High-Precision Position-Specific Isotope Analysis of 13C/ 12C in Leucine and Methionine Analogues

Article abstract of DOI:10.1021/ac0344889

We report an automated method for high-precision position-specific isotope analysis (PSIA) of carbon in amino acid analogues. Carbon isotope ratios are measured for gas-phase pyrolysis fragments from multiple sources of 3-methylthiopropylamine (3MTP) and isoamylamine (IAA), the decarboxylated analogues of methionine and leucine, using a home-built gas chromatography (GC)-pyrolysis-GC preparation system coupled to a combustion-isotope ratio mass spectrometry system. Over a temperature range of 620-900 °C, the characteristic pyrolysis products for 3MTP were CH₄, C ₂H₆, HCN, and CH₃CN and for IAA products were propylene, isobutylene, HCN, and CH₃CN. Fragment origin was confirmed by ¹³C-labeling, and fragments used for isotope analysis were generated from unique moieties with >95% structural fidelity. Isotope ratios for the fragments were determined with an average precision of SD(δ¹³C) < 0.3‰, and relative isotope ratios of fragments from different sources were determined with an average precision of SD(Δδ¹³C) < 0.5‰. Δδ¹³C values of fragments were invariant over a range of pyrolysis temperatures. The Δδ¹³C of complementary fragments in IAA was within 0.8‰ of the Δδ¹³C of the parent compounds, indicating that pyrolysis-induced isotopic fractionation is effectively taken into account with this calibration procedure. Using Δδ ¹³C values of fragments, Δδ¹³C values were determined for all four carbon positions of 3MTP and for C1, C2, and the propyl moiety of IAA, either directly or indirectly by mass balance. Large variations in position-specific isotope ratios were observed in samples from different commercial sources. Most dramatically, two 3MTP sources differed by 16.30‰ at C1, 48.33‰ at C2, 0.37‰ at C3, and 5.36‰ at C(methyl). These PSIA techniques are suitable for studying subtle changes in intramolecular isotope ratios due to natural processes.

Full text of DOI:10.1021/ac0344889

Anal. Chem. 2003, 75, 5495-5503
Hig h -P re c is io n P o s it io n -S p e c ific Is o t o p e An a lys is
1
3
1 2
o f C/ C in Le u c in e a n d Me t h io n in e An a lo g u e s
Ga vin L. Sa c ks a nd J . Thom a s Bre nna *
Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853
We report an automated method for high-precision posi-
tion-specific isotope analysis (P SIA) of carbon in amino
acid analogues. Carbon isotope ratios are measured for
gas-phase pyrolysis fragments from multiple sources of
lies in the development of automated position-specific isotope
analyses (PSIA) for intramolecular analysis.
We have previously suggested that intramolecular isotope
ratios should be sensitive to the physiology of the organism that
synthesizes them and that isotope ratios are potentially of wide
3
-methylthiopropylamine (3MTP) and isoamylamine (IAA),
the decarboxylated analogues of methionine and leucine,
using a home-built gas chromatography (GC)-pyrolysis-
GC preparation system coupled to a combustion-isotope
ratio mass spectrometry system. Over a temperature
range of 6 2 0 -9 0 0 °C, the characteristic pyrolysis prod-
3
utility as markers of physiological state. Intramolecular isotope
ratios should be especially sensitive to shifts in metabolism
because isotope fractionation is localized to carbon sites associated
with the rate-determining step of a particular metabolic process.
Position-specific studies can therefore focus on the atomic posi-
tions that show the greatest susceptibility to isotope fractionation
without dilution from the rest of the molecule. PSIA studies also
offer an extended source of isotopic information since the isotope
ratio at each site is a composite of fractionations associated with
elementary (bio)chemical steps involved in the synthesis and
degradation of molecules.
ucts for 3 MTP were CH
for IAA products were propylene, isobutylene, HCN, and
CH
CN. Fragment origin was confirmed by 1 3 C-labeling,
4 2 6 3
, C H , HCN, and CH CN and
3
and fragments used for isotope analysis were generated
from unique moieties with >9 5 % structural fidelity.
Isotope ratios for the fragments were determined with an
average precision of SD(δ1 3 C) < 0 .3 ‰ , and relative
isotope ratios of fragments from different sources were
determined with an average precision of SD(∆δ1 3 C) <
Over several years, strategies based on isotope ratio mass
4
5
spectrometry (IRMS) and on NMR have been introduced for
high-precision intramolecular analysis. We have previously de-
scribed a system that coupled a tandem gas chromatograph (GC)
0
.5 ‰ . ∆δ1 3 C values of fragments were invariant over a
1
3
range of pyrolysis temperatures. The ∆δ C of comple-
2
to a pyrolysis furnace (Py). Fragments were converted to CO in
mentary fragments in IAA was within 0 .8 ‰ of the ∆δ1 3
C
a combustion furnace, and characterized isotopically by IRMS.
Using this GC-Py-GCC-IRMS system, we have presented results
exploiting pyrolytic fragmentation for PSIA of several classes of
of the parent com pounds, indicating that pyrolysis-
induced isotopic fractionation is effectively taken into
account with this calibration procedure. Using ∆δ1 3
C
4
6
values of fragments, ∆δ1 3 C values were determined for
compounds, including fatty acid methyl esters, fatty alcohols,
7
and n-alkanes. We have shown with labeling studies that open
tube pyrolysis of these compounds does not scramble carbon
positions while also producing high-precision isotope ratios of
stabilized neutral fragments. Yamada et al. have recently extended
this work to PSIA studies of acetate.8
all four carbon positions of 3 MTP and for C1 , C2 , and
the propyl moiety of IAA, either directly or indirectly by
mass balance. Large variations in position-specific isotope
ratios were observed in samples from different com-
mercial sources. Most dramatically, two 3 MTP sources
differed by 1 6 .3 0 ‰ at C1 , 4 8 .3 3 ‰ at C2 , 0 .3 7 ‰ at C3 ,
and 5 .3 6 ‰ at C(methyl). These P SIA techniques are
suitable for studying subtle changes in intramolecular
isotope ratios due to natural processes.
The amino acids are attractive candidates for high-precision
carbon isotope analysis due to their ubiquity as protein building
blocks and their myriad functions in metabolic processes. Carbon
isotope signatures in amino acids have been used to investigate
the origin of organic material in meteorites,9 the symbiotic
High-precision isotope ratio analysis is presently used across
the natural sciences for an increasing array of applications. Bulk
1
(3) Brenna, J. T. Rapid Commun. Mass Spectrom. 2 0 0 1 , 15, 1252-1262.
(
4) Corso, T. N.; Brenna, J. T. Proc. Natl. Acad. Sci. U.S.A. 1 9 9 7 , 94, 1049-
053.
