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Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography—mass spectrometry
Journal of Chromatography, 562 (1991) 555-562 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam CHROMBIO. 5532
Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry RISTO KOSTIAINEN
Food and Environmental Laboratory of Helsinki, Helsing&katu 24, 00530 Helsinki (Finland)
ABSTRACT Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray.are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.
INTRODUCTION
Trichothecenes are significant contaminants in foods and feeds [1-4]. Because of their extreme toxicity [3] and natural occurrence, several identification methods have been developed. Trichothecenes are most often detected as their different types of derivative by gas chromatography (GC) [5,6] or gas chromatography-mass spectrometry (GC-MS) [4,7,8]. The time-consuming derivatization step is not needed in the identification of trichothecenes by liquid chromatography (LC) [9,10], but the specificity and sensitivity of conventional LC detectors are limited. A mass spectrometer provides a nearly ideal detector, but its coupling to LC is more difficult than to GC. However, several interfacing techniques have been developed [11,12]. One of the most commonly used interfaces is thermospray (TSP) [13], which produces with trichothecenes a very abundant ammonium adduct ion with minimal fragmentation [14,15]. TSP LC-MS is suitable for the monitoring of trichothecenes, but the lack of fragmentation makes the structure characterization difficult. The recently introduced interfacing techniques of plasmaspray (PSP) [16,17] and dynamic fast-atom bombardment (FAB) [18,19] provide more energetic ionization processes than TSP and also often more fragmentation. In PSP a differ0378-4347/91/$03.50
©
1991 Elsevier Science Publishers B.V.
556
R. KOSTIAINEN
ential voltage is applied between the thermospray vaporizer and the ion-source block to create a d.c. plasma o f sample and solvent ions [16]. In dynamic FAB an eluent, containing 1-5% glycerol, is b o m b a r d e d continuously by neutral atoms with high kinetic energy [19]. The b o m b a r d m e n t results in sputtering o f molecules and f o r m a t i o n o f positive and negative ions. Both methods have been applied to the analysis o f several c o m p o u n d s [12,16,17]. This study compares the spectra o f six trichothecenes ( D O N , MAS, DAS, TAS, HT-2, and T-2) recorded by TSP, PSP and dynamic F A B L C - M S . EXPERIMENTAL
R#agents All the trichothecenes (Table I) were obtained from Sigma (St. Louis MO, U.S.A.) and dissolved in methanol. TABLE I THE SIX TRICHOTHECENES STUDIED
.......~ " ~ Z ' / ~ : ~ ~ ' " " ' H R4 _- "CH3 t5~H2
R2
R3 Compound
R1
R2
R3
R,,
R5
T-2 Toxin (T-2) HT-2 Toxin (HT-2) Triacetoxyscirpenol(TAS) Diacetoxyscirpenol(DAS) Monoacetoxyscirpenol(MAS) Deoxynivalenol(DON)
OH OH OAc OH OH OH
OAc OH OAc OAc OH H
OAc OAc OAc OAc OAc OH
H H H H H OH
OCOCH2CH(CH3) 2
OCOCHzCH(CHa) 2 H H H --O
Equipment and conditions The three different experimental instrumental set-ups in L C - M S measurements are summarized in Table II. The mass spectrometers were operated in low-resolution (1000 R) mode. In dynamic F A B the matrix (glycerol) in methanol was added by means o f a post-column at a flow-rate o f 250 pl/min. The eluent was split (1:200) before the mass spectrometer by a Jeol pneumatic splitter. Xenon was used in the b o m b a r d m e n t (particle energy 5 keV).
