Reactivity of thermally treated α-dicarbonyl compounds

Article abstract of DOI:10.1021/jf302959k

The degradation reaction of thermally treated 3-deoxy-d-erythro-hexos-2- ulose and methylglyoxal, both key intermediates in Maillard chemistry, was investigated. Different analytical strategies were accomplished to cover the broad range of formed products and their different chemical behavior. These involved HPLC-DAD and accordingly LC/MS analysis of the quinoxaline derivates, GC/MS analysis of the acetylated quinoxalines, and GC-FID analysis of the decyl ester of acetic acid. As a main degradation product of 3-deoxy-d-erythro-hexos- 2-ulose, 5-(hydroxymethyl)furfural could be identified. At alkaline pH values, 3-deoxy-d-erythro-hexos-2-ulose generated various acids but no colored products. In contrast, thermal treatment of methylglyoxal yielded high molecular weight, brownish products. A dimer of methylglyoxal, first precursor for aldol-based polymerization of methylglyoxal, could be clearly identified by GC/MS.

Full text of DOI:10.1021/jf302959k

Article
pubs.acs.org/JAFC
Reactivity of Thermally Treated α‑Dicarbonyl Compounds
Yvonne V. Pfeifer, Paul T. Haase,* and Lothar W. Kroh
Institute of Food Technology and Food Chemistry, Technical University of Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
ABSTRACT: The degradation reaction of thermally treated 3-deoxy-D-erythro-hexos-2-ulose and methylglyoxal, both key
intermediates in Maillard chemistry, was investigated. Different analytical strategies were accomplished to cover the broad range
of formed products and their different chemical behavior. These involved HPLC-DAD and accordingly LC/MS analysis of the
quinoxaline derivates, GC/MS analysis of the acetylated quinoxalines, and GC-FID analysis of the decyl ester of acetic acid. As a
main degradation product of 3-deoxy-D-erythro-hexos-2-ulose, 5-(hydroxymethyl)furfural could be identified. At alkaline pH
values, 3-deoxy-D-erythro-hexos-2-ulose generated various acids but no colored products. In contrast, thermal treatment of
methylglyoxal yielded high molecular weight, brownish products. A dimer of methylglyoxal, first precursor for aldol-based
polymerization of methylglyoxal, could be clearly identified by GC/MS.
KEYWORDS: α-dicarbonyl compounds, 3-deoxy-D-erythro-hexos-2-ulose, methylglyoxal, Maillard reaction
compounds. Furthermore, we intended to expand knowledge of
the formation of methylglyoxal-based melanoidines and
whether methylglyoxal polymers take part in the formation of
color.
INTRODUCTION
■
The term nonenzymatic browning comprises caramelization
and the Maillard reaction as the most important reactions.1,2
The Maillard reaction is a complex sequence of various
reactions, in which amino acids and reducing carbohydrates are
transformed into new components by heating. Because almost
every food contains carbohydrates, amino acids, and proteins,
respectively, this reaction plays a central role for manufacturing,
processing, and storage.3 α-Dicarbonyl compounds are key
intermediates of the Maillard reaction. These are significant for
flavor and color as well as for the stability of food.4,5 For
example, the Amadori compound of D-glucose delivers 1-
deoxyhexodiulose via the Amadori−Heyns rearrangement and
subsequent 2,3-enolization.3,6 This well-investigated compound
is converted to various products, e.g., 1-deoxythreosone or
threosone.7,8 These short-chain α-dicarbonyls are highly
reactive species, and they are essential for color formation
during the Maillard reaction.
3-Deoxy-D-erythro-hexos-2-ulose can be directly generated
from D-glucose or from the Amadori compound via 1,2-
enolization. The formation and reactivity of this α-dicarbonyl
compound is well-known in Maillard literature. Hodge already
postulated 3-deoxy-D-erythro-hexos-2-ulose as being involved in
the formation of HMF (5-(hydroxymethyl)furfural),1 an
important product from, for example, heated honey.9,10
Another reaction pathway of 3-deoxy-D-erythro-hexos-2-ulose
delivers high molecular weight, colored melanoindins. Via
retroaldol reactions, the α-dicarbonyl compound is converted
to methylglyoxal and subsequently to brown polymers.11
Caemmerer et al.5 postulated a mechanism whereby deoxy-
osones react with each other in an aldol-type condensation to
form a basic melanoidin skeleton of amino-branched sugar
degradation products.12 Both postulated melanoidin models are
key products for coloration.
MATERIALS AND METHODS
■
Chemicals. The following chemicals were obtained commercially.
