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KINETICS OF FURAN FORMATION FROM ASCORBIC ACID DURING HEATING UNDER REDUCING AND OXIDIZING CONDITIONS Burçe Ataç Mogol, and Vural Gökmen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Sep 2013 Downloaded from http://pubs.acs.org on September 29, 2013
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Journal of Agricultural and Food Chemistry
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KINETICS OF FURAN FORMATION FROM ASCORBIC ACID DURING
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HEATING UNDER REDUCING AND OXIDIZING CONDITIONS
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Burçe Ataç Mogol, Vural Gökmen*
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Hacettepe University, Department of Food Engineering, 06800 Beytepe, Ankara,
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Turkey
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Corresponding Author:
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Prof. Dr. Vural Gökmen
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Phone: +903122977108 Fax: +903122992123
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e-mail:
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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ABSTRACT
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This study aimed to investigate the effect of oxidizing and reducing agents on the
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formation of furan through ascorbic acid (AA) degradation during heating at
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elevated temperatures (≥100 oC) under low moisture conditions. To obtain these
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conditions, oxidizing agent, ferric chloride (Fe), or reducing agent, cysteine (Cys)
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was added to reaction medium. Kinetic constants, estimated by multiresponse
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modeling, stated that adding Fe significantly increased furan formation rate
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constant, namely 369-fold higher than that of control model at 100oC. Rate-
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limiting step of furan formation was found as the reversible reaction step between
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Intermediate (Int) and diketogluconic acid (DKG). Additionally, Fe decreased
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activation energy of AA dehydration and furan formation steps by 28.6% and
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60.9%, respectively. Results of this study are important for heated foods, fortified
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by ferric ions and vitamins, which targets specific consumers, e.g infant
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formulations.
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Keywords: Furan, ascorbic acid, heat treatment, oxidation-reduction potential,
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multiresponse modeling
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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Furan is a volatile oxygen heterocycle formed in a number of heated foods. It has
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received considerable attention as it is classified ‘possibly carcinogen to humans’
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(Group 2B) by the International Agency for Research on Cancer (IARC).1 Moro et
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al.,2 reported that furan was found to induce hepatocellular adenomas and
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carcinomas in rats and mice, and high incidence of cholangiocarcinomas in rats
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at doses higher than of 2 mg per kg per body weight. Due to its low boiling point,
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furan, formed during thermal processing, easily vaporizes. However, this give
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rise to concern in canned or jarred foods as furan accumulates in the headspace.
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Monitoring furan levels in foods has outlines its occurrence in a broad range of
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products (roasted coffee, bakery products, baby foods, etc.) from none
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detectable levels to 7000 µg/kg.3-12
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AA has been shown as one of the major precursors of furan formed by different
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thermal processes.13-16 Maga reported that the primary source of furans in food is
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thermal degradation of carbohydrates such as glucose, lactose, and fructose.17
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Moreover, US FDA report indicated that variety of carbohydrate/amino acid
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mixtures or protein model systems (e.g., alanine, cysteine, casein) and vitamins
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(AA; dehydroascorbic acid, DHAA; thiamin) have been used to generate furans in
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food.18 Furan could also be formed through oxidation of polyunsaturated fatty
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acids and carotenoids at elevated temperatures.13 Formation of furan has been
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studied in simple model systems containing precursors in order to understand
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their contributions in the formation mechanism. In a previous study, Perez Locas
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and Yaylayan proposed a mechanism describing that AA might be transformed 3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
non-oxidative
pyrolytic
conditions
to
2-deoxyaldotetrose
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under
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intermediate leading directly to furan.13
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Model systems are widely used to set experiments in order to understand the
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fate of reactions. However, foods are complex systems in which different
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constituents create different conditions for the reactions occurring in foods during
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thermal processing. One of these conditions to be considered is the oxidation-
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reduction potential of the food. In a previous study, it was reported that the
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oxidation-reduction potential of the reaction medium significantly affect the rate of
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AA degradation.19
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On the other hand, reaction mechanisms in foods are challenging, as key
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compounds involve in many simultaneous and successive steps. In this sense,
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multiresponse modeling is a useful approach to understand such mechanisms.