13
15
stable isotope analysis (BSIA) of δ C and δ N in complex natural
1
solids and compound-specific isotope analysis (CSIA) of volatile
compounds are automated and routine; the analytical frontier now
(5) Zhang, B. L.; Buddrus, S.; Martin, M. L. Bioorg. Chem. 2 0 0 0 , 28, 1-15.
(6) Corso, T. N.; Lewis, P. A.; Brenna, J. T. Anal. Chem. 1 9 9 8 , 70, 3752-3756.
2
(
7) Corso, T. N.; Brenna, J. T. Anal. Chim. Acta 1 9 9 9 , 397, 217-224.
8) Yamada, K.; Tanaka, M.; Nakagawa, F.; Yoshida, N. Rapid Commun. Mass
Spectrom. 2 0 0 2 , 16, 1059-1064.
(
*
Corresponding author. E-mail: jtb4@cornell.edu. Phone: (607) 255-9182.
Fax: (607) 255-1033.
(9) Engel, M. H.; Macko, S. A.; Qian, Y.; Silfer, J. A. In Life Sciences and Space
Research XXV (4); Cogoli, A., et al., Eds.; Committee on Space Research;
Pergamon: New York, 1994; Vol. 15, pp 99-106.
(
(
1) Asche, S.; Michaud, A. L.; Brenna, J. T. Curr. Org. Chem., in press.
2) Meier-Augenstein, W. J. Chromatogr., A 1 9 9 9 , 842, 351-371.
1
0.1021/ac0344889 CCC: $25.00 © 2003 American Chemical Society
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5495
Published on Web 08/30/2003

the pyrolysis of four different commercial sources of the analogues
and use these values to show intramolecular isotope ratio differ-
13
ences (δ∆ C) between the sources.
EXPERIMENTAL SECTION
Fig u re 1 . Structures of 3MTP (I) and IAA (II).
GC-P y-GCC-IRMS System and Data P rocessing. The on-
line pyrolysis system consists of a home-built GC-pyrolysis-GC
interface coupled to a continuous-flow combustion/ high-precision
IRMS, described in detail elsewhere. Briefly, two commercial
capillary GCs (GC-1, HP 5890, Palo Alto, CA; GC-2, Varian, Inc.
1
0
relationship between plankton and algae, and the role of
4
microbes in soil.11 The earliest PSIA study was performed on
amino acids12 and used off-line decarboxylation to demonstrate
that the carboxyl position of amino acids is enriched with respect
to the rest of the molecule and that the degree of enrichment
depends on the organism from which it is derived.
Pyrolytic decomposition proceeds primarily by free-radical
chain mechanisms, which often lead to fragment patterns funda-
mentally different from the electron impact mass spectra of the
same compounds.13 Hydrocarbon pyrolysis is described well by
modified Rice theory,14 which predicts an initial bond cleavage to
form free radicals, followed by propagation and stabilization by
the loss of neutral olefins to form smaller free radicals, or
termination by the recombination of two free radicals. For
intramolecular comparisons, pyrograms should ideally contain
fragments with well-defined origins. Thus, the pyrolysis temper-
ature is chosen not only to maximize signal level but also to
produce characteristic pyrograms which contain fragments that
arise from specific locations. We use the term structural fidelity
to describe fragments that originate from unique positions in the
parent molecule. Our previous work has shown that methyl
palmitate labeled in the methyl and terminal carbons shows no
pyrolysis-induced rearrangement. However, that work was con-
cerned primarily with long-chain fragments (C , n >3). Small
fragments such as CH may result from either primary or
secondary fragmentation and, therefore, are more likely to be
formed from multiple sites within the parent molecule. Method
development for PSIA involving small fragments requires optimal
chromatography, fragment identification, and confirmation of
structural fidelity.
3
400, Walnut Creek, CA) were linked via a pyrolysis reactor. The
sample was injected, splitless, via an autosampler (Varian 8200)
into GC-1, and components were separated on a 30 m × 0.32 mm
×
5.0 µm DB-1 capillary column (cross-linked dimethylpolysilox-
ane; Agilent Technologies, Palo Alto, CA). The initial GC-1 oven
temperature was 100 °C, held for 5 min, ramped to 200 °C at 20
°C/ min, and held for 20 min. The head pressure was set at 25
psi, resulting in a flow velocity through GC-1 and GC-2 of 30 cm/
s. The GC-1 column was connected to an automated rotary valve
(
Valco, Houston, TX), which led to either (a) a flame ionization
detector (FID), for determining retention times, or (b) the
pyrolysis-GC system. Using the FID retention times, the rotary
valve was programmed to select a single component for pyrolysis
from the eluent stream.
The pyrolysis reactor consisted of a single piece of 0.32-mm
fused-silica capillary tubing positioned such that effluent entered
from the GC-1 rotary valve, traveled sequentially through a heated
transfer line, a 20-cm-long pyrolysis furnace zone, and finally
another heated transfer line into GC-2, where it connected to a
second capillary column. The fused silica was anchored to a
ceramic tube inside the pyrolysis furnace for mechanical support.
The pyrolysis zone was resistively heated by a Fibercraft furnace
(Thermcraft, Winston-Salem, NC), and the temperature was
controlled to (0.5 °C by a CN9000A series temperature controller
(Omega Engineering, Stamford, CT).
Following pyrolysis, fragments were directed to GC-2 for
separation by a 30 m × 0.32 mm × 1.5 µm GS-CarbonPLOT
column (Agilent). The GC-2 oven was held at 30 °C for 2-4 min
past the retention time of the analyte to capture all pyrolysis
products. The oven was ramped to 70 °C at 50 °C/ min, ramped
to 90 °C at 3 °C/ min and held for 5 min, ramped to 140 °C at 50
°C/ min, and finally, ramped to 200 °C at 5 °C/ min and held for 5
min. After the fragments were separated, they were directed by
a second rotary valve for either isotopic or molecular analysis.
For isotopic analysis, the fragments entered a home-built combus-
tion furnace/ Nafion water trap interface coupled via an open split
to a Finnigan-MAT 252 (Bremen, Germany) operating in high-
stability mode for high-precision isotope analysis. The combustion
reactor consisted of a 30-cm-long, 0.5-mm-i.d. ceramic tube, packed
with oxidized Cu, and resistively heated to 950 °C by a second
Fibercraft furnace. For molecular analysis, fragments entered a
Varian Saturn III QISMS ion trap operated in positive ion electron
impact mode. Ion trapping is inefficient at m/ z < 20, so the
4
n
4
Amino acids are nonvolatile and thus incompatible with GC
analysis. Decarboxylation is an attractive alternative to other
derivatization schemes that add exogenous carbon and would
likely interfere with the fidelity of pyrolysis fragments.15 In this
report, we develop a method for PSIA studies for the decarboxy-
lated analogues of two amino acids (Figure 1): 3-methylthiopropyl-
amine (3MTP), the analogue of methionine (I), and isoamylamine
(
IAA), the analogue of leucine (II). We describe the effects of
varying pyrolysis temperatures on the distribution and nature of
pyrolysis fragments and optimize methods for obtaining fragments
representative of specific positions in the parent compound.
