MS Method for the Determination of

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Development of a SIDA-LC-MS/MS Method for the Determination of Phomopsin A in Legumes Svenja Schloß, Matthias Koch, Sascha Rohn, and Ronald Maul J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04792 • Publication Date (Web): 15 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015

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Journal of Agricultural and Food Chemistry

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Development of a SIDA-LC-MS/MS Method for the Determination

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of Phomopsin A in Legumes

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Svenja Schloß†, ‡, Matthias Koch†, Sascha Rohn‡, and Ronald Maul*, §, ‡

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†

6 7

Straße 11, 12489 Berlin, Germany ‡

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BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-

University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany

§

Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany

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*Corresponding author:

Tel: +49-(0)40 42838 7816

13

Fax: +49-(0)40 42838 4342

14

E-mail: [email protected]

1 ACS Paragon Plus Environment


ABSTRACT 15

A novel method for the determination of phomopsin A, 1, in lupin flour, pea flour, and

16

bean flour as well as whole lupin plants was established based on stable isotope

17

dilution assay (SIDA) LC-MS/MS using

18

standard. Artificially infected samples were used to develop an optimized extraction

19

procedure and sample pretreatment. The limits of detection were 0.5 to 1 µg/kg for all

20

matrices. The limits of quantitation were 2 to 4 µg/kg. The method was used to

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analyze flour samples generated from selected legume seeds and lupin plant

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samples which had been inoculated with Diaporthe toxica and two further fungal

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strains. Finally, growing lupin plants infected with D. toxica were investigated to

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simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 µg/kg of 1 were

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found in the pods and 7.2 µg/kg in the stems and leaves.

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N6-1 as an isotopically labeled internal

26 27

Keywords: Diaporthe toxica; stable isotope dilution assay; isotopically labeled internal

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standard; legume contamination


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INTRODUCTION 29

Grain legumes such as beans, peas, and soy are used in vegan or vegetarian diets

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to replace meat products due to their high protein content. The flour and protein

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isolates of lupin grain can be processed to a wide range of food products (e.g. bread,

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sausages, bread spreads, ice cream, sweets).1 Lupins were introduced to the

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European agriculture in the 19th century. As the plant is well adapted to sandy soils, it

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was cultivated intensively to be used as feed for sheep and cows and as a green

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manure crop. An alarming increase of diseases among sheep flocks fed with lupin

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hay was recognized and led to investigations of toxicological aspects. Apart from

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poisonings caused by alkaloids of the bitter lupin, another disease, later referred to

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as lupinosis, was responsible for lethal liver degenerations in the sheep.2

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Although Kühn3 suggested a connection between lupinosis and toxic metabolites of

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saprophytic fungi found on lupins in 1880, the responsible species and its toxic

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potential was not determined until 1970. The final evidence was presented by van

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Warmelo et al.4, who identified the fungus Phomopsis leptostromiformis as the cause

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of lupinosis. P. leptostromiformis was later described as the anamorph of Diaporthe

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toxica. The isolation of the metabolites phomopsin A, 1, (Figure 1) and B by Culvenor

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et al.5 facilitated further investigations of their chemical and biological properties and

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the occurrence in food and feed. Three additional phomopsins (C, D, and E) have

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been identified so far. 1 is regarded as the main toxin with a toxic potential two to five

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times higher than phomopsin B.6 Toxicological studies on 1 have been carried out

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showing liver toxic effects in vivo and cytotoxicity in in vitro assays.6 The acute liver

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toxicity of 1, a hexapeptide consisting of three dehydro amino acids forming a

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macrocycle and a lateral chain of three dehydro amino acids derives from

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antimicrotubular effects.7-10 Moreover, hepatocarcinogenic effects have been 3 ACS Paragon Plus Environment


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detected in rats in a high exposure experiment.11 The occurrence of 1 in feed and

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food is poorly documented and most studies dealing with toxicity refer to livestock or

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laboratory animals and not to human consumption.

