3154 J. Agric. Food Chem., Vol. 48, No. 8, 2000
Zamora et al.
al., 1998). Briefly, the solution of pyrrole in water (800 µL)
was introduced into a 1.5 mL microtube and treated with 128
µL of 2% p-(dimethylamino)benzaldehyde in 3.5 M HCl/ethanol
Ta ble 1. Absor ba n ce Ma xim a a n d Extin ction Coefficien ts
of Eh r lich Ad d u cts Obta in ed fr om Mod el P yr r oles
absorbance maximum (extinction coefficient)a
(
4:1). The tube was closed and heated at 45 °C for 30 min,
compound
at 450-500 nm
at ∼520 nm
at ∼560 nm
557 (37000)
565 (38000)
and, finally, the maximum at 450-600 nm was determined
spectrophotometrically.
III
IV
496 (18000)
Rea ction of 4,5(E)-Ep oxy-2(E)-h ep ten a l w ith Lysin e.
Lysine (41 mg, 0.28 mmol) was dissolved in 7 mL of 0.3 M
sodium phospate buffer, pH 7.4, and treated with 18 mg (0.14
mmol) of 4,5(E)-epoxy-2(E)-heptenal. The reaction was main-
tained at 37 or 60 °C, and, at different intervals of time,
samples (500 µL) were withdrawn for analytical determina-
V
VI
VII
VIII
IX
521 (65000)
523 (56000)
511 (5)
481 (4)
485 (20)
557 (3)
563 (15)
517 (24)
541 (2500)
557 (6800)
553 (100)
563 (800)
X
XI
XII
XIII
XIV
tions. Samples were extracted with 500 µL of CHCl
3
/MeOH
(
2:1) and centrifuged at 2250g for 5 min, and the aqueous
phase was employed for the determination of color, fluores-
cence, and pyrrole content.
The color of the solutions was determined by using the
weighted-ordinate method (Hunter, 1973). Tristimulus values
490 (230)
521 (240)
528 (5900)
a Wavelength in nm; extinction coefficient in M-1.
(X, Y, Z) were calculated from the transmittances (T) obtained
in a Beckman spectrometer. Transmittances were recorded at
constant intervals (10 nm) from 400 to 700 nm using 1 cm
glass cells. These readings were then converted by means of a
computer program into the corresponding tristimulus and
CIELAB L* a* b* color values (CIE, 1978). The difference of
color (∆E) between CIELAB L* a* b* determined at the initial
time and that determined at each time was calculated,
according to Hunter (1973), using the following equation:
peared at 541 nm. In addition, compounds VII and VIII
also exhibited a similar maximum at 511-517 nm, but
the two maxima had very small extinction coefficients.
On the contrary, if there was no proton at the R-position,
the main maximum of the Ehrlich adduct appeared at
521-528 nm. Additionally, some derivatives exhibited
other maxima at wavelengths slightly below 500 nm.
It occurred with the pyrrole and the derivatives con-
taining ketone and nitrile groups. Thus, the pyrrole
exhibited a second maximum at 496 nm, which had an
extinction coefficient lower than the main maximum.
This maximum was also present in compounds VII,
VIII, and XIII but with much smaller extinction coef-
ficients.
2
2
2 1/2
∆
E ) [(∆a*) + (∆b*) + (∆L*) ]
(1)
Fluorescence spectra were recorded on a Perkin-Elmer LS-5
fluorescence spectrometer of 25-50 µL of aqueous phase
diluted to 2.5 mL with 50 mM sodium phosphate buffer, pH
7
.4. A slit width of 5 nm was used, and the instrument was
standardized with quinine sulfate (0.1 µM in 0.1 N H SO ) to
2
4
The highest extinction coefficients were obtained
when only hydrogen or alkyl groups were present as
substituents. These were compounds III)VI, and the
extinction coefficients ranged from 37000 for compounds
III and IV to 60000 for compounds V and VI. The
introduction of a hydroxyalkyl group decreased the
extinction coefficient, and it was smaller if the substitu-
ent was an aldehyde. Nevertheless, the smallest extinc-
tion coefficients were observed when the substituent
was a ketone.
These results suggested that in pyrrole mixtures, for
which the maximum observed is the sum of the maxima
of the different compounds, this maximum would mainly
be the sum of the unsubstituted pyrroles and the
pyrroles substituted only with alkyl groups because
these are the pyrroles with higher extinction coef-
ficients. By using an extinction coefficient of 37000 (for
the maximum at ∼560 nm) or 60000 (for the maximum
at ∼520 nm) in pyrrole mixtures, it is possible to
determine a minimum concentration of pyrroles in the
mixture, which will mainly correspond to the sum of the
unsubstituted and alkyl-substituted pyrroles present.
In the case of pyrrole polymerization, 2-(1-hydroxyal-
kyl)pyrroles are converted to alkyl-substituted polypyr-
roles (Hidalgo and Zamora, 1993), and these last
compounds should contribute significantly to the maxima
of the Ehrlich adducts.
give fluorescence intensity of 100 at 450 nm, when excitation
was at 350 nm. Results are given for 25 µL of sample.
Determination of pyrrole amino acids was carried out as
described previously. The pyrrole content was determined
spectrophotometrically at the maximum at ∼564 nm by using
an extinction coefficient of 37000.
Rea ction of Lin olen ic Acid w ith Lysin e. Lysine (41 mg,
0
.28 mmol) was dissolved in 7 mL of 0.3 M sodium phospate
buffer, pH 7.4, and treated with 39 mg (0.14 mmol) of linolenic
acid. The reaction was maintained at 37 or 60 °C, and, at
different intervals of time, samples (500 µL) were withdrawn
for determination of color, fluorescence, and pyrrole content.
3
Samples were extracted with 500 µL of CHCl /MeOH (2:1) and
centrifuged at 2250g for 5 min. The aqueous phase was
employed for the determinations, which were carried out as
described above. The only difference was the measurement of
pyrrole amino acids, which were determined spectrophoto-
metrically at the maximum at ∼525 nm by using an extinction
coefficient of 60000.
RESULTS
Deter m in a tion of Absor p tion Ma xim a a n d Ex-
tin ction Coefficien ts of Eh r lich Ad d u cts of Mod el
P yr r oles. As a first step in the determination of pyrrole
formation and polymerization using the Ehrlich reagent,
different model pyrroles were derivatized with p-(di-
methylamino)benzaldehyde and studied spectrophoto-
metrically. The reaction of a pyrrole ring with the
Ehrlich reagent always produced at least one main
maximum of absorbance at 500-600 nm. Both the
position and the intensity of this maximum depended
on the number and the type of the substituents in the
pyrrole ring. Table 1 collects the absorption maxima and
extinction coefficients of model pyrroles III)XIV. Most
of the pyrroles that had at least one proton in the
R-position exhibited the main maximum at 553-565
nm. However, for compound IX, this maximum ap-
R ea ct ion of 4,5(E)-E p oxy-2(E)-h ep t en a l w it h
Lysin e. To study how the nonenzymatic browning
produced as a consequence of pyrrole formation and
polymerization may be followed by using the Ehrlich
reagent, the reaction between 4,5(E)-epoxy-2(E)-hepte-
nal with lysine was selected because it has been well
characterized and it is known that the color and
fluorescence produced are a consequence of the forma-
tion of N-substituted 2-(1-hydroxyalkyl)pyrroles and