Spherisorb ODS-2, 3 #m, 10cm × 4.6mmI.D. (Phase Separations, Queensferry, U.K.) HzO + 0.1 MNH4CH3COO CH3OH + 0.1 M NH4CH3COO B; 20-70% for 0-20 min 0.8 ml/min 20 #1 0.8 ml/min VG ZAB-E (Manchester, U.K.) VG plasmaspray 250°C 250°C
1 ml/min Finnigan MAT 90 (Bremen, F.R.G.) Finnigan MAT thermospray 220°C 200°C
Interface Ion-source temperature Vaporizer temperature
Mobile phase A Mobile phase B Run programme HPLC flow-rate Injection volume Split ratio Flow-rate to MS Mass spectrometer
Column
LKB-2249 (Bromma, Sweden)
Perkin Elmer, Series 2 (Ueberlingen, F.R.G.) LiChrosorb RP-18, 5/~m, 15 cm x 4.6 mm I.D. (Merck, Darmstadt, F.R.G.) H20 + 0.1 M NHaCH3COO CH3OH + 0.1 M NH4CHaCOO 65% B isocratic 1 ml/min 20 pl
Chromatograph
Dynamic Fab HPLC-.MS
PSP HPLC-MS
TSP HPLC-MS
SYSTEM DESCRIPTIONS
TABLE II
ODS-I8, 3 #m 15cm × 4.6ram (Nomura Kagaku, Tokyo, Japan) HzO CH3OH B; 30-80% for 0-15 min 1 ml/min 20/~1 l/200 5 #l/rain Jeol JMS-SX 102 (Tokyo, Japan) Jeol frit FAB 50°C
HP 1090 LC, (Palo Alto, CA, U.S.A.)
(3 t~ Z
©
©
558
R. K O S T I A I N E N
R E S U L T S A N D DISCUSSION
All the TSP spectra of the trichothecenes exhibit a very abundant ammonium adduct ion without fragmentation (Table III). The spectra are very similar to ammonia desorption chemical ionization (DCI) spectra recorded in an earlier study [20]. This is because the ionization process in ammonium acetate-buffered TSP is analogous to that in ammonia CI. The proton affinities of the trichothecenes [21,22] are similar to the proton affinity of ammonia (859 kJ/mol) [23], leading to low exothermicity of the ion-molecule reactions between trichothecenes and ammonium ions and thus to the formation of an abundant ammonium adduct ion with minimal fragmentation. Ammonium acetate-buffered TSP provides good selectivity in the identification of the trichothecenes, since compounds with a proton affinity of less than 787 kJ/mol cannot be ionized efficiently and they are transparent in the analysis [24]. Good sensitivity (0.1-1 ng//A) is achieved in the single-ion (ammonium adduct ion) monitoring of the pure trichothecenes, but the lack of fragmentation decreases the reliability of the identification and makes structure characterization very difficult.
T A B L E III T H E R M O S P R A Y S P E C T R A O F T H E SIX T R I C H O T H E C E N E S S T U D I E D
Compound T-2 HT-2 TAS DAS MAS DON
[M + H] +
[M + NH4] +
-297 (46)
484 442 426 384 342 314
(100) (100) (100) (100) (100) (100)
T A B L E IV PLASMASPRAY SPECTRA OF THE TRICHOTHECENES STUDIED
Compound
[M + H] +
[M + NH4] +
Other ions
T-2 HT-2 DAS MAS DON
- 425 367 325 297
484 442 384 342 314
365 323 307 307 284
(100) (I) (3) (89)
(100) (42) (100) (100) (100)
(2), 335 (4), 305 (5), 275 (3), 257 (2), 245 (3), 215 (3) (6), 293 (4), 275 (3), 263 (14), 245 (7), 215 (8) (4) (17), 282 (20), 265 (33), 247 (3) (5), 249 (8)
LC-MS OF TRICHOTHECENES
559
TABLE V DYNAMIC FAST-ATOM BOMBARDMENT SPECTRA OF THE SIX TRICHOTHECENES STUDIED Compound
[M+H] +
[M+H+92] ÷
Other ions
T-2
467 (33)
559 (12)
HT-2
425 (67)
517 (19)
TAS
409 (100)
501 (20)
DAS
367 (97)
459 (39)
MAS DON
325 (17) 297 (100)
417 (25) 389 (32)
449 (12), 407 (5), 365 (96), 323 (I0) 305 (100), 275 (18), 263 (15), 257 (18) 245 (59), 233 (21), 215 (65), 203 (22) 197 (19) 407 (21), 365 (15), 323 (69), 305 (12), 263 (100), 245 (25), 233 (26), 215 (42), 203 (28) 391 (17), 367 (13), 349 (37), 307 (15), 289 (23), 247 (26), 229 (35), 201 (13), 199 (14) 349 (34), 307 (100), 289 (9), 265 (10), 247 (30), 229 (28), 201 (13) 199 (12) 307 (46), 265 (100), 247 (10), 229 (8) 215 (68)
11~ m/z:
314
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!