1
3
Finally, we report δ CPDB values of the fragments resulting from
(
(
(
10) Uhle, M. E.; Macko, S. A.; Spero, H. J.; Engel, M. H.; Lea, D. W. Org.
Geochem. 1 9 9 7 , 27, 103-113.
11) Glaser, B.; Amelung, W. Rapid Commun. Mass Spectrom. 2 0 0 2 , 16, 891-
98.
12) Abelson, P. H.; Hoering, T. C. Proc. Natl. Acad. Sci. U.S.A. 1 9 6 1 , 47, 623-
32.
8
4
identification of CH was confirmed by retention time using a
6
(
(
13) Kelley, J. D.; Wolf, C. J. J. Chromatogr. Sci. 1 9 7 0 , 8, 583-585.
14) Rice, F. O. Collected Papers of F. O. Rice; Catholic University of America
Press: Washington, DC, 1958.
standard. Interpretation of spectra on the QISMS ion trap was
aided by the Wiley mass spectral database (Palisades, Newfield,
NY). For isotope analysis of the parent compounds (CSIA), the
column in GC-2 was replaced with a 1 m × 0.32 mm fused-silica
(
15) Zaideh, B. I.; Saad, N. M. R.; Lewis, B. A.; Brenna, J. T. Anal. Chem. 2 0 0 1 ,
7
3, 799-802.
5496 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

transfer line, and the oven was held at 200 °C. The pyrolysis
furnace was set to 300 °C, well below the threshold for observable
pyrolysis.
Ta b le 1 . S o u rc e s o f 3 MTP a n d IAA Us e d in Th is Wo rk a
source
E
compound/ manufacturer
prep
Consecutive GC-Py-GCC-IRMS runs were performed using
SAXICAB,16 a home-written LabVIEW 6i17 software for continuous-
flow IRMS automation. SAXICAB allows for automated control of
most GC-Py-GCC-IRMS parameters during a run, including
injecting the sample, triggering the GC ovens, switching the GC-1
rotary valve, setting the pyrolysis furnace temperature, and
introducing standard pulses. Data were collected from the IRMS
using high-precision NI435x data acquisition boards (National
3MTP
DL-methionine from J. T. Baker
De-CO2
(
99%; Phillipsburg, NJ)
F
L-methionine from Avocado RC, Ltd. De-CO2
(99%; Heysham, England)
G
H
L
L-methionine from Acros
99%; Geel, Belgium)
3MTP from Acros
97%; Geel, Belgium)
L-leucine from Acros
99%; Geel, Belgium)
IAA from Aldrich
99%; St. Louis, MO)
De-CO2
Used as is
De-CO2
used as is
used as is
(
(
IAA
(
M
N
(
1
3
Instruments; Austin, TX). δ Cpdb values were also calculated by
SAXICAB. We have previously demonstrated that SAXICAB
achieves accuracy and precision identical to that of the proprietary
ISODAT (Finnigan MAT; Bremen, Germany) software.16 Smaller
IAA from Acros
(99%; Geel, Belgium)
a
Abbreviations in the left-most column are used in the rest of the
paper.
4
4
peaks were integrated by curve fitting the three traces ( CO
2
,
45CO , 46CO
) to an exponentially modified Gaussian function. The
2
2
fit was optimized by the Levenberg-Marquardt algorithm using
native LabVIEW functions. Previous work has shown that curve
fitting achieves better accuracy and precision than traditional
summation methods for small18 and overlapping19 peak areas.
Larger peaks often displayed distorted peak shape, which inter-
fered with curve fitting; large peak areas were instead calculated
by the familiar method of summing the ion current over the width
of the peak. The “dynamic” summation algorithm, as outlined by
Reporting Intramolecular Isotope Ratios. The accepted
method for reporting isotope measurements in CSIA and BSIA
studies is to compare the isotope ratio of the analyte to the isotope
ratio of a standard. For carbon studies, analytes are indirectly
calibrated against PeeDee Belemnite (PDB) and expressed in
delta notation:
1
3
Ricci,20 was used to correct the CO
background when the
Rspl - RPDB
Cx
2
1
3
δ C
)
× 1000; R )
(1)
PDB
x
12
summation method was used. For both curve-fitting and summa-
RPDB
[
]
Cx
1
3
21
tion, δ C was calculated using the method of Santrock et al., to
1
7
account for the contribution of the O concentration to the m/ z
)
45 signal.
where Rspl is the isotope ratio of the sample. Isotope ratios are
normally calibrated by analysis of sample followed by analysis of
a working standard calibrated ultimately against PDB. Routinely
Amino Acid Analogue Samples. The amino acid analogues
were acquired commercially either “as is” or as amino acids and
decarboxylated. The latter was necessary due to the scarcity of
commercial sources for either natural abundance or 13C-labeled
amino acid analogues. The decarboxylation procedure was adapted
from Hashimoto et al.22 Briefly, the amino acid (2 g) was added
to 10 mL of a 1% solution of 2-cyclohexen-1-one (95%+, Sigma-
Aldrich; St. Louis, MO) in cyclohexanol (99%, Fluka; Buchs,
Switzerland) and heated to reflux for 5 h. The reaction mixture
was diluted with methanol to form a 2 mg/ mL solution of the
analogue prior to GC-Py-GCC-IRMS analysis, without further
isolation or purification. Yields were >60% for both amino acids.
Four sources of 3MTP (designated E, F, G, H) and three
sources of IAA (L, M, N), shown in Table 1, were studied by GC-
2
the isotopic standard is CO gas admitted to the analysis stream
via a special reference gas injector. Implicitly, this procedure takes
into account fractionation that would be inconvenient to character-
ize, for example, fractionation that occurs in the IRMS ion source
and detectors. In PSIA, the degree of fragmentation in the
pyrolysis furnace provides an additional source of fractionation.
13
δ Cpdb values of fragments are inherently meaningful only in the
case of quantitative pyrolysis, which has been verified in some
cases (e.g., ref 7). However, as shown below, partial pyrolysis is
usually necessary to ensure the fidelity of a fragment’s origin, and
isotopic fractionation always occurs under these circumstances.