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The genus Diaporthe/Phomopsis comprises a large number of plant pathogens,

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endophytes, or saprophytes with an ubiqitous distribution, responsible for several

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diseases of economically important crops. So far, a multitude of species has been

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recognised as producers of interesting enzymes and secondary metabolites. They

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seem to be able to colonise diverse hosts as opportunists, and several different

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species could even appear on the same host leading to different infection patterns.12

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Although D. toxica seems to be specialized on lupins, the huge adaptability of fungi

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to environmental influences and a potential host change should be taken into

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consideration.13

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As only limited data is available about the occurrence of 1 in food and feed, fast and

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sensitive methods are required for contaminant monitoring programs. The necessity

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of such a method has also been stated by the European Commission in their

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mandate M/520 requesting the development of a method for the determination of 1 in

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food and feed.14 Former methods were time consuming (nursling rat bioassay), were

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limited regarding sensitivity (LC-UV), or are no longer available (ELISA).6 Significant

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improvements in specificity and sensitivity of HPLC analysis were achieved by using

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mass spectrometric detection.15-17 However, to date none of these methods are

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implemented in routine analyses despite existing legal limits in Australia and New

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Zealand. The introduction of an isotopically labeled internal standard (ISTD) would

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help to overcome the matrix effects in mass spectrometric analysis as one of the

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major obstacles. However, amongst others co-elution of matrix components

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influences the lowest quantifiable amount of 1 as one crucial parameter in trace 4 ACS Paragon Plus Environment

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analysis. In the work of Suman et al.18 an ion suppression rate of 50% for the

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modified mycotoxin deoxynivalenol-3-glucoside for bread samples was found, which

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is reflected by the greatly decreased slope of the calibration curve. Ion suppression

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or ion enhancing effects in LC-MS-analysis can be compensated by using matrix-

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matched calibration, standard addition experiments, or the use of isotopically labeled

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ISTD improving the accuracy (trueness and precision) in LC-MS/MS analyses. Matrix

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matched standards depend of the availability of blank matrix materials, which is not

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always feasible while standard addition dramatically increases the number of

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samples needed to be measured. Stable isotope dilution assay (SIDA) LC-MS/MS is

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considered as primary ratio method representing a high level of metrology and

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facilitates a more versatile application.19,20 SIDA methods are matrix independent;

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samples with varying matrices can be analyzed using the same calibration. The only

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limitation occurs when matrix compounds share some of the mass transitions as the

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analyte or the ISTD. In this case it is necessary to ensure that the matrix peaks and

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the peaks of the analyte and the ISTD are separated by chromatography. The first

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isotopically labeled ISTD for 1 has been developed at the Federal Institute for

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Materials Research and Testing (BAM), Berlin, Germany. The isotopic purity and

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chemical properties of the standard are suitable for the deployment in a SIDA LC-

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MS/MS method.21

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Although hybrid mass spectrometers allow for the sensitive quantitation of

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contaminants at ultratrace levels without extensive sample clean-up by using either

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multiple reaction monitoring (MRM) or high resolution MS techniques an appropriate

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sample pretreatment improves the method performance and often enhances the

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lower limits of detection and quantitation (LOD and LOQ). For 1, one important

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analytical benchmark to be reached is as low as 5 µg/kg, the regulatory limit for lupin 5 ACS Paragon Plus Environment


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seeds and the products thereof, specified by the Australia-New Zealand Food

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Authority.22 In the EU, the occurrence of 1 is not regulated so far, but a similar

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recommendation for a limit is probable.

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This work describes the development and application of the first SIDA-based LC-

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MS/MS method for the determination of 1 in various legume flours as well as in the

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whole lupin plant as sample matrix. The extraction efficiency was evaluated using

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artificially infected materials. The implementation of an isotopically labeled ISTD is

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supplemented with a fast and convenient sample clean-up technique using one-step

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purification columns. The columns are designed to remove matrix compounds such

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as proteins, fats, pigments, and other matrix compounds with a blend of several

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sorbent materials. The columns act following a one-step principle retaining the matrix

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compounds and allowing the analytes to pass through directly without a separate

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elution step.

MATERIALS AND METHODS 116

Standards and reagents

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1 (purity 83%, determined by NMR) was purchased from Santa Cruz Biotechnology

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Inc. (Heidelberg, Germany) and dissolved in methanol to prepare a stock solution of

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32 mg/kg.

