 18:0, 18:1 and 18:2, unsuitable for accurate CSIA of these three compounds.

It should be noted that the chromatographic isotope

effect is not caused by a vapour pressure effect but
is the result of different solutestationary phase interactions that are dominated by Van der Waals dispersion forces leading to an earlier elution of the heavier
isotopomer [18]. This difference in chromatographic
solutestationary phase interaction is caused by lower
molar volume of the heavier isotopomer. The reason
for the decrease in molar volume is the increased
bond strength and thus shortened bond length between 13 CH and to a lesser degree, 12 C13 C, 12 CH
and 12 C12 C, respectively. If the chromatographic isotope effect were indeed the result of a vapour pressure effect, one would expect the lighter isotopomer
of a given compound to elute more rapidly from the
GC column because of its higher vapour pressure and
hence, lower boiling point as compared to the heavier
isotopomer.
2.1.2. Isotopic calibration during GC/CIRMS
analysis
Owing to its unique design, it is not possible in
GC/CIRMS to calibrate target compounds against a

standard of known isotopic composition, introducing

the standard in exactly the same way as the analyte.
There are only three feasible means of introducing a
standard: (a) addition of reference compounds to the
sample; (b) introduction of reference gas pulses into
the carrier gas stream; or (c) introduction of reference
gas pulses directly into the ion source.
The results of an extensive study into methods of
isotopic calibration by Merritt and co-workers emphasised these demands [21,22]. Comparing the use
of internal reference compounds with the introduction of reference gas pulses directly in the ion source
of the IRMS, Merritt and co-workers found an offset
of >2 between the two methods due to incomplete
combustion and other systematic errors affecting only
the analytes. These systematic errors affected both
the analytes and the co-injected reference compounds
but were of course not compensated for by external
reference gas pulses. Other groups reported similar
observations [13,20,23,24]. In the absence of such
systematic errors, Merritt and co-workers found that
both methods of isotopic calibration gave consistent
results as long as multiple reference peaks were used

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

to permit drift correction. Using only one reference

point, such as methylundecanoate (Me11:0) for internal isotopic calibration of fatty acid isotope analysis
is not adequate to compensate for the influence of
GC parameters, such as analytestationary phase interaction or column temperature on measured isotope
abundance [20].
There are several stages during GC/CIRMS analysis where mass discrimination and hence, isotopic
fractionation can occur. Closer inspection identifies
seven potential sources.
1. Isotopic fractionation during sample injection
(which can be overcome by on-column or timeprogrammed splitless injection).
2. Chromatographic isotope effect.
3. Chromatographic peak distortion (leading and trailing peaks).
4. Combustion process.
5. Peak distortion of CO2 gas peak during passage of
the combustion interface.
6. Changing flow conditions at the open split prior to
the IRMS.
7. The IRMS itself.

Obviously, the external reference gas pulses only

compensate for factor (7), whereas internal reference
compounds reflect all of the aforementioned. Recently,
a method for isotopic calibration was reported that,
provided a combustible gas was used, could reflect
the systematic errors caused by factors (4)(7). This
method combines the convenience and practicability
of external reference gas calibration with the advantage of reflecting the majority of physical influences to
which analytes are subjected in a GC/CIRMS system
[25]. Introducing reference gas pulses this way is the
only way to meet the condition for reference peaks to
match samples peaks in shape and size [13] in a very
practical fashion (Fig. 3).
2.2. Sample treatment by liquid chromatography
One could be forgiven for assuming organic compounds with a molecular weight above 150 amu are
less prone to isotopic fractionation than gases and
volatile organic compounds. However, there is evidence to the contrary. Hofmann et al. observed
a shift of 15 N-values to more negative values of

Fig. 3. Ion chromatogram of mass trace m/z 44 of fatty acids (as FAMEs) from the US National Institutes of Health fatty acid standard
NIH-C. Peaks marked with R are CO2 reference peaks of 13 s pulse widths introduced using the reference gas inlet module described in
reference [25].

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

approximately 2 for amino acids that were isolated by ion-exchange chromatography [26]. Caimi
and Brenna reported that the leading edge of the
HPLC peak of methylpalmitate (Me16:0) showed
13 C-enrichment relative to the parent material, while
the tail of the peak was slightly depleted in 13 C
[24]. These observations show that quantitative peak
collection of the entire LC peak is important for accurate isotope ratio analysis. Hence, LC techniques,
including solid phase extraction, must be applied with
caution when used for sample preparation or sample
clean up of complex mixtures intended for isotope
ratio analysis.
Any LC technique involves a form of two-phase partitioning where differences in solutestationary phase
interaction will lead to mass discrimination and hence,
isotopic fractionation. Very recently, Filer presented
a comprehensive overview of isotopic fraction during
chromatography [27].