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Multiresponse modeling, which is based on measurement of reactants and
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products simultaneously, provides the possibility to estimate parameters more
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accurately than with uniresponse modeling in which only one reactant or only one
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product is analyzed.20 Modeling the formation of compounds derived from kinetic
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data is a great tool to estimate kinetic parameters, which facilitate to understand
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the reaction mechanism. Initially, a proposed reaction mechanism describing the
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change of concentration of key reaction compounds is needed to develop a
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mechanistic model.20
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This study aimed to understand the effect of oxidizing and reducing agents on
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the formation of furan through AA degradation during heating at elevated
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temperatures (≥ 100 oC) under low moisture conditions. Ferric chloride (FeCl3) or 4 ACS Paragon Plus Environment
as
key
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Journal of Agricultural and Food Chemistry
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cysteine (Cys) was added to the model system to create oxidizing or reducing
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conditions, respectively. Both Cys and Fe3+ ions may be naturally present in the
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foods. Ferric ions might also migrate from metal containers used in processing14,
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or foods might be fortified by ferric ions and/or AA targeting specific consumers,
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e.g infant formulations. Multiresponse kinetic modeling was used to obtain more
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information the degradation reaction network of ascorbic acid into furan proposed
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previously13, as well as to determine kinetic constants and rate limiting step.
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MATERIALS AND METHODS
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Chemicals
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Solvents, HPLC-grade water, and methanol used for chromatographic analysis
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were purchased from Sigma-Aldrich (Steinheim, Germany) and formic acid (98%)
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was purchased from J.T. Baker (Deventer, Holland). L-(+)-Ascorbic acid (min
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>99.7%), L-(+)Dehydroascorbic acid, cysteine (min 99%), furan (99.9%), silica
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gel 60GF (for thin-layer chromatography) were obtained from Merck and Fe(III)-
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chloride anhydrous was obtained from Riedel-de Haën (Seelze, Germany). d4-
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Furan (for NMR 99% atom) was purchased from Acros Organics (New Jersey,
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USA). Stock solutions of furan and spiking d4-furan were prepared in methanol at
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a concentration of 1000 mg/ml. All solutions were prepared and kept at 4oC.
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Preparation of model systems
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Three model systems were prepared to monitor furan formation from AA under
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different conditions. Reaction mixtures were prepared containing 100 µmol/ml AA
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in water. Oxidizing or reducing agents were added to the reaction mixtures,
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specifically 10 µmol Fe3+ equivalent ferric chloride or 10 µmol Cys, respectively.
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100 µl of these reaction mixtures, containing 10 µmol AA, 1 µmol Fe3+ or 1µmol
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Cys, mixed with 30 mg silica gel in 20-ml headspace vials, and then covered with
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additional 270 mg of silica gel. The vials were sealed with crimp cap,
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immediately, and then heated in a temperature-controlled oven (Memmert,
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Schwabach, Germany) at 100 oC, 120 oC, and 140 oC for 5, 10, 15, 20, 30, 60,
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120, 180, and 360 min. All reactions were performed in duplicate. The reaction
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conditions and response variables for the model systems used for kinetic
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modeling are given in Table 1.
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Analysis of furan
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After reaction, the vials containing reactants and reaction products were spiked
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through septa with 1.0 nmol of d4-furan. Determination of furan was carried out
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using Agilent 6890N series GC coupled with Agilent 5973 mass selective
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detector. Furan was extracted by 75 mm carboxen-polydimethylsiloxane Solid
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Phase Micro Extraction (SPME) fiber (Supelco, Bornem, Belgium). Before use,
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the fiber was conditioned in the GC injection port under helium flow in
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accordance with the temperature and time recommended by the manufacturer.