D-Glucose, diacetone-D-glucose, o-phenylenediamine, and acetic acid
were purchased from Fluka (Neu-Ulm, Germany). L-Alanine, 5-
(hydroxymethyl)furfural, and tributyltin hydride were purchased from
Aldrich (Steinheim, Germany). Methylglyoxal (40% in H2O),
imidazole, sodium hydride (60% in mineral oil), methyl iodide,
Dowex 50WX8, and 2-deoxy-D-ribose were purchased from Acros
(Geel, Belgium). Pyridine, acetic anhydride, chlorosuccinic acid, decyl
chloroformate, carbon disulfide, PROD (250 UN), and catalase were
purchased from Sigma (Steinheim, Germany). Methanol, diethyl ether,
and ethyl acetate were purchased from Roth (Karlsruhe, Germany).
The quinoxalines of the standard HPLC mix were synthesized as
described by Pfeifer et al.14
Synthesis of 3-Deoxy-D-erythro-2-hexosulose (3-DH).13 3-
Deoxy-D-erythro-2-hexosulose (3-DH) was synthesized largely accord-
ing to the method of Voziyan et al.
1,2:5,6-Di-O-isopropylidene-3-O-(methylthio)thiocarbonyl-α-D-
glucopyranose. To a solution of 1,2:5,6-di-O-isopropylidene-α-D-
glucofuranose (5.20 g, 20.0 mmol) in 100 mL of absol THF were
added imidazole (30 mg) and then NaH (1.00 g, 40 mmol). The
mixture was vigorously stirred for 1 h under Ar atmosphere at ambient
temperature. Carbon disulfide (6.00 mL, 100 mmol) was added, and
stirring was continued for 2 h. Methyl iodide (3.00 mL, 48 mmol) was
then added, and the reaction mixture was stirred for an additional 1 h.
Then the reaction was quenched with 1 M HCl, and the organic layer
was washed with saturated NaHCO3 and brine. The solution was dried
with Na2SO4 and concentrated in vacuo to give the xanthate (6.01 g,
1
86%) as a yellow oil. TLC Rf 0.86 (EtOAc); H NMR (400 MHz,
CDCl3): δ [ppm] 1.41 (s, 12H), 2.00 (s, 3H), 3.83 (t, J = 2.8 Hz, 1H),
3.89 (m, 1H), 3.98 (m, 1H), 4.14 (m, 1H), 4.21 (m, 1H), 4.29 (m,
The aim of this work was to find out more about the
formation of color in 3-deoxy-D-erythro-hexos-2-ulose and
methylglyoxal model systems. We wanted to show that
shorter-chained α-dicarbonyl compounds are responsible for a
greater part in the colorization than the C6-α-dicarbonyl
Received: July 11, 2012
Revised: February 19, 2013
Accepted: February 23, 2013
Published: February 23, 2013
© 2013 American Chemical Society
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1H), 5.82 (d, J = 3.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ [ppm]
19.9, 26.6, 65.9, 73.1, 74.4, 76.6, 81.4, 105.6, 113.5, 215.3.
Water was degassed with helium before samples were prepared:
samples were deaerated with argon before thermal treatment.
Derivatization Reactions. Quinoxaline Derivatives. The method
described in ref 15 was adopted: 500 μL of the sample was spiked with
500 μL of o-phenylendiamine (0.05 M) and kept for 3 h at rt prior to
injection into the HPLC-DAD system. Quinoxalines were monitored
at λM = 320 nm. Quantification was carried out by comparison of peak
areas with those of standard solutions containing amounts of pure,
authentic reference compounds. The standard solution contains
commercially available quinoxalines such as quinoxaline, 2-methyl-
quinoxaline, and 2,3-dimethylquinoxaline and pure authentic quinoxa-
lines synthesized in our working group. With this quinoxaline method,
acids and imidazoles can be identified as well.
Acetylated Derivatives. The method described in ref 16 was
adopted: Samples were extracted twice with n-BuOH. The combined
organic layers were spiked with 5 μL of internal standard (5.5 mg of
diphenylquinoxalin/10 mL of toluene), and the solvent was dried. A
400 μL amount of toluene/pyridine (30:1) and 100 μL of acetic
anhydride were added to the residue and incubated for 30 min at 70
°C. The samples were analyzed by GC/MS in selected ion mode
(SIM). Quantification was carried out by comparison of peak areas
obtained in the TIC with those of standard solutions containing
amounts of pure, authentic reference compounds.
Decyl Chloroformate Derivative of Acetic Acid. The method
described in ref 17 was adopted: Samples (60 μL) were spiked with a
solution of chlorosuccinic acid (50 μg) in water as internal standard,
and 40 μL of pyridine and 50 μL of decyl chloroformate were added.