69

ready-to-use silylation reagents do not achieve complete derivatisation, the addition of a base promoting
the reaction, such as pyridine or triethylamine helps
to achieve quantitative derivatisation.
However, silylation should be regarded as a last
resort when it comes to sample preparation for
GC/CIRMS. In the case of unsubstituted FFAs,
derivatisation with TMS adds three additional carbon
atoms to the molecule of interest, thus changing its
13 C value. That said, unless the true isotopic composition of the target compound(s) must be known,
changes of the 13 C/12 C isotope ratio due to derivatisation are usually a minor consideration. In most
biochemical applications, such as in vivo or in vitro
tracer studies the interest is focused on changes in
isotopic enrichment rather than the absolute values.
This is easily determined since measurements are
compared against control samples, collected prior to
tracer administration, which have undergone the same
sample preparation procedure.

2.3. Sample derivatisation

When derivatisating a sample prior to CSIA, one
should bear in mind the various ways this can influence
the analysis and its results: (i) isotopic fractionation
due to kinetic isotope effects during derivatisation;
(ii) change of isotopic composition in the target compound(s) due to derivatisation agent(s), e.g. tracer
dilution; (iii) gas chromatographic consequences; (iv)
potential adulteration of isotope ratio measurement
caused by non-quantitative sample conversion.
Of course, one way of avoiding the problems with
derivatisation is not to derivatise the sample at all.
For free fatty acids (FFAs), this approach involves the
use of moderately polar to polar stationary phases and
high-temperature GC. However, high-temperature capability of polar stationary phases is limited even when
oxygen free helium is used as carrier gas. In the following, the two most commonly used derivatisation
methods for fatty acids are briefly mentioned.
2.3.1. Trimethylsilylation
Silylation agents still enjoy considerable popularity for derivatisation of polar functionalities, such as
carboxyhydroxyl groups (COOH) of FFAs. Derivatisation introducing TMS groups is usually quantitative, reaction conditions are relatively mild and the
reagents can be used off the shelf. In cases where the

2.3.2. Methylation
Methylation is the standard derivatisation method
for carboxylic acids in general and for fatty acids
in particular. With the commercial availability of
BF3 methanol complex (14% (w/v)), there is little or
no need to use the cumbersome HClmethanol and
HBrmethanol solutions. BF3 is a Lewis acid that
catalyses the esterification with methanol just as well
as HCl or HBr. Its advantage lies in its high volatility and the virtual absence of corrosive properties.
Most importantly, only one carbon atom is added thus
minimising changes to the 13 C value of the parent
compound.
2.4. High resolution capillary gas
chromatography
HRcGC is a prerequisite for CSIA of LCFAs and
is often associated with HTcGC [28,29]. It is often inferred that HTcGC is limited to the use of
cross-bonded apolar stationary phases, such as SE 30
and 52 and therefore, only of limited applicability.
This assumption is mainly based on the maximum
allowable operating temperature (MAOT) given by
GC column manufacturers. However, most stationary phases, except for polyethylene glycol (PEG)
based ones, can be safely used up to 360 C (apolar

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

Table 1
Commonly used combinations of derivatisation and GC methods for CSIA of fatty acids
Derivatisation

Typical GC conditionsa

TMS
TMS
TMS
TMS
Methylation
Methylation
Methylation
Methylation
Methylation
Methylation
Methylation
Methylation
Methylation
Methylation

BP1: 50 C (2 min) to 350 C at 10 C/min

CP-Sil 5CB: 50 C (2 min) to 150 C at 10 C/min, 150300 C at 5 C/min, 300 C (10 min)
HP5: 50 C (2 min) to 180 C at 10 C/min, 180350 C at 4 C/min
HT5: 70 C (2 min) to 150 C at 10 C/min, 150290 C at 5 C/min, 290 C (5 min)
DB1: 50 C (2 min) to 350 C at 10 C/min
CP-Sil 5CB: 40 C (2 min) to 200 C at 10 C/min, 200300 C at 3 C/min, 300 C (15 min)
CP-Sil 8CB: 130 C (2 min) to 300 C at 5 C/min
SP2380: 160 C (1 min) to 230 C at 20 C/min
DB23: 100160 C at 4 C/min, 160195 C at 2 C/min, 195 C (28 min)
BPX70: 80 C (2 min) to 200 C at 5 C/min
BPX70: 80 C (1 min) to 200 C at 4 C/min
BPX70: 40 C (2 min) to 200 C at 4 C/min
BPX70: 130 C (1 min) to 180 C at 3 C/min, 180200 C at 4 C/min, 200210 C at 1 C/min
Supelcowax 10: 80 C (4 min) to 205 C at 25 C/min, 205225 C at 0.3 C/min

a The information given in these columns should be taken as generalised guidelines providing a starting point for the interested reader
to resolve individual analytical problems.