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Fiber was then incubated in headspace of vials in a temperature-controlled oven
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at 30 oC for 30 min. The vials were gently mixed in every 5 min. Thermal
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desorption of analytes was carried out by exposing the fiber in the GC injector
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port at 200 oC for 5 min and splitless injection was used. Separation was
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performed on a 24 m x 0.32 µm, 20 µm HP-PLOTQ column. The MS was
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operated in electron ionization mode. Working conditions were as follows:
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injector 2 mL/min; oven temperature, 100 °C (5 min), with a temperature ramp of
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10 °C/min to 200 °C and held for 15 min. The MS source temperature was 230
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°C, and the MS quad temperature was 150 °C, with a dwell time of 100 ms.
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Furan was detected using single-ion monitoring of the fragments m/z 68 and 39.
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The internal standard d4-furan was detected by monitoring the fragments m/z 72
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and 42. The concentration of furan in the reaction mixtures was calculated by
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means of a calibration curve built in a range of 0-15 nmol (0, 0.01, 0.15, 1.5, 3.0, 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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7.5, 15.0 nmol). The limit of quantification was 0.01 nmol per reaction mixture for
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furan under the stated analytical conditions. All analytical determinations were
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performed in duplicates.
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Analysis of AA, DHAA, and reaction intermediates by high-resolution mass
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spectrometer
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AA, DHAA, and other intermediates were extracted by adding 5 ml of 10 mM
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formic acid in water to vial in two steps (2x2.5 ml). After mixing thoroughly, the
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extracts were transferred to tube, which was then centrifuged at 5000 g for 10
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min. Supernatants were filtered through 0.45 µm nylon filter to HPLC vials.
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An ultra high-performance liquid chromatography (U-HPLC) Accela system
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(Thermo Fisher Scientific, San Jose, CA, USA) consisting of a degasser, a
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quaternary pump, an auto sampler, and a column oven was used. The U-HPLC
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was directly interfaced to an Exactive Orbitrap MS (Thermo Fisher Scientific, San
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Jose, CA, USA). Chromatographic separations were performed on a HIBAR
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Purospher-STAR RP-18e column (150 × 4.6 mm, 5 µm particle size) (Merck,
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Darmstadt, Germany). An isocratic mixture (95:5, v/v) of 0.1% formic acid in
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water and 0.1% formic acid in methanol was used as the mobile phase at a flow
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rate of 500 µl/min at 30 °C. The total run time was 10 min. The Exactive Orbitrap
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MS equipped with a heated electrospray interface was operated in the negative
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mode, scanning the ions in m/z range of 50–300. The resolving power was set to
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100,000 full width at half maximum resulting in a scan time of 0.5 s. Automatic
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gain control target was set into balanced; maximum injection time was 50 ms.
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The interface parameters were as follows: the spray voltage of 4 kV, the capillary 8 ACS Paragon Plus Environment
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voltage of 25 V, the capillary temperature of 350 °C, a sheath gas flow 45 and
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auxiliary gas flow of 20. The instrument was externally calibrated by infusion of a
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calibration solution (m/z 138 to m/z 1822) by means of an automatic syringe
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injector (Chemyx Inc. Fusion 100 T, USA). The calibration solution (Sigma-
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Aldrich) contained caffeine, Met-Arg-Phe-Ala, Ultramark 1621, and acetic acid in
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the mixture of acetonitrile/methanol/water (2:1:1, v/v/v). Data were recorded
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using Xcalibur software version 2.1.0.1140 (Thermo Fisher Scientific). The
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concentrations of AA and DHAA in the reaction mixtures were calculated by
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means of calibration curves (0.0, 0.05, 0.10, 0.25, 0.50, 1.0 µmol). All analytical
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determinations were performed in duplicates.
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Statistical analysis
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Data were analyzed by one-way analysis of variance (ANOVA) using Duncan
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test with the SPSS program (SPSS 16.0). Significance was defined as p
