Anal Bioanal Chem (2013) 405:9375–9383 DOI 10.1007/s00216-013-7365-4
RESEARCH PAPER
Structural screening by multiple reaction monitoring as a new approach for tandem mass spectrometry: presented for the determination of pyrrolizidine alkaloids in plants Anja These & Dorina Bodi & Stefan Ronczka & Monika Lahrssen-Wiederholt & Angelika Preiss-Weigert
Received: 31 July 2013 / Revised: 9 September 2013 / Accepted: 10 September 2013 / Published online: 10 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In tandem mass spectrometry the multiple reaction monitoring (MRM) mode is normally used for targeted analysis but this mode also has the potential to screen for structural similarities of analytes. On the basis of the fact that in general similar molecular structures result in similar fragments or losses of neutrals, this approach was used for pyrrolizidine alkaloid (PA) screening but could also be easily adapted to screen for other compound classes. PA are plant toxins of which several hundred individual compounds have been identified. Our MRM screening approach uses the structural relation and similar core structure of all PA which results in a common and thus predictable mass spectrometric fragmentation behaviour. On this basis a method was developed which screens for PA structures by MRM transitions and allows the detection of each individual PA down to a low microgram per kilogram concentration range. The approach was applied to investigate plants from the families of Asteraceae (several species of Senecio and Eupatorium), Boraginaceae (Echium, Cynoglossum, Borago and Anchusa officinalis as well as Heliotropium europaeum) and Fabaceae (Crotalaria incana) for a complete qualitative and quantitative PA characterisation. All analytes that were detected as possible PA by MRM screening were further investigated by recording product ion spectra. Analytes which exhibited a typical PA fragmentation pattern were either confirmed as PA or otherwise deleted as false positive signals (false positive rate was below 10 %). Sum formulas of confirmed PA were determined by additional measurements applying high Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7365-4) contains supplementary material, which is available to authorized users. A. These (*) : D. Bodi : S. Ronczka : M. Lahrssen-Wiederholt : A. Preiss-Weigert Federal Institute for Risk Assessment, Department Safety in the Food Chain, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany e-mail:
[email protected]
resolution mass spectrometry. In that way 121 unknown PA were identified and for the first time complete PA profiles of different PA plants were delivered. Keywords Pyrrolizidine alkaloids . Plant toxins . Mass spectrometry
Introduction Pyrrolizidine alkaloids (PA) are secondary metabolites of plants and are assumed to act as a plant defence mechanism. It can be assumed that 3 % of flowering plants are able to form PA [1] and they are mainly found in the angiosperm families of Fabaceae (genus Crotalaria), Asteraceae (genus Senecioneae and Eupatorieae) and several genera of Boraginaceae [2–7]. The ingestion of PA may result in acute and chronic effects in man and livestock and human case reports of poisonings have demonstrated that liver and lung are predominantly affected. Acute poisoning with 1,2-unsaturated PA in humans is associated with high mortality and is further characterised by hepatic venoocclusive disease (HVOD), whereas a subacute or chronic onset may lead to liver cirrhosis [2, 4, 8]. The PA investigated are esters consisting of a 1hydroxymethyl-7-hydroxypyrrolizidine (necine base) and aliphatic mono- or dicarboxylic acids (necic acid) (Fig. 1). Depending on the chemical structure of the necine base several PA types are differentiated (Fig. 1) [4, 9, 15]. Of toxicological relevance are PA with an 1,2-unsaturated necine base, namely the retronecine, heliotridine and otonecine types (Fig. 1a–c), as these are able to form hepatotoxic intermediates [4, 10, 11]. Depending on the esterification of one or two hydroxy groups PA occur as mono- or diesters (Fig. 1). Furthermore, PA of the retronecine and heliotridine type can be oxidized at the nitrogen atom to form the respective N-oxide (Fig. 1g–i). As the N-
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A. These et al.