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preparative HPLC. The eluate containing

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solution.21 HPLC-grade acetonitrile and methanol were acquired from Th. Geyer

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GmbH & Co.KG (Renningen, Germany). Formic acid (extra pure, 98 - 100%), acetic

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acid (100%) and n-hexane were acquired from Merck KGaA (Darmstadt, Germany).

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Ammonium formate was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,

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Germany). Ultrapure water, purified with a “Seral” system Seralpur Pro 90 CN (Seral

15

N6-1 (isotopic purity > 95%) was obtained by biosynthesis and 15

N6-1 was used directly as the ISTD


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GmbH, Ransbach-Baumbach, Germany), was used in the preparation of working

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solutions and mobile phase. The purification columns MultiTox MS TC-MT3000 with

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a sample capacity of 2 mL for the clean-up of extracted samples were provided by

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Trilogy Analytical Laboratory Inc. (Washington, MO).

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Fungal cultures

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Vegetative cultures of D. toxica, Phomopsis juniperovora and P. viticola (Leibniz-

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Institute for the German Collection of Microorganisms and Cell Cultures - DSMZ,

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Brunswick, Germany) for the inoculation of sample material were maintained on

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oatmeal agar (Sigma-Aldrich Chemie GmbH) at 24 °C for four weeks.

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Legume Samples

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Lupin seeds (Lupinus angustifolius L.) were purchased from a plant breeder

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(Saatzucht Steinach GmbH & Co KG, Bocksee, Germany). Beans (Phaseolus

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vulgaris L.) and peas (Pisum sativum L.) were bought from local supermarkets.

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Whole lupin seeds, peas, and beans were milled and passed through a 250 µm

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sieve. Whole lupin plants were grown in greenhouse cultures at the Leibniz-Institute

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of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V. (Großbeeren, Germany).

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After ripening of the seeds, the plants were harvested and the stems and as well as

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the pods and seeds were separated. The different plant materials were freeze-dried

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separately and milled to a fine powder. This material was used as blank matrix.

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Freshly harvested whole lupin plants (above-ground plant parts) and the seeds of

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lupins, peas, and beans were inoculated with a 1 producing strain of D. toxica to

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generate contaminated sample material. Five lupin plants were cut into pieces < 5 cm

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and autoclaved for 20 min in 250 mL Erlenmeyer flasks. 30 g of the three seeds,

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moistened with 10 mL pure water were weighed into 250 mL Erlenmeyer flasks and 7 ACS Paragon Plus Environment


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autoclaved for 20 min. The seeds and the plant material were inoculated with D.

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toxica. After incubating four weeks at 24 °C, the cultures were freeze-dried, milled to

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a fine powder and passed through a 250 µm sieve.

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In addition to the saprophytic inoculation of sterilized lupin plants with D. toxica, a set

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of lupin plants was inoculated during their growth period to investigate the toxin

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production of D. toxica infecting a living plant. Five lupin plants were moistened by

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spraying with an aqueous suspension of the mycelium of a D. toxica plate culture.

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The solution was generated by rinsing off the vegetative parts of D. toxica plate

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cultures with tap water. Ten fully grown plates (8.5 cm diameter) were suspended in

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50 mL water and used for spraying. The treatment was performed 3 weeks after the

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germination of the plants and repeated a week later. The plants were harvested 8

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weeks after the first inoculation and the stems and leaves as well as pods and seeds

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were separated. The different plant materials were freeze-dried separately and milled

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to a fine powder.

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Additionally, lupin seeds, beans and peas were inoculated with P. viticola and P.

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juniperovora; two other fungal strains from the genus Diaporthe/Phomopsis which are

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widespread in Europe and also commercial available. After incubating for four weeks

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at 24 °C, the cultures were freeze-dried and milled to a fine powder.

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Extraction and clean-up

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Contaminated sample material artificially infected with D. toxica (lupin flour, pea flour,

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bean flour, and lupin plants) was used to optimize the extraction and clean-up of

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samples. The material was blended with untreated legume flour to reduce the toxin

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level. The true values for the content of 1 of these samples were unknown; the

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extraction procedure leading to maximal signals in all four matrices was considered

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as the optimum and used for the further method development. de Nijs et al.17 8 ACS Paragon Plus Environment

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proposed to extract 2.5 g sample with 20 mL of acetonitrile and water (80:20, v/v)

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acidified with 1% acetic acid. However, the use of purification columns after

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extraction could require different extraction solvents and sample to solvent ratios.

