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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 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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Development of a SIDA-LC-MS/MS Method for the Determination
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of Phomopsin A in Legumes
3
Svenja Schlo߆, ‡, Matthias Koch†, Sascha Rohn‡, and Ronald Maul*, §, ‡
4 5
†
6 7
Straße 11, 12489 Berlin, Germany ‡
8 9 10
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
11 12
*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
Journal of Agricultural and Food Chemistry
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
21
analyze flour samples generated from selected legume seeds and lupin plant
22
samples which had been inoculated with Diaporthe toxica and two further fungal
23
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
25
found in the pods and 7.2 µg/kg in the stems and leaves.
15
N6-1 as an isotopically labeled internal
26 27
Keywords: Diaporthe toxica; stable isotope dilution assay; isotopically labeled internal
28
standard; legume contamination
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Journal of Agricultural and Food Chemistry
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
31
isolates of lupin grain can be processed to a wide range of food products (e.g. bread,
32
sausages, bread spreads, ice cream, sweets).1 Lupins were introduced to the
33
European agriculture in the 19th century. As the plant is well adapted to sandy soils, it
34
was cultivated intensively to be used as feed for sheep and cows and as a green
35
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
37
poisonings caused by alkaloids of the bitter lupin, another disease, later referred to
38
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
42
Warmelo et al.4, who identified the fungus Phomopsis leptostromiformis as the cause
43
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
45
et al.5 facilitated further investigations of their chemical and biological properties and
46
the occurrence in food and feed. Three additional phomopsins (C, D, and E) have
47
been identified so far. 1 is regarded as the main toxin with a toxic potential two to five
48
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
50
toxicity of 1, a hexapeptide consisting of three dehydro amino acids forming a
51
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
Journal of Agricultural and Food Chemistry
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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
58
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
64
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
69
food and feed.14 Former methods were time consuming (nursling rat bioassay), were
70
limited regarding sensitivity (LC-UV), or are no longer available (ELISA).6 Significant
71
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
74
Zealand. The introduction of an isotopically labeled internal standard (ISTD) would
75
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
81
or ion enhancing effects in LC-MS-analysis can be compensated by using matrix-
82
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
84
matched standards depend of the availability of blank matrix materials, which is not
85
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
87
considered as primary ratio method representing a high level of metrology and
88
facilitates a more versatile application.19,20 SIDA methods are matrix independent;
89
samples with varying matrices can be analyzed using the same calibration. The only
90
limitation occurs when matrix compounds share some of the mass transitions as the
91
analyte or the ISTD. In this case it is necessary to ensure that the matrix peaks and
92
the peaks of the analyte and the ISTD are separated by chromatography. The first
93
isotopically labeled ISTD for 1 has been developed at the Federal Institute for
94
Materials Research and Testing (BAM), Berlin, Germany. The isotopic purity and
95
chemical properties of the standard are suitable for the deployment in a SIDA LC-
96
MS/MS method.21
97
Although hybrid mass spectrometers allow for the sensitive quantitation of
98
contaminants at ultratrace levels without extensive sample clean-up by using either
99
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
101
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-
107
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
109
artificially infected materials. The implementation of an isotopically labeled ISTD is
110
supplemented with a fast and convenient sample clean-up technique using one-step
111
purification columns. The columns are designed to remove matrix compounds such
112
as proteins, fats, pigments, and other matrix compounds with a blend of several
113
sorbent materials. The columns act following a one-step principle retaining the matrix
114
compounds and allowing the analytes to pass through directly without a separate
115
elution step.
MATERIALS AND METHODS 116
Standards and reagents
117
1 (purity 83%, determined by NMR) was purchased from Santa Cruz Biotechnology
118
Inc. (Heidelberg, Germany) and dissolved in methanol to prepare a stock solution of
119
32 mg/kg.
120
preparative HPLC. The eluate containing
121
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
123
acid (100%) and n-hexane were acquired from Merck KGaA (Darmstadt, Germany).
124
Ammonium formate was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,
125
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
140
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
144
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
162
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.
165
juniperovora; two other fungal strains from the genus Diaporthe/Phomopsis which are
166
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
169
Contaminated sample material artificially infected with D. toxica (lupin flour, pea flour,
170
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
172
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
174
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
177
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
183
ultrasonication of the reconstituted sample extracts after the removal of the extraction
184
solvent and a short vortexing was tested without ultrasonication and ultrasonication
185
for 15 min and 30 min.
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The sample clean-up was performed with disposable purification columns (similar to
187
SPE columns) requiring only one elution step. The reservoir of the column was filled
188
with 2 mL of the sample extract which was immediately passed through the sorbent
189
bed with the piston. The eluate was collected in 15 mL tubes.
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Sample preparation
191
Aliquots of 0.5 g of the sample material were weighted in 15 mL centrifugation tubes
192
and 100 µL of the ISTD solution (c = 100 µg/L in acetonitrile/water, 50:50, v/v) was
193
added. After drying for 1 h at room temperature, the samples were extracted with 4
194
mL acetonitrile/water (80:20, v/v) for 30 min on an horizontal shaker. The extract was
195
centrifuged (2,135 x g at 20 °C for 10 min), and 2 mL of the supernatant was passed
196
through the purification column. Subsequently, the solvent was removed in a vacuum
197
centrifuge (40 °C, 10 mbar, 3 h) and the residue was reconstituted with 150 µL
198
acetonitrile/water (80:20, v/v). Each sample was prepared and analysed in triplicate.