The CO
2
gas standard is not subject to this fractionation, and thus
13
Py-GCC-IRMS. For the labeling experiments, [C(methyl)- C]-
13
reporting δ C relative to the standard is not inherently meaning-
1
3
3
MTP was generated by decarboxylation of [C(methyl)- C]-
1
3
ful. The solution is to report relative differences (∆δ C) in
intramolecular isotope ratios among comparable positions in a
series of samples. Instead of comparing isotope ratios to a pulse
13
methionine, and [1- C]-IAA was generated by decarboxylation of
2- C]-leucine. Both labeled amino acids were purchased from
1
3
[
Cambridge Isotope Laboratories (99%; Andover, MA).
2
of standard CO gas, the isotope ratios of the analogous fragments
from a sample compound are compared to the fragments from a
standard compound run immediately before or after under
identical conditions. This approach, which we adopt here, is
(
16) Sacks, G. L.; Brenna, J. T.; Sepp, J. T. 49th ASMS Conference on Mass
Spectrometry, Chicago, IL, 2001.
17) National Instruments, Austin, TX, 2000.
18) Goodman, K. J.; Brenna, J. T. J. Chromatogr., A 1 9 9 5 , 689, 63-68.
19) Goodman, K. J.; Brenna, J. T. Anal. Chem. 1 9 9 4 , 66, 1294-1301.
20) Ricci, M. P.; Merritt, D. A.; Freeman, K. H.; Hayes, J. M. Org. Geochem.
(
(
(
(
functionally analogous to calibration of CO
standards.
2 2
samples against CO
1
9 9 4 , 21, 561-571.
21) Santrock, J.; Studley, S. A.; Hayes, J. M. Anal. Chem. 1 9 8 5 , 57, 1444-
448.
22) Hashimoto, M.; Eda, Y.; Osanai, Y.; Iwai, T.; Aoki, S. Chem. Lett. 1 9 8 6 ,
93-896.
Pyrolysis of small molecules causes isotopic fractionation due
to kinetic isotope effects. In GC-Py-GCC-IRMS, the isotope ratio
of the fragment, 13RFragment, is measured and is representative of
(
(
1
8
the isotope ratio at a site or moiety before fragmentation, 13
Moiety
R .
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5497

1
3
This relationship may be written as
and the δ Cpdb of the same fragment/ site from a standard
compound as shown in eq 7. Assuming the sample and standard
are analyzed under identical circumstances, and all values are near
1
3
13
RFragment ) R(T,L,f,...) R
(2)
Moiety
13
natural abundance, ∆δ C should be independent of the pyrolysis
conditions.
where R is the fractionation factor, which is dependent on the
temperature (T), the length of the furnace (L), the flow rate
through the furnace (f), and innumerable other parameters. Note
that no equivalent linear relationship can be written to relate the
isotope fraction, 13F, of the reactants to the products. The ratio of
ratios between the sample (Rspl) and standard (Rstd) isotope ratios
for a fragment is
There is no well-established notation for designation of the site
13
to which specific ∆δ C value refers. As a convention in this report,
the name of the standard is written in subscript, and the identity
of the fragment or site is enclosed in parentheses afterward. Here
we arbitrarily choose source N (for IAA) and source H (for 3MTP)
13
as standards. As an example, the expression “∆δ C
3.22‰ for source E”, means that the HCN fragment generated
H
(HCN) )
1
1
3
13
Rspl
RSpl Rspl
from E is enriched by 13.22‰ with respect to the H standard. By
)
‚
(3)
13
1
3
observed
pyrolysis
fragments
13
fragments in
unpyrolyzed
compound
definition, ∆δ CStd(X) ) 0 for any fragment or site, X, produced
from a standard, Std.
[
]
RStd
[
]
Rstd
Rstd
13
The variance of δ C for the standard and the source were
assumed to be independent. Propagation of errors for ∆δ C was
calculated through standard methods. Statistical analysis to
1
3
If the sample and the standard are run under identical pyrolysis
conditions, then their respective fractionation factors are equal.
Equation 3 may be rewritten:
13
13
compare ∆δ C and δ C from different conditions was performed
by the two-tailed t-test at the 95% confidence level (p < 0.05), using
standard statistical tables.
1
3
13
R (fragment)
R (original site)
spl
spl
) 13
(4)
1
3
R (fragment)
R (original site)
std
std
RESULTS AND DISCUSSION
GC-Py-GCC-IRMS of 3MTP was performed at pyrolysis tem-
peratures of 620, 660, 700, 740, 780, 820, 860, and 900 °C and on
IAA at 700, 740, 780, 820, 860, and 900 °C to establish optimal
fragmentation conditions. Once molecular structure was deter-
mined, the optimal pyrolysis temperature was established for each
fragment such that the signal was maximal while the fragments
were derived with high structural fidelity. Because a PLOT column
was chosen for GC-2 to optimize resolution of the low molecular
weight fragments, larger fragments and the 3MTP parent eluted
only at very high temperatures among a background of high
column bleed and were not analyzed isotopically.
Thus, the ratio of isotope ratios for a fragment from a sample
source with respect to a fragment from a standard source remains
invariant with changes in pyrolysis conditions and is therefore
appropriate for studying intramolecular isotopic variation. Because
CSIA and BSIA data are reported in terms of δ Cpdb, we find it
more useful to express eq 4 using the “per mil” (‰) framework:
1
3
1
3
Rspl
13
13
-
1 × 1000 ) δ C (spl) - δ Cpdb(std) +
13
)
pdb
(
Rstd
1
3
Rspl
13
Fragmentation P atterns and Fragment Origin. Pyrograms
-
1 × δ Cpdb(std) (5)
(
13
)
Rstd
2 4 3
of IAA and 3MTP show some similar fragments (C H , HCN, CH -
CN), as well as other fragments specific to each amino acid
analogue. Representative IRMS pyrograms are shown in Figure
The 13R’s in eq 5 may refer to a fragment, a site, or the total
2
. The distribution of fragments for both analogues at increasing
compound. If the sample and standard isotope ratios are near
natural abundance, then the final term will tend toward zero, and
we can write
pyrolysis temperatures is shown in Figure 3. The plot is in terms
of “% molar conversion”, the molar ratio of fragment detected to
parent compound injected. This value will range from 0 to 100%
for fragments generated with ideal structural fidelity but can be
1
3
R (fragment)
1
3
spl
∆
δ C(spl) )
- 1 × 1000
(6)
(7)
2 4
greater than 100% for fragments such as C H that are generated
(
13
)
R (fragment)
std
from multiple locations. Generally, fragmentation does not occur
below 600 °C, increases markedly in the range of 700-780 °C,
and plateaus or decreases above 780 °C. This decrease at high
pyrolysis temperatures is likely due to decomposition of the
fragments to graphite and hydrogen.24
1
3
13
13
∆
δ C(spl) ) δ C (spl) - δ C (std)
pdb
pdb
13
∆
δ C is therefore the per mil (‰) difference of analogous sites
To assess the origin of fragments, 3MTP was labeled at the
C(methyl) site, and IAA was labeled at the C1 position, according
to numbering shown in Figure 1. 13C-Labeled standards at three
or fragments from two different sources of the same compound.