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The optimum relation of sample size and extraction volume was assessed by using

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three different sample sizes (0.5, 1.0, and 2.0 g) and four different extraction volumes

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(2, 4, 6, and 8 mL). The initial composition of the extraction solution was varied by

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using 4 mL acetonitrile/water (80:20, v/v) + 1 mL n-hexane, 4 mL acetonitrile/water

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(80:20, v/v) + 1% acetic acid and 4 mL acetonitrile/water (50:50, v/v). The use of

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ultrasonication of the reconstituted sample extracts after the removal of the extraction

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solvent and a short vortexing was tested without ultrasonication and ultrasonication

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for 15 min and 30 min.

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The sample clean-up was performed with disposable purification columns (similar to

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SPE columns) requiring only one elution step. The reservoir of the column was filled

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with 2 mL of the sample extract which was immediately passed through the sorbent

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bed with the piston. The eluate was collected in 15 mL tubes.

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Sample preparation

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Aliquots of 0.5 g of the sample material were weighted in 15 mL centrifugation tubes

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and 100 µL of the ISTD solution (c = 100 µg/L in acetonitrile/water, 50:50, v/v) was

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added. After drying for 1 h at room temperature, the samples were extracted with 4

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mL acetonitrile/water (80:20, v/v) for 30 min on an horizontal shaker. The extract was

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centrifuged (2,135 x g at 20 °C for 10 min), and 2 mL of the supernatant was passed

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through the purification column. Subsequently, the solvent was removed in a vacuum

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centrifuge (40 °C, 10 mbar, 3 h) and the residue was reconstituted with 150 µL

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acetonitrile/water (80:20, v/v). Each sample was prepared and analysed in triplicate.



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LC-MS/MS analysis

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Chromatographic separation was carried out on a 1100 Series HPLC system from

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Agilent Technologies Deutschland GmbH & Co. KG (Waldbronn, Germany). Several

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different columns were tested during the LC-MS/MS method development: 150 mm x

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2 mm i.d., 3 µm, Gemini NX-C18 (Phenomenex Ltd., Aschaffenburg, Germany), 100

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mm x 2 mm i.d., 2.5 µm, Synergi Hydro RP (Phenomenex Ltd.), and 150 mm x 4.6

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mm i.d., 2.7 µm Ascentis Express RP-Amide (Sigma-Aldrich Chemie GmbH). In the

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final LC-MS/MS method, a pentafluorophenylpropyl column, 4.6 mm x 150 mm i.d., 5

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µm, Ascentis Express F5 with a 5 mm x 4.6 mm i.d. guard column of the same

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material (Sigma-Aldrich Chemie GmbH) was used, which was kept at 40 °C. Eluent A

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was water, eluent B was acetonitrile/water (90:10, v/v), both eluents containing 5 mM

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ammonium formate. An aliquot of 10 µL was injected into the LC-MS system.

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Gradient elution was performed at a flow rate of 0.4 mL/min, starting with 95% eluent

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A for 0.5 min, then a linear gradient to 40% eluent B at 3 min was applied. This was

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kept for 4 min, then a linear gradient to 100% eluent B at 1 min was applied. This was

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kept for 2 min before the initial conditions were restored. The LC column was

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equilibrated for 8 min before the next injection. Electrospray ionization tandem mass

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spectrometry (ESI-MS/MS) data were acquired on an API 4000 triple-quadrupole

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MS/MS system (AB Sciex Germany GmbH, Darmstadt, Germany), operating at +5KV

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in positive ionization mode. The optimization of the MS/MS parameters was carried

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out by infusing a standard solution of 1 (100 µg/L in acetonitrile/water (50:50, v/v)

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containing 0.1% formic acid) with a syringe pump at 10 µL/min. The mobile phase

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was later modified with ammonium formate instead of formic acid. Optimized

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parameters

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obtained.16,17,21 The MS/MS parameters were transferred to corresponding mass

similar

to

those already

described in


previous

studies

were

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N6-1 as derived from the fragmentation pattern.21 The mass

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transitions of the

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transitions used for quantitation and qualification are displayed in Table 1. Data

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acquisition and handling was done using Analyst 1.6.2 software (AB Sciex Germany

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GmbH, Darmstadt, Germany).

