HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Article abstract of DOI:10.1080/10715762.2018.1517260

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.

Full text of DOI:10.1080/10715762.2018.1517260

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
HPLC study on Fenton-reaction initiated oxidation
of salicylic acid. Biological relevance of the
reaction in intestinal biotransformation of salicylic
acid
Eszter Nyúl, Mónika Kuzma, Mátyás Mayer, Sándor Lakatos, Attila Almási &
Pál Perjési
To cite this article: Eszter Nyúl, Mónika Kuzma, Mátyás Mayer, Sándor Lakatos, Attila Almási &
Pál Perjési (2018): HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological
relevance of the reaction in intestinal biotransformation of salicylic acid, Free Radical Research,
DOI: 10.1080/10715762.2018.1517260
To link to this article: https://doi.org/10.1080/10715762.2018.1517260
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Sep 2018.
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HPLC study on Fenton-reaction initiated oxidation
of salicylic acid. Biological relevance of the reaction
in intestinal biotransformation of salicylic acid.
Eszter Nyúl1, Mónika Kuzma1, Mátyás Mayer2,
Sándor Lakatos1, Attila Almási1, Pál Perjési1*
1Institute of Pharmaceutical Chemistry, University of Pécs, H-7624 Pécs, Rókus str. 2, Hungary
2Department of Forensic Medicine, University of Pécs, H-7624 Pécs, Szigeti str. 12, Hungary
Running title: In vitro - in vivo hydroxilation of salicylic acid.
*Corresponding author:
Pál Perjési, PhD
E-mail: pal.perjesi@aok.pte.hu
Postal address: H-7624 Pécs, Rókus str. 2., Hungary
Phone: +36 72 503 626
Fax: +36 72 503 627
Keywords
Fenton reaction
Reactive oxygen species (ROS)
Salicylic acid
Non-steroidal anti-inflammatory agents (NSAIDs)
Diabetes
Abbreviations:
HPLC: high performance liquid chromatography
UV-Vis: ultraviolet-visible
MS: mass spectrometry
l: microliter
µ
mM: millimol/liter
M: micromol/liter
µ
ROS: reactive oxygen species
CAT: catechol
SA: salicylic acid
2,3-DHBA: 2,3 dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid

2,5-DHBA: 2,5-dihydroxybenzoic acids
2,6-DHBA: 2,6-dihydroxybenzoic acid
4-NP: 4-nitrophenol
STZ: streptozotocin
NADPH: reduced nicotinic acid adenine dinucleotide phosphate
Abstract:
Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic
acid was investigated by HPLC-UV-Vis method. By means of the developed HPLC method
salicylic acid, catechol and all the possible monohydroxylated derivatives of salicylic acid can be
separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar
ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples
were analyzed at different time points of the reactions. The biological effect of elevated reactive
oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by
an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence
of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence of non-enzyme catalyzed
intestinal hydroxylation of xenobiotics.

1. Introduction
There is an increasing number of lines of epidemiological evidence that reactive oxygen (ROS)
and nitrogen (RNS) species are implicated in the pathogenesis of several chronic diseases, such
as cancer, cardiovascular and neurodegenerative diseases [1-3]. These reactive species can react
with sensitive endogenous and exogenous molecules to form characteristic products. It is well
documented in the literature that several acute and chronic diseases are accompanied by elevated
level of reactive oxygen species [4]. Among others, diabetes and acut or chronic inflammations
are those ones that can cause increased non-enzymatic oxidation of both endogenous compounds
and xenobiotics [5].
A large number of drug molecules containing aromatic rings are administered in the above
disorders in which involvement of oxidative damage has been suggested. Anti-inflammatory
drugs have been reported to scavenge hydroxyl radicals generated in solution at almost
diffusion-controlled rates (rate constants about 1010 M−1 s−1) [6]. Based on the latter reactivity,
aromatic hydroxylation can be used for measuring in vitro hydroxyl radical production [7].
Acetylsalicylic acid (aspirin) is a widely used medication to treat pain, fever, and inflammation.
Salicylic acid (SA) is the main primary metabolite of acetylsalicylic acid (ASA) [8]. The
hydrolytic transformation is rather fast; the halflife of ASA is about 20 minutes [9, 10].
Accordingly, pharmacological effects of aspirin substantially involve those of salicylic acid
[8]. The major route of metabolism of ASA and SA in humans is conjugation with glycin and
glucoronic acid [10, 11]. Two oxidative metabolites of salicylic acid are 2,3-dihydroxybenzoic
acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA) [10, 12, 13]. These products can
be attributed to oxidation by cytochrome P450 enzymes as well as Fenton-type non-enzymatic
hydroxylation. Attack by hydroxyl radicals has been reported to form the 2,3-DHBA and is
speculated to form some of the 2,5-DHBA product as well [5, 7, 12-15].
In our earlier study, in vivo intestinal absorption and oxidative metabolism of salicylic acid (SA)
was investigated in rats. In the intestinal perfusate low levels of 2,3-DHBA and 2,5-DHBA
were detected and identified by HPLC-MS [16]. As a continuation of our earlier study here we
report on the similar investigations performed with hyperglycemic rats. Hyperglycemia is one
of the leading factor of diabetic complications. It can result oxidative stress and accompanied
by increased formation of reactive oxygen species (ROS) [17].
Fenton reaction is a tool for modeling oxidative stress by forming hydroxyl radicals in a reaction
of hydrogen peroxide and some transitional metals. The key features of the reaction are believed
to be reaction conditions, such as reagent concentrations, pH and temperature [18]. The exact
nature of the oxidizing intermediates involved in Fenton reaction is still to be identified, but
there are several views about the mechanism and the nature of intermediate products which

