Stable Nitrogen Isotope Analysis of Amino Acid Enantiomers by Gas Chromatography/Combustion/ Isotope Ratio Mass Spectrometry

Article abstract of DOI:10.1021/ac960956l

The analysis of the stable nitrogen isotope compositions of individual amino acid stereoisomers through the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is presented. Nitrogen isotopic compositions of single amino acids or of their enantiomers is possible without the labor-intensive and time-consuming preparative-scale chromatographic procedures required for conventional stable isotope analysis. Following hydrolysis and derivatization, single-component isotope analysis is accomplished on nanomole quantities of each of the stereoisomers of an amino acid, utilizing the effluent stream of gas chromatographic separation. Nitrogen isotope fractionation is minimal during acylation of the amino acid, with no additional nitrogen being added stoichiometrically to the derivative. Thus, the isotopic composition of the nitrogen in the derivative is that of the original compound. Replicate stable nitrogen isotope analyses of 11 amino acids, and their trifluoroacetyl (TFA)/isopropyl (IP) ester derivatives, determined by both conventional isotope ratio mass spectrometry (IRMS) and GC/C/IRMS, indicate that the GC procedure is highly reproducible (standard deviations typically 0.3-0.4‰) and that isotopic differences between the amino acid and its TFA/IP derivative are, in general, less than 0.5‰.

Full text of DOI:10.1021/ac960956l

Anal. Chem. 1997, 69, 926-929
Stable Nitrogen Isotope Analysis of Amino Acid
Enantiomers by Gas Chromatography/Combustion/
Isotope Ratio Mass Spectrometry
Stephen A. Macko* and Maria E. Uhle
Department of Environmental Sciences, The University of Virginia, Charlottesville, Virginia 22903
Michael H. Engel and Vladimir Andrusevich
School of Geology & Geophysics, The University of Oklahoma, Norman, Oklahoma 73019
stable nitrogen isotope measurements are the many modes of
fractionation of nitrogen.1 Furthermore, important information at
the molecular level is obscured in bulk analyses, a consequence
of differences in the biosynthetic pathways of individual compo-
nents10,11 and diagenesis.12,13 Attempts have been made to isolate
individual components of complex mixtures for off-line combustion
and stable nitrogen isotope analysis by conventional IRMS.14,15
However, the preparative chromatographic steps required to
isolate sufficient milligram quantities of a component for conven-
tional 15N analysis are labor intensive and often are complicated
by column bleed and isotopic fractionation resulting from incom-
plete recovery of the entire component. Thus, numerous samples
of interest remain unanalyzed owing to the complexities of this
type of approach, and available data sets of δ15N values of
individual compounds are quite limited.
The analysis of the stable nitrogen isotope compositions
of individual amino acid stereoisomers through the use
of gas chromatography/ combustion/ isotope ratio mass
spectrometry (GC/ C/ IRMS) is presented. Nitrogen iso-
topic compositions of single amino acids or of their
enantiomers is possible without the labor-intensive and
time-consuming preparative-scale chromatographic pro-
cedures required for conventional stable isotope analysis.
Following hydrolysis and derivatization, single-component
isotope analysis is accomplished on nanomole quantities
of each of the stereoisomers of an amino acid, utilizing
the effluent stream of gas chromatographic separation.
Nitrogen isotope fractionation is minimal during acylation
of the amino acid, with no additional nitrogen being added
stoichiometrically to the derivative. Thus, the isotopic
composition of the nitrogen in the derivative is that of the
original compound. Replicate stable nitrogen isotope
analyses of 1 1 amino acids, and their trifluoroacetyl
(TFA)/ isopropyl (IP ) ester derivatives, determined by both
conventional isotope ratio mass spectrometry (IRMS) and
GC/ C/ IRMS, indicate that the GC procedure is highly
reproducible (standard deviations typically 0 .3 -0 .4 ‰ )
and that isotopic differences between the amino acid and
its TFA/ IP derivative are, in general, less than 0 .5 ‰ .