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m/z:342
k
AS
5&
m/z:384 58
1~
m/z|426
_ ~ T - 2
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m/z:442
jf~AS
18~58m/z:484 2~8~
,18~
'
G188
'
8:88
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'
18:80 I
"
,
"
Fig. 1. Mass chromatograms of ammonium adduct ions of the pure trichothecenes (10 ng/#l; 20-#1 injection) recorded by TSP LC-MS (isocratic).
560
R. K O S T I A I N E N
4.
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at
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Fig. 2. M a s s c h r o m a t o g r a m s o f a m m o n i u m a d d u c t ions of the trichothecenes (10 ng/#l; 20-pl injection) recorded by P S P L C - M S (gradient).
The recently introduced plasmaspray (PSP) LC-MS method provides a more energetic ionization process than TSP, owing to a differential voltage applied between the thermospray vaporizer and the ion-source block to create a d.c. plasma of sample and solvent ions (H30 + and CH3OH+). The lower proton
L C - M S OF T R I C H O T H E C E N E S
561
affinities of the solvent ions than that of ammonia result in a more exothermic ionization of the sample [23]. All the PSP spectra of the trichothecenes exhibit an abundant ammonium adduct ion (Table IV). The trichothecenes with two or more hydroxy groups in the molecule (DON, MAS and HT-2) produce more abundant fragment ions than those with one hydroxy group (DAS and T-2). DON, MAS, and HT-2 can reliably be identified by multiple-ion monitoring, whereas DAS and T-2 must be identified by single-ion monitoring, which decreases the reliability of the identification. In dynamic FAB LC-MS the eluent was bombarded continuously by xenon atoms. In the bombardment the trichothecenes produced an abundant protonated molecule, a glycerol adduct ion and several abundant fragment ions formed by loss of functional groups as neutral species in various combinations (Table V). The ionic species were described in a previous report [25]. The greater fragmentation obtained with FAB than with TSP and PSP indicates that the ionization process with FAB is more energetic than with TSP and PSP. The formation of abundant fragment ions by FAB allows reliable identification of the trichothecenes by multiple-ion monitoring and makes the structure characterization easier than with TSP and PSP. The detection limits of the pure trichothecenes (1-10 ng/#l following 20-#1 injection), with dynamic FAB are only one order of magnitude worse than with TSP, although the eluent was split with the Max 3 9 3 . 1 6 4
. . . . . . . . .
~ . . . . . . . . .
UV 210 ~
i~ . . . . . . . . .
R.
iF
r.~.
~ HT-2
A
A
425 a67 B65 263 409
• "A
JA\
~07
265
100
150
200
258
380
350
488 Sean
Fig. 3. Mass chromatograms of protonated molecules and some fragment ions of the trichothecenes (10 ng//~l; 20-/11 injection) recorded by dynamic FAB L C - M S (gradient).