phases up to 450 C) if certain precautions are observed. (i) To avoid damage to the stationary phase
at high temperatures, the carrier gas must be free
of oxygen and moisture traces. Considering the cost
for replacing a GC column, the investment in a high
capacity gas purifier will soon have paid off. (ii) It
is a remarkably little known fact that polar stationary phases are light sensitive [30]. Even exposure to
indirect daylight or light from fluorescent tubes will
lead to column deterioration. The first sign of this
happening is usually a dramatic increase in column
bleed. (iii) Conditioning the column properly prior
to high-temperature usage will minimise normal column bleed (e.g. at 4 C/min to 300 C; maintain at
300 C for 5 h; repeat but program to 360 C and
maintain at 360 C for 2 h). (iv) If a polar phase is to
be used for HTcGC, a polysiloxane-based phase with
a high cyanopropyl content should be chosen since
cyanopropyl-substituted polysiloxane phases are more
stable and inert than phenyl substituted phases. Grob
suggested that this might be caused by high surface
tension of phenyl substituted phases, which in turn
causes problems with film stabilisation during column
coating [30].
To obtain the sharp peak shapes associated with
HRcGC, consideration should be given to the following practises. As mentioned before, due to the risk of
mass discrimination during injection, samples should
be injected in splitless mode. However, to avoid peak

broadening and peak tailing, the split should be opened

10 s after injection. Use of a retention gap (also referred to as pre- or guard-column) will prolong the lifetime of the GC column and improve chromatographic
performance. The retention gap (a deactivated piece of
fused silica capillary tubing, typically 0.63 m long)
re-focuses the injected sample thus leading to sharper
peaks.
Typical GC conditions for the analysis of fatty acids
are given in Table 1 and details of GC stationary
phases and commercial GC columns are summarised
in Table 2.
3. 13 C isotope analysis of fatty acids at natural
abundance level
High-precision CSIA of 13 C isotopic abundance at
both natural abundance and low enrichment levels
yields measurements of 13 C values with a precision
of 0.3 or better. Due to this high precision, even
minute changes in the 13 C isotopic composition can
be reliably detected. These minute changes are caused
by kinetic isotope effects associated with enzyme mediated biochemical reactions. The resulting variation
in the natural abundance of 13 C can be as high as
0.1 AME [1]. This wide range reflects the varying degree of mass discrimination associated with the different pathways of carbon assimilation and fixation but

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

71

Table 2
GC columns and stationary phases used for fatty acid analysis
Commercially available GC columns

Corresponding stationary phasesa and composition

Suitable for

DB-1, CP-Sil 5CB, BP1,

Rtx-1, HP Ultra-1,
DB-5, CP-Sil 8CB, BP5, Rtx-5,
HP Ultra-2, HP5, HT5
DB-1701, CP-Sil 19CB, BP10,
Rtx-1701, HP-1701
DB-225, CP-Sil 43CB, BP225,
HP-225, Rtx-225
DB-Wax, CP-Sil 52CB, BP20,
HP-Wax, HP-20M,
Stabilwax
DB-FFAP, CP-Wax 58CB,
BP21, HP-FFAP
BPX70

SE 30: apolar 100% polymethylsiloxane,

0% other functional groups
SE 52 (SE 54)b : apolar 5% phenyl

LCFAs, both as methyl esters or

TMS-derivatised
LCFAs, both as methyl esters or
TMS-derivatised
FAMEs

CP-Sil 88CB, Supelcowax 10

OV-1701: moderately polar 7%

cyanopropyl, 7% phenyl
OV-225: moderately polar 25%
cyanopropyl, 25% phenyl
Carbowax: polar PEG

OV-351: polar PEG nitroterephthalic

acid ester
Silar 7CP: highly polar 75%
cyanopropyl, 25% phenyl
Silar 10C: highly polar 100% cyanopropyl

FAMEs
FAMEs

FFAs and FAMEs

FAMEs
FAMEs

a Stationary phases are listed in order of increasing polarity from top to bottom. For polymethylsiloxane-based stationary phases, only
the percentage of functional groups other than methyl is given.
b Strictly speaking, SE 54 is not the same as SE 52. However, since there are no commercially available columns coated with 5%
phenyl, 1% vinyl polymethylsiloxane, it is usually listed together with SE 52 equivalents (5% phenyl polymethylsiloxane).

can also reflect different growing conditions due to

climatic and geographic differences.
Differences in 13 C values were reported for total
leaf tissue, total surface lipid extracts and individual
n-alkanes isolated from plants utilising either C3 , C4
and Crassulacean acid metabolism (CAM) pathways
for carbon fixation [31,32]. The average 13 C values
obtained from C3 plant tissues are typically 1015
lower than the 13 C values of C4 and CAM plants. Although the vast majority of plants (>300,000) belongs
to the C3 group with 13 C values of <24, differences in enzyme kinetics of biochemical pathways
(mainly caused by environmental factors, such as climate and geographical location) result in subtle variations in the 13 C signature at natural abundance level
of bioorganic compounds, such as fatty acids. The information locked into organic molecules due to these
kinetic isotope effects provides valuable information
on origin, authenticity and food webs, to name but a
few, and can only be accessed by GC/CIRMS using
HRcGC techniques.
3.1. Authenticity and origin
High-quality, single source vegetable oils are a
target for fraudulent adulteration, i.e. partial or total

