Determination of Exposure to the Alternaria Mycotoxin Tenuazonic

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Article

Determination of the Exposure to the Alternaria Mycotoxin Tenuazonic Acid and its Isomer allo-Tenuazonic Acid in a German Population by Stable Isotope Dilution HPLC-MS 3

Yannick Hövelmann, Sebastian Hickert, Benedikt Cramer, and Hans-Ulrich Humpf J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02735 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 26, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Determination of the Exposure to the Alternaria Mycotoxin Tenuazonic Acid and its Isomer

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allo-Tenuazonic Acid in a German Population by Stable Isotope Dilution HPLC-MS3

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Yannick Hövelmann†, Sebastian Hickert†, ‡, Benedikt Cramer†, Hans-Ulrich Humpf*†, ‡

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48149 Münster, Germany,

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*Corresponding author (Tel: +49 251 83 33391; Fax: +49 251 83 33396; E-mail:

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[email protected])

Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45,

NRW Graduate School of Chemistry, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany.

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ABSTRACT

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The content of the Alternaria toxin tenuazonic acid and its isomer allo-tenuazonic acid was

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quantitated in urine of a German cohort (n = 48) using a newly developed and successfully

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validated solid phase extraction based stable isotope dilution HPLC-MS3 method. Tenuazonic

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acid was detected in all of the samples and quantifiable in 97.9% of these samples in a range

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of 0.16 – 44.4 ng/mL (average: 6.58 ng/mL) or 0.07 - 63.8 ng/mg creatinine (average:

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8.13 ng/mg creatinine). allo-tenuazonic acid was for the first time detected in human urine

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(95.8% of the samples positive) and quantitated in 68.8% of the samples in a range of 0.11 –

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5.72 ng/mL (average: 1.25 ng/mL) or 0.08 – 10.1 ng/mg creatinine (average: 1.52 ng/mg

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creatinine), representing 3.40 – 25.0% of the sum of both isomers (average: 12.4%). Food

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frequency questionnaires were used to document food consumption of study participants in

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order to correlate mycotoxin exposure to nutritional habits. Though no statistically

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significant correlation between consumption of a specific food and urinary excretion of

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tenuazonic acid could be determined, a trend regarding elevated intake of cereal products

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and higher excretion of tenuazonic acid was evident. Based on these results, a provisional

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mean daily intake (PDI) for both tenuazonic acid and allo-tenuazonic acid was calculated,

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being 0.183 µg/kg body weight and 0.025 µg/kg body weight, respectively. A combined

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mean PDI for both isomers amounts to 0.208 µg/kg body weight with the highest individual

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PDI for one of the participants (1.582 µg/kg body weight) slightly exceeding the threshold of

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toxicological concern assumed for tenuazonic acid by the European Food Safety Authority of

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1.500 µg/kg body weight. This is the first study which investigated the tenuazonic acid

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content in human urine of a larger sample cohort enabling the calculation of PDIs for

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tenuazonic acid and allo-tenuazonic acid.

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Keywords: mycotoxin, alternaria, mass spectrometry, stable isotope dilution assay, liquid

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chromatography, exposure assessment, MRM3, HPLC-MS/MS

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INTRODUCTION

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Tenuazonic acid, 1 (Figure 1) is a mycotoxin predominantly produced by fungi of the genus

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Alternaria, whereas Pyricularia oryzae1 and Phoma sorghina2 are also known to

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biosynthesize 1. Apart from its acute toxicity towards rodents (LD50 for mice:

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81 - 186 mg/kg body weight; LD50 for rats: 168 - 180 mg/kg body weight)3 and chicken

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embryos (LD50 = 0.55 mg/egg),4 1 showed adverse effects in several animal feeding trials,5-7

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for instance causing acute vomiting, bloody diarrhea and death after 8 - 18 days after oral

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application of 10 mg/kg body weight per day to dogs.5 1 has also been associated with

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Onyalai, a haematologic disorder occurring in black populations of Africa2. Furthermore, 1

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exhibits cytotoxic, antibacterial and antiviral activity8 as well as phytotoxic effects.9

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Upon treatment with bases10-12 as well as under acidic conditions13 epimerization of 1 has

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been observed, yielding the isomer allo-tenuazonic acid, 2 (Figure 2). As the configuration of

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various moieties of 1 and its analogues seems to be particularly important for toxic effects in

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different test systems,8,

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separately for an accurate risk assessment. Mixtures of 1 and 2 have been reported to be

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phytotoxic independent of the ratio of both isomers9, whereas 2 showed no toxic effects on

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HT-29 cells in contrast to 1 significantly reducing cell viability14. Further toxicological data

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concerning 2 as well as data on the chronic toxicity of both isomers in general are lacking.