This is similar to the concept of “∆” used by Rieley to describe
1
3
changes in δ C as a result of fractionation during the derivatiza-
13
nominal enrichments (δ Cpdb ) 0, 200, and 400‰) were analyzed
to identify the origin of fragments from within the parent molecule,
where the δ value refers to the enrichment at the labeled site.
The observed isotope ratio of a fragment can be expressed as a
2
3
tion of organic compounds.
To summarize, the relative isotope ratio of a fragment or site
13
from a sample compound can be accurately reported as ∆δ C,
1
3
the difference between the δ Cpdb of the sample fragment/ site
(
23) Rieley, G. Analyst 1 9 9 4 , 119, 915-919.
498 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003
(24) Sofer, Z. Anal. Chem. 1 9 8 6 , 58, 2029-2032.
5

Fig u re 2 . Representative GC-Py-GCC-IRMS pyrograms of (a) IAA at 780 °C and (b) 3MTP at 880 °C. Key: A ) CH4, B ) C2H2, C ) C2H4,
D ) C2H6, E ) HCN, F ) propylene, G ) CH3CN, H ) isobutylene, and I ) CS2.
Fig u re 3 . Pyrolysis efficiencies of (a) IAA and (b) 3MTP over a range of temperatures. The yields are expressed as the molar ratio of fragment
produced divided by parent compound injected on column. Yields may be greater than 100% for fragments that can be produced from multiple
locations in the parent.
weighted sum of the isotope ratios from each carbon position:
1
3
13
Robs
)
R [Ì ]
(8)
∑i
i
i
where 13Robs is the observed isotope ratio of the fragment, 13
the isotope ratio of the ith carbon position in the parent molecule,
and [Ì ] is the molar fraction that the ith position contributes to
the fragment. If a fragment is generated completely from the ith
carbon, then [Ì ] ) 1.00; if [Ì ] ) 0.00, then the ith carbon makes
i
R is
i
i
i
no contribution to the fragment.
We can separate out the contribution of the labeled carbon
from the rest of the summation:
Fig u re 4 . Representative plot of measured 13R of pyrolysis frag-
ments vs 13R(C-methyl) of three standards of 3MTP enriched at
different levels at the methyl position (0.0108, 0.0137, 0.0164) and
pyrolyzed at 700 °C. Best-fit equations: methane (O) y ) 0.92x +
1
3
13
13
Robs
)
R [Ì ] +
R [Ì ]
(9)
lab
lab
∑
i
i
i,i*lab
0
0
.0008; acetylene (0) y ) 0.17x + 0.009; ethylene (4) y ) 0.09x +
.01; ethane (b) y ) 0.93x + 0.0006; HCN (9) y ) 0.02x + 0.0107;
A plot of 13Robs versus 13
Rlab should yield a straight line with
CH3CN (2) y ) 0.04x + 0.0102; The slope of the best-fit line is equal
slope [Ìlab], the mole fraction contribution of the labeled carbon.
Plots of this nature were generated for the major pyrolysis
fragments of each amino acid analogue over a range of temper-
atures. A representative plot for these labeling experiments is
shown for the pyrolysis of 3MTP at 700 °C in Figure 4. The slope
of the best-fit line for a given fragment is equal to the percent
to the mole fraction that the C(methyl) site contributes to the observed
fragment; e.g., 92% of the carbon in CH derives from the C(methyl).
4
contribution of the labeled site to the fragment. For example, 92%
4
of the CH is derived from the C(methyl) site. The percent
contributions of labeled sites to pyrolysis fragments of IAA and
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5499

Ta b le 2 a
observed from both analogues at temperatures above 780 °C.
Labeling experiments confirm that the HCN fragment is primarily
temperature (°C)
generated from the C1 carbon by simple cleavage of the CH
2
-
fragment 620 660 700 740 780 820 860 900
CH NH bond. There is a negligible contribution of the labeled
2
2
3
MTP
CH4
C2H2
C2H4
C2H6
HCN
CH3CN
C2H4
99
97
92
17
9
93
2
86
25
9
89
1
80
31
12
87
1
76
34
13
85
1
71
35
14
85
1
68
34
15
84
1
C(methyl) site of 3MTP, as expected. Labeling experiments on
IAA show 94-98% contribution of the C1 carbon to the HCN
fragment over a temperature range of 780-900 °C.
4
99
6
96
2
3
Both analogues produce a modest yield of CH CN at temper-
3
4
25
4
24
4
4
4
4
atures above 780 °C. This fragment is primarily formed by
IAA
22
99
0
0
50
17
94
0
0
48
16
95
0
0
48
17
95
0
0
48
HCN
cleavage between the C2 and C3 carbons. The C1 position of IAA
n-C3H6
i-C4H8
CH3CN
0
0
0
0
49
contributes ∼50% to the CH
3
CN fragment at 780 °C, which is
expected if the fragment was formed solely by single bond
cleavage. However, in the pyrolysis of 3MTP, the C(methyl) site
a
All values are percentages. Top shows contribution of C1 position
shows a steady 4% contribution to the CH
3
CN fragment over a
to IAA pyrolysis fragments as a function of temperature. Bottom shows
contribution of C(methyl) of 3MTP to each fragment as a function of
pyrolysis temperature.
wide range of temperatures. The mechanism for this is likely a
•
•
recombination between CH
In IAA, the formation of the HCN and CH
results in the generation of corresponding isobutylene (C
propylene (C ) fragments in very good yield (20 and 60%,
3
and CH
2
NH
2
free radicals.
CN fragments
) and
3
4 8
H
3
H
6
3MTP are reported in Table 2. For IAA presented in Table 2, HCN
respectively, at 860 °C). The C1 carbon did not contribute to either
fragment over a wide range of temperatures, so both fragments
are likely generated by simple homolytic cleavage.
originates with 99% fidelity from the C1 site at 780 °C, while
propylene and isobutylene are uncontaminated with C1 carbon
at all temperatures. CH
3
CN is ∼50% C1 at all temperatures, as
expected if there were no contamination from other sites. All three
of these fragments should therefore have isotope ratios repre-
sentative of their respective positions in the original molecule once
Pyrograms of 3MTP do not show evidence of CH or
3
SC
2
H
5
CH SCH , the corresponding fragments of HCN and CH CN. If
3
3
3
aliphatic S-containing radicals are formed, further cleavage is
probably favored over recombination with hydrogen or other free
radicals due to the fragile nature of the C-S bonds. Sulfur is
properly calibrated. Similarly, Table 2 shows that CH
CH CN will provide representative fragments for C1 analysis in
MTP.