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Evaluation of the method

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To evaluate the whole analytical process, which includes sample extraction, clean-up

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and LC-MS/MS analysis, recoveries and RSDs for spiking experiments were

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determined by applying the SIDA technique developed. Four different matrices (lupin,

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pea, bean, and lupin plant) were spiked in quadruplicate at 5, 25, and 50 µg/kg,

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corresponding to the regulatory limit for lupin seeds and the products thereof, and

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increased levels (five- and tenfold) to evaluate a wider range. An aliquot of 125 mg

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spike solution (20, 100, and 200 ng/g 1, corresponding to 2.5, 12.5, and 25 ng 1 per

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sample) and 100 µL of ISTD solution were added to 0.5 g of each sample prior to any

237

treatment. The amount of ISTD was adjusted to a signal to noise ratio of

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approximately 50 in matrix samples to assure a proper intergration and was used

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identically for all samples and calibration points. Quantitation was performed with an

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external calibration. The solvent calibration standards contained 0.5, 2.5, 5.0, 10.0,

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15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 ng 1 absolute, dissolved in 4 mL

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acetonitrile/water (80:20, v/v). Each standard was spiked with 100 µL of ISTD

243

solution (corresponding to 95 ng approx.).

RESULTS AND DISCUSSION 244

Extraction and clean-up

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With regard to the signal intensity the optimal parameters were found to be a sample

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size of 0.5 g and an extraction volume of 4 mL. The extraction efficiency achieved by 11 ACS Paragon Plus Environment


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the different extraction solvents in combination with the four matrices is shown in

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Figure 3A. For each matrix, extraction with acetonitrile/water (80:20, v/v) without any

249

modification led to the highest signals in the subsequent LC-MS/MS analysis. The

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corresponding peak areas of the ISTD are shown in Figure 3B. The signal intensities

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of 1 and ISTD are influenced by adsorption effects, matrix suppression and losses

252

during clean-up. The remaining difference between the extraction solvents after ISTD

253

correction describes the extraction efficiency. The use of an extraction solvent with a

254

high proportion of water (acetonitrile/water 50:50, v/v) led to the co-extraction of polar

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matrix compounds, thus, decreasing the signal intensity for both, 1 and ISTD. As the

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ISTD is spiked before the extraction process it should not be subject to any major

257

biological matrix binding effect, any decrease in signal intensity is based on matrix

258

suppression or adsorption effects and not on a reduced extraction. Even if these

259

effects are compensated by the SIDA technique, a decreased signal of the analyte

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has an adverse impact on the LOD and LOQ. On the contrary, an extraction with

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80% acetonitrile increases the signal intensity of native 1 and ISTD due to a

262

decreased extraction of

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suppression.23,24 The addition of n-hexane was intended to remove very nonpolar

264

molecules but had neutral effects regarding the signal intensities of 1 and the ISTD.

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When the pH of the extraction solvent is lowered with 1% acetic acid, molecules with

266

amino groups such as 1 are protonated, become more polar and are therefore less

267

soluble in the nonpolar extraction solvent. The use of ultrasonication had no effects

268

on the recovery of 1.

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In order to establish and apply a SIDA based quantitation method for 1, the

270

applicability of one step purification columns was assessed initially. These

271

investigations have shown that the loss of 1 during the clean-up of sample extracts 12

polar matrix compounds that may cause matrix

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using purification columns is negligible. The columns were able to remove matrix

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compounds, which became visible by a pronounced decolorization of the sample

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solution (Figure 2A). The mass loss of purified sample extracts was determined by

275

weighing the residues of the untreated samples and the purified samples of lupin,

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pea, bean, and lupin plant extracts after evaporation of the solvent. The average

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residue mass of purified samples decreased by 42% in relation to crude extracts. The

278

removal of co-extracted compounds facilitated the concentration step of sample

279

solutions due to a faster evaporation of the extraction solvent and the enhanced

280

solubility of the residues. As shown in Figure 2B, the peak area of 1 could be

281

increased by a factor of 4 (lupin plant) to 12 (pea) after the concentration and

282

reconstitution.