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There are many reports about the occurrence of 1 in food commodities, particularly in

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cereals and products thereof15-17 as well as in tomatoes and respective processed products.

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17-19

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and infant food.22 According to the recent opinion of the European Food Safety Authority

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(EFSA) on the risks for public health related to 1 in food, one of the most important

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it is essential to determine the exposure towards 1 and 2

1 has further been detected in fruit juices, 15, 16, 18 beer,20 wine,21 edible oils,15 spices16

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contributors to the exposure towards 1 are grain and grain products23. Recently, 1 was for

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the first time quantitated in human urine of six volunteers as part of a study in southern

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Germany24. The presence of 2 in foods was only shown in tomato products so far.14

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Quantitative analysis of 1 is usually based on high performance liquid chromatography

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(HPLC). Due to high acidity and strong metal-chelating properties causing irreproducible

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chromatographic behavior, chromatography of 1 can be quite demanding. Improving

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retention by addition of modifiers to the mobile phase (e.g. Zn(II)SO4) or by application of

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ion chromatography techniques25 often excludes mass spectrometric (MS) detection, since

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most of the used additives are either not volatile or suppress ion intensity. One possibility to

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achieve better chromatographic behavior on reversed phase columns and at the same time

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allowing sensitive MS detection was presented by Siegel et al.26 in 2009 based on

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derivatization of 1 with 2,4-dinitrophenylhydrazine (DNPH) prior to HPLC-MS/MS analysis. A

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drawback of this approach as well as most published methods dealing with determination of

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1 in food commodities and physiological samples is that no chromatographic separation of 1

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and 2 is achieved. Since 2 has been shown to exhibit no cytotoxic effects in contrast to 1,14

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determination of both isomers as a sum parameter might result in overestimating the

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potential health risk. In a recent study a QuEChERS (quick, easy, cheap, effective, rugged and

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safe) based stable isotope dilution method was developed for detection and quantitation of

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1 and 2 in tomato products, without laborious derivatization and including chromatographic

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separation of both toxins on a high carbon load column.14 The same type of high carbon load

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column was also employed by Siegel et al.13 for the chromatographic separation of 1 and 2.

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Based on these findings, the objective of this study was the development of a sensitive

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method for the determination of 1 as well as 2 in human urine and its application to the

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analysis of both toxins in urine samples collected as part of a recently conducted human 5 ACS Paragon Plus Environment

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study.27 Since urine is a rather complex matrix, quantitation of analytes is often hampered by

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interfering matrix compounds. Therefore, the multiple reaction monitoring with multistage

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fragmentation (MRM3) technique was applied in order to achieve an increase in sensitivity

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compared to conventional MRM. The difference between both experiments is that for MRM3

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a subsequent second fragmentation step is implemented in Q3, which is operated as a linear

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ion trap. Thus a specific fragment ion generated due to collision of a precursor in Q2 is

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subjected to another fragmentation and the “granddaughter” ion is finally used for detection

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and quantitation. The second stage fragmentation increases selectivity, resulting in higher

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sensitivity and better signal-to-noise ratios. Lastly, correlations between nutritional habits

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and urinary excretion of 1 were investigated based on a food frequency questionnaire (FFQ).