The mixture was sonicated for 10 min, and afterward the decyl esters
were extracted with 200 μL of hexane. The organic layer was analyzed
by GC-FID. Quantitative results were obtained by internal calibration
using commercially available acetic acid. (Data obtained with GC-FID
showed standard deviations <20 mmol/mol methylglyoxal and
resulting coefficients of variation <3.5%. Coefficient of determination
was always >0.97).
Chromatography. Thin-layer chromatography (TLC) was
performed on silica gel 60 F254 plates (Merck, Darmstadt, Germany).
Preparative column chromatography was performed on silica gel 60,
40−63 μm (Merck, Darmstadt, Germany). All solvents were of
chromatographic grade.
1,2:5,6-Di-O-isopropylidene-3-deoxy-α-D-glucofuranose. Tribu-
tyltin hydride (4.00 mL, 13.7 mmol) was dissolved in 50 mL absol
toluene and refluxed. Xanthate (3.50 g, 10 mmol) dissolved in 60 mL
of toluene was added by a dropping funnel. Refluxing was continued
overnight, and the solution was concentrated in vacuo. Acetonitrile (40
mL) and petroleum ether (40 mL) were added and stirred vigorously
for 15 min. The two phases were separated, and the petroleum ether
phase was washed with acetonitrile twice. The combined acetonitrile
phase was evaporated. Purification via flash chromatography on silica
(EtOAc:petroleum ether, 1:1) afforded pure 1,2:5,6-di-O-isopropyli-
dene-3-deoxy-α-D-glucofuranose (2.11 g) in 67% yield. TLC Rf 0.75
(EtOAc); 1H NMR (400 MHz, CDCl3): δ [ppm] 1.41 (s, 12H), 1.80
(m, 1H), 2.04 (m, 1H), 3.82 (t, J = 2.8 Hz, 1H), 4.11−4.14 (m, 3H),
4.76 (t, J = 8.4 Hz, 1H), 5.82 (d, J = 3.6 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ [ppm] 26.7, 35.2, 67.2, 78.6, 80.4, 105.6, 109.6, 111.3.
3-Deoxy-D-glucose. 1,2:5,6-Di-O-isopropylidene-3-deoxy-α-D-glu-
cofuranose (1.50 g, 6.14 mmol) was dissolved in 120 mL of 0.1%
H2SO4, and the reaction mixture was refluxed for 1 h. After cooling,
−
the solution was treated with Dowex 1X8 (HCO3 ) to adjust the pH
to 7. The mixture was filtered through a folded filter and concentrated
in vacuo to give 3-deoxy-D-glucose (1.01 g) in quantitative yield. TLC
Rf 0.05 (EtOAc).
3-Deoxy-D-erythro-hexos-2-ulose. 3-Deoxy-D-glucose (500 mg,
3.04 mmol) was dissolved in 500 mL of double distilled H2O and
stirred with aeration at 25 °C. PROD (250 IU) and catalase (10 000
IU, 0.2 mg, Sigma) were added while the pH was maintained at 7.0
with periodic additions of 0.01 N NaOH. The reaction was completed
after 5 h. The enzyme was filtered off through a membrane filter (0.2
μm filter) and rinsed with H2O. The solvent was removed at 30 °C in
vacuo. Purification via ionic exchange chromatography on Dowex
50X8 (200−400 mesh) in the Ca2+ form afforded pure 3-deoxy-D-
erythro-hexos-2-ulose (492 mg) in 99% yield. TLC Rf 0.2 (EtOAc); 1H
NMR (400 MHz, D2O): δ [ppm] 1.99 (s, 0.2H), 2.35−2.43 (q, J = 6
Hz, 2.5H), 2.58−2.59 (m, 1H), 2.62−2.64 (m, 1.5H), 2.67−2.68 (d, J
= 3.6 Hz, 0.5H), 3.05 (d, J = 7.2 Hz, 0.3H), 3.60−3.63 (m, 2.5H),
3.75−3.78 (m, 2H), 3.84−3.87 (m, 2H), 4.02−4.06 (m, 2.5H), 4.26−
4.29 (m, 1.5H), 4.41 (d, J = 2.8 Hz, 1.5H), 4.55−4.60 (m, 0.5H),
5.03−5.05 (m, 0.5H), 5.19 (s, 0.1H), 5.3 (q, J = 9.6 Hz, 0.3H), 8.22 (s,
1H), 8.29 (s, 0.2H), 8.45 (s, 1H). 13C NMR (100 MHz, D2O) δ
[ppm] 40.1, 40.3, 61.2, 62.7, 65.5, 69.3, 69.7, 71.9, 74.6, 164.3, 171.1,
179.5. GC/MS (3-DH-quinoxaline after acetylation): tR 27.99 min; m/
z 360 (0.2%, M+), 301 (7), 300 (9), 258 (12), 241 (49), 199 (33), 181
(24), 157 (34), 144 (100), 102 (11), 43 (89).