substitution of minor quality and hence, cheaper

oils for the high quality product. In a blind study,
Woodbury et al. were able to detect the adulteration
of maize germ oil with oils of C3 plant origin down
to a level of 5% (w/w) [33]. They found the saturated
16:0 fatty acid in maize oil to be more depleted in 13 C
than the unsaturated fatty acids (18:1 and 18:2) from
the same oil. In addition, consistent differences were
observed for 13 C values of vegetable oils from the
same plant species but from different geographical
regions. For example, the major fatty acids in maize
oil (16:0, 18:1 and 18:2) from Argentinean maize
showed on average +2 higher 13 C values than
those from maize grown in Italy. In subsequent work,
Woodbury et al. determined fatty acid composition
and 13 C values of the major fatty acids of more than
150 vegetable oils [34], thus establishing a database
that provides isotopic information for authenticity
control of vegetable oils. Variability in 13 C values
could be related to geographical origin, year of harvest and the particular variety of oil. Their findings
suggest that ultimately 13 C values of fatty acids are
determined by a combination of environmental and
genetic factors. Employing position-specific hydrolysis with pancreatic lipase to study fatty acids from
different positions on the glycerol backbone, they

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

could also demonstrate 13 C-isotopic identity of fatty

acids irrespective of their position on the glycerol
backbone [35].
Kelly et al. investigated authenticity of single seed
vegetable oils of C3 plant origin, such as groundnut,
palm, rapeseed and sunflower oils [36]. They found
that the 13 C values for the authentic vegetable oil
fatty acids fell within a narrow range of 27.6 to
32.1. Employing canonical discriminant analysis,
13 C data from sunflower oil could be separated from
other oils, exploiting small yet significant differences
in 13 C values within the oil varieties. To detect adulteration of olive oils, Angerosa et al. compared 13 C
values of the aliphatic alcoholic oil fractions and found
those of the adulterant pomace oil to be significantly
more negative than those of virgin and refined olive
oils [37]. In a subsequent study, Angerosa et al. employed both 13 C and 18 O isotope analysis to determine
geographical origin of olive oils according to climatic
regions from different Mediterranean countries, such
as Greece, Morocco and Spain [38].
Apart from blending high quality vegetable oils
such as virgin olive oil with olive oil of lower quality (such as pomace olive oil) or lower cost oils (such
as rapeseed oil), thermally induced degradation due
to deodorisation or steam washing can alter the 13 C
isotopic signature of the whole oil and its key fatty
acid components [39]. Other factors found to influence both 13 C isotopic signature and relative fatty
acid composition of olive oils are vintage (year of
production), storage period (oxidation of unsaturated
fatty acids), botanical species and maturity at harvest
[40,41].
In a study to characterise wild and farmed salmon,
fish oils and lipid extracts from fish muscle were subjected to a statistical analysis of their respective fatty
acid compositions. In addition, overall 2 H/1 H (D/H)
and 13 C/12 C isotope ratios and molar fractions of the
isotopomeric deuterium clusters were determined to
distinguish the different groups of salmons studied
[42].
3.2. Origin in the context of archaeology and diet
Measuring 13 C/12 C isotope ratios has become an
increasingly important tool to glean information on
prehistoric diet and lifestyle from organic residues
preserved in archaeological artefacts. Employing both

HTcGCMS and GC/CIRMS to identify chemical

structures and measure 13 C values, respectively,
Evershed et al. showed that lipid extracts (C25 C33
alkanes and a C29 ketone, nonacosain-15-one) from
organic residues found in archaeological potsheds
were derived from Brassica species (wild-type cabbage) [43]. Later work on organic residues from
archaeological pottery vessels revealed C31 , C33 and
C35 ketones with 13 C values that were up to 10 less
depleted than those found for the C29 ketones from
wild-type Brassica species. Based on HTcGCMS and
GC/CIRMS data, Evershed et al. formed the hypothesis that a precursorproduct relationship may exist between C35 ketones and fatty acids, and corresponding
triacylglycerols, such as tripalmitin and tristearin from
animal fats [44]. Studying pyrolysis reactions of acyl
lipids and monitoring their products by HTcGCMS
and GC/CIRMS, they confirmed that the C31 , C33
and C35 mid-chain ketones found in archaeological
pottery vessels indeed derived from a mixture of FFAs
[28]. Comparing carbon number distributions of triacylglycerols and 13 C values of 16:0 and 18:0 acyl
moieties of lipid compounds from extracts of neolithic
vessels with modern reference animal fats, Evershed
et al. could identify animal fat residues found in vessels dated circa 4200 BP as being close to reference
pig adipose fats, whereas residues found in vessels
dated circa 4500 BP were closer to reference ruminant
fat [45].
Another insight into prehistoric life was gleaned
from the 13 C analysis of adsorbed lipids preserved
in the fabric of Minoan lamps and conical cups. The
13 C value of palmitic acid together with HTcGCMS
profiles of saturated fatty acids 24:0 to 34:0 identified
beeswax as the illuminant burned in prehistoric Aegan lamps rather than olive oil as hitherto supposed
[46]. Through investigation of lipid residues in ceramic vessels via GC, GCMS and GC/CIRMS, saturated carboxylic acids in the range C12 C18 were
detected (with an unusually high abundance of C12 )
from vessels from the Nubian site of Qasr Ibrim [47].
This finding was mirrored in the saturated fatty acid
distributions detected from the kernels of modern and
ancient date palm (Phoenix dactylifera L.) and dom
palm (Hyphaena thebaica L. Mart.). These results provided the first direct evidence for the exploitation of
palm fruit in antiquity and the use of pottery vessels
in its processing.