562
R. KOSTIAINEN
ratio of 1:200 in the dynamic FAB. This suggests the possibility that the ionization process with FAB is more efficient than with TSP. Fig. 1-3 show the mass chromatograms of the trichothecenes obtained by TSP, PSP and dynamic FAB LC-MS with multiple-ion monitoring. The mass chromatograms show that the trichothecenes were eluted with adequately symmetric and narrow peaks under the chosen gradient LC-MS conditions (PSP and dynamic FAB). All the trichothecenes can be detected under the chosen isocratic conditions (TSP), although the peaks of T-2 and HT-2 became wider. The wider peaks in dynamic FAB than in PSP are due to the post-column addition of glycerol. ACKNOWLEDGEMENTS
Dr. Ernst Schr6der from Finnigan MAT, Dr. Nick Ordsmith and Dr. Chris Harbach from VG Analytical, and Dr. Matsuura from Jeol Ltd. are acknowledged for the operating of the instruments. Financial support from Magnus Ehrnrooth and Oskar Oflund foundations is acknowledged. REFERENCES 1 Y. Ueno, in K. Miller (Editor) Toxicological Aspects o f Food, Elsevier, Essex, 1987, p. 150. 2 Y. Ueno, in P. Krogh (Editor) Mycotoxins in Food, Academic Press, London, 1988, pp. 123-147 and 217-249. 3 Y. Ueno (Editor), Developments in Food Science 4; Trichothecenes; Chemical, Biological and Toxicological Aspects, Kodansha and Elsevier, Tokyo, 1983, p. 195. 4 R. Kostiainen and S. Nokelainen, J. Chromatogr., 513 (1990) 31. 5 C. E. Kientz and A. Verweij, J. Chromatogr., 355 (1986) 229. 6 R. M. Black, R. J. Clarke and R. W. Read, J. Chromatogr., 388 (1987) 365. 7 R. Kostiainen and A. Rizzo, Anal. Chim. Acta, 204 (1988) 233. 8 C. J. Mirocha, S. V. Pathre, R. J. Pawlosky, and D. W. Hewetson, in R. J. Cole (Editor), Modern Methods in the Analysis and Structural Eludiation of Mycotoxins. Academic Press, Orlando, FI, 1986, p. 353. 9 A. Visconti and A. Bottalico, Chromatographia, 17 (1983) 97. 10 P. Kuronen, Arch. Environ. Contain. Toxicol., 18 (1989) 336. 11 T. R. Covey, E. D. Lee, A. P. Bruins and J. D. Henion, Anal. Chem., 58 (1986) 1451A. 12 R. M. Caprioli, Biochemistry, 27 (1988) 513. 13 C. R. Blakey, M. L. Vestal, Anal. Chem., 55 (1983) 750. 14 R. D. Voyksner, W. M. Hagler Jr. and S. W. Swanson, J. Chromatogr., 394 (1987) 183. 15 P. Sakkers, E. Rajakyl/i and K. Laasasenaho, J. Chromatogr., 384 (1987) 391. 16 K. Rollins and V. Gould, VG Masslab Application Sheet, Contact 18, 1988. 17 G.A. Mills, V. Walker, M. R. Clench and V. C. Parr, Biomed. Environ. Mass Spectrom., 16 (1988) 259. 18 Y. Ito, T. Takeuchi, D. Ishii, M. Goto and T. Mizuno, J. Chromatogr., 346 (1985) 161. 19 Y. Ito, T. Takeuchi, D. Ishii, M. Goto and T. Mizuno, J. Chromatogr., 391 (1987) 296. 20 R. Kostiainen and A. Hesso, Biomed. Environ. Mass Spectrom., 15 (1988) 79. 21 R. Kostiainen, Biomed. Environ. Mass Spectrom., 18 (1988) 116. 22 R. Kostiainen, Thesis, University of Helsinki, Helsinki, 1989. 23 D. H. Aue and M. T. Bowers, in M. Bowers (Editor), Gas Phase Ion Chemistry, Academic Press, New York, vol. 2, 1979, p. 1. 24 T. Kenough and J. Stefano, Org. Mass Spectrom., 16 (1981) 527. 25 R. Kostiainen, K. Matsuura and K. Nojima, J. Chromatogr., in press.