Nuclear Magnetic Resonance Spectroscopy (NMR). NMR
spectra were recorded on a Bruker AC 400 instrument (Rheinstetten,
Germany). Chemical shifts are given in parts per million relative to
residual nondeuterated solvent as an internal reference.
High Performance Liquid Chromatography (HPLC/DAD).
Instrumentation: Degasser, Gegasys DG-13000 (Knauer); pump,
Shimadzu LC-10 AT; thermostat, 30 °C, Shimadzu CT0-6A; guard
column, Nucleosil 120-5 C18 Macherey-Nagel; column, Nucleosil 5
C18 (250 mm × 4.6 mm); injection volume, 40 μL; eluent, methanol/
water gradient, 1 mL/min.
D-Glucose Model Reactions. In a typical experiment an aqueous
solution of D-glucose (180 mg, 1 mmol) with or without L-alanine (89
mg, 1 mmol) in 10 mL of H2O or phosphate buffer (pH 5) was
adjusted to a starting pH of 5 with 3 N HCl or to a pH of 8 with 3 N
NaOH. The model solutions were heated in sealed ampules at 100
1
High Performance Liquid Chromatography (HPLC/MS).
Instrumentation. Degasser: Agilent 1100; pump: Agilent 1100;
thermostat: 30 °C; guard column: Nucleosil 120-5 C18 Macherey-
Nagel; column: Nucleosil 5 C18 (250 mm × 4.6 mm); injection
volume: 40 μL; eluent: methanol/water gradient, 1 mL/min; detector:
Quattro LC, Waters.
°C or at 130 1 °C for up to 300 min in a thermoblock (Behr Labor
Technik, behrotest ET2). After a defined reaction time, 500 μL of the
samples was trapped with 500 μL of 0.05 M o-phenylenediamine
solution to intercept the α-dicarbonyl compounds as quinoxalines.
After 3 h at 25 °C, quinoxalines were analyzed by HPLC-DAD and
GC/MS after acetylation.
Gas Chromatography (GC/MS). Gas chromatograph: Finnigan
GCQ; capillary column: BPX 5 (SGE, 30 m, 0.25 mm ID, 0.5 μm film
thickness); carrier gas: helium 4.6; detector: Finnigan ion trap mass
analyzer GCQ; injection temperature: 270 °C; temperature program:
initial temperature 95 °C, hold 1 min, 95 °C to 200 °C at 15 °C/min,
200 °C for 1 min, 200 °C to 280 °C at 3 °C/min, 280 °C for 5 min,
280 °C to 300 °C at 5 °C/min, 300 °C for 5 min.
α-Dicarbonyl Model Reactions. In a typical experiment an
aqueous solution of an α-dicarbonyl compound (methylglyoxal, 3-
deoxy-D-erythro-hexos-2-ulose, 0.1 mmol) with or without L-alanine
(89 mg, 1 mmol) in 10 mL of H2O or phosphate buffer (pH 5) was
adjusted to a pH of 5 with 3 N HCl or to a pH of 8 with 3 N NaOH.
The model solutions were heated in sealed ampules at 100 1 °C or
at 130
1 °C for up to 300 min in a thermoblock (Behr Labor
Gas chromatography (GC/MS) was used for the oligomeric
compounds of MGO. Gas chromatograph: Agilent 5979; mode:
electron ionization (EI), 70 eV; capillary column: fused silica capillary
column Optima-5, (30 m, ID 25 mm, FD 1.0 μm Macherey-Nagel);
carrier gas: helium 4.6; injection temperature: 220 °C; temperature
program: initial temperature 50 °C isotherm, 50 °C to 300 °C at 120
°C/min, 300 °C for 2.4 min.
Technik, behrotest ET2). After a defined reaction time, 500 μL of the
samples was trapped with 500 μL of 0.05 M o-phenylenediamine
solution to intercept the α-dicarbonyl compounds as quinoxalines.
After 3 h at 25 °C, quinoxalines were analyzed by HPLC-DAD and
GC/MS after acetylation.
Deaerated Incubations. Degradation of α-dicarbonyl compounds
under deaerated conditions was carried out using bidistilled water.
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Figure 1. Degradation of 3-deoxy-D-erythro-hexos-2-ulose (3-DH) resulting in formation of 3,4-dideoxyglucoson-3-ene (3,4-DDH), methylglyoxal
(MGO), 5-(hydroxymethyl)furfural (HMF), and diacetyl (DA) in model systems thermally treated at 130 °C at a pH of 5 over a 300 min time
period measured as quinoxalines via HPLC.
