The most frequently used methods are based on high-
performance liquid chromatography (HPLC) using various types
derived Amadori compounds.24 However, the analysis of pentose-
derived Amadori compounds by the techniques mentioned above
has not been achieved so far.
9
,12,14-18
of stationary phases and detectors.
However, HPLC tech-
niques require time-consuming cleanup procedures to isolate the
Amadori compounds from complex matrixes prior to chromatog-
raphy. Furthermore, the majority of the methods developed offer
neither sufficient resolution nor satisfactory sensitivity. The most
efficient HPLC method was developed by Eichner et al. separating
up to 16 Amadori compounds on a DEAE-Si column and monitor-
ing them by UV detection at 480 nm after postcolumn derivati-
zation with triphenyltetrazolium chloride. This method offers both
The aim of this work was to develop a high-performance ion
exchange chromatography method that would be compatible with
MS detection and also suitable for rapid analysis of Amadori
compounds derived from both hexose and pentose sugars.
EXPERIMENTAL
Materials. The following compounds were obtained com-
mercially:
Darmstadt, Germany);
-isoleucine, -valine,
D
-glucose and sodium dihydrogen phosphate (Merck,
-xylose, glycine, -phenylalanine, -leucine,
-proline, -methionine, -alanine, and 2,3,5-
1
2,16
good separation and sensitivity.
D
L
L
Recently, a method based on high-performance anion exchange
chromatography (HPAEC) coupled with an electrochemical or
diode array detector has been reported as a powerful analytical
technique for the detection and monitoring of known Amadori
L
L
L
L
L
triphenyl-2H-tetrazolium chloride (TTC) (Aldrich/Fluka, Buchs,
Switzerland); sodium hydroxide 46/48% solution (Fisher Scientific,
Pittsburgh, PA). The glucose-derived Amadori compounds N-(1-
1
9-21
compounds.
The method permits sensitive and relatively
deoxy-
-proline (Fru-Pro), and N-(1-deoxy-
(Fru-Glu) were prepared using already reported procedures.
D
-fructos-1-yl)glycine (Fru-Gly), N-(1-deoxy-
D
-fructos-1-yl)-
selective detection of hexose-derived Amadori compounds, but
also simultaneous analysis of their precursors and some degrada-
tion products. However, unequivocal identification of Amadori
compounds can hardly be achieved by this method. In addition,
the chromatographic conditions for the analysis of hexose-derived
Amadori compounds are not suitable for the more labile pentose-
derived analogues due to their rapid on-column decomposition.
L
D-fructos-1-yl)-
L-glutamic acid
22,25,26
The solutions and eluents were prepared using ultrapure deionized
water (specific resistivity g18.2 MΩ‚cm) from a Milli-Q system
(Millipore, Bedford, MA). The poly(vinylidene fluoride) (PVDF)
syringe filters (0.45 µm) were from Supelco (Bellefonte, PA). All
other reagents were of analytical grade and were used without
further purification.
The quantification of the Amadori compound N-(1-deoxy-
fructos-1-yl)glycine was also achieved by fast atom bombardment
FAB) tandem mass spectrometry (MS).22 Its particular advantage
D-
Synthesis of N-(1-Deoxy-D-xylulos-1-yl)glycine (Xyl-Gly).
(
The Amadori compound Xyl-Gly was prepared as described in
the literature using some modifications.27 Xylose (270 g) and water
(45 mL) were placed in a round-bottom flask and heated in a
boiling water bath. When the temperature of the solution reached
90 °C, glycine (33.8 g) and sodium disulfite (40.5 g) were added
and the reaction mixture was heated at 90 °C for 20 min. After
cooling, the reaction mixture was dissolved in an ethanol/water
mixture (70 + 30, v/v, 1650 mL) and the solution was passed
through a column (5 × 40 cm) filled with a Dowex 50WX4 ion
is the simple sample preparation procedure, as the samples are
directly introduced into the ion source. However, simultaneous
analysis of several Amadori compounds cannot be achieved by
this method.