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

ODonoghue et al. reported 13 C values in the range

of 25.4 to 29.2 for the principal fatty acids (16:0
to 24:1) of radish seed found in a sixth century a.d.
storage vessel [48]. Composition and 13 C values of
fatty acids found in the ancient radish seeds matched
closely those found in modern radish seeds. For this
work, an aliquot of the total lipid extract was methylated at 70 C for 15 min using BF3 methanol. The
fatty acid methyl esters (FAMEs) were extracted into
n-hexane and were separated on a GC capillary column
coated with a highly polar stationary phase (BPX70;
Table 2).
Using porcine bone tissue from animals in a controlled feeding study, Stott et al. measured 13 C values of FAMEs Me14:0, Me16:0, Me16:1, Me18:0,
Me18:1 and Me18:2 to monitor the routing of dietary
and biosynthesised lipids. The 13 C signature of the
fatty acids extracted from bone tissue provided an accurate reflection of the C3 and C4 plant material composition of the respective diets fed to the animals [49].
The virtual identity of 13 C values for the essential
fatty acid 18:2 in both diets and tissues for both C3
and C4 fed animals confirmed direct incorporation of
dietary 18:2 into bone tissue without significant isotopic fractionation occurs in pigs. By CSIA of six
serum fatty acids from human subjects on controlled
fat diets, Rhee et al. determined their range of natural
isotope abundance and demonstrated the levelling effect of a well-controlled diet [50]. At baseline, mean
13 C values for 16:0, 16:1, 18:0, 18:1, 18:2n-6 and
20:4n-6 were 24.1, 21.7, 21.6, 25.6, 29.6 and
25.0, respectively (average standard deviation was
1.9). Most 13 C values decreased during the diet
period and appeared to have stabilised by week 5 at
25.3, 21.9, 22.3, 26.5, 30.1 and 24.5, respectively. Except for 18:2, the 13 C values of experimental diets were lower than that of serum fatty acids,
consistent with observations made in animals.
3.3. Environment and food webs
High-precision CSIA of fatty acids by GC/CIRMS
has been used to record the 13 C values of individual biomarker compounds (C20 C30 LCFA) extracted
from terrestrial and marine sediment to assess vegetation changes [51]. Ficken et al. compared lipid
biomarker distributions and their 13 C values with
those of eleven species of living plant dominant at the

73

contemporary surface of a blanket bog in the Scottish

Cairngorm Mountains. The observed lipid stratigraphy
showed only partial agreement with that calculated
using macrofossil abundance data and the lipid distributions for the living taxa, a result which reflected
the inherent uncertainties in both the lipid biomarker
and the macrofossil approaches to palaeoenvironmental stratigraphy [52].
SIA of lipid biomarkers from sediments was
also used to gain direct information about biogeochemical processes involving micro-organisms
[53,54]. Using 13 C-labelled precursors, Boschker
et al. showed acetate was predominantly consumed by sulphate-reducing bacteria similar to the
Gram-positive Desulfotomaculum acetoxidans and
not by a population of the more widely studied
Gram-negative Desulfobacter spp. Furthermore, experiments with 13 C-labelled methane suggested type
I methanotrophic bacteria dominating methane oxidation [53]. In another study, a new pulse-chase
experiment employing long-term enrichment with
[12 C]-CH4 followed by short-term exposure to
[13 C]-CH4 was used to isotopically label methanotrophs in a soil from a temperate forest. Analysis of
labelled phospholipid fatty acids (PLFAs) provided
unambiguous evidence of methane assimilation at
true atmospheric concentrations (1.83.6 ppm). High
proportions of 13 C-labelled C18 fatty acids and the
co-occurrence of a labelled, branched C17 fatty acid
indicated that a new methanotroph, similar at the
PLFA level to known type II methanotrophs, was
the predominant soil micro-organism responsible for
atmospheric methane oxidation [54].
CSIA of muscle tissue and storage lipid fatty acids
found in shrimps living in hydrothermal vent areas
of the mid-Atlantic ridge show high proportions of
PUFAs, such as 16:2, 18:2, 20:5 and 22:6. The associated 13 C signatures suggest that PUFAs 16:2 and
18:2 found in muscle tissue are derived from dietary
sources since their 13 C values of 11 to 13 are
typical of chemosynthetically fixed carbon as found
in hydrothermal vent bacteria. PUFAs 20:4 and 22:6
from neutral lipid, however, exhibit 13 C values of
15.8 to 27.1 typical for carbon of photosynthetic
origin thus indicating storage of lipids from an early
planktotrophic life stage of these shrimps [5558].
Mono-unsaturated (n-8) fatty acids in tissue samples from hydrothermal vent mussels confirmed the