We previously reported a GC/ C/ IRMS method for the on-line
stable carbon isotope analysis of complex mixtures of individual
amino acid enantiomers at nanomole levels.16 More recently, the
feasibility of determining δ15N values of amino acids by GC/ C/
IRMS has been demonstrated.17-20 However, these initial GC/
C/ IRMS methods are not amenable for determining the δ15N
values of individual D- and L-enantiomers of amino acids in complex
mixtures at natural abundance levels with a sufficiently high
degree of precision. The ability to make such measurements is
important for documenting the origin(s) and diagenesis of amino
Stable nitrogen isotope analysis of bulk organic materials is a
well-established method for tracing biosynthesis1 as well as the
sources and history of organic matter in the geosphere.2 For
example, nitrogen isotopes have been used to assess trends in
early diagenesis,3-5 to elucidate conditions on the early Earth,6
and to assess the origins of organic nitrogen in extraterrestrial
materials7 as well as to establish trophic orders in modern and
fossil food chains.8,9 Often complicating the interpretation of bulk
(7) Epstein, S.; Krishnamurthy, R. V.; Cronin, J. R.; Pizzarello, S.; Yuen, G. U.
Nature 1 9 8 7 , 326, 477-479.
(8) Macko, S. A.; Engel, M. H. Phil. Trans. R. Soc. London B 1 9 9 1 , 333, 367-
374.
(9) Ostrom, P. H.; Macko, S. A.; Engel, M. H.; Russell, D. A. Geology 1 9 9 3 ,
21, 491-494.
(10) Macko, S. A.; Estep, M. L. F.; Hare, P. E.; Hoering, T. C. Isotope Geosci.
1 9 8 7 , 65, 79-92.
(11) Hare, P. E.; Fogel, M. L.; Stafford, T. W., Jr.; Mitchell, A. D.; Hoering, T. C.
J. Archaeol. Sci. 1 9 9 1 , 18, 277-292.
(12) Silfer, J. A.; Engel, M. H.; Macko, S. A. Isotope Geosci. 1 9 9 2 , 15, 211-221.
(13) Macko, S. A.; Engel, M. H.; Qian, Y. Chem. Geol. 1 9 9 4 , 114, 365-379.
(14) Bidigare, R. R.; Kennicutt, M. C.; Keeney-Kennicutt, W. L.; Macko, S. A.
Anal. Chem. 1 9 9 1 , 63, 130-133.
(1) Fogel, M. L.; Cifuentes, L. A. In Organic Geochemistry, Principles and
Applications; Engel, M. H., Macko, S. A., Eds.; Plenum: New York, 1993; p
73.
(2) Macko, S. A.; Engel, M. H.; Parker, P. L. In Organic Geochemistry, Principles
and Applications; Engel, M. H., Macko, S. A., Eds.; Plenum: New York,
1993; p 211.
(3) Wada, E. In Isotope Marine Chemistry; Goldberg, E. D., Horibe, Y., Sarahashi,
J. J., Eds.; Uchida Rokakuho Publ.: Tokyo, 1980; p 375.
(4) Altabet, M. A. Deep-Sea Res. 1 9 8 8 , 35, 535-554.
(5) Qian, Y.; Engel, M. H.; Macko, S. A. Isotope Geosci. 1 9 9 2 , 15, 201-210.
(6) Schidlowski, M.; Hayes, J. M.; Kaplan, I. R. In Earth’s Earliest Biosphere:
Its Origin and Evolution; Schopf, J. W., Ed.; Princeton Univ. Press: Princeton,
NJ, 1983; p 149.
(15) Engel, M. H.; Goodfriend, G. A.; Qian, Y.; Macko, S. A. Proc. Natl. Acad.
Sci. U.S.A. 1 9 9 4 , 91, 10475-10478.
(16) Silfer, J. A.; Engel, M. H.; Macko, S. A.; Jumeau, E. J. Anal. Chem. 1 9 9 1 ,
63, 370-374.
(17) Merritt, D. A.; Hayes, J. M. J. Am. Soc. Mass Spectrom. 1 9 9 4 , 5, 387-397.
(18) Brand, W. A.; Tegtmeyer, A. R.; Hilkert, A. Org. Geochem. 1 9 9 4 , 21, 585-
594.
(19) Preston, T.; Slater, C. Proc. Nutrit. Soc. 1 9 9 4 , 53, 363-372.
(20) Metges, C. C.; Petzke, K. J.; Hennig, U. J. Mass Spectrom. 1 9 9 6 , 31, 367-
376.