Over the past decade, FAB has been almost completely
replaced by electrospray ionization (ESI). This technique, offering
very “soft” ionization and easy coupling to on-line separation tools
as particular advantages, has recently been used in combination
with hydrophilic interaction liquid chromatography (HILIC-ESI-
MS) to identify five glucose-derived and three maltose-derived
+
exchange resin (H form). The resin was washed with an ethanol/
water mixture (70 + 30, v/v, 5 L) and then with water (1.5 L)
23
28
Amadori compounds in wheat gluten hydrolysates. The coupling
of tandem mass spectrometry to capillary electrophoresis (CE-
MS/MS) has successfully been applied to discriminate six glucose-
until the eluent was negative to the TTC test. Xyl-Gly was eluted
with ammonium hydroxide (0.2 mol/L) collecting 100-mL frac-
tions. Each fraction was tested using the TTC and Elson-Morgan
test.29 The fractions positive in both tests were collected, then
charcoal was added, and the filtrate was freeze-dried. Xyl-Gly was
obtained as an amorphous white powder (19 g, 22% yield) with a
purity of 98% by NMR. The product is hygroscopic and must be
stored under argon. A ternary mixture of the R, â, and open-chain
form was obtained in a ratio of 30/40/30 (Figure 1) based on the
(
14) van den Ouwerland, G. A. M.; Peer, H. G.; Tjan, S. B. In Flavour of Foods
and Beverages; Charalambous, G., Ed.; Academic Press: New York, 1978;
pp 131-143.
(
(
(
15) Moll, N.; Gross, B.; Vinh, T.; Moll, M. J. Agric. Food Chem. 1982, 30, 782-
7
86.
16) Reutter, M.; Eichner, K. Z. Lebensm. Unters. Forsch. 1989, 188, 28-35 (in
German); Chem. Abstr. 1989, 110, 211066d.
17) Debrauwer, L.; Vernin, G.; Metzger, J.; Siouffi, A. M.; Larice, J. L. Bull. Soc.
integration of H-C
1
and H-C
3
NMR signals.
Chim. Fr. 1991, 128, 244-254 (in French); Chem. Abstr. 1991, 115,
1
2
2
R-Xyl-Gly: H NMR (360 MHz, H
2
O, δ/ppm) 3.31 (d, 1H, J )
), 3.61-3.66 (m, 2H,
), 4.24 (m, 1H,
O, δ/ppm) 49.8 or
1
14893s.
2
(
(
18) Huyghues-Despointes, A.; Yaylayan, V. A. Food Chem. 1994, 51, 109-117.
19) Davidek, T.; Clety, N.; Aubin, S.; Blank, I. J. Agric. Food Chem. 2002, 50,
12.9 Hz, C
C1′), 3.90 (m, 1H, C
); 4.26 (m, 1H, C
1
), 3.38 (d, 1H, J ) 12.9 Hz, C
1
3
5
), 4.13 (d, 1H, J ) 5.5 Hz, C
3
5
472-5479.
20) Davidek, T.; Clety, N.; Devaud, S.; Robert, F.; Blank, I. J. Agric. Food Chem.
003, 51, 7259-7265.
21) Blank, I.; Davidek, T.; Devaud, S.; Clety, N. In The Maillard Reaction in
Food Chemistry and Medical Science: Update for the Postgenomic Era;
Horiuchi, S., Taniguchi, N., Hayase, F., Kurata, T., Osawa, T., Eds.;
International Congress Series 1245; Elsevier Science: Amsterdam, 2002;
pp 263-267.
13
2
C
5
4
); C NMR (90 MHz, H
2
(
(
2
(24) Hau, J.; Devaud, S.; Blank, I. Electrophoresis 2004, 2077-2083.
(25) Beksan, E.; Schieberle, P.; Robert, F.; Blank, I.; Fay, L. B.; Schlichtherle-
Cerny, H.; Hofmann, T. J. Agric. Food Chem. 2003, 51, 5428-5436.
(26) Blank, I.; Devaud, S.; Matthey-Doret, W.; Robert, F. J. Agric. Food Chem.
2003, 51, 3643-3650.
(
(
22) Staempfli, A. A.; Blank, I.; Fumeaux, R.; Fay, L. B. Biol. Mass Spectrom.
(27) Hodge, J. E.; Fisher, B. E. Methods Carbohydr. Chem. 1963, 2, 99-107.
(28) Horn, M. J.; Lichtenstein, H.; Womack, M. J. Agric. Food Chem. 1968, 16,
471-475.
1
994, 23, 642-646.
23) Schlichtherle-Cerny, H.; Affolter, M.; Cerny, C. Anal. Chem. 2003, 75, 2349-
354.
2
(29) Elson, L. A.; Morgan, W. T. J. Biochem. J. 1933, 27, 1824-1828.
Analytical Chemistry, Vol. 77, No. 1, January 1, 2005 141