may have effect on the outcome of the reaction [19]. One of the important processes during
the reaction is the iron(II)/iron(III) redox cycling because the iron(II) regeneration is the rate
limiting step of the Fenton system [20]. The optimal pH for this reaction and for the formation of
hydroxyl radicals is in the acidic range, near pH 3 [21]. In the presence of salicylic acid complex
formation between iron ions and salicylic acid may also influence the quality and the quantity
of the reaction products [22-24].
To identify and quantify the largest number of possible oxidative products of salicylic acid
under the investigated conditions, an HPLC-UV-Vis coupled with an HPLC-MS method was
developed. Earlier, identification and quantitation of the hydroxylated derivatives has been
accomplished by GC [25-27] and RP-HPLC methods [13, 28-32]. None of the published
methods can be applied, however, for separation of the parent salicylic acid and all the previously
known salicylic acid oxidation products.
In our present experiments Fenton-oxidation of salicylic acid was carried out in an acidic
medium (pH 3.0) with two reagent (molar) ratios: (1) salicylic acid: iron(II): hydrogen peroxide
1:3:1, and (2) salicylic acid: iron(II): hydrogen peroxide 1:0.3:1. Under the first condition large
excess of iron(II) was used, which can act as a hydroxyl radical scavenger in the absence of
organic substrates [33]. The latter conditions (iron(II): hydrogen peroxide appr. 1:3) is like
that one reported to be the most effective oxidative conditions [34]. Under such conditions all
possible oxidative derivative of salicylic acid is expected to be formed.
In the in vivo experiments, we aimed to investigate how the experimental diabetes affects the
intestinal elimination and metabolism of salicylic acid. To receive information about the possible
role of hyperglycemia-induced ROS formation in biotransformation of phenolic xenobiotics,
jejunal perfusion of streptozotocin (STZ) -treated animals with sodium salicylate was performed
and its elimination and oxidative metabolism was studied to compare the results with those
obtained in the non STZ-treated rats [16].
2. Materials and methods
2.1. Chemicals
2,3-Dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA) standards
were purchased from Sigma-Aldrich (Budapest, Hungary). Salicylic acid (SA), 2,4-
dihydroxybenzoic acid (2,4-DHBA)and 4-nitrophenol (4-NP) standards were obtained
from Fluka (Budapest, Hungary).Sodium sulfate, potassium chloride, magnesium sulfate,
calcium chloride dihydrate, glucose, mannitol and tris(hydroxymethyl)aminomethane (TRIS)
were purchased from Spektrum-3D Kft. (Debrecen, Hungary). HPLC-grade methanol
and tert-butyl methyl ether were obtained from VWR International Kft. (Debrecen,

Hungary). HPLC-grade formic acid was purchased from Sigma-Aldrich (Budapest, Hungary).
Iron(II)sulfateheptahydrate, catechol (CAT) and 2,6-dihydroxybenzoic acid (2,6-DHBA) were
purchased from Reanal (Budapest, Hungary). Sulfuric acid and concentrated hydrogenperoxide
(30 % v/v) were purchased from Molar Chemicals (Budapest, Hungary). Distilled water was
purified in the Department of Pharmaceutical Chemistry, University of Pécs by use of a Purelab
Option Q7 Water System.
2.2. Fenton incubations
In order to examine the outcome of the Fenton reaction with different iron (II) ion concentrations
and with different incubation time intervals 1.00 mL samples were prepared. Each incubate
contained 700 µL of sulfuric acid (pH 3.0), 100 µL of 10 mM salicylic acid solution, 100 µL
of 30 mM or 3 mM iron(II) solution and 100 µL of 10 mM hydrogen peroxide solution. The
components were mixed in the respective order and the reaction mixtures were placed in a 37°C
water bath. The samples were analyzed after „0”, 1, 5, 10 and 30 minutes of incubation. Before
analysis the incubates were concentrated with liquid-liquid extraction.
2.3. Quantum chemical calculations
Quantum Chemical calculations were performed using Chem 3D Ultra 10.0 (Cambridge
Scientific Computing, Inc.) molecular modeling and analysis software product. Mulliken atomic
charges were calculated by means of the MOPAC interface on the MM2-minimized ground state
Table 1
structures. Mulliken atomic charge data are summarized in
.
2.4. Animal experiments
Diabetic male Wistar rats (n=5; average bodyweight: 234.7 g; average blood glucose
concentration: 23.1 mmol/l) were treated according to the experimental protocol of Bojcsev et al.
[35].The experimental diabetes was induced by 65 mg/kg intravenous streptozotocin injection
pretreatment one week before the intestinal perfusion. The animals were anesthesized by 1.2
g/kg intraperitoneal urethane injection, then a 10 cm long jejunal segment was isolated and
cannulated. The intestinal perfusion was carried out by a peristaltic pump (flow rate: 13 ml/min)
with 250 M sodium-salicylate dissolved in isotonic perfusion medium. During the 90 minute
µ
time-course of the perfusion, samples with the volume of 250 l were collected in previously
µ
determined time points. The chromatographic analysis of the perfusion samples was performed
immediately after the sample collection and sample preparation.
Animals were kept according to standard protocols controlled by institutional regulations,
which adhere to the Act of Animal Care issued by the Hungarian Ministry of Economy and
Environmental Protection which is based on appropriate EU regulations.
2.5. Sample preparation method