necic acid
R
2
O
necine base
R
1
R R
O H 1
7R
1
R
2
R
O
O
O
H
O
O
1
7S
1
2
O
O
2
2
N
N
N CH3
a) retronecine-type b) heliotridine-type c) otonecine-type O R H
O
O
1
R
2
R
O
R
O
O
H
1
R
H
O
O O
O
H
O
N
1
2
N N
d) monoester
e) open chained diester f) cyclic diester
O R H
O
1
O R
O H
2
R O
O
O
1
R R
N
-
O
g) monoester-NO
O H
+
+
O
O O
O
N
1
2
H
-
h) open diester-NO
N
+
O
-
i) cyclic diester-NO
Fig. 1 Categorization of PA structure types depending on necine base (a–c), type of esterification (d–f) and oxidation of the nitrogen (g–i)
oxide can be reduced to the free base during passage through human gut they have to be included in the determination of total toxicity [12, 13]. The exact number of naturally occurring PA can only be estimated, but up to now several hundred have been identified [9, 14]. Only 19 of them are commercially available as analytical standards. Owing to the difference between the number of PA described in the literature and the number of PA available as standards, the determination of PA content and thus the assessment of the total toxicity are difficult. Two approaches are mainly applied for PA detection [15]. The first utilizes an indirect determination of the total PA content by transferring all individual PA into a common necine base structure which is determined subsequently. Requirements for a quantitative determination of PA content are (a) all PA relevant for toxicity have to be transferred into the common structure and (b) the transfer of individual PA has to be quantitative or transfer rates have at least to be comparable. As the available indirect methods do not fulfil these requirements their results are semiquantitative. The second detection approach is the direct determination of individual PA followed by summation of each determined PA content. So-called targeted analysis uses specific methods which were optimized on the basis of analytical standards. The disadvantage of this approach is that PA, other than available standards, that may also be present in the sample would remain undetected. Therefore, there is a need for alternative analytical approaches without losing specificity or sensitivity. A high specificity accompanied by a sufficient sensitivity can be achieved by a detection using high mass resolution or multiple
reaction monitoring (MRM) transitions. To benefit from high mass resolution the exact mass of the analyte of interest has to be known and thus this procedure is a classical targeted analysis. This also holds true for detection applying MRM transitions, as specific fragments of an analyte will be monitored and thus only PA with known structures and fragmentation pattern can be detected. The objective of our study was to assess the holistic PA content in a sample without losing structural information and without losing signal intensity. We accomplished this by an MRM screening based on the fact that individual PA toxins with an 1,2-unsaturated necine base indeed differ in the structure of their necic acids but still share a similar core structure. This similarity manifests in a common fragmentation pattern resulting in similar product ions or similar losses of neutrals. Extracts of different plant samples were investigated for PA contents by MRM screening and suspicious signals were checked by recording product ion spectra. If product ion spectra confirmed the presence of a PA molecule as well as the type of PA structure (monoester, diester etc.) the sum formulas were identified subsequently by determination of exact masses by Orbitrap technology. Postulating a comparable mass spectrometric response of PA molecules belonging to the same type of PA structure, as for instance monoester, open-chained diester etc., we estimated the contents of the identified PA using the MS response of structurally comparable and available PA reference standards. This procedure allowed an effective screening of numerous plant samples and the complete assessment of PA contents.
Experimental Chemicals and standards Ammonium formate, formic acid (analytical grade) and methanol (MeOH; hypergrade) were obtained from Merck (Darmstadt, Germany). The following PA standards were purchased from Phytolab (Vestenbergsgreuth, Germany): monocrotaline (Mc), monocrotaline N-oxide (McN), intermedine (Im), lycopsamine (La), retrorsine (Re), trichodesmine (Td), retrorsine N-oxide (ReN), seneciphylline (Sp), heliotrine (Hn), heliotrine N-oxide (HnN), seneciphylline N -oxide (SpN), senecionine (Sc), senecionine N-oxide (ScN), echimidine (Em), senkirkine (Sk), lasiocarpine (Lc), lasiocarpine N-oxide (LcN), europine (Eu) and europine N-oxide (EuN). Plant extraction The plant material was ground into a particle size of 500 μm. A 2-g portion of sample was extracted with 25 mL of 25 %
Structural screening by multiple reaction monitoring
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B was 95 % MeOH and 5 % water containing 0.1 % formic acid and 5 mM ammonium formate. The following gradient elution was adopted: 0–0.5 min, 95 % A/5 % B; 7.0 min, 50 % A/50 % B; 7.5 min, 20 % A/80 % B; 7.6 min, 0 % A/100 % B; 9.0 min, 0 % A/100 % B; 9.1–15 min, 95 % A/5 % B. A flow rate of 300 μL/min was applied and 10 μL was injected.
MeOH in 2 % formic acid by vortex mixing. The supernatant was decanted after centrifugation. Extraction was repeated one more time and the collected supernatants were made up to 50 mL. Samples were filtered using a 0.2-μm membrane (VWR, Germany).