Identification of trichothecenes by thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry RISTO KOSTIAINEN
Food and Environmental Laboratory of Helsinki, Helsing&katu 24, 00530 Helsinki (Finland)
ABSTRACT Thermospray, plasmaspray and dynamic fast-atom bombardment liquid chromatography-mass spectrometry are compared for the identification of six trichothecenes. Thermospray spectra of the trichothecenes exhibit only a very abundant ammonium adduct ion. Plasmaspray, which provides a more energetic ionization process than thermospray, produces some fragment ions in addition to an abundant ammonium adduct ion. The spectra obtained by dynamic fast-atom bombardment exhibit a protonated molecule, a glycerol adduct ion and numerous fragment ions formed by the losses of functional groups as neutrals in various combinations. Thermospray and plasmaspray.are suitable only for monitoring of the trichothecenes, whereas dynamic fast-atom bombardment is suitable for monitoring and for structure characterization.
INTRODUCTION
Trichothecenes are significant contaminants in foods and feeds [1-4]. Because of their extreme toxicity [3] and natural occurrence, several identification methods have been developed. Trichothecenes are most often detected as their different types of derivative by gas chromatography (GC) [5,6] or gas chromatography-mass spectrometry (GC-MS) [4,7,8]. The time-consuming derivatization step is not needed in the identification of trichothecenes by liquid chromatography (LC) [9,10], but the specificity and sensitivity of conventional LC detectors are limited. A mass spectrometer provides a nearly ideal detector, but its coupling to LC is more difficult than to GC. However, several interfacing techniques have been developed [11,12]. One of the most commonly used interfaces is thermospray (TSP) [13], which produces with trichothecenes a very abundant ammonium adduct ion with minimal fragmentation [14,15]. TSP LC-MS is suitable for the monitoring of trichothecenes, but the lack of fragmentation makes the structure characterization difficult. The recently introduced interfacing techniques of plasmaspray (PSP) [16,17] and dynamic fast-atom bombardment (FAB) [18,19] provide more energetic ionization processes than TSP and also often more fragmentation. In PSP a differ0378-4347/91/$03.50
©
1991 Elsevier Science Publishers B.V.
556
R. KOSTIAINEN
ential voltage is applied between the thermospray vaporizer and the ion-source block to create a d.c. plasma o f sample and solvent ions [16]. In dynamic FAB an eluent, containing 1-5% glycerol, is b o m b a r d e d continuously by neutral atoms with high kinetic energy [19]. The b o m b a r d m e n t results in sputtering o f molecules and f o r m a t i o n o f positive and negative ions. Both methods have been applied to the analysis o f several c o m p o u n d s [12,16,17]. This study compares the spectra o f six trichothecenes ( D O N , MAS, DAS, TAS, HT-2, and T-2) recorded by TSP, PSP and dynamic F A B L C - M S . EXPERIMENTAL
R#agents All the trichothecenes (Table I) were obtained from Sigma (St. Louis MO, U.S.A.) and dissolved in methanol. TABLE I THE SIX TRICHOTHECENES STUDIED
.......~ " ~ Z ' / ~ : ~ ~ ' " " ' H R4 _- "CH3 t5~H2
R2
R3 Compound
R1
R2
R3
R,,
R5
T-2 Toxin (T-2) HT-2 Toxin (HT-2) Triacetoxyscirpenol(TAS) Diacetoxyscirpenol(DAS) Monoacetoxyscirpenol(MAS) Deoxynivalenol(DON)
OH OH OAc OH OH OH
OAc OH OAc OAc OH H
OAc OAc OAc OAc OAc OH
H H H H H OH
OCOCH2CH(CH3) 2
OCOCHzCH(CHa) 2 H H H --O
Equipment and conditions The three different experimental instrumental set-ups in L C - M S measurements are summarized in Table II. The mass spectrometers were operated in low-resolution (1000 R) mode. In dynamic F A B the matrix (glycerol) in methanol was added by means o f a post-column at a flow-rate o f 250 pl/min. The eluent was split (1:200) before the mass spectrometer by a Jeol pneumatic splitter. Xenon was used in the b o m b a r d m e n t (particle energy 5 keV).