Figure 2. Degradation of 3-deoxy-D-erythro-hexos-2-ulose (3-DH) resulting in formation of 2-ribonic acid, 3-deoxyhexonic acid, pyruvate,
methylglyoxal (MGO), and diacetyl (DA) in model systems thermally treated at 130 °C at a pH of 8 over a 300 min time period measured as
quinoxalines via HPLC.
Gas Chromatography (GC/FID). Gas chromatograph: HP 6890N
chromatograph equipped with a flame ionization detector. Capillary
column: BPX 5 (SGE, 30 m, 0.25 mm ID, 0.5 μm film thickness);
carrier gas: helium 4.6; injection temperature: 250 °C; split ratio: 1:10;
detector: 270 °C.
could be identified were assigned to o-phenylendiamine, 3-
deoxy-D-erythro-hexos-2-ulose, 3,4-dideoxyglucoson-3-ene,
methylglyoxal, diacetyl, and 5-(hydroxymethyl)furfural. As
excepted o-phenylenediamine applied as derivatization reagent
in large excess gave the highest signal. The degradation of 3-
deoxy-D-erythro-hexos-2-ulose (at a pH of 5) and the formation
of generated products are shown in Figure 1. Apparently, 3-
deoxy-D-erythro-hexos-2-ulose degradation proceeded quite fast.
After 30 min of thermal treatment at 130 °C, nearly 50% of the
α-dicarbonyl compound was decomposed, and after 180 min, it
could not be detected anymore. Obviously, 5-(hydroxymethyl)-
furfural was formed rather fast within the first 30 min;
afterward, the maximum was exceeded and the 5-
(hydroxymethyl)furfural concentration decreased. As minor
RESULTS AND DISCUSSION
■
The degradation of 3-deoxy-D-erythro-hexos-2-ulose and
methylglyoxal in the absence or presence of an amino acid in
model systems, which were thermally treated at 130 °C at a pH
of 5 or a pH of 8 for a defined time, was studied.
3-Deoxy-D-erythro-hexos-2-ulose Model Systems. The
degradation of 3-deoxy-D-erythro-hexos-2-ulose leads to several
peaks, which could be qualified and quantified as quinoxalines
via HPLC/MS or acetylated via GC/MS. Important signals that
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Figure 3. Degradation of methylglyoxal (MGO) resulting in formation of 3-deoxytetrosulose (3-DT) and diacetyl (DA) in model systems thermally
treated at 130 °C at a pH of 5 over a 300 min time period measured as quinoxalines via HPLC.
Figure 4. Degradation of methylglyoxal (MGO) resulting in formation of acetic acid and diacetyl (DA) in model systems thermally treated at 130 °C
at a pH of 8 over a 300 min time period measured as quinoxalines via HPLC.
compounds, 3,4-dideoxyglucoson-3-ene, methylglyoxal, and
later diacteyl could be identified. 3,4-Dideoxyglucoson-3-ene
is a precursor of 5-(hydroxymethyl)furfural and was generated
within the first 60 min and subsequently converted into 5-
(hydroxymethyl)furfural. Methylglyoxal and diacetyl could be
detected but only in very low concentrations.
Figure 2 shows the degradation of 3-deoxy-D-erythro-hexos-2-
ulose at an alkaline pH value. In comparison to slightly acidic
reaction conditions, the observed product is completely
different. Important signals that could be identified were
assigned to 3-deoxy-D-erythro-hexos-2-ulose, 2-deoxyribonic
acid, 4,5,6-trihydroxy-2-oxohexanoic acid (in the following
referred to as 3-deoxyhexonic acid), pyruvate, methylglyoxal,
and diacetyl. What is remarkable here are the very high
amounts of 2-deoxyribonic acid, which could already be
detected in the unheated standard. 3-Deoxy-D-erythro-hexos-2-
ulose underwent oxidative cleavage at room temperature and
yielded formic acid and 2-deoxyribonic acid. After thermal
treatment at 130 °C for 30 min, 3-deoxy-D-erythro-hexos-2-
ulose could not be detected anymore. A further degradation
product of 3-deoxy-D-erythro-hexos-2-ulose, which was gen-
erated in relatively high amounts, was 3-deoxyhexonic acid.
Obviously, this compound was formed rather slowly and did
not pass a maximum concentration after 300 min of thermal
treatment. Short-chain compounds such as methylglyoxal,
pyruvate, an oxidation product of methylglyoxal, and diacetyl
could be detected but only as minor decomposition products.
The Maillard model system 3-deoxy-D-erythro-hexos-2-ulose/
L-alanine (not shown within this work) afforded a quite similar
product spectrum. The only difference was that no 5-
(hydroxymethyl)furfural could be detected but rather the
pyrrole derivative. In all 3-deoxy-D-erythro-hexos-2-ulose model
systems, but especially in the alkaline model systems,
formaldehyde could be detected as a precursor for the oxidative
formic acid formation.