HPLC Tandem mass spectrometry Analyses were performed on a Thermo Fisher Accela LC system in combination with an HTS PAL autosampler (PalSystem, Zwingen, Germany). Chromatography was carried out on a 150×2.1 mm, particle size 1.9 μm C18 Hypersil Gold column with guard protection (Thermo Fisher, Germany). Eluent A was 100 % water containing 0.1 % formic acid and 5 mM ammonium formate and eluent
Prod_HnN_330_CE27eV RT: 7.1 NL: 6.88E5 + c ESI Full ms2 330.200 [50.000 -350.000]
Prod_Hn_314_CE27eV RT 6.8 NL:5.62E5 + c ESI Full ms2 314.200: [50.000 -350.000] 100 138
HO
O
H
H3C
120
65 60 55 50 45 40 35
N
156 + CH2
+ CH2
HO
120
m/z 138
m/z 120
15 10 5 0
314 60
80
100
prod_Lc_412_CE28eV
120
140
160
180
200 m/z
220
240
260
280
300
-
+
CH2
CH3 CH3
O
138
H3C O
OH
O HO H3C
H
O 120
CH3
N
330
30 25 20
220
80
138 156
412
HO
CH3 O
H3C O 120
O
CH3 H
- 28 amu
O 138
N
138
308
96
60
153 107 122
82 100
120
140
160
290
220
156 80
200 m/z
220
240
260
280
300
180
200 m/z
220
240
260
280
300
320
340
340
CH3 O CH3 CH3
O
138 O
O HO H3C
H
OH CH3
+
O
-
136 136 138
410
352
120
428 338
94 237 154 172 190 218
118 100
150
80 75 70 65 60 55 50
f) ScN
200
328
250
m/z
300
350
400
450
352 HO
CH3
CH3
O
O H3C O
O
- 28 amu
H
138
120
120
+
N
136
45 40 35
500
NL: 1.85E5
118
O
94
-
136 220
138 106
15 10 5 0
Δ 28
320
N
30 25 20
30 25 20
68
180
+ c ESI Full ms2 352.260 [50.000 -380.000]
120
15 10 5 0
160
H3C
Prod_ScN_352_CE29eV RT: 7.5
Relative Abundance
Relative Abundance
e) Sc
94
140
120
100 95 90 85
336
45 40 35
120
m/z 136
O
+ c ESI Full ms2 336.160 [50.000 -350.000]
65 60 55 50
100
m/z 118
138 136 155
d) LcN
80 75 70 65 60 55 50 45 40 35
50
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 m/z
prod_Sc_336_CE29eV RT: 7.4 NL: 3.32E5
80 75 70
80
N
254
15 10 5 0
336
238
100 95 90 85
111
94
30 25 20
60
CH2
N
100 95 90 85
O
94
+
HO
Prod_LcN_428_CE29eV RT:9.9 NL:4.25E5 + c ESI Full ms2 428.200 [50.000 -500.000]
45 40 35
15 10 5 0
+
60
CH3
c) Lc
80 75 70 65 60 55 50
CH3
H3C
N
340
RT:9.5 NL:2.05E5
+ c ESI Full ms2 412.200 [50.000 -450.000] 100 120 95 90 85
320
O
H
O 136
15 10 5 0
108
82
OH O
138
HO
30 25 20
N
N
i-Pr O 172
45 40 35
96
30 25 20
Relative Abundance
CH3
172
b) HnN
80 75 70 65 60 55 50
O
138
Relative Abundance
80 75 70
OH
O
156
100 95 90 85
Relative Abundance
Relative Abundance
i-Pr
a) Hn
95 90 85
Electrospray ionization tandem mass spectrometry (ESI-MS/ MS) data were acquired on a TSQ Vantage (Thermo Scientific, San Jose, CA, USA). PA were analysed in the positive ion mode using a spray voltage of +3,500 V, an S-lens voltage of 140 Vand a vaporizer temperature of 300 °C. Q1 and Q3 were set at unit resolution. Dwell time for MRM transitions was set
324
153
246
178
Δ 28
248
236 60
80
100
120
140
160
180
200
220 m/z
240
260
280
300
320
340
360
Fig. 2 Product ion spectra of a monoester, b N-oxide monoester, c open-chained diester, d N-oxide open-chained diester, e cyclic diester and f N-oxide of a cyclic diester. Fragments used for MRM screening are indicated and structure proposals of fragment ions are included
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A. These et al.
Table 1 Ion intensities of typical PA fragments shown in Fig. 2 PA structure type
[M+H]+ → 120
[M+H]+ → 138
Monoester
***
*****
Open-chained diester
*****
**
Cyclic diester
*****
***(*)
*
**(*)
**(*)
Monoester N-oxide
[M+H]+ → 136
[M+H]+ → Δ28 amu
[M+H]+ → 156
[M+H]+ → 172
[M+H]+ → 150
[M+H]+ → 168
***
*****
**** *
Open-chained diester N-oxide
****(*)
****(*)
****(*)
Cyclic diester N-oxide
*****
****
****(*)
***
* *****
**(*)
Otonecine
Ratios between fragments were used for MRM screening and the subsequent classification into different PA structure types (Fig. 1d–i). Ion ratios might depend on individual MS conditions; therefore the respective laboratory MS conditions have to be checked *****Base peak in product ion spectrum ****Second highest peak ***,**,*Peaks of descending order of intensity in product ion spectra
to 20 ms resulting in a cycle time of 1.2 s. Collision energy (CE) was set to 27 eV for [M+H]+ → 120, [M+H]+ → 138, [M+H]+ → 136, [M+H]+ → 156, [M+H]+ → 172, [M+H]+ → 150, [M+H]+ → 168 and 23 eV for [M+H]+ → (M+H− 28). Product ion spectra were recorded using a CE of 27 eV for monoesters, 28 eV for open-chained diesters and 29 eV for cyclic diesters.
Results and discussion Fragmentation patterns of PA and postulations for MRM screening
High resolution mass spectrometry High resolution mass spectrometry (hRMS) data were acquired on an Orbitrap Exactive system (Thermo Fisher, Bremen, Germany). The mass spectrometer was operated at +3, 700 V using an S-lens voltage of 140 V. Two scan events were recorded in parallel by permanent switching. Firstly, a full scan mass range of m /z 160–500, applying a resolving power
Table 2 List of injections and methods for MRM screening of a single sample Injection no.