Both the in vitro and the in vivo samples were concentrated and purified by the following liquid-
liquid extraction method.Before extraction, 10 µL of 1 mg/mL 4-nitrophenol solution as an
internal standard was added to the samples. Afterwards, the samples were acidified with 10 µL
of 2 M sulfuric acid and vortex mixed for 30 seconds. Then 500 µL of tert-butyl methyl ether
was added and vortex mixed for 30 seconds. The upper phase was separated and 300 µL of
formic acid solution (pH 2.6) was added in order to purify it. After 30 seconds of vortex mixing
the purified upper phase was separated and was evaporated under nitrogen. The dry residue was
dissolved in 50 µL chromatographic mobile phase (pH 2.6 formic acid/methanol; 77/23; v/v).
3. Instrumentation and chromatographic conditions
3.1. Agilent 1100 HPLC system
Analysis of the extracted incubates and identification of the formed products was performed by
an Agilent 1100 HPLC system.
The high performance liquid chromatography system was equipped with a quaternary HPLC
pump (G1311A), a degasser (G1379A), an autosampler (G1313A), a thermostated column
compartment (G1316A) and a diode-array detector (G1315B). Data were recorded and
evaluated by Agilent ChemStation software (Rev.B.03.02-SR2).
Chromatographic separation of the hydroxylated products was performed through a reversed-
phase chromatographic column (Poroshell 120 EC-C18, 4.6 mm x 150 mm, 2.7 µm particle
size). The mobile phase consisted of formic acid solution (pH 2.6) and methanol (77:23, v/v).
Runtime was 30 min. The isocratic separation was performed at 0.600 mL/min flow rate at 45
°C temperature. The injected volume was 5 µL. Detection was performed at 237 nm.
3.2. Jasco HPLC/Waters MS system
The integrated high performance liquid chromatography system (Jasco) was equipped with an
intelligent HPLC pump (PU-980), a degasser, a manual injector (RHEODYNE 7725i) with
a 5 μL loop, a column oven and a diode-array detector (MD-2010). Data were recorded and
evaluated by ChromNav software (ver. No. 1.21). Jasco LC-system is connected with a Waters
Xevo TQ-S Mass Detector. MS data were recorded and evaluated by MassLynx V4.1 software.
Separation of compounds was performed on a 4.6 mm x 150 mm, 2.7 µm particle size,
PowerShell 120 EC-C18 analytical column. The mobile phase consisted of formic acid solution
(pH 2.6) and methanol (77/23, v/v). Chromatography was performed at 45 °C, the flow rate
was 0.6 mL/min. The injected volume was 5 μL. MS measurement was performed in negative
ion mode. The ESI source was operated with a spray voltage of 3.00 kV, cone voltage was 30
V. Desolvation gas was delivered at 500 L/hour and temperatured at 500 °C. Cone gas was
delivered at 150 L/hour. Collision gas flow was 0.13 mL/min. Full-range mass spectra (80–400
m/z) were collected under the optimal conditions. During daughter scan measurement (20-420
m/z) of the selected parent ion the collision energy was 20 eV.