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Methylglyoxal Model Systems. To compare the reaction
behavior of 3-deoxy-D-erythro-hexos-2-ulose, a α-dicarbonyl
compound, which is stabilized by formation of many isomers,
the same experiments were accomplished with methylglyoxal, a
short-chain α-dicarbonyl compound. Figure 3 shows the
thermal treatment at 130 °C at a pH of 5 of a methylglyoxal
model system under aerated conditions. Important signals that
could be identified were assigned to methylglyoxal, 3-
deoxytetrosulose, and diacetyl. Obviously, methylglyoxal
degradation was relative slow; after a thermal treatment at
130 °C for a 300 min time period, the α-dicarbonyl compound
still could be measured in concentrations of 20% of the starting
value. 3-Deoxytetrosulose could be detected as a α-dicarbonyl
compound, too. This product could be generated from
methylglyoxal and formaldehyde. Formaldehyde was formed
in high amounts due to a α-dicarbonyl cleavage of
methylglyoxal. The LC/MS experiments as well as HPTLC
measurements (data not shown) showed that most of the
decomposed methylglyoxal is converted to a dimer of
methylglyoxal. This compound was generated in concentrations
almost up to 50% of the methylglyoxal starting value.
deoxyhexonic acid (Figure 5). As minor components, the
autoxidation product pyruvate and the α-dicarbonyl com-
At alkaline pH values, the concentration of methylglyoxal
decreased relatively fast (see Figure 4). The thermal treatment
of methylglyoxal at 130 °C at a pH of 8 afforded a completely
different product spectrum in comparison to experiments at a
pH of 5. As a main degradation product, acetic acid could be
identified. Acetic acid is an autoxidation product and could not
be measured under deaerated conditions. Another α-dicarbonyl
compound, which could be analyzed as a minor degradation
product, was diacetyl.
In Maillard model system methylglyoxal/L-alanine the
degradation of proceeded faster. At slightly acidic pH values a
quite similar product spectrum could be observed, and at
alkaline pH values, the amino acid had influence on the
generated products. Whereas in pure methylglyoxal model
systems acetic acid is one of the main degradation products, in
the Maillard model systems with L-alanine, pyruvate could be
measured in high amounts (e.g., after a thermal treatment over
a 60 min time period, 60% of the starting value was converted
to pyruvate). On the basis of all these results, the product
formation during the degradation of 3-deoxy-D-erythro-hexos-2-
ulose depends on the adjusted pH value. Whereas at slightly
acidic pH values one of the main degradation products was 5-
(hydroxymethyl)furfural, at alkaline pH values the formation of
autoxidation products such as 2-deoxyribonic acid and 3-
deoxyhexonic acid was preferred. Methylglyoxal yielded at pH 5
as main components the aldehydes formaldehyde and
acetaldehyde and a dimer of methylglyoxal. At alkaline pH
values, again autoxidation products such as acetic acid and
pyruvat are generated predominantly.
Figure 5. Alkaline degradation pathway of 3-deoxy-D-erythro-hexos-2-
ulose (3-DH) via oxidative processes yielded 2-deoxyribonc acid, 3-
deoxyhexonic acid, and pyruvate.
pounds methylglyoxal and diacetyl could be detected. These α-
dicarbonyl compounds are typical degradation products
reported in Maillard literature. Yaylayan and co-workers18,19
found that a C3/C4-cleavage of 3-deoxy-D-erythro-hexos-2-ulose
produced methylglyoxal and glyceraldehyde that subsequently
underwent amino acid-mediated chain elongation to form
diacetyl.
2-Deoxyribonic acid was generated via an oxidative α-
dicarbonyl cleavage. Direct autoxidation of 3-deoxy-D-erythro-
hexos-2-ulose afforded the oxidation product 3-deoxyhexonic
acid. One explanation for the very high amounts of 2-
deoxyribonic acid could be the rise of entropy (Figure 5).
Due to the formation of various small molecule acids at alkaline
pH values as main degradation products of 3-deoxy-D-erythro-
hexos-2-ulose, no coloration of the reaction solution could be
detected.
Having demonstrated that color formation is not based on 3-
deoxy-D-erythro-hexos-2-ulose in Maillard reaction systems, we
turned our focus on the investigation of methylglyoxal-derived
melanoidins. Thermal treatment of methylglyoxal model
solutions is well investigated in Maillard literature. Fiedler
and co-workers11 postulated a high molecular weight
melanoidin skeleton based on methylglyoxal units (Figure 6).