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

presence of methanotrophic bacterial endosymbionts

with the highest level of mono-unsaturated fatty acids
found in gill tissues. Corresponding CSIA of fatty
acids gave results in the range of 24.9 to 34.9,
typical for the range expected for methanotrop-based
nutrition [59]. CSIA of fatty acids isolated from
mussels found in two Newfoundland estuaries indicated a dominant marine phytoplankton dietary
source [60]. The dominance of 16:0, 16:1, 20:5 and
22:6 fatty acids was consistent with the 13 C isotopic
data.
Fatty acids isolated from subcutaneous fat samples
of Texas redhead ducks, which had consumed a diet
comprised primarily of roots and rhizomes of a particular sea-grass for at least 1 month prior to sampling,
exhibited 13 C values of fatty acids more positive than
those obtained for identical fatty acids in the sea-grass.
This discrepancy in 13 C values seems to indicate that
fatty acids in ducks are synthesised from molecules
with more positive 13 C values, such as carbohydrates
and/or proteins, rather than by direct incorporation of
fatty acids from the diet [61].
CISA of fatty acids was used in conjunction
with fatty acid profiling to study symbiotic and
non-symbiotic brittlestars from Oban Bay (Scotland).
Higher percentages of 16:17 and, to a degree,
18:17 were found in brittlestar species containing
symbiotic bacteria. However, no relationship could
be established based on 13 C signatures of the fatty
acids alone [62]. Borobia et al. investigated possible
differences in the diet or metabolism of sympatric
finback and humpback whales in the Gulf of St.
Lawrence through analysis of their blubber fatty acids
[63]. They found that chemical and isotopic differences observed were consistent with a slightly lower
trophic position for humpbacks compared to finbacks
in the Gulf of St. Lawrence, reflecting a difference in
long-term, average diet. Applying CSIA of fatty acids
and fatty acid profiling, Smith et al. could distinguish
between populations of fresh- and salt-water harbour
seals [64]. The blubber of freshwater harbour seals
contained higher levels of 18:2n-6, 18:3n-3, 20:4n-6
and C18 PUFAs than blubber from marine populations with mean 13 C values of fatty acids lower (i.e.
more 13 C depleted) than those from the marine seals.
These findings were consistent with the hypothesis
that freshwater harbour seals mainly subsist on a diet
derived from freshwater species.

4.

13 C

analysis of fatty acids in tracer studies

The high sensitivity of GC/CIRMS has also been

increasingly exploited in metabolic studies investigating turnover, incorporation and biosynthetic processes
in vivo that previously could have only been investigated with stable isotope tracers at high enrichment
level using GCMS, for reasons of excessive tracer
dilution in the various metabolic pools and/or low incorporation rates. Although in these cases radioactive
tracers can provide an alternative, their use is associated with certain risks that are nowadays regarded as
ethically unacceptable especially when dealing with
members of the paediatric age group [65,66].
4.1. Tracer studies with selectively
or naturally enriched fatty acids

13 C-labelled

Using [U-13 C]-glucose as precursor (via acetate)

for palmitic acid derived from di-saturated surfactant phospholipids, fractional synthesis rate (FSR) of
palmitate from acetate in premature infants suffering
from respiratory stress syndrome was calculated to be
5.2% per day. An increase in the 13 C isotopic composition of palmitate was first detected 12.3 h after
the tracer infusion and the 13 C-enrichment increased
in a linear fashion to reach a plateau after 47 h [67].
Infusing 13 C-labelled 16:0 and 18:2, surfactant kinetics were studied in two groups of pre-term infants
with respiratory distress syndrome, with infants in
group 1 meeting treatment criteria and thus receiving exogenous surfactant, whereas infants in group
2 did not. FSRs of fatty acid phosphatidylcholines
were similar in both patient groups. However, FSR
and half-life for phosphatidylcholine palmitate were
approximately two-fold higher than those measured
for phosphatidylcholine linoleate [68].
CSIA of fatty acid by GC/CIRMS proved to be
a decisive tool in determining if and to what extent
neonates and premature infants of very low birth
weight could biosynthesise arachidonic acid, which
is essential for their growing tissues, from dietary
fatty acids. Feeding full term neonates on a formula
diet with all fat derived from maize oil, it could be
shown that on average 23% of free plasma arachidonic acid on study day 4 originated from infantile
linoleic acid conversion [54]. 13 C content of linoleic
acid and arachidonic acid in 0.250.5 ml serum was