Investigated PA structure type
Mass range (m/z)
1.
Monoester, diestera
298–322
2.
Monoester, diestera
324–348
3.
Monoester, diestera
350–374
4.
Monoester, diestera
376–400
5.
Monoester, diestera
402–426
6.
Monoester, diestera
428–452
7.
a
Monoester, diester
454–478
8.
Monoester, diestera
480–500
9.
Otonecineb
350–424
10.
Otonecineb
426–500
Monitored transitions: [even-numbered M+H]+ → 120; → 138; → 136; → Δ28 amu; → 156; → 172 a
b
of 50,000 and secondly, fragments were generated by an HDC 30 Vexperiment and acquired in a mass range of m /z 100–500 using a resolving power of 25,000.
Monitored transitions: [even-numbered M+H]+ → 150; → 168
Fragmentation patterns have been studied by recording product ion spectra for 19 commercially available PA standards and for more than several hundred compounds extracted from PA-containing plant material. Figure 2 shows product ion spectra of different PA structure types represented in Fig. 1 (data for otonecines are not shown). Figure 2 shows the fragmentation pattern for monoesters, namely heliotrine (a) and heliotrine N-oxide (b); open-chained diesters, lasiocarpine (c) and lasiocarpine N-oxide (d); and cyclic diesters, senecionine (e) and senecionine N-oxide (f). These product ion spectra are shown as examples, but fragmentation patterns of other PA molecules belonging to the same PA structure type follow the same fragmentation rules. PA of the heliotridine and retronecine type are diastereomers at the position C-7. Owing to the same fragmentation pattern exhibited by both these types (Fig. 1a, b) they can not be distinguished by mass spectrometry and are not distinguished further. Observed fragmentation pathways in our studies were also intensively discussed in other studies in which typical diagnostic ions as well as ion clusters of PA were reported [16–19]. From product ion data the following statements can be made: beside compound-specific fragments all monoesters, cyclic and open-chained diesters exhibit typical fragments of m/z 94, 120 and 138 (Fig. 2a, c, e) [16–22]. These three fragments represent the carbon core structure of the necine
Structural screening by multiple reaction monitoring
base of 1,2-unsaturated PA shown in Fig. 1 and were detected for all PA structure types irrespective of the necic acid ester linked to the necine base. For a given collision energy the signal ratios of these three fragments are even relatively constant within the respective ester type. That means, for instance, that diesters, cyclic as well as open-chained diesters, show fragments of m/z 94, 120 and 138 and the ratio of intensities of these three fragments is typically around 10:100:10 for the group of open-chained diesters (Fig. 2c) and typically around 75:100:75 for cylic diesters (Fig. 2e). The typical relative ion intensities of PA fragments observed in product ion spectra of different PA structure types are compiled in Table 1. The signal to noise ratio of the m/z 94 ion is rather low in MRM chromatograms of plant extracts and was not considered for further investigations. A first postulation for the MRM screening method derived from product ion spectra can be stated as follows: each PA exhibits the fragments of m /z 120 and m/z 138 and, consequently, each analyte that is detected by MRM transitions of [M+H]+ → 120 and [M+H]+ → 138 within the ion intensities described in Table 1 is suspected to be a 1,2-unsaturated PA. The detected ion intensity ratios are also criteria for a classification into a certain PA structure type. Furthermore, PA structure types concerning esterification and oxidation (Fig. 1d–i) can also be distinguished owing to their typical fragmentation patterns. Cyclic diesters show a neutral loss of CO (−28 amu) (Fig. 2e) which is not present in product ion spectra of monoor open-chained diesters (Fig. 2a, c) [18]. The intensity of the fragment ions resulting from neutral loss of CO is typically around one fifth of the m /z 120 fragment. Therefore, the fragmentation of a cyclic diester and the resulting MRM transitions to be screened for can be stated as follows: in addition to fragments of m/z 120 and m/z 138, cyclic diesters will exhibit a fragment resulting from neutral loss of 28 amu. The respective relative ion intensities of these three fragments are summarized in Table 1. That means an analyte can be identified as a cyclic diesters PA if three MRM transitions, namely [M+H]+ → 120, [M+H]+ → 138 and [M+H]+ → (M+H−28 amu), are detected and the required ion intensities given in Table 1 are fulfilled. Fragmentation patterns of N-oxides of diesters are comparable to those of the non-oxidized form but additional fragments occur. The oxygen at the nitrogen atom can be easily cleaved by the neutral loss of water (−18 amu); thus, in parallel to m/z 120 and 138, fragments of m/z 136 and 118 will be formed (Fig. 2d, f) [18, 19, 22]. Monoesters exhibit the fragment of m/z 156 and its intensity is comparable to or even higher than the signal of m/z 120 (Fig. 2a) [18, 20, 22]. The fragments of m/z 156 were also observed for certain diesters (Fig. 2c, e) but the fragment intensity of m/z 156 for diesters is very low compared to that for monoesters. Thus, monoesters can be identified by the fragments m/z 120 and m/z 138 and the additional monitoring of m/z 156. This