Due to the conjugated melanoidin structure derived from
methylglyoxal polymerization, the model solutions are highly
embrowned. The aldol condensation reactions which lead to
the melanoidins proceed very efficiently at alkaline pH values,
which explains the even more intensive coloration at pH 8.
Our investigations showed that methylglyoxal reacts under
slightly acidic conditions via two main degradation pathways
(Figure 6). Short-chained reactive products are generated via an
aldol reaction of methylglyoxal with formaldehyde to 3-
deoxytetrosulose or by dimerization of acetaldehyde and a
subsequent oxidation to yield diacetyl. The other pathway leads
to high molecular weight, brownish compounds via a dimer,
which affords in further aldol condensation the melanoidin
Although mechanistic studies of the HMF formation from D-
glucose, respectively, 3-deoxy-D-erythro-hexos-2-ulose, are well
investigated, we could show in our work that 3-deoxy-D-erythro-
hexos-2-ulose did not produce colored products such as in
Maillard literature-postulated melanoidines5 but preferentially
acidic cleavage products. Thermal treatment of a 3-deoxy-D-
erythro-hexos-2-ulose solution in the absence or presence of an
amino acid at 130 °C over a 300 min time period did not
embrown the solution, and the maximal absorption (measured
at 420 nm) was 0.1 (control: double distilled water).
At alkaline pH values, the product spectrum derived from 3-
deoxy-D-erythro-hexos-2-ulose is more complex. Main products
are the autoxidation products 2-deoxyribonic acid and 3-
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Figure 6. Main degradation pathways of thermally treated methylglyoxal under slightly acidic conditions yielded short-chain α-dicarbonyl
compounds such as 3-deoxytetrosulose (3-DT), diacetyl, and high molecular weight melanoidins.
Figure 7. The GC/MS chromatogram of MGO and its polymers: the dimer at 2.67 min and the trimer at 3.45 min.
postulated by Fiedler.11 The dimer could be identified by LC/
MS for the first time, was further investigated by GC/MS, and
is a good choice for the postulated melanoidin structures.
Therefore, an aqueous solution of methylglyoxal was stored for
seven days at 0 °C and subsequently measured and interpreted
by GC/MS without derivatization. Two of the main resulting
products were a dimer of methylglyoxal with a mass/charge
ratio of 146.06 g/mol [m/z = 145 → 115 → 73 → 59 → 43]
and a trimer with a mass/charge ratio of 216.06 g/mol [m/z =
215 → 187 → 159 → 115 → 73 → 59 → 43] (MS
fragmentation in the negative mode in square brackets) (Figure
7).
Thermal treatment of the Maillard model system methyl-
glyoxal/L-alanine leads to deeper embrowned solutions, e.g.,
thermal treatment at 130 °C over a 300 min time period at a
pH of 5 gave an absorption of 0.8 (measured at 420 nm,
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(10) Fallico, B.; Arena, E.; Zappala, M. Degradation of 5-
Hydroxymethylfurfural in Honey. J. Food Sci. 2008, 73, C625−C631.
(11) Fiedler, T.; Moritz, T.; Kroh, L. W. Influence of alpha-
Dicarbonyl Compounds to the Molecular Weight Distribution of
Melanoidins in Sucrose Solutions, Part 1. Eur. Food Res. Technol. 2006,
223, 837−842.
(12) Caemmerer, B.; Kroh, L. W. Investigation on the Influence of
Reaction Conditions on the Elementary Composition of Melanoidins.
Food Chem. 1995, 53, 55−59.
(13) Chetyrkin, S. V.; Zhang, W.; Hudson, B. G.; Serianni, A. S.;
Voziyan, P. A. Pyridoxamine Protects Proteins from Functional
Damage by 3-Deoxyglucosone: Mechanism of Action of Pyridoxamine.
Biochemistry 2008, 47, 997−1006.
(14) Pfeifer, Y. V.; Kroh, L. W. Investigation of Reactive α-
Dicarbonyl Compounds Generated from the Maillard Reactions of L-
Methionine with Reducing Sugars via Their Stable Quinoxaline
Derivatives. J. Agric. Food Chem. 2010, 58, 8293−8299.