3.3. Preparation of working standard solutions and HPLC-MS stock solutions
10 mM (0.0138 g/10.0 mL) stock solution of SA was prepared in distilled water. 10 mM
hydrogen peroxide solution was obtained by dilution of 11.3 µL concentrated hydrogen peroxide
(30 % v/v) with distilled water to 10.0 mL. 30 mM (0.0834 g/10.0 mL) iron(II) sulfate stock
solution was prepared in sulfuric acid (pH 3.0). The 3 mM iron(II) sulfate solution was prepared
by tenfold dilution of 1.0 mL of the 30 M stock solution with sulfuric acid. For analytical method
development 1 mg/ml acetonitrile solutions were prepared from CAT, 2,3-DHBA, 2,4-DHBA,
2,5-DHBA, 2,6-DHBA, SA and 4-nitrophenol. The working standard solutions were always
prepared freshly before the experiments; the stock solutions were stable at least for 1 month,
stored at -8°C.
4. Validation of the chromatographic method
4.1. Linearity
Linearity was studied by preparing standard acetonitrile solutions of CAT, 2,3-DHBA, 2,4-
DHBA, 2,5-DHBA, 2,6-DHBA and SA at seven different concentration points between 0.5 g/
µ
ml and 50 g/ml (0.5; 0.75; 1; 5; 10; 25; 50 g/mL). Data were obtained from three parallel
µ
µ
injections of two independent weighings (6 injections altogether) applied at each concentration
level. Calibration curves were created by plotting the theoretical concentrations against the peak
areas. Linearity was determined by least-squares regression. Linearity data are summarized in
Table 2
.
4.2. System suitability
System suitability data were extracted from chromatograms of the standard solutions of CAT,
2,3-DHBA, 2,4-DHBA, 2,5-DHBA, 2,6-DHBA and SA, 10 g/mLeach. Results were obtained
µ
Table 3
from five parallel injections. System suitability data are summarized in
.
4.3. Precision
Precision was studied by investigating repeatability and intermediate precision. Repeatability
was determined by measuring intra-day data of 3 parallel injections of 3 parallel dilutions of 2
independent weighings at three different concentration levels (1; 10; 50 μg/mL). Intermediate
precision was determined by measuring inter-day (by injection of the samples over three
consecutive days) data of 3 parallel injections of 3 dilutions from two weighings at three different
concentration levels (1; 10; 50 μg/mL).Evaluation was based on relative standard deviation
Tables 4 and 5
(RSD %). Data for precision were summarized in
.
4.5. Determination of LOD and LOQ
Limit of detection (LOD) was determined experimentally, and taken as the concentration
producing a detector signal that could be clearly distinguished from the baseline noise (3 times
baseline noise). The limit of quantification (LOQ) taken as the concentration that produced a
detector signal tentimes greater than the baseline noise. The LOQ values for 2,3-DHBA, 2,4-

DHBA, 2,5-DHBA, 2,6-DHBA and SA was 0.5 μg/mL and for CAT, were 1.0 μg/mL. The LOD
values for for 2,3-DHBA, 2,4-DHBA, 2,5-DHBA, 2,6-DHBA and SA was 1.0 μg/mL and for
CAT, were 5.0 μg/mL.
4.6. Specificity
Specificity was defined as the ability of the method to differentiate and quantify the analytes
Figure 6.
in the presence of endogenous constituents of the perfusate sample.
representative HPLC chromatograms of the 0 minute perfusate containing only the isotonic
Figure 6a
depicts the
perfusion medium, 250 μM sodium salicylate and the internal standard, 4-NP (
) and
the perfusate generated in rat small intestine luminal perfusion experiments at the 45th minute of
Figure 6b
the perfusion period ( ). There are no endogenous peaks in the regions of the retention
times of CAT, 2,3-DHBA, 2,4-DHBA, 2,5-DHBA, 2,6-DHBA, SA and 4-NP.
4.7. Precision of liquid–liquid extraction
Precision of liquid-liquid extraction was also studied by preparing standard solutions of CAT,
2,3-DHBA, 2,4-DHBA, 2,5-DHBA and SA at five different concentrations ranging from 500 to
10000 ng/mL (500, 750, 1000, 5000 and 10000 ng/mL) using the chromatographic mobile phase
(pH 2.6 formic acid/methanol; 77/23; v/v) as solvent. Each solution had a known concentration
(10 g/mL) of 4-NP as internal standard. Data were obtained from three parallel injections of two
independent weighings applied at each concentration level. Calibration curves were generated
by plotting the theoretical concentrations against the peak areas related to the internal standard.
Linearity was determined by least-squares regression calculated from six injections. The method
was linear with respect to each analyte over the examined range, where y is the ratios of the
peak areas of the examined compound and the internal standard and x is the concentration of
the examined compound expressed in ng/mL (CAT: y = 0.00001x - 0.00039, r2= 0.9978; 2,3-
DHBA: y = 0.00003x - 0.00188, r2= 0.9992; 2,4-DHBA: y = 0.00005x + 0.00248, r2= 0.9999;
2,5-DHBA: y = 0.00005x + 0.00005, r2=0.9995; SA: y = 0.00006x - 0.00570, r2=0.9996).
4.8. Recovery of liquid–liquid extraction
Dilution series of CAT, 2,3-DHBA, 2,4-DHBA, 2,5-DHBA and SA using pH 3.0 sulfuric acid
as solvent were prepared at three levels of concentration (1000, 500 and 100 ng/mL). Each
solution had a known concentration (10 g/mL) of 4-NP as internal standard. Application of the
above liquid–liquid extraction (‘Extraction method’) five parallel extractions were performed
at each concentration level. After dissolution of the dry residues, correlation between the
concentration of the standards and the measured peak areas related to that of the internal standard
was determined. The 1000 ng/ml samples resulted in 47.95%, 84.6%, 51.14%, 54.35% and
53.99 % mean recoveries for CAT, 2,3 DHBA, 2,4-DHBA, 2,5 DHBA and SA, respectively.
-
-
Similar data of the 500 ng/ml samples were 50.84%, 78.75%, 58.7%, 61.14% and 54.0%
for CAT, 2,3 DHBA, 2,4-DHBA, 2,5 DHBA and SA, respectively. Reliable direct recovery
-
-
determination at the lowest investigated concentration level (100 ng/mL) was not possible,