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

determined before and during 4 days after the infants

diet contained maize oil. Baseline 13 C values for
linoleic acid and arachidonic acid were 31.5 1.1
and 30.1 1.2, respectively; after 4 days, changes
in 13 C values over baseline were +12.7 0.7 and
+2.7 0.7, respectively [69].
Carnielli et al. added linoleic acid and linolenic
acid, both 13 C-labelled, to the formula diet which
was administered continuously for 48 h (birth weight
1.17 0.12 kg; gestational age 28.4 1.3 weeks)
[70]. They could show that both tracers were rapidly
incorporated into plasma phospholipids and that their
metabolic products including arachidonic acid and
docosahexaenoic acid became highly enriched with
13 C. Incorporation of 13 C-octanoic acid into plasma
triglycerides (10% of the enrichment of the diet),
noticeably into myristic and palmitic acid, by very
low-weight pre-term infants was reported earlier by
the same group [71].
A similar observation was made during a study with
an entirely different objective. Using GC/CIRMS,
Koziet et al. demonstrated that ethanol itself may be
used as a substrate for lipogenesis, although only to
a small extent [72]. They calculated that <10% of
fatty acids contained in very-low-density lipoprotein
(VLDL) triglycerides were derived from this pathway,
with ethanol predominantly being incorporated into
myristic and palmitic acids. In a more recent study
based on the application of [1,2,3,4-13 C]-palmitate and
[1-13 C]-ethanol, it was determined that in spite of a
rise in the fractional contribution from de novo lipogenesis (DNL) to VLDL-triacylglycerol palmitate from
2 to 30%, the absolute rate of DNL represented <5%
of the ingested alcohol dose [73].
The majority of 13 C tracer studies published have
dealt with various aspects of fatty acid biochemistry,
such as metabolism of triglycerides [74,75], transport
and turnover of free saturated [7679] and unsaturated
fatty acids [8083].
4.2. Tracer studies with uniformly
fatty acids

13 C-labelled

Unlike GCMS, where increasing the amount of

label has no general effect on detection limits, in
GC/CIRMS, increasing label enrichment in precursor
compounds produces significantly improved detection
limits. In a recent review of high-precision CF-IRMS,

75

Brenna discussed theoretical and practical considerations of this approach to study fatty acid and lipoprotein metabolism in man [84]. High-precision CSIA in
these type of tracer studies has been shown to possess
advantages over organic GCMS for stable isotopic
tracer detection and to be superior to radio-isotopic
tracer methods in terms of dose size and analysis
efficiency [85]. Employing this approach, the bioequivalence of dietary -linolenic acid (18:3) and docosahexaenoic acid (22:6) as substrates for brain and
retinal n-3 fatty acid accretion in neonatal and foetal
baboons has been demonstrated [53,86,87]. Similarly,
using doses of uniformly 13 C-labelled PUFAs, it was
also shown that recycling of 18:2, 18:3 and 22:6 into
saturated and monounsaturated fatty acids is a major metabolic pathway in chow-fed Rhesus monkeys
in the perinatal period [88]. Huang et al. employed
[U-13 C]18:3n-3 in an in vitro system to measure kinetics of biosynthesis and incorporation of n-3 long-chain
PUFAs in Y79 human retinoblastoma cells [89].
Using [U-13 C]--linolenic acid, Sheaff et al. were
able to demonstrate that conversion of -linolenate
into docosahexaenoate was not depressed by high
dietary levels of linoleic acid [90]. Administering physiological doses of [U-13 C]--linolenate to
mother-reared 6-day-old rat pups, 13 C-enrichment
data were obtained for brain cholesterol, brain palmitate and brain docosahexaenoate indicating that carbon from -linolenate is not exclusively conserved
for synthesis of docosohexaenoate. Due to a high
rate of -oxidation and carbon recycling, carbon
from -linolenate is a readily accessible source for
DNL during early brain development in the suckling rat [91,92]. While studying the metabolism of
13 C-labelled PUFAs by 13 C-NMR, using GC/CIRMS
Cunnane et al. found low levels of 13 C-labelled
-linolenic acid in brain phospholipids of suckling rat
pups that could not be detected by 13 C-NMR [93].
Using bile duct-ligated rats as physiological model
and 13 C-labelled linoleic acid as tracer, Minich et al.
could demonstrate that impaired linoleic acid status
in choleastic liver disease might be mainly due to
decreased net absorption and not to alterations in
post-absorptive metabolism [94].
Hughes et al. compared lipoprotein metabolism
in normolipidemic men using 13 C-labelled palmitic
and stearic acids as tracers. The results of their study
indicated that saturated fatty acids were metabolised

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W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

in unique ways thus being not metabolically equivalent or similar [95]. An interconversion of saturated
dietary fatty acids (e.g. 18:0) into unsaturated fatty
acids (e.g. 18:1) in human plasma of about 14% was
observed by Rhee et al. [96] and simultaneous measurement of desaturase activities using [U-13 C]16:0
and [U-13 C]18:2n-6 as tracers was reported by Su
and Brenna [97]. Administering [U-13 C]-linoleic acid
as 3-oleyl-1,2-[U-13 C]-linoleyl glycerol, Scrimgeour
et al. showed that a diet rich in trans--linolenic
acid did not inhibit the conversion of linoleic acid to
dihomo--linolenic and arachidonic acid in healthy
middle-aged men [98]. A single bolus of 45 mg of
[U-13 C]-linoleic acid (dissolved in olive oil) was sufficient to monitor oxidation but not conversion of
linoleic acid into longer-chain PUFAs. Due to the
low tracer/tracee ratio for arachidonic acid, it was
concluded that studying linoleic acid conversion into
longer-chain PUFAs would require a higher dose [99].
Contribution of direct dietary uptake of long-chain
PUFAs as well as their endogenous conversion into
other PUFAs to the total PUFA secretion into milk
of lactating women has been studied by employing
[U-13 C]18:2 and [U-13 C]22:6 in small doses (e.g.
1 mg/kg body weight) [100102]. Fatty acid composition of milk and their corresponding 13 C-enrichment
was assessed by GC/CIRMS. It has thus been shown
that 30% of milk linoleate is directly transferred from
the diet, whereas only a small amount (0.11.2%)
of milk arachidonic acid originates directly from
endogenous conversion of dietary 18:2.