9379
means that an analyte can be identified as a PA monoester if three MRM transitions, namely [M+H]+ → 120, [M+H]+ → 138 and [M+H]+ → 156, are detected within the relative ion intensities given in Table 1. Product ion spectra of monoester N-oxides exhibit a fragment of m /z 172, which has a high intensity, instead of m/z 156 but m /z 120 is not formed (Fig. 2a, b) [22]. The requirements for identification of monoester N-oxides are therefore the detection of three MRM transitions, namely [M+H]+ → 136, [M+H]+ → 138 and [M+H]+ → 172 within given relative intensities (Table 1). For PA of the otonecine type typical fragments of m /z 122, 150 and 168 can be determined [17–19] (data not shown). As the fragment of m /z 122 is also formed by the non-toxic platyphyllines only m/z 150 and 168 were recorded as specific fragments and set as requirements for detection of otonecines. If the MRM intensity of [M+H]+ → 168 is around one third higher compared to that of [M+H]+ → 150 the molecule can be identified as an otonecine (Table 1). Setting MRM transitions for screening and data processing At first, the Q1 mass range of interest has to be restricted. Most molecular masses for PA described in the literature range
a)
b)
c)
d) e) f)
Fig. 3 Data processing done by the automatic peak identification software ToxID, illustrated for m/z 352 that is the Q1 mass of retronecine (cyclic diester) and seneciphyllin N-oxide (cyclic diester N-oxide). Transition a 352 → 120 and b 352 → 138 reflect PA core structure fragmentation and the identification criteria as PA. Transition c 352 → 324 (loss of CO) is a criterion of cyclic diesters identification and d 352 → 136 is used for the identification as PA N-oxide. No signals were detected for transitions of 352 to 156 and 172 (e, f; in grey) which are criteria for monoesters and their N-oxides. Thus these transitions were not displayed by ToxID but are included here for completeness
17
Crotalaria incana
Anchusa officinalis
16 Seeds
Seeds
Stem spadix
Seeds
Seeds
Flowers
Whole
Flowers
Seeds
Roots
Whole
Whole
Whole
Seeds
Whole
Whole
Whole
Plant parta
2.11
Targeted
Targeted –b
0.06 0.01
Screening
–b
2.25
7.45 4.68
Screening Targeted
0.40
Screening Targeted
Targeted Dresden, SN, DE
0.08 0.07
Screening
7/20/2010
–b
3.68 2.75
Screening
–b
3.08
Targeted
8.98
Screening
ARG
0.04
2.79
2.41
6.78
8/15/2010
Screening Targeted
7/29/2011
Targeted
Berlin, BE, DE
Screening
ARG
Targeted 8/15/2010
4.65 2.56
Screening
–b
0.96 0.52
Screening Targeted
8/15/2010
1.57
1.85
2.22
3.24
1.64
ARG
Screening Targeted
6/22/2010
Targeted
Berlin, BE, DE Berlin, BE, DE
Screening
4/15/2011
Screening Targeted
11/25/2010 Berlin, BE, DE
0.46 0.19
Screening Targeted
–b
3.41 2.01
Screening Targeted
7/25/2010
0.65
1.85
0.43
0.74
PA content (%)c
Berlin, BE, DE
Screening Targeted
8/14/2010 Alps, BY, DE
Screening Targeted
7/23/2010
Method
Mechow, BB, DE
Collection date and location (place, federal state, country)
– –
– – 4.64
0.02
0.03
–
7.34
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
– –
–
– –
–
–
–
0.35
0.37
0.85
0.88
1.14
1.21
0.68
0.74
0.09
0.12
1.24
1.97
0.43
0.99
0.15
0.27
Cyclic diester N-oxide (%)
0.17
0.17
0.72
0.72
0.90
1.58
0.79
0.80
0.10
0.21
0.61
0.95
0.21
0.69
0.27
0.44
Cyclic diester (%)
0.45
0.01
– –
0.02 –
– – –
– –
–
–
0.02
–
–
0.09
0.14
0.18
0.38
0.04
0.08
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.01 –
– –
–
–
–
–
–
0.03
0.01
0.02
0.43
1.11
–
0.33
–
0.25
–
–
–
–
–
–
– –
–
0.20 –
– –
– –
0.18
0.17
– –
– –
–
0.30
–
–
– –
–
–
–
–
–
–
–
–
–
Openchained diester Noxide (%)
Openchained diester (%)
0.17
0.01
0.03
0.01
0.03
Otonecine (%)
0.02
0.06
0.01
0.01
0.40
0.72
0.03
0.03
1.41
1.43
2.89
3.52
–
0.10
2.03
2.21
2.56
2.60
–
0.14
–
0.10
–
0.04
–
0.07
–
–
–
0.01
–
–
–
–
Monoester (%)
–
0.01
–
0.05
–
1.48
0.03
0.03
0.82
1.01
0.01
4.74
–
2.36
0.38
4.57
–
2.05
–
0.26
–
0.12
–
0.20
–
0.04
–
0.13
–
0.18
–
0.14
–
–
Monoester N-oxide (%)
Kindly provided by the botanical garden of Goettingen
“Whole plant” indicates total plant above ground
15
6
14
18
70
79
35
25
11
53
27
47
66
37
46
46
42
Detected PAd
0
0
0
0
0
4
0
0
0
6
2
5
8
3
6
4
6
Detected chlorinated PAd
3
0
2
4
37
44
12
7
4
21
13
21
28
15
11
16
8
Number of PA not listed in literature
d
c
Including isomers
“Screening” illustrates the PA content obtained by summation of all PA detected by MRM screening and “targeted” illustrates the content obtained by analysis of 19 standards. The difference between screening and targeted indicates the underestimation of the real PA content by targeted analysis focused on 19 PA standards
b
a
Complete data on detected sum formulas and respective PA amounts etc. for each investigated plant material is given in the Electronic Supplementary material
Fabaceae
Borago officinalis
15
Eupatorium boniifolium
10
Borago officinalis
Eupatorium purpureum
9
14
Senecio madagascariensis
8
Heliotropium europaeum
Senecio vulgaris
7
13
Senecio vernalis
6
Cynoglossum sp.