Table 6
however, regarding the linearity data and the RSD% values (see
reproducible at that concentration level as well.
), the extraction was
5. Results
The validated HPLC-DAD method was applied for monitoring the jejunal elimination of
Figure
salicylic acid in samples generated in rat small intestine luminal perfusion experiments.
th
5
shows the amount of salicylic acid which is continuously decreased from the 15 minutes until
the end of the perfusion experiment.
When hydroxyl radicals react with SA, it may follow one of the three paths: (1) hydrogen
atom abstraction, (2) addition to the aromatic ring and (3) radical substitution [36]. All the
previously published primary Fenton-oxidation products of SA (CAT, 2,3-DHBA, 2,4-DHBA
and 2,5-DHBA) can be considered as a result of hydrogen atom abstraction or addition reactions
of the hydroxyl radicals and the aromatic ring.
The developed HPLC method was suitable for separation of SA, CAT, 2,3-DHBA,
2,4-DHBA, 2,5-DHBA and 2,6-DHBA, all the possible primary oxidation products of Fenton
Figure 1)
reaction of SA. (
The products in the incubates were identified based on their
Table 3
chromatographic retention times (see
) and their structure was verified by means of mass
spectrometric measurements. The Fenton oxidation of SA under the investigated conditions
resulted in formation of 2,3-DHBA, 2,4-DHBA, 2,5-DHBA and CAT. 2,6-DHBA could not be
Figures 2a and 2b
found in the samples. (
)
After evaluation of the chromatograms it could be ascertained that 2,3-DHBA and 2,5-
DHBA were formed in the biggest amount as monohydroxylated derivatives of SA. In smaller
amounts CAT and 2,4-DHBA could also be identified in the incubates. At tR= 4.876 min a
peak appeared, which was not identical to either of the standards used in the present analytical
method. MS measurements identified the compound as trihydroxybenzoic acid with 169 m/z
Figures 3a and 3b
( ).
For determination of relative amount of the products, relative ratios of the peak areas to
that of the internal standard was calculated. To assign, how iron(II) concentration and incubation
time affected the product distribution, the relative amounts of SA and its hydroxylated products
was plotted as a function of the incubation time. The product distributions in the different sets
Figure 4a and 4b
of experiments can be seen in
.
In the samples containing 1 mM SA, 3 mM iron(II) and 1 mM hydrogen peroxide, the
amount of SA only slightly changed during the incubation time. The amount of 2,5-DHBA
continuously decreased while concentration of 2,3-DHBA remained unchanged during the time
of the experiment.
In the incubates containing 1 mM SA, 0.3 mM iron(II) and 1 mM hydrogen peroxide,
the initial concentration of the unreacted SA was twice as high over the first fifth minutes as

in those with the 3 mM iron(II) concentration. At the later sampling times the amount of SA
was comparable to those measured in the samples using 3 mM iron(II). The area of 2,5-DHBA
showed a similar kinetic pattern to that one observed in the experiment using 3 mM iron(II); it
continuously decreased during the incubation time. The amount of 2,3-DHBA increased until
the fifth minute of incubation, after that, a “steady-state” concentration could be observed with
no significant change.
In vivo intestinal elimination and oxidative metabolism of SA was investigated by
the newly developed HPLC-UV-Vis method applying the same experimental protocol as used
earlier with non-hyperglycaemic rats [16]. Based on the HPLC-UV-Vis studies, continuous
decrease of the amount of SA could be detected in the small intestinal perfusates from the 15th
minute of the experiment. The time course of the intestinal elimination of the compound in
Figure 5
the hyperglycaemic animals (
) showed no significant difference in comparison to that
one observed in the non-STZ-treated rats [10]. Besides the parent compound (and the internal
standard) two more peaks corresponding to more polar compounds with tR= 6.45 and 7.84 min
Figure 6b
). By means of mass spectrometric
retention times appeared in the chromatograms (
analysis they could be identified as 2,5-DHBA and 2,3-DHBA, respectively.
6. Discussion
Our experimental results support the earlier observations that in in vitro Fenton
reaction of SA 2,3-DHBA and 2,5-DHBA are produced in the largest amount as primary
monohydroxylated products [30]. It was also noticed that 2,3-DHBA was formed in the bigger
amount. This observation is in accordance with the calculated atomic charges showing the C3
Table 1
.) The result also
and C5 atoms of SA possessing the two highest electron densities. (
strengthens the earlier observations that increased amount of 2,3-DHBA is a diagnostic value
of non-enzymatic hydroxylation of SA [5,7]. Furthermore, formation of trihydroxybenzoic acid
Figure 3a and
was detected and identified as a result of further oxidation of the DHBAs (
3b
). This latter observation draw attention to further oxidative transformation of the primary
dihydroxybenzoic acid metabolites. Further oxidative transformations are supported by the
Table 1
electron densities calculated for the four isomeric DHBAs. (
) Such transformations can
occur under physiological conditions (e.g., at the sites of inflammation) and can be important
from both chemical and biological points of views [37].
For evaluation of the results, it is important to note that hydrogen peroxide was added to
the mixture of SA and iron(II) ions. Under such conditions it can be assumed that in the samples
containing 0.3 mM iron(II) most of the iron(II) ions are complexed wit SA [38]. On the contrary,
the greater proportion of the iron(II) ions are in non-complexed form in the samples with 3 mM
iron(II) concentration.