283

Development of the SIDA-LC-MS/MS method

284

For chromatographic separation, several stationary phases were tested. Two

285

conventional C18 columns (150 mm x 2 mm i.d., 3 µm, Gemini-NX C18 and 100 mm

286

x 2 mm i.d., 2.5 µm, Synergi Hydro RP) with full porous phase material were limited

287

with regard to peak shape. The polarity of 1 requires aqueous starting conditions,

288

resulting in a longer equilibration period. By using a pentafluorophenyl column or a

289

RP-amide column with fused-core particles (150 mm x 4.6 mm i.d., 5 µm, Ascentis

290

Express F5, and 150 mm x 4.6 mm i.d., 2.7 µm, Ascentis Express RP-Amide),

291

sharper peaks and a shorter equlilibration time were achieved.

292

Figure 4 illustrates four MRM chromatograms of lupin flour samples, blank samples

293

and samples spiked with 1 and

294

the mass transitions of 15N6-1, but did not affect the analysis, at all. However, addition

295

of ammonium formate instead of formic acid as modifier as well as a flat gradient

296

profile are necessary to achieve the separation of the interfering peaks and the ISTD.

15

N6-1. Matrix peaks appeared in all four matrices for



Page 14 of 32

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Evaluation of the method

298

The quantitation of 1 in the spiked samples was performed with external calibration

299

standards. The calibration function y = 0.9784 x + 0.0002, R2 = 0.9999 was

300

generated by using the SIDA technique.

301

In order to evaluate the suitability of the biosynthesized labeled standard in LC-

302

MS/MS analysis for the compensation of matrix effects and losses during sample

303

preparation, spiked samples with and without ISTD correction were compared (Figure

304

5A and B). When SIDA technique is applied, the influence of different matrices on the

305

signal intensity of 1 can be compensated by SIDA using the biosynthesized

306

standard. The amount of 1 recovered was compared to the spiked amount of 1. The

307

average recovery rates and the relative standard deviations (RSD) of 1 obtained from

308

the different spiking levels are summarized in Table 2. Recoveries of approximately

309

100% were achieved for 1 quantitated in each matrix by SIDA. The data calculated

310

for the recovery of 1 also corresponds to the provision in EU regulation 657/2002 (> 1

311

µg/kg to 10 µg/kg: 70% - 110%). LOD (S/N = 3) and LOQ (S/N = 10) were derived

312

from signals for 1 in the samples spiked in a range from 0.5 to 5 µg/kg and blank

313

samples. LOD was determined as 1 µg/kg for lupin flour, pea flour and lupin plant and

314

0.5 µg/kg for bean flour. LOQ was set at 3 µg/kg for lupin flour, 4 µg/kg for pea flour

315

and lupin plant, and 2 µg/kg for bean flour. With respect to the reliable identification of

316

1 and

317

the relative ion ratio of the qualifier ion versus the quantifier ion for the solvent

318

standards and spiked samples. In comparison with the solvent standards, the ion

319

ratio of 1 and the ISTD remains stable for all samples spiked.

320

The developed SIDA-LC-MS/MS method for the determination of 1 in lupin flour, pea

321

flour, bean flour and whole lupin plants based on the isotopically labeled standard

15

15

N6-1

N6-1 in the samples, the stability of ion ratios was evaluated. Figure 6 shows


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15

323

advantage of the SIDA-technique. The use of the ISTD

324

quantitation in terms of accurary (trueness and precison) and simplifies the process

325

as no matrix-matched calibration is necessary. The method sensitivity meets the

326

current maximum level requirement of 5 µg/kg of the Australia-New Zealand Food

327

Authority and may, thus, be considered as suitable for future monitoring programs of

328

1.22,25 Future efforts should focus on the optimization of the extraction columns in

329

order to improve the sensitivity of the method.