926 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
S0003-2700(96)00956-0 CCC: $14.00 © 1997 American Chemical Society

acid stereoisomers in fossil systems (e.g., refs 8 and 15), as well
as for establishing the origin of chirality in our solar system prior
to life’s origin (e.g., ref 21) and, perhaps, for confirming the
existence of life on other planetary bodies such as Mars.22 Here
we report a new GC/ C/ IRMS method for the stable nitrogen
isotope analysis of individual amino acid enantiomeric components
of complex mixtures.
sample. Details of the EA method have been previously re-
ported.23 Differences between the two methods for the assess-
ment of the 15N were minimal and within the precisions of the
measurements reported previously (∼0.1‰, one standard devia-
tion). Additionally, the results of the 13C EA analyses were in
excellent agreement with previously reported values for the same
amino acid standards.16 Stable nitrogen isotope values are
reported using the standard convention
EXPERIMENTAL SECTION
δ15N(‰) ) [Rsample/ Rstandard - 1] × 103
Standards and Reagents. Standard solutions of individual
amino acid stereoisomers and of racemic mixtures of amino acids
were prepared following the methodology of Silfer et al.16 Briefly,
crystalline amino acids (Sigma Chemical, St. Louis, MO) were
dissolved in distilled 0.1 N HCl (final concentration of 0.05 M)
and stored at 4 °C until further use. 2-Propanol (commonly
isopropanol, IP; HPLC grade, Fisher Scientific, Fairlawn, NJ) was
acidified to 2.8 M by the addition of 250 µL (per milliliter of IP)
of acetyl chloride (99+%, Aldrich, Milwaukee, WI). Trifluoroacetic
anhydride (TFAA, 99+%, Pierce Chemical Co., Rockford, IL) was
used without further purification. All reagents were of the same
lot numbers for the derivatization procedures described below.
Derivatization P rocedures. Two-hundred microliter aliquots
of a 0.05 M solution of each amino acid (50 µmol) were placed in
an ashed screw-cap vial and evaporated to dryness at 40 °C under
a stream of N2. Approximately 0.5 mL of acidified IP was added
to each sample vial. The vials were sealed with Teflon-lined caps
and heated at 100 °C for 1 h. The reaction was quenched by
placing the vials in a freezer at -5 °C. The residual IP was
removed under a gentle stream of N2. To remove excess water
and IP, 0.25 mL of distilled CH2Cl2 was added to each vial in two
successive aliquots, each of which was evaporated under a gentle
stream of N2 at room temperature. The resultant amino acid
esters were acylated by the addition of 0.5 mL of TFAA and 0.5
mL of distilled CH2Cl2. The vials were sealed and heated at 100
°C for 10 min. The vials were then placed in an ice bath, where
the excess TFAA and CH2Cl2 was removed under a gentle stream
of N2. To remove residual TFAA and CH2Cl2, 0.25 mL of distilled
CH2Cl2 was added to each vial, and this was again removed at 0
°C under a gentle nitrogen stream.
Conventional IRMS. The individual amino acid enantiomers
and aliquots of their respective TFA/ IP derivatives were converted
to gases of suitable purity for isotope analysis using one of two
procedures. In some instances, following the procedure of Macko
et al.,10 crystalline amino acids or the derivatives were added to
purified granular copper oxide (850 °C; Mallinckrodt Baker,
Phillipsburg, NJ) and copper wire (Alpha Resources, Racine, WI)
inside an ashed (550 °C, 1 h) quartz tube. Following evacuation,
sealing, and combustion at 850 °C, the resulting gases were
cryogenically purified, and the isolated N2 was isotopically
analyzed on a PRISM (Micromass Instruments, Manchester, UK)
stable isotope ratio mass spectrometer. Alternatively, certain of
the bulk isotope measurements were made on a Micromass
Optima instrument which had been interfaced to a Carlo Erba
elemental analyzer (EA). In this case, the resulting gases were
introduced directly into the source of the mass spectrometer,
following chromatographic separation and chemical trapping of
water, with both the 13C and the 15N being determined on a single
where R is the 15N/ 14N of the standard or sample. The standard
for 15N analysis is atmospheric N2 and is defined to be 0.0‰. For
routine analysis, the samples are analyzed against a laboratory
reference tank of high-purity molecular nitrogen (99.999%, Linde
Specialty Gases, Union Carbide, Danbury, CT) which has been
calibrated against atmospheric nitrogen as well as other interna-
tional standards.