Spherisorb ODS-2, 3 #m, 10cm × 4.6mmI.D. (Phase Separations, Queensferry, U.K.) HzO + 0.1 MNH4CH3COO CH3OH + 0.1 M NH4CH3COO B; 20-70% for 0-20 min 0.8 ml/min 20 #1 0.8 ml/min VG ZAB-E (Manchester, U.K.) VG plasmaspray 250°C 250°C
1 ml/min Finnigan MAT 90 (Bremen, F.R.G.) Finnigan MAT thermospray 220°C 200°C
Interface Ion-source temperature Vaporizer temperature
Mobile phase A Mobile phase B Run programme HPLC flow-rate Injection volume Split ratio Flow-rate to MS Mass spectrometer
Column
LKB-2249 (Bromma, Sweden)
Perkin Elmer, Series 2 (Ueberlingen, F.R.G.) LiChrosorb RP-18, 5/~m, 15 cm x 4.6 mm I.D. (Merck, Darmstadt, F.R.G.) H20 + 0.1 M NHaCH3COO CH3OH + 0.1 M NH4CHaCOO 65% B isocratic 1 ml/min 20 pl
Chromatograph
Dynamic Fab HPLC-.MS
PSP HPLC-MS
TSP HPLC-MS
SYSTEM DESCRIPTIONS
TABLE II
ODS-I8, 3 #m 15cm × 4.6ram (Nomura Kagaku, Tokyo, Japan) HzO CH3OH B; 30-80% for 0-15 min 1 ml/min 20/~1 l/200 5 #l/rain Jeol JMS-SX 102 (Tokyo, Japan) Jeol frit FAB 50°C
HP 1090 LC, (Palo Alto, CA, U.S.A.)
(3 t~ Z
©
©
558
R. K O S T I A I N E N
R E S U L T S A N D DISCUSSION
All the TSP spectra of the trichothecenes exhibit a very abundant ammonium adduct ion without fragmentation (Table III). The spectra are very similar to ammonia desorption chemical ionization (DCI) spectra recorded in an earlier study [20]. This is because the ionization process in ammonium acetate-buffered TSP is analogous to that in ammonia CI. The proton affinities of the trichothecenes [21,22] are similar to the proton affinity of ammonia (859 kJ/mol) [23], leading to low exothermicity of the ion-molecule reactions between trichothecenes and ammonium ions and thus to the formation of an abundant ammonium adduct ion with minimal fragmentation. Ammonium acetate-buffered TSP provides good selectivity in the identification of the trichothecenes, since compounds with a proton affinity of less than 787 kJ/mol cannot be ionized efficiently and they are transparent in the analysis [24]. Good sensitivity (0.1-1 ng//A) is achieved in the single-ion (ammonium adduct ion) monitoring of the pure trichothecenes, but the lack of fragmentation decreases the reliability of the identification and makes structure characterization very difficult.
T A B L E III T H E R M O S P R A Y S P E C T R A O F T H E SIX T R I C H O T H E C E N E S S T U D I E D
Compound T-2 HT-2 TAS DAS MAS DON
[M + H] +
[M + NH4] +
-297 (46)
484 442 426 384 342 314
(100) (100) (100) (100) (100) (100)
T A B L E IV PLASMASPRAY SPECTRA OF THE TRICHOTHECENES STUDIED
Compound
[M + H] +
[M + NH4] +
Other ions
T-2 HT-2 DAS MAS DON
- 425 367 325 297
484 442 384 342 314
365 323 307 307 284
(100) (I) (3) (89)
(100) (42) (100) (100) (100)
(2), 335 (4), 305 (5), 275 (3), 257 (2), 245 (3), 215 (3) (6), 293 (4), 275 (3), 263 (14), 245 (7), 215 (8) (4) (17), 282 (20), 265 (33), 247 (3) (5), 249 (8)
LC-MS OF TRICHOTHECENES
559
TABLE V DYNAMIC FAST-ATOM BOMBARDMENT SPECTRA OF THE SIX TRICHOTHECENES STUDIED Compound
[M+H] +
[M+H+92] ÷
Other ions
T-2
467 (33)
559 (12)
HT-2
425 (67)
517 (19)
TAS
409 (100)
501 (20)
DAS
367 (97)
459 (39)
MAS DON
325 (17) 297 (100)
417 (25) 389 (32)
449 (12), 407 (5), 365 (96), 323 (I0) 305 (100), 275 (18), 263 (15), 257 (18) 245 (59), 233 (21), 215 (65), 203 (22) 197 (19) 407 (21), 365 (15), 323 (69), 305 (12), 263 (100), 245 (25), 233 (26), 215 (42), 203 (28) 391 (17), 367 (13), 349 (37), 307 (15), 289 (23), 247 (26), 229 (35), 201 (13), 199 (14) 349 (34), 307 (100), 289 (9), 265 (10), 247 (30), 229 (28), 201 (13) 199 (12) 307 (46), 265 (100), 247 (10), 229 (8) 215 (68)
11~ m/z:
314
H
!