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MATERIALS AND METHODS

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Chemicals and Reagents

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All solvents used were of gradient grade and purchased from VWR (Darmstadt, Germany), if

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not stated otherwise. ASTM type 1 water was produced with a Purelab Flex 2 system from

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Veolia Water Technologies (Celle, Germany). Equimolar mixtures of 1 and 2 as well as of the

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labeled standards (13C2-1/13C2-2) were synthesized in our working group19, including

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confirmation of the respective equimolar composition by 1H-NMR19 and HPLC-MS/MS.14

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Preparation of standard solutions

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The equimolar mixtures of 1 and 2 as well as

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C2-1 and

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C2-2 were both dissolved in

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acetonitrile and the exact concentrations determined by UV spectroscopy on a V-700 series

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spectrometer (Jasco Labor- u. Datentechnik GmbH, Groß-Umstadt, Germany) using the

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molar absorptivity value of 1.427 ± 0.016 x 104 L/mol/cm at 276 nm in acetonitrile.14 As the

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spectra for 1 and 2 show no significant differences,8 the sum concentration of both isomers

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can be determined by UV spectroscopy.

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Stock solutions of 395.5 ± 1.09 µg/mL 1/2 and 54.30 ± 2.70 µg/mL

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obtained. The solutions were stored at -20 °C in the dark prior to usage to ensure stability

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over the course of the experiment.13

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13

C2-1/13C2-2 were

Calibration

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The stock solutions of 1 and 2 as well as of the respective 13C2-labeled standards were both

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equimolar mixtures containing the same concentration for the two isomers. Starting from

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the stock solution, eight calibration solutions were prepared in the range between 2 –

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200 ng/mL for unlabeled 1 and 2, respectively, by dilution with mobile phase of the starting

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composition of the HPLC run. The calibration solutions were spiked with

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obtain the same concentration of both labeled isomers in each calibration solution

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(100 ng/mL).

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13

C2-1/13C2-2 to

Sample collection

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Urine samples (n = 48) were randomly picked out of the sample pool of a recently conducted

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study in our working group27 with a total of 101 volunteers (44 males, 57 females),

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predominantly university students in the age group of 20 – 30 years. The study deals with

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the assessment of mycotoxin exposure (neither 1 nor 2 was investigated) in Germany and its

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design was approved by the research ethical committee of the University Hospital Münster,

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Germany (File reference: 2012-378-f-S). Participants filled out an FFQ providing information

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on food consumption over the last 24 h, the last 30 days as well as on age, body weight and

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height to determine possible correlations between nutritional habits and biomarker

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excretion. To normalize biomarker concentrations to ng/mg creatinine the urinary creatinine 7 ACS Paragon Plus Environment

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levels were measured for each sample using the Jaffe method (Siemens Healthcare

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Diagnostics, Eschborn, Germany). Measurements were carried out at the central laboratory

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of the University Hospital, Münster, Germany. The samples were stored at -80 °C and

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thawed directly before sample preparation.

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Sample preparation

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An aliquot of the urine sample (9.89 mL) was precisely transferred in a 50 mL polypropylene

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tube. After addition of 100 µL formic acid the sample was spiked with labeled standard

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(10 µg/mL 13C2-1/2; 10 µL = 5 ng/mL for both isomers). The sample was shaken on a vortex

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shaker for 10 s and used for solid phase extraction.

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Solid phase extraction

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For solid phase extraction a 6 mL PS/DVB column (200 mg, 46.2 µm, 170 Å, Bond Elut Plexa,

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Agilent Technologies, Waldbronn, Germany) was used. After conditioning with 3 mL iso-

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propanol and 3 mL water at a flow rate of about 2 drops/s by applying positive pressure

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using a syringe, the urine sample (4 mL) was passed through the column at a flow rate of

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about 1 drop/s. Washing was carried out with 3 mL of methanol/water (20:80, v/v), followed

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by drying the cartridge by continuously applying positive pressure for 2 min. Afterwards, the

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analytes were eluted with 2 mL (2 x 1 mL) methanol containing 1% formic acid at a flow rate

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of about 1 drop/s. Subsequent to evaporation to dryness at 40 °C in a nitrogen stream, the

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remainder was reconstituted in 200 µL acetonitrile/water/formic acid (5:95:1, v/v/v), leading

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to an almost twentyfold concentration of the analytes. After filtration through a 0.45 µm

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PTFE Membrane (Phenomenex, Aschaffenburg, Germany) 20 µL were injected into the HPLC-

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MS/MS system. Each sample was worked up in duplicate. Samples exceeding the calibration

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range regarding the concentration of 1 were diluted with water prior to the sample

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preparation described above.