GC/ C/ IRMS. The individual stereoisomers, as well as several
racemic mixtures of amino acids, were analyzed directly for their
stable nitrogen isotope compositions using a Micromass Instru-
ments Optima GC/ C/ IRMS system. This system is a modification
of that which has been previously reported for the analysis of
molecular carbon isotope abundances.16 The GC/ C/ IRMS system
for 15N analysis of individual molecular components consists of a
Hewlett Packard 5890 gas chromatograph interfaced to an Optima
stable isotope ratio mass spectrometer, through a combustion
system consisting of an oxidation furnace composed of braided
nichrome/ copper oxide wire and a reduction furnace of granulated
copper, followed by a H2O/ CO2 trap at liquid N2 temperature
(Figure 1). Other details of the system hardware and software
are similar to those reported previously by Freedman et al.24 The
separation of the amino acid stereoisomers was accomplished
using a 50 m × 0.25 mm i.d. fused silica capillary column bonded
with an optically active stationary phase (Chirasil-Val, Alltech
Associates, Deerfield, IL) which is capable of baseline resolution
of TFA/ IP esters of amino acid enantiomers. The GC conditions
are as follows: splitless injection (∼1-2 nmol of each enantiomer
derivative was injected, combusted, and subsequently introduced
directly into the source of the MS); the carrier gas was ultrapure
He (99.9999%) at a head pressure of 80 kPa; the injector
temperature was 200 °C, and the temperature of the interface
between the GC and the oxidation furnace was 350 °C; the GC
temperature program was 45 °C for 3 min, 45-90 °C at 45°/ min,
90 °C isothermal for 15 min, 90-190 °C at 3 °C/ min, and then
190 °C isothermal for 30 min. The solvent (ethyl acetate) peaks
were removed from the effluent of the GC through a heart split
valve which is open to the FID at the time of injection. The valve
is programmed to close at 1500 s to allow the column effluent to
be directed to the oxidation furnace. Calibration of the stable
nitrogen isotope composition of each component is accomplished
by comparison to three reference gas pulses (each of 30 s
duration) introduced at the start of the run and following the
opening of the heart split valve at the end of each run, i.e., after
4500 s. Unlike conventional IRMS, which requires the combustion
of ∼1 mg of an amino acid for isotope analysis, the N2 effluent
(21) Engel, M. H.; Macko, S. A.; Silfer, J. A. Nature 1 9 9 0 , 348, 47-49.
(22) McKay, D. S.; Gibson, E. K., Jr.; Thomas-Keprta, K. L.; Vali, H.; Romanek,
C. S.; Clemett, S. J.; Chillier, X. D. F.; Maechling, C. R.; Zare, R. N. Science
1 9 9 6 , 273, 924-930.
(23) Fry, B.; Brand, W.; Mersch, F. J.; Tholke, K.; Garritt, R. Anal. Chem. 1 9 9 2 ,
64, 288-291.
(24) Freedman, P. A.; Gillyon, E. C. P.; Jumeau, E. J. Am. Lab. (Fairfield, Conn.)
1 9 8 8 (June), 114-119.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 927

Figure 1. Schematic of the GC/C/IRMS system.
Figure 2. Chromatogram of the TFA/IP esters of the racemic mixtures of amino acids. The trace at the top is the instantaneous ratio of mass
29 to mass 28 ion currents. The trace at the bottom is of the mass 29 and mass 28 ion currents as a function of time. The y-axis is the amplifier
response for the minor (mass 29 ion).
from the reduction furnace of the GC/ C/ IRMS system is intro-
duced directly into the ion source (5 × 10-6 mbar) of the Optima,
allowing for analysis of each component at the low nanomole
concentration of injection.
underivatized values (Table 1). As in the case of carbon,16 small
discrepancies may, in part, reflect impurities of unknown stable
isotope compositions either in the reagents used for derivatization
or in the bulk amino acid standards.
GC/ C/ IRMS. It is apparent that the chromatographic separa-
tion employed in GC/ C/ IRMS eliminates impurites from the bulk
sample derivatives. The δ15N values of the amino acid derivatives
determined by GC/ C/ IRMS were, within the analytical error of
the methods, almost identical to the δ15N values of their respective,
underivatized amino acids (Table 1). In fact, average replicate
values for seven of the amino acids determined by GC/ C/ IRMS
were within 0.1‰ of their underivatized values. These results
RESULTS AND DISCUSSION
Conventional IRMS. We previously reported16 a stable
carbon isotope fractionation during amino acid acylation that likely
resulted from the preferential incorporation of 12C during bond
rupture and formation. In general, however, the δ15N values of
the TFA/ IP amino acid derivatives determined by conventional
IRMS were within 0.5‰ of the δ15N values of their respective
928 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997

Table 1. δ15N Values of Amino Acids Determined by
IRMS and GC/C/IRMS
Table 2. δ15N Values for Racemic Mixtures
(GC/C/IRMS)
amino acid
δ15Na
δ15N derivativeb
δ15N GC/ C/ IRMSc
amino acid
δ15Na
amino acid
δ15Na
D-Ala
L-Ala
â-Ala
D-Val
L-Val
Ivald
12.5
-6.3
-3.6
4.6
7.8
9.7
1.2
2.8
-5.3
-1.7
5.1
11.8
-6.3
-4.0
4.4
7.2
9.3
1.3
2.8
-4.8
-2.0
4.7
12.4
-5.9
-3.7
4.2
7.7
10.0
1.3
D-Ala
L-Ala
D-Val
L-Val
D-Leu
L-Leu
4.29
4.27
18.39
18.44
7.87
D-Asp
L-Asp
D-Glu
L-Glu
-2.58
-2.77
17.86
18.80
7.63
Gly
a
D-Leu
D-Asp
D-Phe
R-Aibae
2.7
Values are averages of nine GC/ C/ IRMS runs. The standard
deviation for each analysis is e(0.4‰.