The major fragments appear to result from cleavage of the
weakest bonds in both analogues, in accordance with Rice theory.
is the dominant fragment at high temperatures. In our
previous PSIA studies of alcohols and n-alkanes, we observed both
and C as common “end-point” fragments. The same
4
, HCN, and
3
3
2 2
detected in the pyrograms in the form of CS and H S. The origin
of the carbon in the CS fragment is unclear, and therefore, the
2
fragment is not useful for PSIA studies.
C
2
H
4
In summary, each analogue produced four pyrolysis fragments
of acceptable yield and fidelity to be useful for PSIA. For 3MTP,
C
2
H
4
2 2
H
these fragments are CH
the C(methyl) group; HCN at 780-900 °C, which derives from
the C1 position; CH CN at 780-900 °C, which derives 50% from
4 2 6
and C H at 680 °C, which derive from
phenomenon is seen in the thermolytic cracking of aliphatic
hydrocarbons. The labeling data above show that the contribution
of the C(methyl) site of 3MTP and of the C1 site in IAA to the
3
C1, 46% from C2 position, and 4% from C(methyl). These
C
2
H
4
and C
2
H
2
fragments varies with temperature. Therefore,
13
fragments enable the calculation of ∆δ C at every position in
these fragments probably do not represent the isotopic nature of
any specific location in 3MTP and thus are not useful for PSIA.
3
MTP via the following equations:
There is a modest yield of CH
MTP but negligible amounts of these fragments in the pyrolysis
of IAA. Table 2 shows that C(methyl) contributes >95% to CH
and C at low temperatures (660 °C and below). The C-S bonds
of 3MTP are highly labile; the bond dissociation energy of the
CH
-S bond in methionine is estimated to be 71 kcal/ mol, ∼10
kcal/ mol less than the aliphatic CH is
-C bond of IAA.25 CH
therefore formed by simple homolytic cleavage of the CH -S
bond, and C is a result of the recombination of methyl free
4
and C
2
H
6
in the pyrolysis of
13
13
∆
δ C(C1) ) ∆δ C(HCN)
(10a)
3
4
13
13
13
∆
∆
δ C(C2) ) (∆δ C(HCN) - 0.04∆δ C(C1) -
2 6
H
13
0
.5∆δ C(C - methyl))/ 0.46 (10b)
1
3
13
13
13
13
3
4
δ C(C3) ) ∆δ C(Total) - (∆δ C(C1) + ∆δ C(C2) +
3
1
3
∆
δ C(C - methyl))/ 4 (10c)
1
2 6
H
radicals. As the temperature increases, the contribution of
C(methyl) decreases, indicating increased contamination of the
13
13
∆δ C(C
- methyl) ) ∆δ C(C
H
)
(10d)
1
2
6
4 2 6
CH and C H peaks by secondary fragmentation. We chose a
pyrolysis temperature of 680 °C for these two fragments as a
compromise between signal size and structural fidelity.
Gas-phase pyrolysis of aliphatic primary amines is known to
produce short-chain nitriles,26 and a significant yield of HCN is
For IAA, useful fragments are HCN above 780 °C, which
derives from the C1 position; CH CN, which derives from the
3
C1and C2 position; propylene, which derives from the propyl
group; and isobutylene, which derives from the C2 position and
(
(
25) McMillen, D. F.; Golden, D. M. Annu. Rev. Phys. Chem. 1 9 8 2 , 33, 493-
32.
26) Lovas, F. J.; Clark, F. O.; Tiemann, E. J. Chem. Phys. 1 9 7 5 , 62, 1925-1931.
13
the propyl group. These fragments enable the calculation of ∆δ C
for the C1 and C2 positions and the propyl moiety of IAA by the
5
5500 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

Ta b le 3 . Ab s o lu t e (δ1 3 CP d b ) a n d Re la t ive (∆δ1 3 CN) Is o t o p e Ra t io s o f 3 MTP P yro lys is Fra g m e n t s (T ) 8 8 0 °C)
fragment
source
E
CH4
C2H6
HCN
CH3CN
CSIAa
1
3
δ Cpdb
-49.18 ( 0.58
2.04 ( 0.70
-46.20 ( 0.53
5.02 ( 0.65
-46.42 ( 0.46
4.79 ( 0.60
-51.21 ( 0.38
0
-53.43 ( 0.06
2.75 ( 0.31
-50.82 ( 0.43
5.36 ( 0.53
-51.33 ( 0.31
4.86 ( 0.46
-56.18 ( 0.31
0
-29.16 ( 0.39
13.22 ( 0.42
-26.08 ( 0.14
16.30 ( 0.22
-26.14 ( 0.11
16.24 ( 0.20
-42.37 ( 0.16
0
-24.76 ( 0.22
25.03 ( 0.27
-19.19 ( 0.16
30.60 ( 0.22
-19.85 ( 0.06
29.94 ( 0.17
-49.79 ( 0.16
0
-24.07( 0.17
13.96 ( 0.33
-20.43 ( 0.12
17.59 ( 0.31
-20.71 ( 0.12
17.32 ( 0.31
-38.03 ( 0.28
0
1
3
∆
δ CH
1
3
F
δ Cpdb
13
∆
δ CH
1
3
G
H
δ Cpdb
13
∆
δ CH
1
3
δ Cpdb
1
3
∆δ CH
a
13
13
CSIA refers to δ C and ∆δ CH of the total compound.
Ta b le 4 . Ab s o lu t e (δ1 3 Cp d b ) a n d Re la t ive (∆δ1 3 CN) Is o t o p e Ra t io s o f IAA P yro lys is Fra g m e n t s (T ) 7 8 0 °C)
fragment
source
L
n-C3H6
i-C4H8
HCN
CH3CN
CSIAa
1
3
δ Cpdb
-29.59 ( 0.06
-8.28 ( 0.09
-20.64 ( 0.19
0.67 ( 0.19
-21.31 ( 0.07
0
-27.98 ( 0.31
-5.68 ( 0.20
-22.11 ( 0.10
0.20 ( 0.27
-22.31 ( 0.25
0
-29.24 ( 0.40
-9.69 ( 0.69
-36.62 ( 0.21
-17.06 ( 0.61
-19.55 ( 0.57
0
-19.43 ( 0.17
-0.55 ( 0.17
-27.94 ( 0.44
-9.06 ( 0.44
-18.88 ( 0.44
0
-25.49 ( 0.19
-5.66 ( 0.27
-29.39 ( 0.19
-3.90 ( 0.27
-31.14 ( 0.20
0
1
3
∆
δ CN
1
3
M
N
δ Cpdb
13
∆δ CN
1
3
δ Cpdb
1
3
∆δ CN
a
13
13
CSIA refers to δ C and ∆δ CN of the total compound.
following equations:
source, but instead synthesized by a nonnatural method. Unfor-
tunately, manufacturers generally consider information about
sources proprietary, and they were not able to discuss with us
the origin of their sources in detail.