Senecio inaequidens
5
12
Senecio jacobaea
4
Echium vulgaris
Senecio jacobaea
3
11
Senecio jacobaea
2
Boraginaceae
Senecio jacobaea
1
Asteraceae
Plant species
Sample no.
Plant family
Table 3 List of investigated plants
9380 A. These et al.
Structural screening by multiple reaction monitoring
9381
purposes peak areas of positively identified PA were manually transferred into an Excel spreadsheet. The amounts of respective PA were calculated by a onepoint calibration assuming the same response for all PA and PA N-oxides belonging to the same structure types (Fig. 1d–i).
350
400
450
500
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 4.7 2.6 0.0 0.0 0.0 0.0 0.0 1.7 4.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 1.7 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 2.7 4.5 2.9 0.0 0.0 0.0 0.0 0.0 0.0 4.6 0.0 3.6 0.0 0.0 0.0 0.0 2.4 0.0 3.3 0.0 0.0 0.0 2.0 2.4 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 4.3 2.5 0.0 0.0 0.0 0.0 0.0 1.4 3.2 0.0 3.7 1.9 1.6 0.0 0.0 0.0 1.7 3.6 1.4 0.0 0.0 2.9 0.0 0.0 0.0 2.2 3.8 3.3 0.0 3.1 0.0 0.0 0.0 0.0 2.6 0.0 0.0 1.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.7 0.0 0.0 0.0 0.0 0.9 1.8 1.1 0.0 0.0 0.0 0.0 1.6 0.0 0.0 0.0 0.0 0.0 0.0 1.9 1.7
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Senecio inaequidens
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.3 5.1 0.0 0.0 0.0 0.0 0.0 0.0 4.3 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.7 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.6 3.5 0.0 0.0 0.0 0.0 0.0 2.4 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.8 2.4 1.4 0.0 0.0 0.0 0.0 0.0 0.9 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.1 4.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1 0.0 0.0 0.0 1.5 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Senecio vernalis
2.3 2.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6 0.0 0.0 0.0 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.3 4.6 0.4 0.0 0.0 0.0 0.0 0.0 0.7 4.2 3.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.5 4.5 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 0.0 0.4 0.0 0.0 0.0 0.0 2.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 2.3 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Senecio vulgaris
Fig. 4 3D map for some investigated samples (Table 3): detected m/z values are displayed on the y-axis and the x-axis is subdivided into columns of cyclic diesters and their N-oxides, otonecines, open-chained
Plant screening
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.8 4.0 0.0 0.0 0.0 0.0 0.0 0.0 4.6 3.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.7 0.0 0.0 2.6 1.9 0.0 2.0 0.0 0.0 2.8 0.0 3.7 0.0 0.0 0.0 0.0 0.0 2.0 0.0 0.0 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.4 4.1 0.0 0.0 0.0 0.0 0.0 0.0 4.6 3.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 1.7 0.0 0.0 0.0 0.0 2.4 0.0 4.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.6 3.0 0.0 0.0 0.0 0.0 0.0 0.0 2.3 3.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Senecio jacobaea
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6 0.0 0.0 0.0 0.0 0.0 0.0 2.5 3.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.2 1.3 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 3.9 1.3 1.0 0.0 0.0 0.0 0.0 0.0 4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.6 1.8 2.8 -0.3 0.0 0.0 2.0 0.0 0.0 2.1 4.0 0.0 0.0 0.0 0.0 1.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.4 0.0 3.5 0.0 0.0 0.0 0.0 2.2 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 1.1 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.7 4.0 1.0 2.0 0.0 0.0 0.0 0.0 1.3 4.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.7 0.0 1.7 0.0 0.0 0.0 0.0 0.0 3.9 0.0 0.0 0.0 0.0 2.0 0.0 0.0 1.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4 0.4
3.0 5.5 0.0 0.0 0.0 0.0 0.0 0.0 4.0 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Cynoglossum sp.