-5.2
-1.5
5.0
acids were, within the current limits of the method, identical
(Table 2). The larger discrepancy observed for D- and L-glutamic
acid may result from a minor contribution of column bleed, as
these two components elute at a higher temperature at the end
of the run.
a
The δ15N values are for underivatized amino acids determined by
conventional IRMS; standard deviation (0.1‰. b The δ15N derivative
values were determined by conventional IRMS; standard deviation
(0.1‰. c The GC/ C/ IRMS values are averages of nine runs, with an
average standard deviation for each amino acid of e(0.4‰. d Ival,
isovaline. e R-Aiba, R-aminoisobutyric acid.
CONCLUSIONS
It is now possible to determine δ15N values of amino acid
enantiomers by GC/ C/ IRMS. Within experimental error, δ15N
values of amino acid derivatives obtained by GC/ C/ IRMS are
indistinguishable from δ15N values of the underivatized amino
acids determined by conventional IRMS. This new GC/ C/ IRMS
approach is superior in that (1) it permits the analysis of much
smaller samples (nanomoles as opposed to milligram quantities
required for IRMS) and (2) it will enable researchers to monitor
stable nitrogen isotope variation of individual components within
bulk samples, thus providing a better understanding of isotope
fractionations associated with biosynthetic pathways and the
diagenesis of organic matter in the geosphere. Furthermore,
stable nitrogen isotope analysis of amino acid enantiomers by GC/
C/ IRMS will provide an independent verification of the molecular
stable carbon isotope method currently used to establish the
indigeneity of these compounds in fossil15 and extraterrestrial21
materials.
clearly indicate that stable nitrogen isotope fractionation during
derivatization is minimal.
As a further test of the method, replicate GC/ C/ IRMS analyses
were performed on a mixture of racemic amino acids. Excellent
resolution of the
D- and L-enantiomers of the respective amino
acids was achieved with the chromatographic column employed
(Figure 2). Unlike carbon,25 the instantaneous 29/ 28 ratio for N2
clearly indicates a normal chromatographic discrimination (Figure
2), in which molecules containing the lighter nitrogen isotope (m/ z
28) elute prior to molecules containing the heavier isotope (m/ z
29). This isotope effect is the reverse of that observed for 13C-
GC/ C/ IRMS25 and is indicative of the lack of influence of the
chromatographic separation on the 15N component of the deriva-
tive. As previously documented for carbon,26 the lack of shoulders
on the peak inflections of the 29/ 28 ratios can be used as an
independent check of compound purity. Coeluting compounds
are likely to have at least slight differences in isotope compositions,
which would be observed as minor abberations (shoulders) on
the 29/ 28 ratios.
ACKNOWLEDGMENT
This work was supported by the National Science Foundation
(Grant EAR-9504618). We thank T. Brockwell (Micromass, Inc.)
for his assistance with the GC/ C/ IRMS analyses.
The δ15N values for individual amino acid enantiomer deriva-
tives in this racemic mixture had an average standard deviation
of e(0.4‰. Thus, with the exception of glutamic acid, the δ15N
values for the respective stereoisomers of the individual amino
Received for review September 18, 1996. Accepted
December 3, 1996.X
(25) Hayes, J. M.; Freeman, K. H.; Popp, B. N.; Hoham, C. H. Org. Geochem.
1 9 9 0 , 16, 1115-1128.
AC960956L
(26) Lichtfouse, E.; Freeman, K. H.; Collister, J. W.; Merritt, D. A. J. Chromatogr.
1 9 9 1 , 585, 177-180.
X Abstract published in Advance ACS Abstracts, January 15, 1997.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 929