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Method performance

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Determination of the limit of detection (LOD) and limit of quantitation (LOQ) as well as of

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the recovery rate was carried out for 2 only, since no urine sample free of 1 could be

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obtained. In order to determine LOD, blank urine without detectable amounts of 2 was

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spiked with 2 at four different concentrations (0.05 ng/mL; 0.30 ng/mL; 0.50 ng/mL;

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0.75 ng/mL) in duplicate and treated as described above. Based on the resulting calibration

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curve, the LOD was calculated as the corresponding concentration to the response of blank

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urine (in quadruplicate) plus three times the standard deviation of the respective response.

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The LOQ equals three times the calculated value for LOD.

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The recovery rate was also calculated for 2 only for the above-mentioned reason. Therefore

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blank urine was spiked with 2 at three levels within the calibration range (2.5 ng/mL;

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5.0 ng/mL; 7.5 ng/mL) and underwent the sample preparation described above. Experiments

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were performed in duplicate and the recovery rate was determined by averaging the single

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recovery rates obtained for each spiked sample.

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As the above-mentioned almost twentyfold concentration of the samples bears the risk of

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signal suppression due to coeluting matrix compounds, the signal suppression/enhancement

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(SSE) factor was estimated. To that end calibration solutions for 2 were prepared in the same

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concentration range in HPLC mobile phase as well as in blank urine matrix that underwent

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the described sample preparation, yielding two separate calibration curves. The respective

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slopes

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suppression/enhancement.

of

the

two

calibration

curves

were

used

to

calculate

the

signal

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Reproducibility of the method was determined by analyzing the urine sample of one of the

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participants (2.40 ng/mL 1; 0.42 ng/mL 2) at three different days in duplicate during a course

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of two weeks.

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Calculation of the tenuazonic acid and allo-tenuazonic acid content in urine samples

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To evaluate the recorded data, Analyst software, ver. 1.6.2, (Sciex, Darmstadt, Germany) was

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used. The peak areas of the quantifier MRM3 for 1 and 2 in the calibration solutions were

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divided by the peak areas of the respective

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ratios were plotted against the concentrations of 1 and 2 to obtain two separate calibration

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curves. All calibration solutions were prepared in duplicate and the peak areas of both

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measurements averaged. The slope and intercept of these curves were used to calculate the

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concentrations of 1 and 2. The sample volume and concentration factor resulting from

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sample preparation were taken into account; the values were not corrected by recovery

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rates as these were found to be close to 100%.

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C2-labeled substances. Resulting peak area

Correlation of specific food intake and urinary excretion of tenuazonic acid

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In order to identify correlations between consumption of a specific food item and urinary

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excretion of 1, FFQs provided by the participants were compared with their respective

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urinary excretion of 1. To that end ingested amounts of the foods of interest were plotted

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against the excretion of 1 for all the study participants. Participants were also divided into

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percentiles representing low, medium, increased and high intake of a specific food item,

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whereupon the averaged excretion of 1 for the respective food groups were compared.

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HPLC-MS/MS settings

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A QTRAP 6500 mass spectrometer (Sciex) equipped with a 1260 Infinity LC System (Agilent,

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Waldbronn, Germany) was used for the analysis. Electrospray ionization was applied in 10 ACS Paragon Plus Environment

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positive ion mode. The source temperature was set to 500 °C, the curtain gas to 30 psi and

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the ion source gases 1 and 2 were set to 35 and 45 psi, respectively. Ion spray voltage was

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set to 4500 V and the collision gas parameter set to “high”. Optimization of declustering

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potential (DP) as well as collision energy (CE) for 1 and 2 and the respective labeled

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standards was carried out by direct infusion of these substances into the MS system using a

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syringe pump. For MRM3 Q3 was operated in LIT mode with the scan rate set to 10000 Da/s.

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Various ion trap fill times (30 ms, 60 ms, 90 ms, 120 ms, 150 ms, 200 ms) were investigated,

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whereas a linear increase of signal intensity with increasing fill time was observed.

