Gaisberger and Solar
395
97%), sodium hydroxide (Fluka, 98%), K3Fe(CN)6 (Fluka,
98%), and phosphoric acid (Aldrich, 98%). Solutions were
made with triple-distilled water. To obtain oxidizing condi-
tions, the solutions were saturated with high purity N2O or
oxygen (5.0, Messer Griessheim) prior to irradiation.
Introduction
Hydroxybenzoic acids and their derivatives are ubiquitous
in plant material. The phenolic groups of these compounds
are often methoxylated or present as glycoside (1–3). Upon
irradiation of aqueous aromatic compounds, the main pri-
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mary process is the electrophilic addition of OH -radicals to
Gamma radiolysis
activated ring positions, which can eventually result in
hydroxylation of the initial material. Such radiolytically
formed hydroxylated compounds can be used as markers for
irradiated food, as is the case with o-tyrosine, originating
from phenylalanine in protein rich food (4–7). The detection
of hydroxylation products from phenolic acid-food compo-
nents was proposed in 1989 as a promising chemical method
for the detection of irradiation treatment of fruits and vegeta-
bles (8, 9), but no marker compounds have been found so
far.
γ-Radiolysis experiments (Gammacell 220, MDS Nordion
International Inc., Kanata, Ontario, Canada) were carried out
at a dose rate of 160 Gy min–1, which was determined by
Fricke dosimetry using a radiation chemical yield of G-
value2 G (Fe3+) = 15.6 = 1.617 µmol J–1 (16).
Product analysis
Product analysis was carried out by high-performance liq-
uid chromatography (HPLC) using a Hewlett–Packard 1050
chromatograph (column: 125 × 4 mm Spherisorb ODS 2 RP-
18 (5 µm), Lichrosorb precolumn, flow rate 1.0 ml min–1)
equipped with a multiple wavelength detector. The mobile
phase consisted of water (Millipore® filtered) and methanol
(Promochem, HPLC grade), the binary gradient was water–
methanol 10–35%. (The gradient was 1–5 min: H2O:CH3OH =
90:10, 5–10 min: H2O:CH3OH = 85:15, 10–20 min:
H2O:CH3OH = 65:35 per volume). As for 3-HBA the mobile
phase was H2O:CH3OH = 90:10. The individual compounds
were detected by measuring their absorptions at 210, 235,
and 255 nm. Concentrations were determined by calibration
with standard solutions. The identity of the products was
confirmed by comparing the retention time and the UV spec-
tra to that of the corresponding standards. Irradiations were
carried out at least in duplicate, the HPLC analyses were re-
producible within 10%.
For methoxybenzoic acids, it has been reported that upon
irradiation in the presence of N2O and at pH ≤ 3, radical cat-
ions and phenoxyl radicals are generated. Radical cations are
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formed from the nonipso OH -adducts by an acid-catalysed
water elimination. The phenoxyl radicals result from metha-
nol elimination from the ipso adduct carrying the methoxyl
group (10, 11). The work of these authors concentrated on
the detection of these radical intermediates by pulse
radiolysis and on the determination of methanol. There are,
however, no data concerning the formation of the phenolic
products from methoxylated benzoic acids. Upon radiolysis
of ortho- and para-hydroxybenzoic acid, the formation of
phenolic products has been found to be the major reaction
path (12–15).
This study focuses on a comparison of γ-radiation-
induced degradation and hydroxylation processes of benzoic
acids with free as well as methoxylated OH-groups, and its
importance for the detection of radiation-chemical changes
of phenolic components in food of plant origin. Priority was
given to the influence of oxygen on the product distribution.
Results and discussion
Radiolysis in the presence of N2O
In the first step of the investigations, the seven products
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obtained as a consequence of the OH -radical reactions with
Experimental
the isomers of MBA (anisic acids) were studied. These com-
pounds were used as model substrates for derivatives of
hydroxybenzoic acids having the phenol group linked with
other residues. 3-HBA was investigated for comparison.
Exposure of water or dilute aqueous solutions to γ-radia-
Chemicals and preparation of the solutions
All chemicals were of the highest purity commercially
available: 4-methoxybenzoic acid (4-MBA) (p-anisic acid)
(Aldrich, 99%), 3-MBA (m-anisic acid) (Aldrich, 99%), 2-
MBA (o-anisic acid) (Fluka, 99%), 2-hydroxybenzoic acid
(2-HBA) (Merck, 98%), 3-HBA and 4-HBA (Merck, 98%),
3,4-dihydroxybenzoic acid (3,4-DHBA) (Aldrich, 97%), 2,3-
DHBA (Aldrich, 99%), 2,5-DHBA (Aldrich, 97%), 4-
hydroxy-3-methoxybenzoic acid (vanillic acid (VA))
C
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tion leads to the primary species e −aq, OH , H , H2, and
H2O2. In the presence of N2O, the e −aq are converted into
C
C
OH (eq. [1]), yielding a G (OH ) = 5.5, which corresponds
to a concentration of 0.57 µmol J–1.
−
−
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[1]
e aq + N2O → OH + OH + N2
(Aldrich,
97%),
3-hydroxy-4-methoxybenzoic
acid
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By addition of OH to methoxylated benzoic acids, iso-
meric methoxyhydroxycyclohexadienyl radicals (demon-
strated for 4-MBA in eq. [2]) are formed, which after several
(isovanillic acid (iso-VA)) (Sigma, 97%), 2-hydroxy-3-
methoxybenzoic acid (3-methoxysalicylic acid (3-MSA))
(Aldrich, 97%), 4-MSA (Aldrich, 97%), 5-MSA (Aldrich,
[2]
2 G-value is the number of molecules reacting per 100 eV of absorbed energy. Conversion into SI-units: a G-value of 1 molecule per 100 eV
corresponds to a radiation-chemical yield of 0.10364 µmol J–1.
© 2001 NRC Canada