Journal of Agricultural and Food Chemistry
Article
Figure 1. Possible complexes of copper with glucose and alanine.
The separation was performed using a fused silica DB-5MS column
(50 m length × 0.2 mm inner diameter × 33 μm film thickness). The
GC method used for the analysis of the volatiles was as follows: The
GC column flow rate was regulated by an electronic flow controller
(EFC) and set at a pressure pulse of 55 psi for the first 3 min, then
decreased to 32 psi at the rate of 300 psi/min, and finally, increased to
70 psi at a rate of 1.23 psi/min for the rest of the run. The GC oven
temperature was set at −5 °C for first 5 min using CO2 as the
cryogenic cooling source and then increased to 50 °C at a rate of 50
°C/min. Then, the oven temperature was again increased to 270 °C at
a rate of 8 °C/min and kept at 270 °C for 5 min. The samples were
detected using an ion-trap mass spectrometer. The MS transfer-line
temperature was set at 250 °C; the manifold temperature was set at 50
°C; and the ion-trap temperature was set at 175 °C. The ionization
voltage of 70 eV was used, and electron multiplier voltage (EMV) was
set at 2000 V. The generated data was analyzed using the AMDIS_32,
version 2.69, computer software, and peak identification will be
performed using the National Institute of Standards and Technology
(NIST), version 2.0, mass spectral research program.
Liquid Chromatography−Mass Spectrometry (LC−MS) Anal-
ysis of the Amino Acid−Copper Synthetic Adducts. The dry
samples were dissolved in deionized water (Millipore, Billerica, MA) to
a concentration of 1 mL/mg. The sample was then diluted 10-fold in
10% methanol prior to analysis by LC−MS. The LC−electrospray
ionization (ESI)−MS analysis comprised of a Dionex Ultimate 3000
RS liquid chromatograph (Dionex, Germering, Germany) coupled to a
Bruker Maxis Impact quadruple-time-of-flight mass spectrometer
(Bruker Daltonics, Bremen, Germany) in positive mode. A total of 1
μL of the sample was injected directly into LC−MS. The electrospray
interphase settings were the following: nebulizer pressure, 0.6 bar;
drying gas, 8 L/min; temperature, 180 °C; and capillary voltage, 4500
V. The scan range was from m/z 100 to 1000. The data were analyzed
using Bruker Compass DataAnalysis software, version 4.1.
MATERIALS AND METHODS
■
Materials and Reagents. L-Alanine (99%), L-methionine (99%),
L-leucine (99%), L-glycine (98%), copper(II) chloride (CuCl2)
(99.9%), copper(I) chloride (CuCl) (97%), calcium chloride
(CaCl2), zinc chloride (ZnCl2) (98%), chloral hydrate (98%), 2-
methylpyridine (98%), potassium hydroxide, D-glucose, and acetalde-
hyde (99.5%) were purchased from Sigma-Aldrich Chemical Co.
(Oakville, Ontario, Canada). [13C-1]Alanine (98%), [13C-2]alanine
(98%), [13C-3]alanine (98%), [15N]alanine, and [13C-1, 13C-2]glycine
(98%) were purchased from Cambridge Isotope Laboratories
(Andover, MI).
Preparation of Model Systems. Model systems comprising of
amino acids alone, amino acids with metal salts (in a 2:1 relative molar
ratio), or synthetic amino acid metal complexes (see Table 1) were
Table 1. Composition of the Reactants in the Model Systems
a
amino acid
metal salt
synthetic metal complex
alanine
CuCl2/CuCl
FeCl2/FeCl3
ZnCl2
(Gly)2Cu
glycine
(Ala)2Cu
leucine
(Gly)Cu(Ala)
methionine
CaCl2
a
All of the listed amino acids were studied in the presence and absence
of metal ions. The amino acids alanine and glycine were studied with
all of the metal salts listed in the table and as synthetic metal
complexes. Leucine and methionine were studied only with CuCl2.
homogenized and mixed using a microscale porcelain mortar and
pestle. After thorough homogenization, approximately 0.5 mg of the
individual reactants or their mixtures were weighed into a quartz tube
(0.3 mm thickness), plugged at both ends with glass wool (Supelco,
Bellefonte, PA), inserted inside the coil probe, and pyrolyzed at 250
°C for 20 s under a helium atmosphere, as described below. All
samples were analyzed in duplicate.
Synthesis of Amino Acid−Metal Adducts. Alanine−copper and
glycine−copper complexes or mixed complexes were prepared by
dissolving alanine (0.89 g), glycine (0.75 g), [13C-1, 13C-2]glycine, or
an equimolar mixture of alanine and glycine at room temperature in
methanol (10 mL) in the presence of KOH (0.05 g) and stirred until
all completely dissolved, followed by the addition of 0.5 mol of CuCl2.
The dark blue (with CuCl2) precipitates were filtered, washed with
methanol, and dried.
Structural Identification. Specific reaction products (listed in
Table 2) were identified by comparison of their retention times and
mass spectra to commercial or in-situ-generated standards in addition
to NIST library matches and stable isotope labeling data (Table 2).
Elemental composition of selected metal complexes was based on their
accurate mass determination by ESI−QqTOF analysis [alanine−
copper complex at m/z 261.9985 [M + Na] (calculated for
C6H12CuN2NaO4 with an error of −1.4 ppm and glycine copper
complex at m/z 233.9665 [M + Na] (calculated for C4H8N2O4CuNa
with an error of 5.443 ppm].
Pyrolysis−Gas Chromatography/Mass Spectrometry (Py−
GC/MS). A Varian CP-3800 gas chromatograph coupled to a Saturn
2000 ion-trap detector interfaced to a CDS Pyroprobe 2000 unit
through a valved interface (CDS 1500) was used for Py−GC/MS
analysis of model systems shown in Table 1. The samples were packed
in the quartz tube inserted inside the coil probe and pyrolyzed at 250
°C for 20 s under a helium atmosphere.
RESULTS AND DISCUSSION
■
Under the Maillard reaction conditions, independent degrada-
tion reactions of amino acids play an important role in the
generation of amino-acid-specific products that result from
thermal decarboxylation or oxidative decarboxylation (Figure
8519
dx.doi.org/10.1021/jf502751n | J. Agric. Food Chem. 2014, 62, 8518−8523