1
3
13
∆
δ C(C1) ) ∆δ C(HCN)
(11a)
1
3
13
13
∆
δ C(C2) ) 2(∆δ C(CH CN) - 0.5∆δ C(C1)) (11b)
3
13
We calculated ∆δ C values as described in eq 7. Source N
(
IAA) and source H (for 3MTP) were arbitrarily chosen as
1
3
13
∆
δ C(propyl) ) ∆δ C(propene)
(11c)
13
13
standards. ∆δ C
fragments are reported. By definition, ∆δ C
N equals zero for any fragment. The average precision for the
H N
(Table 3) and ∆δ C (Table 4) of the pyrolysis
13
13
H
N
of H and ∆δ C of
Comparison of Fragment ∆δ1 3 C from Multiple Sources.
δ Cpdb values calibrated against CO
fragments, generated at 680 °C, and the HCN and CH
fragments, generated at 880 °C, are reported for the four sources
of 3MTP in Table 3. δ Cpdb values of the C
CH CN fragments, all generated at 780 °C, are reported for the
three sources of IAA in Table 4. The δ Cpdb of the total carbon in
each source, as measured by GCC-IRMS, is also reported. As
was discussed earlier, the δ C values of the observed fragments
calibrated against CO gas may not be equal to the isotope ratios
of the corresponding position(s) on the original source due to
13
13
1
3
∆δ C measurements was SD(∆δ C) < 0.5‰ for both IAA and
3
2
gas for the CH
4
and C
2 6
H
13
MTP. This is slightly worse than the precision for the δ C values,
3
CN
due to propagation of errors.
13
13
1
3
The utility of the ∆δ C notation over δ C is evident when we
3 6 4 8
H , C H , HCN, and
13
study the invariance of ∆δ C to changes in pyrolysis temperature.
We determined δ C
fragments of all 3MTP sources at 800, 840, and 880 °C, and δ Cpdb
N 3 3 6
and ∆δ C for the HCN, CH CN, and C H fragments for all IAA
sources at 780, 800, 820, and 840 °C. Plots of the dependency of
∆ 13
5 for two of the fragments, HCN and propylene from the IAA
3
13
13
1
3
pdb 3
and ∆δ CH for the HCN and CH CN
13
13
1
3
2
13
δ C and δ C with respect to temperature are shown in Figure
1
3
pyrolysis-induced fractionation. Thus, ∆δ C are also calculated
from δ Cpdb to emphasize relative isotope ratios.
13
1
3
parent. In all cases, ∆δ C is less dependent on temperature than
δ C. The average range of ∆δ C for a fragment over the
measured temperature range was only 0.5‰. Eleven of the 12
13
13
The average precision for fragments from both 3MTP and IAA
1
3
was SD(δ C) < 0.3‰. Even allowing for a sizable fractionation
factor of 5-10‰, the CH fragment, and hence the terminal
C(methyl) carbon, is clearly depleted in all 3MTP sources (δ Cpdb
-45 to -51‰). In sources derived from methionine (E, F, G),
C(methyl) is markedly depleted with respect to the total carbon
13
fragments did not show a significant variation (p < 0.05) in ∆δ C
values over the observed temperature ranges. In contrast, the
4
13
13
)
average range of δ Cpdb was 1.6‰, and only one fragment did
not show a significant variation (p < 0.05) in δ Cpdb as temperature
was varied.
13
1
3
of the source (δ Cpdb ∼ -20‰). If our commercial source of
methionine is from a biological source, this may reflect fraction-
ation of the terminal methyl as a result of methionine’s role in
the one-carbon pool. The disparity between the C(methyl) carbon
and the other fragments is much smaller in source H. This may
indicate that the commercial 3MTP is not derived from a biological
Isotope ratios of complementary fragments of IAA further
13
indicate that ∆δ C is well suited for reporting intramolecular
isotope values. Complementary fragments, introduced in a PSIA
studies of hydrocarbons,7 are defined as two fragments that
contain all the carbon positions in the parent molecule. Two pairs
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5501

1
3
13
13
13
Fig u re 5 . Dependence of (b) δ C and (a) ∆δ C of HCN, and (d) δ C and (c) ∆δ C of propylene fragments, from IAA on pyrolysis temperature.
13
13
Relative isotope ratios (∆δ CN) are much more invariant to changing pyrolysis temperature than absolute isotope ratios (δ C). Key to sources:
L ([); M (×); N (0).
Fig u re 6 . (a) ∆δ13CN and (b) δ13C values of total carbon of IAA reconstructed from complementary fragments, compared to total carbon as
13
measured by GCC-IRMS. Error bars for δ C values are mostly within the data points. Key: closed square, HCN + isobutylene; open square,
CH3CN + propylene; crossed square, total measured by CSIA.
of complementary fragments exist for IAA: (CH
HCN, C ). If the complementary fragments are formed without
competing pathways, then the weighted averages of the ∆δ C
3
CN, C
3
H
6
) and
is within 0.8‰ of the CSIA value for all four pairs, and the
(
4
H
8
differences were not significant (p < 0.05).
13
13
N
Figure 6b shows raw δ C of the total carbon in IAA as
1
3
values should add to make the ∆δ C
N
values of the total
measured by CSIA and as reconstructed from complementary
compound by the following equation:
13
fragments generated at 780 °C. In contrast to the ∆δ C results,
13
the reconstructed δ C values were enriched by 3-5‰ with
respect to the CSIA value and were significant (p < 0.05) for all
complementary fragments. Changing the pyrolysis temperature
did not improve this agreement.
1
3
13
13
∆
δ C (Total) ) F ∆δ C (A) + F ∆δ C (B);
N A N B N
F + F ) 1 (12)
A
B
The labeled 3MTP studies indicate that CH
4 2 6
and C H derive
where F
x
is the mole fraction contribution of complementary
their carbon primarily from the C(methyl) group. Therefore, we
fragment X. If the complementary fragments are formed without
fractionation, then the weighted averages of the δ Cpdb values
should be additive:
13
13
expect that ∆δ C
H 4 H 2 6
(CH ) and ∆δ C (C H ) should be the same
1
3
for any one sample. At a pyrolysis temperature of 680 °C, the
1
3
13
difference between ∆δ C
.71‰ for E to 0.07‰ for G (Table 4), and the differences are not
significant. Because CH and C derive from the same carbon
H 4 H 2 6
(CH ) and ∆δ C (C H ) ranges from
1
3
13
13
0
∆
δ C (Total) ) F δ C (A) + F ∆δ C(B);
A B
4
2 6
H
F + F ) 1 (13)
A
B
site, the difference between their isotope ratios is invariant at
constant pyrolysis conditions, although their absolute isotope
13
13
13
ratios ((δ C) differ by 4-5‰. We conclude that ∆δ C are robust
Figure 6a shows ∆δ C of the total carbon in IAA as measured
by CSIA and reconstructed from complementary fragments
representations of relative carbon isotope ratios.