o.J
m/z:342
k
AS
5&
m/z:384 58
1~
m/z|426
_ ~ T - 2
188' t5ff
m/z:442
jf~AS
18~58m/z:484 2~8~
,18~
'
G188
'
8:88
"
'
18:80 I
"
,
"
Fig. 1. Mass chromatograms of ammonium adduct ions of the pure trichothecenes (10 ng/#l; 20-#1 injection) recorded by TSP LC-MS (isocratic).
560
R. K O S T I A I N E N
4.
41p
8oI DON
i00. 1 60
Injection
at
7 ~utel
~ ~ ~
40 2O 0
"12;0"0 1433
816
3. 9 1 6
"1"4;0"0' 1672
"£6;00" "1'8~60 1811
2150
20,00 2389
2"210"0" 2628
2'410"0" 2867
'2"6;0"0" 3106
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Fig. 2. M a s s c h r o m a t o g r a m s o f a m m o n i u m a d d u c t ions of the trichothecenes (10 ng/#l; 20-pl injection) recorded by P S P L C - M S (gradient).
The recently introduced plasmaspray (PSP) LC-MS method provides a more energetic ionization process than TSP, owing to a differential voltage applied between the thermospray vaporizer and the ion-source block to create a d.c. plasma of sample and solvent ions (H30 + and CH3OH+). The lower proton
L C - M S OF T R I C H O T H E C E N E S
561
affinities of the solvent ions than that of ammonia result in a more exothermic ionization of the sample [23]. All the PSP spectra of the trichothecenes exhibit an abundant ammonium adduct ion (Table IV). The trichothecenes with two or more hydroxy groups in the molecule (DON, MAS and HT-2) produce more abundant fragment ions than those with one hydroxy group (DAS and T-2). DON, MAS, and HT-2 can reliably be identified by multiple-ion monitoring, whereas DAS and T-2 must be identified by single-ion monitoring, which decreases the reliability of the identification. In dynamic FAB LC-MS the eluent was bombarded continuously by xenon atoms. In the bombardment the trichothecenes produced an abundant protonated molecule, a glycerol adduct ion and several abundant fragment ions formed by loss of functional groups as neutral species in various combinations (Table V). The ionic species were described in a previous report [25]. The greater fragmentation obtained with FAB than with TSP and PSP indicates that the ionization process with FAB is more energetic than with TSP and PSP. The formation of abundant fragment ions by FAB allows reliable identification of the trichothecenes by multiple-ion monitoring and makes the structure characterization easier than with TSP and PSP. The detection limits of the pure trichothecenes (1-10 ng/#l following 20-#1 injection), with dynamic FAB are only one order of magnitude worse than with TSP, although the eluent was split with the Max 3 9 3 . 1 6 4
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562
R. KOSTIAINEN