Non-complexed iron(II) ions are readily oxidized to iron(III) which form very stable
complex with SA. (The violet color of the reaction mixtures that could be observed after mixing
the reactants are in agreement with this reaction.) Since non-complexed iron(II) ions react with
hydroxyl radicals in a very fast reaction (Fe2+ + .OH = OH- + Fe3+) [39] this reactivity can be the
reason why the amount of SA only slightly changed during the incubation time in the samples
containing 1 mM SA and 3 mM iron(II). 2,3-DHBA is a powerful iron-chelating molecule
[40,41] while 2,5-DHBA has been reported a powerful radical scavenger [40]. This latter activity
can be the reason for its decreasing concentration in the incubations.
In the case of incubations containing 1 mM SA, 0.3 mM iron(II) and 1 mM hydrogen
peroxide, most of the iron(II) ions are complexed with SA [38]. Under such conditions the initial
salicylate concentrations were found to be much higher than those measured in the incubations
containing 3 mM iron(II). This observation is in accordance with the low Fenton-type reactivity
of salicylate-complexed iron(II) ions [38]. Change in 2,3-DHBA and 2,5-DHBA concentrations
showed similar patterns to those observed in the case of experiments using 3 mM iron(II).
Furthermore, it is reasonable to suppose thatin the incubations containing 0.3 mM
iron(II) less (if any) non-complexediron(III) ions -formed in the Fenton oxidation - could
participate in the redox cycle which is the driving force of the reaction and responsible for the
continuous production of the oxidizingintermediates[42]. The amount of the reaction products
also supports this presumption because in the incubate with 3 mM iron(II) the reaction yielded
significantly more oxidizedproducts than in that with 0.3 M iron(II).
m
The validated HPLC-UV-Vis method was applied for monitoring the jejunal elimination
Figure 5
of SA in samples generated in rat small intestine luminal perfusion experiments.
shows
the amount of salicylic acid which is continuously decreased from the 15th minutes until the
end of the perfusion experiment. Although the timecourse of elimination of SA is very much
the same as that observed in similar experiment wit non-hyperglycaemic rats, the amount of
eliminated salicylate (appr. 80 % of the initial concentration) was found to be much higher
than that (appr. 60%) found in the non STZ-treated rats [10]. Recent studies revealed that ASA
causes small intestinal mucosal injury at a considerably higher rate than previously believed.
The first step of NSAID-induced enteropathy is the increase of small intestinal epithelial cell
permeability [43]. It has also been reported that ASA also increases intestinal epithelial cell
permeability as well as conventional NSAIDs in human studies [44]. Based on these literature
data and the observed increased elimination of SA from the small intestine, it is reasonable to
presume that SA, like ASA, can also increase permeability of epithelial cells. Level of SA in
the perfusate, however, is the net result of the kinetics of twodirectional transport of the drug
between the lumen and the epithelial cells [45].
HPLC-UV-Vis measurements indicated that the absorbed salicylate can be metabolized
in the proximal jejunal loop and its two hydroxylated metabolites (2,5-DHB and 2,3-DHB)
Figure 6b
appeared in the intestinal lumen ( ).

In the small intestine of untreated rats CYP1A1, 2B1, 2C6, 2C11, 2D1-4 and 3A1 were
found to be expressed [46]. Among the CYP isoforms reported to metabolize SA, however,
only the CYP3A1 (homologue of the human CYP3A4) expressed in the rat small intestine,
which showed catalyzing almost exclusive formation of 2,5-DHBA [47]. Accordingly, the
increased amount of hydroxylated metabolites – 2,3-DHBA, in particular - are formed in non-
CYP catalyzed, hydroxyl radical initiated hydroxylation reaction. This finding indicates that
non-enzymatic hydroxylation plays an important role in the biotransformation of phenolic
xenobiotics.
One of the major source of ROS formation in diabetic patients are NADPH oxidases
of the Nox family [48]. Nox1 is particularly highly expressed in the epithelial cells of the
gastrointestinal tract [49]. Another source of ROS could be the increased myeloperoxidase
activity of the intestinal mucosa caused by the local inflammation caused by the relatively high
SA level [10]. As the consequence of the hydroxyl radical scavenger effect of SA, its aromatic
hydroxylation can occur at the site of formation of the reactive oxygene species [50]
Our earlier results indicated low levels of 2,3- and 2,5-DHBA in the same experiments
performed with non STZ-treated rats. In those experiments small amounts of 2,3- and 2,5-
DHBAs could be detected and identified by HPLC-MS. The levels of the formed compounds
did not reach the LOQ of the analytical method (0,5 g/mL and 0.75 g/mL, respectively) [16].
The respective LOQ of the present method is 1 g/ml for both DHBAs and the quantitated
amounts of the compounds in the 90 minute perfusate are 1.076 μmol/L and 0.242 μmol/L ,
respectively. Accordingly, hyperglycaemia increased the amount of hydroxyl radical mediated
intestinal biotransformation of salicylic acid.
7. Summary
Non-enzymatic in vitro and in vivo oxidation of SA was investigated by a newly
developed HPLC-UV-Vis method. The method was used to analyze product composition
of oxidation products of SA (CAT and isomeric dihydroxybenzoic acids) under different
experimental conditions. It was found that the primary hydroxylated metabolites of SA
can be further metabolized under the Fenton’s conditions. In vivo experimental results with
hyperglycaemic rats also indicated non-enzyme catalyzed hydroxylation of salicylate in the
small intestine of hypertglycaemic rats. The results give further evidence for involvement of in
vivo hydroxyl radical mediated biotransformation of salicylates at the sites where high hydroxyl
radical level could orrur.
8. Conflict of interest
The authors confirm that this article content has no conflict of interest.