(15) Hollnagel, A.; Kroh, L. W. Degradation of Oligosaccharides in
Nonenzymatic Browning by Formation of α-Dicarbonyl Compounds
via a “Peeling Off” Mechanism. J. Agric. Food Chem. 2000, 48, 6219−
6266.
dilution factor 100) in the absence of L-alanine and an
absorption of 1.3 in the presence of L-alanine. An explanation
for this could be the formation of the melanoidin structure
postulated by Tressl and co-workers (Figure 6).20
At alkaline pH values, the product spectrum derived from
methylglyoxal showed the same phenomenon of preferred
oxidation processes as already observed for 3-deoxy-D-erythro-
hexos-2-ulose model systems. Under caramelization conditions,
methylglyoxal reacted preferentially via an oxidative α-
dicarbonyl cleavage to formic acid and acetic acid. Under
Maillard conditions, thermal treatment of methylglyoxal yielded
pyruvate as one main product. Because of the relatively high
amounts of generated acids, the pH value decreased, and after a
short reaction time, the conditions were approaching those of
the pH 5 models. The increased colorization at alkaline pH
values could be explained with the preferred aldol reactions
under basic conditions. In contrast to acidic pH values the
conjugated melanoidins were built up faster and furnished deep
brown model solutions.
We showed that 3-deoxy-D-erythro-hexos-2-ulose and its
thermally formed degradation products are not directly
involved in coloration during the Maillard reaction. Main
degradation products are 5-(hydroxymethyl)furfural and
subsequently formed levulinic acid in the absence of amino
acids and pyrrole derivatives in presence of amino acids.
Furthermore, we validated the formation of brownish
melanoidins based on methylglyoxal units by LC/MS
investigations. Our results emphasize that color in Maillard
systems is based on short-chained α-dicarbonyl compounds and
its degradation products rather than on those with a C6-frame.
(16) Nedvidek, W.; Ledl, F.; Fischer, P. Detection of 5-
Hydroxymethyl-2-methyl-3(2H)-furanone and of alpha-Dicarbonyl
Compounds in Reaction Mixtures of Hexoses and Pentoses with
Different Amines. Z. Lebensm. Unters. Forsch. A 1992, 194, 222−228.
(17) Husek, P. Amino-Acid Derivatization and Analysis in 5 Minutes.
FEBS Lett. 1991, 280, 354−356.
(18) Yaylayan, V. A.; Keyhani, A. Origin of 2,3-Pentandione and 2,3-
Butandione in D-Glucose/L-Alanine Maillard Model Systems. J. Agric.
Food Chem. 1999, 47, 3280−3284.
(19) Yaylayan, V. A.; Keyhani, A. Origin of Carbohydrate
Degradation Products in L-Alanine/D[13C]-Glucose Model Systems.
J. Agric. Food Chem. 2000, 48, 2415−2419.
(20) Tressl, R.; Wondrak, G. T.; Garbe, L.-A.; Kruger, R.-P.; Rewicki,
̈
D. Pentoses and Hexoses as Sources of New Melanoidin-like Maillard
AUTHOR INFORMATION
■
Polymers. J. Agric. Food Chem. 1998, 46, 1765−1776.
Corresponding Author
*E-mail: paul.haase@tu-berlin.de, phone: +49 30 314 72 806,
fax: +49 30 314 72 585.
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Hodge, J. E. Chemistry of Browning Reactions in Model Systems.
J. Agric. Food Chem. 1953, 1, 928−943.
(2) Maillard, L. C. C. R. Acad. Sci. Ser. 1912, 154, 1554.
(3) Ledl, F.; Schleicher, E. Die Maillard-Reaktion in Lebensmitteln
und im menschlichen Korper - neue Ergebnisse zu Chemie, Biochemie
̈
und Medizin. Angew. Chem. 1990, 102, 597−626.
(4) Perez-Martinez, M.; Caemmerer, B.; Pena, M. D.; Kroh, L. W.
Influence of Brewing Method and Activity Regulators on the
Antioxidant Capacity of Coffee Brew. J. Agric. Food Chem. 2010, 58,
2958−2965.
(5) Caemmerer, B.; Kroh, L. W. Intact Carbohydrate Structures as
Part of the Melanoidin Skeleton. J. Agric. Food Chem. 2002, 50, 2083−
2087.
(6) Glomb, M. A.; Tschirnich, R. Detection of α-Dicarbonyl
Compounds in Maillard Reaction Systems and in Vivo. J. Agric. Food
Chem. 2001, 49, 5543−5550.
(7) Voigt, M.; Glomb, M. A. Reactivity of 1-Deoxy-D-erythro-hexo-
2,3-diulose: A Key Intermediate in the Maillard Chemistry of Hexoses.
J. Agric. Food Chem. 2009, 57, 4765−4770.
(8) Voigt, M.; Smuda, M.; Pfahler, C.; Glomb, M. A. Oxygen-
Dependent Fragmentation Reactions during the Degradation of 1-
Deoxy-D-erythro-hexo-2,3-diulose. J. Agric. Food Chem. 2010, 58,
5685−5691.
(9) Anet, E. F. L. J. 3-Deoxyglycosuloses (3-deoxyglycosones) and
the Degradation of Carbohydrates. Adv. Carbohydr. Chem 1964, 19,
181−218.
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