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Therefore, the maximum fill time of 200 ms was applied in the final method for each of the

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MRM3 transitions with the excitation time set to 25 ms. Substance specific MRM3

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parameters were tuned with a standard solution containing the respective compounds using

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chromatographic runs. In that process different excitation energies (AF2) were evaluated.

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Best results regarding signal intensities were achieved with the excitation energy set at

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0.06 V for all MRM3 transitions. MRM transitions were also recorded with a dwell time of

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10 ms for comparative reasons.

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Separation of 1 and 2 was achieved on a 100 mm x 2.1 mm i.d., 3 µm, Hypercarb column

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equipped with a 10 mm x 2.1 mm guard column of the same material (Thermo Scientific,

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Braunschweig, Germany). A binary gradient consisting of acetonitrile (A) and water (B), both

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containing 1% formic acid, was applied with the column temperature set to 80 °C. 20 µL of

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sample were injected under starting conditions of the HPLC run at 5% A at a flow rate of

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250 µL/min, which were held constant for 1.5 min. After linearly increasing the percentage

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of A to 70% and the flow rate to 325 µL/min at 14.5 min, the content of A was further

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increased to 95% with the same flow rate at 16 min. These conditions were kept constant for

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2.5 min, followed by 8 min of equilibrating the column under starting conditions prior to the 11 ACS Paragon Plus Environment

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next injection. A diverter valve was integrated into the method discarding the first 10 min

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and the last 6.5 min of each HPLC run to avoid unnecessary contamination of the MS system

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by matrix components.

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RESULTS AND DISCUSSION

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Method development

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In this study, a quick and effective SPE based stable isotope dilution HPLC-MS3 method was

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developed, successfully validated and applied to the determination of both tenuazonic acid,

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1 and allo-tenuazonic acid, 2 in human urine, without requiring laborious derivatization for

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sample preparation. Frequently occurring difficulties associated with chromatography of 1

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are overcome by using a high carbon load column, enabling almost complete

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chromatographic baseline separation of both isomers (Figure 2).

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Method performance

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The determination of the limit of detection (LOD) and the limit of quantitation (LOQ) as well

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as recovery rates was carried out for 2 only, since no urine sample free of 1 was available.

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The determined parameters for 2 are applicable for 1 as well due to the same mass

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spectrometric behavior of both isomers. A similar approach was recently applied for the

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determination of method performance characteristics for ochratoxin A (OTA) and its isomer

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2'R-OTA in blood samples.28 The LOD and LOQ for 2 were found to be 0.04 ng/mL and

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0.11 ng/mL, respectively, showing an increased sensitivity compared to a previously

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published method for the determination of 1 in urine with an LOD of 0.2 ng/mL and an LOQ

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of 0.6 ng/mL.24 The recovery rate was determined in duplicate at three different spiking

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levels and was calculated to be 94.1 ± 2.6% at urine concentrations for 2 of 2.5 ng/mL,

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5.0 ng/mL and 7.5 ng/mL, respectively. Due to the lack of legal limits for 1 and 2 in the 12 ACS Paragon Plus Environment

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European Union, specific criteria for analytical methods for both toxins are missing.

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According to the guideline CEN/TR 16059:201029 recovery rates between 50 and 120% for

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unregulated mycotoxins are acceptable in food analysis. Applying this criterion for the

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analysis of human urine as well, it is met by the newly developed method regarding the

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calculated recovery rate of 94.1 ± 2.6%. Regression coefficients (R2) of >0.99 were obtained

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for both analytes in their respective calibration range, indicating sufficient linearity. The

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signal suppression/enhancement factor for 2 was calculated to be 88.2%, which is

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satisfactory regarding the almost twentyfold concentration of the samples during sample

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preparation. Reproducibility was determined by analysis of one of the urine samples at three

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different days in duplicate and the respective relative standard deviation was found to be

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9.7% for 1 and 13.7% for 2.

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Application of MRM3 technology to mycotoxin analysis

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The newly developed method is the first application of multiple reaction monitoring with

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multistage fragmentation (MRM3) to mycotoxin analysis in physiological samples. MRM3 is a

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technology not commonly applied to the analysis of low molecular weight molecules ([1+H]+

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= m/z 198). Application of MRM3 usually deals with larger molecules, such as peptides in

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complex biological matrices30,

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MRM3 to the quantitative analysis of smaller molecules, such as retinoic acid in complex

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matrices and metanephrines, was recently demonstrated.33, 34 Regarding mycotoxin analysis,

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Lim et al.35 compared the application of MRM3 and MRM to the determination of

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trichothecenes in grains and found comparable results concerning method performance

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criteria.