9. Acknowledgements
The present scientific contribution is dedicated to the 650th anniversary of the foundation of the
University of Pécs, Hungary. This study was supported by the European Union, co-financed by
the European Social Fund EFOP-3.6.1.-16-2016-00004).
(

10. Figure captions:
Figure 1:
HPLC-UV-Vis chromatogram (λ=237 nm) of the system suitability solution
containing catechol (CAT), 2,3-dihydroxybenzoic acid (2,3-DHBA), 2,4-dihydroxybenzoic
acid (2,4-DHBA), 2,5-dihydroxybenzoic acid (2,5-DHBA), 2,6-dihydroxybenzoic acid (2,6-
DHBA), salicylic acid (SA) (25 g/mL for each compound) and 4-nitrophenol as an internal
µ
standard (10 g/mL)
µ
Figure 2a:
HPLC-UV-Vis chromatogram (=237 nm) of the 10 min Fenton incubate of salicylic
acid with 3 mM iron(II)
Abbreviations: SA: salicylic acid; 2,3-DHBA: 2,3-dihydroxybenzoic acid; 2,4-DHBA: 2,4-
dihydroxybenzoic acid; 2,5-DHBA: 2,5-dihydroxybenzoic acid; CAT: catechol
Figure 2b:
HPLC-UV-Vis chromatogram (=237 nm) of the 10 min Fenton incubate of salicylic
acid with 0.3 mM iron(II)
Abbreviations: SA: salicylic acid; 2,3-DHBA: 2,3-dihydroxybenzoic acid; 2,4-DHBA: 2,4-
dihydroxybenzoic acid; 2,5-DHBA: 2,5-dihydroxybenzoic acid; CAT: catechol
Figure 3a:
Figure 3b:
Figure 4a:
Figure 4b:
The mass spectrum belonging to the peak at t=4.876 min.
The daughters of the m/z 169 parent ion at t=4.876 min.
Product distribution in Fenton incubations with 3 mM iron(II) ions
Product distribution in Fenton incubations with 0.3 mM iron(II) ions
Intestinal elimination of sodium salicylate in diabetic rat during the course of a 90-
Figure 5:
minute intestinal perfusion. The results were obtained from 5 independent measurements.
Figure 6a:
HPLC-UV-Vis chromatogram (=237 nm) of the 0 min intestinal perfusion sample
.
of diabetic rat with 250 µM sodium salicylate
Figure 6b:
HPLC-UV-Vis chromatogram (=237 nm) of the 45 min intestinal perfusion sample
of diabetic rat with 250 M sodium salicylate.
µ
Abbreviations: SA: salicylic acid; 2,3-DHBA: 2,3-dihydroxybenzoic acid; 2,5-DHBA: 2,5-
dihydroxybenzoic acid, 4-NP: 4-nitrophenol (IS).
Table 1:
Table 2:
Mulliken atomic charges
Linearity data
Table 3
Table 4
: System suitability data
: Data for intra-day precision
Table 5
Table 6
: Data for inter-day precision
: Data for precision of liquid-liquid extraction

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Table1.
Mulliken atomic charges
Compound
SA
C1
C2
C3
C4
C5
C6
- 0.3004
- 0.2823
- 0.3114
- 0.2920
- 0.3446
0.4262
0.3693
0.4395
0.4135
0.4446
- 0.2437
0.3791
- 0.2084
- 0.2117
0.4125
- 0.2580
- 0.2484
- 0.2497
0.3713
- 0.1573
- 0.1755
- 0.1545
- 0.1704
0.4622
2,3-DHBA
2,4-DHBA
- 0.2943
- 0.2329
- 0.2611
2,5-DHBA
- 0.2610
- 0.2003
2,6-DHBA
- 0.2592
SA: salicylic acid
2,5-DHB: 2,5-dihydroxybenzoic acid
2,6-DHB: 2,6-dihydroxybenzoic acid
2,4-DHB: 2,4-dihydroxybenzoic acid
2,3-DHB: 2,3-dihydroxybenzoic acid
Table 2.
Linearity data.
Compound
CAT
Regression equation
y=0.0032x-0.5131
R2
0.9998
0.9996
0.9993
0.9996
0.9992
0.9997
2,5-DHB
2,6-DHB
2,4-DHB
2,3-DHB
SA
y=0.017x-4.4739
y = 14.158x – 6.9466
y=0.0168x-4.6572
y=0.0114x-5.9960
y=0.0204x-7.1844
CAT: catechol
2,5-DHBA: 2,5-dihydroxybenzoic acid
2,6-DHBA: 2,6-dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid
2,3-DHBA: 2,3-dihydroxybenzoic acid
SA: salicylic acid