13
generated at 780 °C. There is excellent agreement between the
H
We used ∆δ C values of fragments from and mass balance
13
13
reconstructed and CSIA measurements; the reconstructed ∆δ C
H
to calculate the ∆δ C values of individual carbon sites, presented
5502 Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

the name of the manufacturer, it is difficult to draw conclusions
on why these differences exist. However, the three sources of
IAA are clearly distinguishable.
Experimental and Theoretical Limits of Sample Size. The
13
typical lower limit of analyte require to yield precision of SD(δ C)
<
0.3‰ is ∼10 ng of C on-column in continuous-flow IRMS using
the conventional summation algorithm for data reduction. Using
curve fitting to improve data reduction, we routinely achieve
13
precision of SD(δ Cpdb) < 0.5‰ for 500 pmol of C on column.
Pyrolysis efficiency in GC-Py-GCC-IRMS is much less than 100%
for some fragments, so the fragment formed in the poorest yield
limits the minimum sample size. For 3MTP, the limiting fragment
Fig u re 7 . ∆δ13CH for all four carbon positions of 3MTP at T ) 880
C. ∆δ13CH ) δ C(Sample) - δ C(Source H) for a given fragment.
13
13
°
By definition, ∆δ 3CH ) 0 for all fragments and positions from source
1
H.
is C
2
H
6
, which is formed with a pyrolysis efficiency of ∼5% at 680
°C. A yield of 500 pmol of CH
4
requires 10 nmol, or 1 µg, of 3MTP
on column. In our own experimental work, we operated within a
factor of 2 of this limit. The limiting fragment in the pyrolysis of
IAA is CH
3
CN, which is formed with a yield of ∼6%. Since every
mole of CH
pmol of CH
of IAA.
3
CN produces 2 mol of C, we should need only 250
CN postpyrolysis; this requires 4.2 nmol, or 0.4 ng,
3
CONCLUSIONS
We have demonstrated that on-line pyrolysis of two amino acid
analogues, 3MTP and IAA, generates characteristic fragments with
reproducible 13C/ C isotope ratios (SD(δ Cpdb) < 0.3‰). Relative
12
13
Fig u re 8 . ∆δ13CN for C1, C2, and propyl moiety of IAA at T ) 780
13
C. ∆δ13CN ) δ C(Sample) - δ C(Source N) for each a given
13
13
isotope ratios, ∆δ C, were calculated either directly or by mass
balance for all four carbon positions of 3MTP and for two of the
carbon positions and the propyl group of IAA. Our PSIA studies
highlight the intramolecular variability of isotope ratios and also
indicate that a great deal of information is lost in compound-
specific analyses. For example, two unique sources of 3-methyl-
propylamine differed by 17.3‰ by CSIA, but our PSIA work on
the same two sources shows a difference of 48.3‰ at C2 and 1‰
at C3. The system is well suited to study small, biologically
relevant, kinetic isotope effects at specific sites of small molecules
that would otherwise be obscured by CSIA studies. These
techniques may be useful for correlating subtle changes in
intramolecular isotope ratios to physiological events or stresses.
°
13
fragment. By definition, ∆δ CN ) 0 for all fragments and positions
from source N.
in Figure 7. The variation at each position is greater than is
normally seen in, for instance, C3/ C4 photosynthetic fractionation.
Most striking, the C2 position is enriched by +48.33‰ in source
F compared to H. In fact, all three of the 3MTP sources derived
from methionine were enriched by at least 39‰ at the C2 position.
This difference is even more remarkable considering that no other
position shows a difference of >20‰ compared to source H. In
1
3
H
fact, ∆δ C (C3) is not significantly different from 0‰ for any of
the three sources. The enhanced utility of PSIA over CSIA is very
evident in these data. Using CSIA, we could determine only that
the three sources of 3MTP derived from methionine (E, F, G)
were enriched by 14-18‰ with respect to H. With PSIA, we see
that this enrichment is concentrated primarily in the C2 position
13
Our data provide strong evidence that ∆δ C is conserved
throughout pyrolysis, as expected on theoretical grounds. In our
work, ∆δ C values of fragments are invariant for a given set of
pyrolysis conditions. The ∆δ C values measured by CSIA were
identical to those reconstructed from the ∆δ C values of comple-
mentary fragments, and different fragments formed from the same
site within a parent compound have identical ∆δ C values.
Sample size in our work is limited primarily by fragment
pyrolysis efficiencies as low as 5% and therefore requires 1-2
orders of magnitude more sample than CSIA on the same
compound. In future refinements, pyrolysis efficiency might be
raised while still minimizing the degree of secondary fragmenta-
tion. This could be accomplished by altering the residence time
of the analyte within the heated pyrolysis zone or by turning to
new methods of fragmentation such as laser-induced pyrolysis.
13
13
13
(
39-48‰), with minor enrichment in the C1 position (13-16‰)
1
3
H
and C(methyl) position (3-5‰). The similarity of ∆δ C values
13
for F and G at each position suggests that the methionine
purchased from Avocado and Acros Organics was drawn from
the same source.
1
3
Analogously, we used ∆δ C
δ C
N
N
of the fragments to calculate the
values of individual carbon sites in IAA (Figure 8).
Unfortunately, because the CH fragment was not formed in
significant yield, we could not distinguish the isotope ratio of the
1
3
∆
4
13
two CH
3 N
groups from the C3 position; instead, we report ∆δ C -
(
propyl), the intramolecular isotope ratio of the propyl fragment.
The variation is not as dramatic as between the sources of 3MTP.
M is depleted by ∼-3.6‰ compared to N in CSIA. However, PSIA
shows that M is enriched at C(propyl) by 0.7‰, and is depleted at
C1 by -17.1‰, compared to the N standard. A similar trend is
ACKNOWLEDGMENT
This work was funded by NIH Grant GM49209.
13
13
Received for review May 8, 2003. Accepted July 21, 2003.
AC0344889
seen with source L, where ∆δ C
N
(C1) ) -9.7‰ and ∆δ C
N
(C2)
)
8.6‰. Without knowing the origins of each source, other than
Analytical Chemistry, Vol. 75, No. 20, October 15, 2003 5503