3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.7 5.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.4 0.0 0.0 0.0 0.0 0.0 0.0 2.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.4 0.0 0.0 3.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
Seventeen plant materials, belonging to the families of Asteraceae, Boraginaceae and Fabaceae were screened for PA using the described MRM approach. Details of the investigated samples, including date of sampling, PA content of each ester type, number of detected PA, and detected unknowns, are compiled in Table 3. A detailed compilation of detected sum formulas, retention times, mass error of hRMS detection and respective PA amounts for each investigated plant material is given in the Electronic Supplementary Material.
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
300
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
m/z
cyclic diester cyclic diester NO otonecine op. ch.. diester op. ch.. diester NO monoester monoester NO
between 298 and 500 Da [2, 4, 14] and therefore the Q1 mass range used for screening was selected as m /z 298–500. Applying the nitrogen rule for molecules containing one nitrogen atom, it can be stated that PA form even-numbered [M+H]+ values in the positive ion mode. Consequently, every even Q1 mass in the range from 290 to 500 Da was screened for the eight transitions summarized in Table 1, resulting in more than seven hundred MRM transitions to be acquired. In order to secure sufficient dwell time per MRM transition and in order to obtain sufficient data on a chromatographic peak the mass range was subdivided into several reduced mass ranges resulting in ten sub-methods (Table 2). The conditions for mass spectrometric analysis, such as CE, were adopted from those obtained by MS optimization for commercially available PA standards. Data processing was done by the automatic peak identification program ToxID (Thermo Fisher). This program displays MRM transitions exclusively in case of a signal detected above a certain threshold limit (Fig. 3). For quantification
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.0 2.0 3.6 2.0 0.0 0.0 0.0 0.0 0.0 0.0 3.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 0.0
0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
µg/kg 100,000 75,000 10,000 7,500 1,000 750 100 75 25 2.5
< LOD
Echium vulgare
diesters and their N-oxides, monoesters and their N-oxides. Determined amounts of individual PA are displayed in colour
9382
A. These et al.
Determined PA contents in samples of Asteraceae ranged from 0.5 to 6.8 % of dry mass and in samples of Boraginaceae from 0.1 to 9.0 % (Table 3). A content of 7.5 % was detected in the sample of Fabaceae reflecting the high potential of PA formation within all these investigated plant families. There was no correlation between the PA content of a sample and the number of detected individual PA. For instance, in samples dominated by open-chained diesters like Heliotropium europaeum a PA content of 3.7 % was determined resulting from 70 detected individual PA (Table 3). In the sample of the monoester-dominated Eupatorium purpureum a comparable PA content of 4.7 % was determined, whereas only 11 PA were detected (Table 3). As already described in the literature PAproducing plants differ in their qualitative PA pattern as plants of a certain genus predominantly form either cyclic or openchained diesters. Within the family of Asteraceae two tribes are known to form PA: Eupatorieae and Senecioneae. Both of them were investigated and samples of Eupatorium contained mainly monoesters, whereas samples of Senecio predominantly contained cyclic diesters and minor amounts of monoesters. Open-chained diesters were almost exclusively detected in the family of Boraginaceae; however, all species that contained open-chained diesters also contained monesters but in much higher amounts (Table 3). In general, open-chained diesters were
determined in considerably lower amounts than monesters or cyclic diesters (Fig. 4). Otonecines were only detected in Senecio (and Tussilago farfara, data not shown) and amounts determined ranged between trace amounts in seeds of Senecio jacobaea and very high amounts of 0.45 % of dry mass in Senecio inaequidens (Table 3, Fig. 4). This sample contained 29 different otonecines; to the best of our knowledge, 13 of them are not described in the literature. Four of these otonecines were chlorinated. Chlorinecontaining PA were almost exclusively detected in species of Senecio (except trace amounts in Cynoglossum) as can be seen in Table 3. In this study around 10 % of PA molecules detected in Senecio contained one Cl atom. In order to compare the qualitative and quantitative PA pattern of different samples a presentation of results was chosen that displays the PA content of individual detected PA by means of colours. The y-axis displays the molecular weight and the x-axis is divided into the PA structure type. Figure 4 shows the qualitative and quantitative PA profile of the investigated Senecio samples. The application of the MRM screening revealed the underestimation of PA content by the targeted analysis of the 19 analytical standards (Table 3). In most of the investigated plant materials only around 50 % of the PA amount was determined by the targeted analysis compared to the PA content determined using the screening approach.
