for the recently described N-pivaloylsopropyl (NPP) group13 per
mole of amino acid. Since amino acids range from 2 to 11 mol of
C/ mol of amino acid for glycine and tryptophan, respectively,
extraneous derivative C contributes a substantial amount of all C
in the analyzed product.
EXPERIMENTAL SECTION
Amino Acids, Solvents, and Reagents. All amino acids were
analytical grade, >98% purity and were obtained from Sigma
Chemical Co. (St. Louis, MO). Ethanol, methanol, and methylene
chloride of highest available purity and technical grade dichlo-
romethane (DCM) were obtained from Fisher Scientific Co. (Fair
Lawn, NJ). Tetrahydrofuran (THF) and NaBH4 were from Aldrich
Chemical Co. (Milwaukee, WI); I2 was from Mallinckrodt Chemi-
cal Works.
Reduction of Amino Acids. Alanine (Ala), valine (Val),
leucine (Leu), isoleucine (Ile), methionine (Met), and phenylala-
nine (Phe) were tested for reduction by the NaBH4/ I2 method.
Two grams of each was reduced to yield ample product for isotopic
analysis by elemental analyzer-IRMS (EA-IRMS), which requires
milligram quantities of sample per analysis, and to conveniently
assess purity by scanning infrared spectroscopy (IR).
Carbon isotope analysis of derivatized compounds in gas
chromatography continuous-flow (GCC)-IRMS presents two
problems. First, analytes are combusted immediately after emerg-
ing from the GC column to produce CO2 analysis gas. Extraneous
C from the derivative group is indistinguishable from analyte C
in the IRMS, and the δ13C of the analyte must be calculated using
the mass balance equation6 after an independent assessment of
the C isotope contribution from the derivative C. In addition to
possible biases in determination of the derivative C, imprecision
in the final result is unavoidable due to propagation of errors. In
general, this imprecision is larger when the contribution of
derivative carbon to the derivatized compound is a large fraction
of the derivatized analyte, which is usually the case in compounds
with less than 10 carbons, such as amino acids. A second issue is
the possibility of kinetic isotope fractionation of the analyte if the
reaction is not quantitative and if the procedure involves analyte
C bonds.18 This leads to bias that may not be detected with
standards.
Amino acids were reduced as described previously22 with slight
modification. Amino acid (2.0 g) was added to a stirred suspension
of NaBH4 (2.5-fold molar excess compared to amino acids) in THF
(50 mL). The flask was immersed in an ice bath, and a solution
of I2 (equimolar with amino acid) in 10 mL of THF was added
dropwise over a period of 15 min to maintain the temperature
below 20 °C, resulting in vigorous evolution of H2. After addition
of the I2 was complete and gas evolution had ceased, the reaction
mixture was refluxed for 15 h and then cooled to room temper-
ature. Methanol was added carefully until the mixture became
clear to exhaust excess NaBH4. After 45 min, the solvent was
evaporated under an N2 stream leaving a white paste, which was
subsequently dissolved by addition of 33 mL of 20% (w/ w) KOH.
The solution was stirred for 4 h. After three extractions with 50
mL of methylene chloride, the organic extract was dried over
sodium sulfate and the solvent was removed by rotary evaporation.
To verify that the reaction could be scaled down, the reaction was
repeated with six amino acids (Ala, Val, Leu, Ile, Met, Phe) at 2
mg each. The purity of the extracted amino alcohols was evaluated
by infrared spectroscopy. Yields were determined by weighing
the amino acids before reduction and amino alcohols after
extraction and correcting for molecular weights.
GCC-IRMS. The on-line isotopic analysis system consisted
of a Hewlett-Packard 5890 GC equipped with flame ionization
detector (FID) and coupled to a Finnigan-MAT 252 (Bremen,
Germany) high-sensitivity, high-precision IRMS via an in-house-
built combustion/ water trap/ open split interface. The combustion
furnace is made of deactivated fused silica filled with oxidized Cu
wire and Pt catalyst and held at 850 °C. The water trap was of the
Nafion type (DuPont, Wilmington, DE), which eliminates H2O
while retaining CO2. Amino alcohols dissolved in water were
injected in split mode onto a CP-sil 5 CB fused-silica capillary
column (50 m × 0.53 mm; 5-µm film thickness) (Chrompack Inc.)
with He carrier gas. The oven temperature program was increased
from 100 to 150 °C at 10 °C/ min, held at that temperature for 2
min, increased at 10 °C/ min to 250 °C, and finally held at that
temperature for 10 min. The injector and detector temperatures
were 240 and 250 °C, respectively. After separation, the amino
alcohols were either sent to a FID for methods development or
to the combustion interface via a rotary valve (Valco Instruments,
Houston, TX) for isotope analysis. High-precision isotope analyses
are expressed in δ13C notation, which is defined as
An alternative strategy is to modify the chemical properties of
the analyte without introducing extraneous C, which was imple-
mented successfully for conversion of fatty acid methyl esters to
fatty alcohols.19 An analogous approach is to convert amino acids
to amino alcohols by chemical reduction of the carboxyl group.
This approach is most likely to succeed for nonpolar amino acids
since most homologous amino alcohols are sufficiently volatile
for GC analysis. A number of methods have been reported for
chemical reduction of amino alcohols;20-23 however, most suffer
from one or more disadvantages such as excessive refluxing time
and expensive or relatively toxic reagents. A recent approach for
direct reduction of amino acids employs NaBH4 as the reducing
21
agent and an electrophilic catalyst such as H2SO4 or I2.22 We
chose the NaBH4/ I2 procedure because it is a one-step reaction
using inexpensive reagents.22
In this paper, we report an evaluation of this strategy for
reduction of nonpolar amino acids to their corresponding amino
alcohols, demonstrate a suitable GC method to produce sharp
peak shapes from amino alcohols, and evaluate any isotopic effects
of the procedure against bulk analysis of the amino acids before
and after treatment. Data are reported for isotopic analysis of 2 g
of analyte, and the chemical and chromatography results were
verified for 2 mg of analyte.
(15) Yarasheski, K. E.; Smith, K.; Rennie, M. J.; Bier, D. M. Biol. Mass Spectrom.
1 9 9 2 , 21, 486-90.
(16) Calder, A. G.; Garden, K. E.; Anderson, S. E.; Lobley, G. E. Rapid Commun.
Mass Spectrom. 1 9 9 9 , 13, 2080-3.
(17) Chen, Z.; Landman, P.; Colmer, T. D.; Adams, M. A. Anal. Biochem. 1 9 9 8 ,
259, 203-11.
(18) Rieley, G. Analyst 1 9 9 4 , 119, 915-9.
RSPL - RPDB
(19) Corso, T. N.; Lewis, B. A.; Brenna, J. T. Anal. Chem. 1 9 9 8 , 70, 3752-6.
(20) Kanth, J. V. B.; Periasamy, M. J. Org. Chem. 1 9 9 1 , 56, 5964-5.
(21) Abiko, A.; Masamune, S. Tetrahedron Lett. 1 9 9 2 , 33, 5517-8.
(22) McKennon, M. J.; Meyers, A. I.; Drauz, K.; Schwarm, M. J. Org. Chem. 1993,
58, 3568-571.
δ13C )
× 103
[
]
RPDB
(23) Anand, R. C.; Vimal Tetrahedron Lett. 1 9 9 8 , 39, 917-8.
where R is the [13C]/ [12C] ratio and SPL and PDB refer to sample
800 Analytical Chemistry, Vol. 73, No. 4, February 15, 2001