5. Outlook
In recent years, the research efforts of different
groups working in the field of GCIRMS have focused
on extending the scope of on-line CSIA towards the
measurement of 18 O/16 O and 2 H/1 H isotope ratios
of organic compounds. In addition, hyphenated hybrid
systems have been developed that enable CSIA while
at the same time recording a conventional mass spectrum of the target compound to aid its unambiguous
identification [103106].
Another hyphenated technique for position-SIA
(PSIA) of 13 C-labelled Me16:0 using an on-line pyrolysis system was described in detail for the first
time by Corso and Brenna [107]. They coupled a GC

(GC-1) for sample separation prior to pyrolysis to the

GC (GC-2) separating pyrolytic products of the selected sample compound. Furthermore, they installed
a valve into GC-2 to permit separated pyrolysis fragments to be admitted to an organic MS for structure
analysis of these fragments.
Extending the potential of using stable isotopic signatures, Breas et al. studied the relationship between
18 O isotopic abundance in fatty acids from various
vegetable oils and their geographical origin [108].
In a first step towards on-line measurement of 2 H
isotope signatures of organic compounds, Prosser and
Scrimgeour coupled a high mass dispersion IRMS to a
GC via a pyrolysis interface including a 5 molecular
sieve PLOT column to achieve CSIA for 2 H of fatty
acids [109]. Employing this instrumental set-up, 2 H
values for 16:0, 18:1 and 22:6 fatty acids (as methyl
esters) from tuna oil were reported as 148.5 4.1,
155.3 1.0 and 147.7 1.2 (versus VSMOW),
respectively [110]. This GC/PyIRMS system has also
been applied to the measurement of lipid synthesis in
humans, using deuterium oxide (D2 O) incorporation
into fatty acids. Administering safe deuterium enrichments (<0.5 APE), the labelled fatty acids did not contain more than one deuterium per molecule of fatty
acid and show similar chromatographic behaviour to
natural abundance samples. Following overnight incorporation of D2 O, plateau palmitate enrichments
were measured by GC/PyIRMS with a relative standard deviation of 0.5% [110].
Undoubtedly, with the commercial availability of
CF-IRMS instruments capable of on-line sample conversion for 2 H and 18 O isotope analysis, 2 H and 18 O
measurements of fatty acids will experience the same
uptake as 13 C measurements 10 years ago when the
first instruments for on-line 13 C isotope analysis came
on the market. According to the Science Citation
Index, in 1991 seven papers were published dealing
with isotope analysis of fatty acids. Within 8 years,
the annual number of such publications had increased
by 500%.
A similar trend can be expected for 2 H and 18 O
measurements of fatty acids although one area of applications in which 13 C measurements have been
such a success will for the time being not benefit from
the new technology. The area in question is the field
of multi- or uniformly 2 H-labelled fatty acids. Due to
the strong chromatographic isotopic effect, strongly

W. Meier-Augenstein / Analytica Chimica Acta 465 (2002) 6379

deuterated fatty acids elute many seconds before the

undeuterated homologue, an effect current software
packages used for peak detection and calculation of
isotope peak ratios are not equipped to cope with. Furthermore, productprecursor relationship studies with
perdeuterated precursors will lead to a product comprised of more than just two isotopomers thus complicating the detection, measurement and calculation of a
single 1 H/2 H ratio for a given compound even further.
Due to these at present insurmountable complications, 2 H measurements of fatty acids will be
restricted to determining differences at natural abundance level and to tracer studies in which the labelled
fatty acids will not contain more than one or two deuterium per molecule of fatty acid. However, even at
this level such measurements will increase the already
high discriminatory power of compound specific SIA
still further by enabling scientists to characterise a
given fatty acid using three different two-dimensional
isotopic plots plus one three-dimensional plot.
6. Conclusions

[2]
[3]
[4]
[5]

[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]

High-precision CSIA of fatty acids by GCIRMS

at natural abundance and low enrichment levels is a
powerful tool that provides quantitative information,
such as bioavailability, assimilation, turnover, incorporation and metabolism in biological and ecological
systems. Provided care is taken to avoid all potential sources of mass discrimination, GCIRMS yields
insights into complex biological systems offering answers to biochemical, physiological and environmental questions that cannot be obtained with any other
analytical instrumentation.

[19]

[20]
[21]
[22]
[23]
[24]

Acknowledgements
[25]

The author gratefully acknowledges financial

support through the Dietmar-Hopp-Foundation for
Medical Research and Studies (Walldorf, Germany).
Many thanks are due to Dr. Helen F. Kemp for critical
reading of the manuscript.

[26]

[27]
[28]

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