Table 4 Compilation of new identified PA. The subdivision into monoesters, cyclic or open-chained diesters and otonecines was made by recording product ion spectra. The free base and the corresponding N-oxide are connected by lines monoesters m/z PA PANO 298 306 308 312 324 328 328 338 340 340 342 344 344 352 356 356 358 360 366 368 370 370 372 372 374 376 380 382 384 386 386 386 388 398 400 402 402 410 414 418 426 436 462 462 492 sum:
a
no. of isomersa 2 1 1 4 1 1 3 1 1 1 1 1 1 2 3 1 1 1 1 4 1 1 1 3 1 1 1 2 2 1 1 1 1 1 1 3 1 1 1 3 1 1 3 1 1 45
sum formula C15H24O5N C17H24O4N C17H26O4N C16H26O5N C17H26O5N C17H30O5N C16H26O6N C18H28O5N C17H26O6N C17H26O6N C17H28O6N C16H26O7N C17H30O6N C18H26O6N C18H30O6N C17H26O7N C17H28O7N C17H30O7N C18H24O7N C18H26O7N C18H28O7N C19H32O6N C18H30O7N C18H30O7N C17H28O8N C17H30O8N C16H27O7NCl C18H24O8N C18H26O8N C18H25O6NCl C18H28O8N C19H32O7N C18H30O8N C20H32O7N C18H26O9N C18H25O7NCl C18H28O9N C20H28O8N C20H32O8N C19H32O9N C20H28O9N C22H30O8N C21H36O10N C24H32O8N C22H38O11N
plant numberb 6, 10, 12 7 7 13, 14 8 9, 13 13, 14 6 1, 2 6 9 9 13 3, 4, 7 5, 8, 10, 17 2 10, 13 15 4 1, 2, 3, 4, 6, 7 2 5, 8 13 5, 8, 10, 12 10, 13 15 12 2, 3, 4 2, 4 2, 6, 7 2, 4 5, 8, 13 11 4 3, 4, 6, 7 12 2 11 6 2, 3, 7 6 12 12 13
open chained diesters m/z no. of PA PANO isomersa 322 2 324 1 328 1 338 2 340 3 356 2 356 4 362 2 372 2 378 2 380 2 380 2 382 1 384 2 386 1 388 1 396 1 396 1 396 3 398 1 1 400 400 1 402 1 412 1 416 1 422 1 424 2 432 2 438 2 444 2 444 2 448 1 448 1 462 2 450 1 454 1 470 1 498 2 498 1 500 1
sum:
40
sum formula C17H24O5N C17H26O5N C16H26O6N C17H24O6N C17H26O6N C18H30O6N C17H26O7N C20H28O5N C18H30O7N C20H28O6N C20H30O6N C20H30O6N C19H28O7N C19H30O7N C18H28O8N C18H30O8N C20H30O7N C21H34O6N C20H30O7N C19H28O8N C20H34O7N C19H30O8N C18H28O9N C21H34O7N C20H34O8N C22H32O7N C22H32O7N C20H34O9N C22H32O8N C20H30O10N C21H34O9N C19H27O9NCl C20H34O10N C21H36O10N C23H32O8N C23H36O8N C23H36O9N C24H36O10N C24H36O10N C24H38O10N
plant numberb 12, 13 12 9 12, 13 11 13 11, 12, 13 13 13 12, 13 12, 13 12 11 12 12 13 11 13 12, 13 11 11 12 12 13, 14 11 12 12 12 12 12 13 8 12 13 12 13 14 8 12, 13 12
cyclic diesters m/z PANO PA 338 372 378 378 380 394 394 396 402 404 412 420 450 452 464 466 468 482 484 484
sum:
no. of isomersa 1 1 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1
20
sum formula C17H24O6N C18H30O7N C19H24O7N C20H28O6N C19H26O7N C19H24O8N C20H28O7N C19H26O8N C18H25O7NCl C18H27O7NCl C19H26O9N C18H27O8NCl C23H32O8N C23H34O8N C24H34O8N C24H36O8N C23H34O9N C24H36O9N C23H34O10N C20H30O11N
plant numberb 17 7 2, 5 6 5, 7, 8, 17 2, 5 5, 6, 5, 6, 7, 8 3 5, 8 4 5, 8 4 8 4 8 8 8 7 7
otonecine m/z no. of isomersa PA 380 1 386 1 398 1 408 1 416 2 416 2 418 1 422 1 428 1 434 1 440 1 470 1 472 1 474 1 498 1 500 1
sum:
retention times and concentrations of all isomers are listed in the supplementary material, b investigated plants (Table 3)
16
sum formula C20H30O6N C19H32O7N C19H28O8N C21H30O7N C19H27O7NCl C19H30O9N C19H29O7NCl C21H28O8N C20H30O9N C19H29O8NCl C21H30O9N C22H32O10N C21H30O11N C22H33O8NCl C23H32O11N C23H34O11N
plant numberb 5 5 1, 3, 5, 8 5, 6 1, 3, 6 5, 8 1, 3, 5, 8 2 3 5, 8 5 5 3, 5 5 5 5
Structural screening by multiple reaction monitoring
Conclusion This approach allows the screening of samples for structural properties and was used as an effective tool for the identification of individual PA in plant material in a very low concentration range. With this method detection of each single PA and determination of the whole PA content is possible without the necessity for analytical standards of each PA. Furthermore, 121 unknown PA were identified in the investigated plant materials (Table 4). This approach could be adapted to other natural toxins which occur in a high individual number but sharing either structural similarities or similar functional groups. To date the most critical part of this approach is the data processing step as available software for triple quadrupoles is restricted to targeted analysis. Software tools allowing automatic peak identification without prescribing retention times and combining signals obtained in different MRM transitions would offer the routine use of a very attractive application field for tandem mass spectrometry. Acknowledgments We thank Christina Radach for her careful laboratory work, especially the skilful preparation and analysis of samples.
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