Table 3.
System suitability data.
Standard
tR
A
Asym
0,87
0,89
0,33
0,91
0,96
0,99
R
N
CAT
6,410
6,652
7,143
8,815
9,567
124,535
779,779
548,893
540,52
502,857
23542
23977
7186
2,5-DHBA
2,6-DHBA
2,4-DHBA
2,3-DHBA
SA
1,42
1,93
5,99
3,29
11,89
25475
26497
21826
25,944 734,257
tR: retention time
A: peak area
Asym: peak symmetry
R: resolution
N: number of theoretical plates
A: average peak area
CAT: catechol
2,5-DHBA: 2,5-dihydroxybenzoic acid
2,6-DHBA: 2,6-dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid
2,3-DHBA: 2,3-dihydroxybenzoic acid
SA: salicylic acid
Table 4.
Data for intra-day precision

ConcentratioMn easurement A(2,5-
A(2,6-
A(2,4-
A(2,3-
A(CAT)
A(SA)
(µg/ml)
DHBA)
897.343
898.666
898.004
0.766
DHBA)
DHBA)
DHBA)
50
1
2
820.864
802.553
811.709
1.595
847.282
867.546
857.414
1.826
890.861
863.364
877.113
2.427
157.867 1107.669
158.409 1112.370
158.138 1110.020
average*
RSD%**
10
1.157
31.382
31.908
31.645
2.605
3.526
3.766
3.646
4.215
0.586
213.199
201.298
207.248
3.194
1
2
162.752
156.363
159.557
2.831
150.160
156.363
153.262
2.862
170.655
170.520
170.588
1.361
156.590
139.351
147.971
6.468
average*
RSD%**
1
1
2
14.402
14.384
14.393
3.997
14.335
13.999
14.167
1.678
18.653
20.699
19.676
6.300
9.905
19.729
20.280
20.004
2.686
8.956
average*
RSD%**
9.430
6.847
*calculated from 9 parallel injections
**calculated from 9 parallel injections
A: average peak area
CAT: catechol
2,5-DHBA: 2,5-dihydroxybenzoic acid
2,6-DHBA: 2,6-dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid
2,3-DHBA: 2,3-dihydroxybenzoic acid
SA: salicylic acid
Table 5.
Data for inter-day precision

Concentration Day No
(µg/ml)
A(2,5-
DHBA)
A(2,6-
DHBA)
A(2,4-
DHBA)
A(2,3-
DHBA)
A(CAT)
A(SA)
50
1
2
3
923.180
898.004
802.497
805.542
832.608
857.414
945.144
882.968
5.929
894.127
877.113
966.843
916.283
4.955
159.443 1119.334
158.138 1110.020
181.963 1243.851
167.590 1164.139
1075.637 816.846
average*
RSD%**
10
973.708
8.685
808.295
0.935
7.264
26.130
31.645
37.367
31.688
15.968
2.971
5.585
183.542
207.248
253.062
214.717
14.871
21.653
20.004
25.753
1
2
3
148.650
159.557
207.972
173.058
16.315
16.926
14.393
20.352
130.047
144.891
169.531
148.156
13.461
15.299
14.167
16.769
136.417
170.588
192.693
166.217
15.362
15.320
19.676
19.183
144.012
147.971
185.501
159.519
13.428
15.950
14.167
17.210
average*
RSD%**
1
1
2
3
3.646
3.789
average*
RSD%**
17.559
16.004
15.412
8.465
18.002
13.831
14.743
24.132
3.460
12.576
22.795
12.545
*calculated from 18 parallel injections
**calculated from 18 parallel injections
A: average peak area
CAT: catechol
2,5-DHBA: 2,5-dihydroxybenzoic acid
2,6-DHBA: 2,6-dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid
2,3-DHBA: 2,3-dihydroxybenzoic acid
SA: salicylic acid

Table 6.
Precision of liquid–liquid extraction.
Concentration
(ng/mL)
CAT
2,3-DHBA
2,4-DHBA
2,5-DHBA
SA
1000
500
1.098 (*RSD%) 3.185 (*RSD%) 2.275 (*RSD%) 1.386 (*RSD%) 1.250 (*RSD%)
3.116 (*RSD%) 2.230 (*RSD%) 1.395 (*RSD%) 4.851 (*RSD%) 4.017 (*RSD%)
4.578 (*RSD%) 3.756 (*RSD%) 2.439 (*RSD%) 2.575 (*RSD%) 1.232 (*RSD%)
100
Equation
y=0.00002x
+0.00100
y=0.00011x
+0.00020
y=0.00013x y=0.00013x-0.00148y=0.00013x
+0.00100
+0.00227
r2
0.9897
0.9999
1.0000
0.9999
0.9998
CAT: catechol
2,3-DHBA: 2,3-dihydroxybenzoic acid
2,4-DHBA: 2,4-dihydroxybenzoic acid
2,5-DHBA: 2,5-dihydroxybenzoic acid
SA: salicylic acid
*RSD%: relative standard deviation calculated from ratios of the peak areas of the examined
compound and the internal standard (from three parallel extractions)
12