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or complex food matrices.32 However, the application of

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As shown in Figure 3, the second fragmentation step leads to significantly reduced

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background signals compared to conventional MRM technique. Further improvement of the

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signal-to-noise levels was achieved by extension of the linear ion trap fill time to 200 ms. The

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introduction of a further fragmentation step apparently does not influence the method

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performance criteria. The long fill time does not lead to a saturation of the ion trap, as the

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calibration curves show excellent linearity in the respective calibration range. Since the Q3 is

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operated in a full scan mode, which is possible due to the fast scan rate of 10,000 Da/s, all

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possible secondary fragments are recorded simultaneously. This allows to use any of the

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secondary fragments for quantitation retrospectively in the unlikely case of interfering

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signals for the chosen quantifier or qualifier MRM3 transitions. Lastly, the complete

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secondary product ion spectrum adds additional information to assure the correct

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identification of analytes.

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Determination of tenuazonic acid and allo-tenuazonic acid in urine samples

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The developed stable isotope dilution HPLC-MS3 method was applied to the analysis of 1 and

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2 in urine samples of 48 volunteers. The urine samples were randomly picked out of a larger

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sample pool (n = 101) without any regard to information given in the FFQs or previous

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analytical results. As shown in Figure 4, all samples tested positive for 1 with 97.9% of the

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samples being above the LOQ and in the range between 0.16 – 44.4 ng/mL or 0.07 –

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63.8 ng/mg creatinine with average concentrations of 6.58 ng/mL or 8.13 ng/mg creatinine.

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The results regarding the content of 1 in urine are roughly in the range of these obtained in a

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recently published study24 in which 1 was quantitated in the 24 h-urine of six volunteers in

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the range of 1.3 – 17.3 ng/mL or 2.3 – 10.3 ng/mg creatinine. Furthermore, in the present

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study 2 was for the first time detected in human urine with 95.8% of the samples being

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above the LOD and 68.8% above the LOQ in the range of 0.11 - 5.72 ng/mL or 0.08 – 14 ACS Paragon Plus Environment

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10.1 ng/mg creatinine. Average values of 2 are 1.25 ng/mL or 1.52 ng/mg creatinine. The

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high levels of 2 found in the analyzed urine samples, representing 3.40 – 25.0% of the sum of

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both isomers (average: 12.4%), demonstrate the relevance of 2 as a mycotoxin. Until now, 2

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has only been detected in tomato products.14 Since 1 has been shown to epimerize under

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acidic conditions in aqueous solutions yielding the isomer 2,13 epimerization of 1 during

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gastric digestion might also be a source for the detected amount of 2 in the analyzed urine

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samples. Furthermore, 2 is not produced by Alternaria species under alkaline or neutral

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conditions but is detectable in acidic cultures14 supporting the hypothesis of an acidic-

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catalyzed epimerization of 1 to 2. In conclusion, it remains unclear whether the amount of 2

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in human urine is solely attributed to the consumption of food samples contaminated with 2

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or to epimerization of 1 during gastric digestion. In that regard a human or animal trial is

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necessary to further investigate the origin of 2 in human urine and should consequently be

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addressed in the future.

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Correlation of specific food intake and urinary excretion of tenuazonic acid

301

To correlate consumption of a specific food group and changes in urinary excretion of 1,

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FFQs provided by the participants regarding certain food intake were compared with their

303

respective urinary excretion of 1. Depending on the answers given in the food frequency

304

questionnaires, study participants were divided into percentiles representing low, medium,

305

increased and high intake of the respective food group. Special emphasis was put on

306

consumption of cereal products and 24-h dietary recalls, since 1 has been shown to

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commonly occur in cereals and products thereof23 as well to be almost completely excreted

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after 24 h.24 In case of plotting the single data points for cereal consumption against urinary

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excretion of 1, no correlation between the two was evident (R2