330

Analysis of samples

331

The content of 1 of the sample material inoculated with D. toxica was determined

332

after blending samples free of 1 with highly contaminated material in order to obtain

333

sample material with a contamination level in the range that may be expected for real

334

samples mainly according to Australian data.22,26 The content of 1 in the blended

335

samples was 37 µg/kg for lupin flour, 28 µg/kg for pea flour, 25 µg/kg for bean flour,

336

and 32 µg/kg for lupin plants. The calculated content of 1 in the original fresh material

337

prior to freeze-drying and blending was 220 mg/kg for lupin flour, 440 mg/kg for pea

338

flour, 320 mg/kg for bean flour, and 300 mg/kg for lupin plants.

339

In all four matrices the toxin levels reached approx. 200 – 400 mg/kg. This

340

demonstrates the high toxin producing potential of D. toxica under unfavorable

341

conditions. The fungus was able to grow saprophytically on dead plant material

342

without competing microorganism(s). However, a small nest of infection which can

343

develop during the storage of legume seed could led to significant toxin levels once

344

the seeds are milled.

345

Moreover, the fungus does not only grow on lupins, described as its main host so far,

346

but also on peas and beans, producing up to double the amount of 1 compared to

N6-1 combines a simple and fast clean-up method for samples containing 1 with the


15

N6-1 improves the


347

lupin matrix. Consequently, D. toxica infections and the resulting contamination with 1

348

may not be limited to lupins. As legumes other than lupin represent good substrates

349

for the formation of 1 further investigations regarding the infection of other legumes

350

appear to be advisable and could be evaluated by the presented matrix-independent

351

quantitation method.

352

The samples derived from the lupin plants inoculated with D. toxica on the field were

353

analyzed to determine the content of 1. The sample material which was inoculated

354

with P. viticola and P. juniperovora, and control samples spiked with 5 µg/kg were

355

also analyzed to determine the content of 1. The results are shown in Table 3. No 1

356

was detected in the seeds of the inoculated plants. The pods and the green parts of

357

the plant were tested positive for 1 with levels above the regulatory limit of 5 µg/kg

358

specified by the Australia New Zealand Food Authority.22,25 The legume seeds

359

inoculated with P. viticola and P. juniperovora were all tested negative for 1, but the

360

sample material was heavily altered by the fungi during the growth period, and

361

represented a complex diversified matrix. Recoveries of 97%-103% in the samples

362

spiked with 5 µg/kg in four different matrices using the same external calibration

363

standards proved the versatility of the developed method.

ABBREVIATIONS USED 364

SIDA, stabe isotope dilution assay; ISTD, internal standard; MRM, multiple reaction

365

monitoring

ACKNOWLEDGEMENTS 366

We would like to thank Franziska Genzel and Angelika Fandrey (Leibniz-Institute of

367

Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Großbeeren, Germany) for

368

the planting and raising of the lupin plants. We also like to thank Julie Brunkhorst 16 ACS Paragon Plus Environment

Page 16 of 32

Page 17 of 32


369

(Trilogy Analytical Laboratory, Washington, MO), Ronald Niemeijer and Michael

370

Fijnenberg (R-biopharm AG, Darmstadt, Germany) for technical advice and kindly

371

providing us with the purification columns.

SUPPORTING INFORMATION 372

This material is available free of charge via the Internet at http://pubs.acs.org.

373

Figure S1, LC-MS/MS chromatograms obtained from sample extracts containing 1

374

using four different columns; Figure S2, calibration curve of 1 applying

375

internal standard

376


15

N6-1 as


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1.

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378

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presence of phomopsins in feed and food. EFSA Journal 2012, 10, 2567.

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van Warmelo, K. T.; Marasas, W. F. O.; Adelaar, T. F.; Kellerman, T. S.; van

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hexapeptide mycotoxin produced by Phomopsis leptostromiformis. J. Chem. Soc.,

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Phomopsis leptostromiformis. Tetrahedron 1989, 45, 2351-2372.

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causative agent of lupinosis, interacts with microtubules in vivo and in vitro. Eur. J.

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Cell Biol. 1984, 35, 156-164.

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induced in rats by phomopsin. Pathology 1990, 22, 213-222.

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Crous, P. W., Diaporthe: a genus of endophytic, saprobic and plant pathogenic fungi.

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Persoonia 2013, 31, 1-41.

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Colegate, S. M., Plant-associated toxins in animal feed: Screening and confirmation

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assay development. Anim. Feed Sci. Technol. 2005, 121, 5-21.