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LC-MS/MS analysis
200
Chromatographic separation was carried out on a 1100 Series HPLC system from
201
Agilent Technologies Deutschland GmbH & Co. KG (Waldbronn, Germany). Several
202
different columns were tested during the LC-MS/MS method development: 150 mm x
203
2 mm i.d., 3 µm, Gemini NX-C18 (Phenomenex Ltd., Aschaffenburg, Germany), 100
204
mm x 2 mm i.d., 2.5 µm, Synergi Hydro RP (Phenomenex Ltd.), and 150 mm x 4.6
205
mm i.d., 2.7 µm Ascentis Express RP-Amide (Sigma-Aldrich Chemie GmbH). In the
206
final LC-MS/MS method, a pentafluorophenylpropyl column, 4.6 mm x 150 mm i.d., 5
207
µm, Ascentis Express F5 with a 5 mm x 4.6 mm i.d. guard column of the same
208
material (Sigma-Aldrich Chemie GmbH) was used, which was kept at 40 °C. Eluent A
209
was water, eluent B was acetonitrile/water (90:10, v/v), both eluents containing 5 mM
210
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
212
A for 0.5 min, then a linear gradient to 40% eluent B at 3 min was applied. This was
213
kept for 4 min, then a linear gradient to 100% eluent B at 1 min was applied. This was
214
kept for 2 min before the initial conditions were restored. The LC column was
215
equilibrated for 8 min before the next injection. Electrospray ionization tandem mass
216
spectrometry (ESI-MS/MS) data were acquired on an API 4000 triple-quadrupole
217
MS/MS system (AB Sciex Germany GmbH, Darmstadt, Germany), operating at +5KV
218
in positive ionization mode. The optimization of the MS/MS parameters was carried
219
out by infusing a standard solution of 1 (100 µg/L in acetonitrile/water (50:50, v/v)
220
containing 0.1% formic acid) with a syringe pump at 10 µL/min. The mobile phase
221
was later modified with ammonium formate instead of formic acid. Optimized
222
parameters
223
obtained.16,17,21 The MS/MS parameters were transferred to corresponding mass
similar
to
those already
described in
10 ACS Paragon Plus Environment
previous
studies
were
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Journal of Agricultural and Food Chemistry
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N6-1 as derived from the fragmentation pattern.21 The mass
224
transitions of the
225
transitions used for quantitation and qualification are displayed in Table 1. Data
226
acquisition and handling was done using Analyst 1.6.2 software (AB Sciex Germany
227
GmbH, Darmstadt, Germany).
228
Evaluation of the method
229
To evaluate the whole analytical process, which includes sample extraction, clean-up
230
and LC-MS/MS analysis, recoveries and RSDs for spiking experiments were
231
determined by applying the SIDA technique developed. Four different matrices (lupin,
232
pea, bean, and lupin plant) were spiked in quadruplicate at 5, 25, and 50 µg/kg,
233
corresponding to the regulatory limit for lupin seeds and the products thereof, and
234
increased levels (five- and tenfold) to evaluate a wider range. An aliquot of 125 mg
235
spike solution (20, 100, and 200 ng/g 1, corresponding to 2.5, 12.5, and 25 ng 1 per
236
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
238
approximately 50 in matrix samples to assure a proper intergration and was used
239
identically for all samples and calibration points. Quantitation was performed with an
240
external calibration. The solvent calibration standards contained 0.5, 2.5, 5.0, 10.0,
241
15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 ng 1 absolute, dissolved in 4 mL
242
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
245
With regard to the signal intensity the optimal parameters were found to be a sample
246
size of 0.5 g and an extraction volume of 4 mL. The extraction efficiency achieved by 11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
247
the different extraction solvents in combination with the four matrices is shown in
248
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
250
corresponding peak areas of the ISTD are shown in Figure 3B. The signal intensities
251
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
255
matrix compounds, thus, decreasing the signal intensity for both, 1 and ISTD. As the
256
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
260
has an adverse impact on the LOD and LOQ. On the contrary, an extraction with
261
80% acetonitrile increases the signal intensity of native 1 and ISTD due to a
262
decreased extraction of
263
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.
265
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.
269
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
273
compounds, which became visible by a pronounced decolorization of the sample
274
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,
276
pea, bean, and lupin plant extracts after evaporation of the solvent. The average
277
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
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Page 14 of 32
297
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
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N6-1 improves the
Journal of Agricultural and Food Chemistry
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
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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
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N6-1 as
Journal of Agricultural and Food Chemistry
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W. F. O.; Steyn, P. S.; Vleggaar, R.; Wessels, P. L., Structure elucidation and
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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
Gomes, R. R.; Glienke, C.; Videira, S. I. R.; Lombard, L.; Groenewald, J. Z.;
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van Egmond, H. P.; Mol, H. J., Development and validation of an LC-MS/MS method
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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)
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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
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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
spike level 25 µg/kg
spike level 50 µg/kg
lupin plant a b
RSD = relative standard deviation SIDA = stable isotope dilution assay
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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)
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FIGURES
Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6.
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TABLE OF CONTENTS GRAPHIC
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