ratio of 1:200 in the dynamic FAB. This suggests the possibility that the ionization process with FAB is more efficient than with TSP. Fig. 1-3 show the mass chromatograms of the trichothecenes obtained by TSP, PSP and dynamic FAB LC-MS with multiple-ion monitoring. The mass chromatograms show that the trichothecenes were eluted with adequately symmetric and narrow peaks under the chosen gradient LC-MS conditions (PSP and dynamic FAB). All the trichothecenes can be detected under the chosen isocratic conditions (TSP), although the peaks of T-2 and HT-2 became wider. The wider peaks in dynamic FAB than in PSP are due to the post-column addition of glycerol. ACKNOWLEDGEMENTS
Dr. Ernst Schr6der from Finnigan MAT, Dr. Nick Ordsmith and Dr. Chris Harbach from VG Analytical, and Dr. Matsuura from Jeol Ltd. are acknowledged for the operating of the instruments. Financial support from Magnus Ehrnrooth and Oskar Oflund foundations is acknowledged. REFERENCES 1 Y. Ueno, in K. Miller (Editor) Toxicological Aspects o f Food, Elsevier, Essex, 1987, p. 150. 2 Y. Ueno, in P. Krogh (Editor) Mycotoxins in Food, Academic Press, London, 1988, pp. 123-147 and 217-249. 3 Y. Ueno (Editor), Developments in Food Science 4; Trichothecenes; Chemical, Biological and Toxicological Aspects, Kodansha and Elsevier, Tokyo, 1983, p. 195. 4 R. Kostiainen and S. Nokelainen, J. Chromatogr., 513 (1990) 31. 5 C. E. Kientz and A. Verweij, J. Chromatogr., 355 (1986) 229. 6 R. M. Black, R. J. Clarke and R. W. Read, J. Chromatogr., 388 (1987) 365. 7 R. Kostiainen and A. Rizzo, Anal. Chim. Acta, 204 (1988) 233. 8 C. J. Mirocha, S. V. Pathre, R. J. Pawlosky, and D. W. Hewetson, in R. J. Cole (Editor), Modern Methods in the Analysis and Structural Eludiation of Mycotoxins. Academic Press, Orlando, FI, 1986, p. 353. 9 A. Visconti and A. Bottalico, Chromatographia, 17 (1983) 97. 10 P. Kuronen, Arch. Environ. Contain. Toxicol., 18 (1989) 336. 11 T. R. Covey, E. D. Lee, A. P. Bruins and J. D. Henion, Anal. Chem., 58 (1986) 1451A. 12 R. M. Caprioli, Biochemistry, 27 (1988) 513. 13 C. R. Blakey, M. L. Vestal, Anal. Chem., 55 (1983) 750. 14 R. D. Voyksner, W. M. Hagler Jr. and S. W. Swanson, J. Chromatogr., 394 (1987) 183. 15 P. Sakkers, E. Rajakyl/i and K. Laasasenaho, J. Chromatogr., 384 (1987) 391. 16 K. Rollins and V. Gould, VG Masslab Application Sheet, Contact 18, 1988. 17 G.A. Mills, V. Walker, M. R. Clench and V. C. Parr, Biomed. Environ. Mass Spectrom., 16 (1988) 259. 18 Y. Ito, T. Takeuchi, D. Ishii, M. Goto and T. Mizuno, J. Chromatogr., 346 (1985) 161. 19 Y. Ito, T. Takeuchi, D. Ishii, M. Goto and T. Mizuno, J. Chromatogr., 391 (1987) 296. 20 R. Kostiainen and A. Hesso, Biomed. Environ. Mass Spectrom., 15 (1988) 79. 21 R. Kostiainen, Biomed. Environ. Mass Spectrom., 18 (1988) 116. 22 R. Kostiainen, Thesis, University of Helsinki, Helsinki, 1989. 23 D. H. Aue and M. T. Bowers, in M. Bowers (Editor), Gas Phase Ion Chemistry, Academic Press, New York, vol. 2, 1979, p. 1. 24 T. Kenough and J. Stefano, Org. Mass Spectrom., 16 (1981) 527. 25 R. Kostiainen, K. Matsuura and K. Nojima, J. Chromatogr., in press.