Mackay, M. F.; Vandonkelaar, A.; Culvenor, C. C. J., The X-ray structure of

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Tönsing, E. M.; Steyn, P. S.; Osborn, M.; Weber, K., Phomopsin A, the

Peterson, J. E., Biliary hyperplasia and carcinogenesis in chronic liver damage

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459 460



FIGURE CAPTIONS Figure 1. Structure of phomopsin A, 1 Figure 2. A. Lupin plant extract before (left) and after clean-up (right) with MultiTox MS TC-MT3000 purification columns; B. Peak area of phomopsin A in lupin plant extract directly after clean-up (sample volume 1.5 mL) and after concentration step (sample volume 0.15 mL) Figure 3. Peak area of A. phomopsin A and B. the internal standard (ISTD) in four matrices using different extraction solvents: samples were extracted for 30 min by shaking and 15 min ultrasonication (n = 3) Figure 4. MRM chromatograms obtained by recording the quantifier and qualifier mass transitions of 15N6-phomopsin A (A. unspiked sample; B. sample spiked with 20 µg/kg 15N6-phomopsin A) and phomopsin A (C. unspiked sample; D. sample spiked with 5 µg/kg phomopsin A) in lupin flour. Figure 5. Calibration for phomopsin A in lupin flour, pea flour, bean flour and lupin plant A. without and B. with 15N6-phomopsin A (ISTD) correction. Figure 6. Ratios of qualifier to quantifier ions for spiked samples, average ion ratio in solvent standards for phomopsin A (dashed line) and internal standard (ISTD) (dotted line)


Page 22 of 32

Page 23 of 32


TABLES Table 1. MRMa Transitions for Native Phomopsin A / 15N6-Phomopsin A (Dwell Time: 80 ms, DPb: 85 V, EPc: 10 V) phomopsin A Q1d [m/z]

Q3e [m/z]

CEf [V]

CXPg [V]

789.3

226.0

55

18

789.3

323.0

39

20

15

N6-phomopsin A

Q1 [m/z]

Q3 [m/z]

CE [V]

CXP [V]

795.3

227.0

55

18

795.3

325.0

39

20

a

MRM = multiple reaction monitoring DP = declustering potential c EP = entrance potential d Q1 = precursor ion e Q3 = fragment ion f CE = collision energy g CXP = cell exit potential b



Table 2. Recovery and RSDa for the Determination of Phomopsin A in Various Matrices by SIDAb (n=4) average recovery [%]

RSD [%]

lupin flour

100.5

2.8

bean flour

99.6

2.1

pea flour

102.7

3.0

lupin plant

103.1

2.6

lupin flour

99.1

1.2

bean flour

98.4

1.7

pea flour

101.9

0.7

lupin plant

100.8

1.3

lupin flour

99.3

0.7

bean flour

100.6

2.2

pea flour

100.4

1.2

99.5

0.2

spike level 5 µg/kg



lupin plant a b

RSD = relative standard deviation SIDA = stable isotope dilution assay


Page 24 of 32

Page 25 of 32


Table 3. Phomopsin A Content of Lupin Plants Inoculated with D. toxica During Growth Perioda phomopsin A [µg/kg]

phomopsin A [µg/kg]

(dry matter)

(wet matter)

matrix

D. toxica seeds pods stems and leaves

< LOD

< LOD

8.6 ± 1.2

7.2 ± 1.0

14.8 ± 1.3

10.1 ± 0.9 average recovery [%]

matrix

phomopsin A [µg/kg] (spike level 5 µg/kg)

P. juniperovora lupin seeds

< LOD

99.7 ± 1.9

bean seeds

< LOD

96.7 ± 3.0

pea seeds

< LOD

98.6 ± 0.8

lupin seeds

< LOD

102.4 ± 4.9

bean seeds

< LOD

101.1 ± 1.8

pea seeds

< LOD

102.9 ± 2.4

P. viticola

a

Freeze-dried samples and content of the wet material in reference to the drying process, and phomopsin A content of legume seeds inoculated with P. juniperovora and P. viticola, samples and the average recovery of spiked control (n = 3)



FIGURES

Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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Figure 6.



TABLE OF CONTENTS GRAPHIC


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MS Method for the Determination of

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