oo40-4020/89
Tetrahedron Vol. 45, No. 8, PP. 2251 lo 2262, 1989 Printed in Great Britain.
Analysis
of Trichothecene
Mycotoxins
by Mass
Spectrometry
and Tandem
Mass
Spectrometry Ronald
D. Plattner,
Marian
Northern
N. Beremand
Regional
and Richard
Research
G. Powell
Center
USDA-ARS 1815 N. University Peoria,
St.
IL 61604
(Received in South Africa 16 June 1988) abstract To illustrate tandem mass spectrometry (MS/MS) as an alternative to GC/MS for the analysis of trichothecenes in (DAS) levels were solid and liquid cultures, diacetoxyscerpenol GC/MS had the measured by GC/MS and by three MS/MS protocols. lowest detection limit and better precision than the MS/MS procedures; however, GC/MS required 20-30 minutes per MS/MS was more rapid, requiring less than two analysis. DAS could be measured accurately at low minutes per analysis. nanogram levels directly from filtrates of liquid cultures. Extraction of culture filtrates simplified the identification by effectively removing an interfering matrix component which Use had an ion at the same m/z as the protonated DAS molecule. of the GC as an inlet for MS/MS was an effective way to minimize physical manipulations necessary for sample All three MS/MS procedures had similar detection introduction. Occurrence levels of three limits and precisions. and DAS were measured trichothecenes, T-2 toxin, neosolaniol, in a single analysis by sequential selection of parents for each trichothecene. INTRODUCTION mass
Historically, of trichothecene technical
high
instrumentation. improvement cost
spectrometry
mycotoxins.
difficulties
the relatively
of choice mycotoxins.
to identify
they
often
Analysis mycotoxins when
of trichothecenes
produce
called
"chemical
interfering
is the limiting
of the
noise".
a rapid reductions
spectrometer.
the high target
factor
Today
mycotoxins
in the
MS is often
and quantitation selectivity
An alternative
is complicated
of
and sensitivity
at the levels
in
signals
MS is generally the target
mycotoxin,
prior
to ionization
method
of reducing
with
chemical
these
in the
signals
are
not sensitivity,
mycotoxin
by MS.
gas chromatography
or a derivative,
in the mass
2251
noise,
a particular
coupled
matrix.
constituents
spectrum:
chemical
of
sample
extraneous
in the mass cases,
by the fact that these
in a complex
spectrometer,
in the analysis
to separate
sample
MS
components
In most
in practice,
(GC) in order rest
by
only trace
into the mass
sample
Therefore,
MS offers
and to
the necessary
has witnessed
for the identification
and quantitate
to trichothecenes
and significant
a mass
for analysis
to various
occur.
are often
introduced
decade
used
both
and maintaining
capabilities
and supporting
trichothecene required
the past
been
in part,
the technique
of obtaining
However,
the technique
which
(MS) has rarely
This was due,
in applying
cost
in instrument
of obtaining
s3.w+.oo
Pergamon Press plc
from the
spectrometer.
noise,
when
a mycotoxin
is
2252
R. D. PLATTNERet al. not amenable to GC, is tandem mass spectrometry (MS/MS). MS/MS has rapidly gained acceptance in the analytical community over the past few years, and instruments with MS/MS capabilities are now available in many laboratories. The MS/MS technique had its roots in structure elucidation problems and in studies of ion structure using metastable decompositions. However, the rapid rise in popularity of MS/MS has been primarily due to recognition that the technique is particularly well suited for providing sensitive and selective analyses of complex mixtures. There are several excellent reviews describing MS/MS and its potential for the determination of trace components in complex mixtures (l-5). In a typical MS/MS procedure, a crude mycotoxin-containing sample is introduced into the mass spectrometer source and ionized from a solids probe. Mass spectra of all components present in the crude sample are thus generated simultaneously. The mycotoxin of interest is separated from most other components in the sample matrix by selection of a structurally significant ion of the target compound with the first mass filter. This selected ion is then induced to decompose by collision with a neutral target gas (usually argon) in a collision cell. Daughter ions produced by these conditions may then be mass analyzed with the second mass filter, and the resulting daughter spectrum can be used to identify or to quantitate the target mycotoxin. The signal recorded for a MS/MS daughter ion is inherently of lower intensity than would be recorded for the parent ion in conventional MS, however, interference from chemical noise in the MS/MS scan is generally greatly reduced. Because the detection limit for MS analysis with the solids probe is often limited by chemical noise, overall sensitivity, defined as signal-to-noise ratio for the analysis, is frequently improved using MS/MS. This is particularly true in cases where the mycotoxin in question is only a trace component in the sample analyzed. Detection of the mycotoxins zearalenone and deoxynivalenol at sub part-per-million levels in Fusarium-infected grains using probe inlet MS/MS has been reported (6). The only sample clean-up involved in the analysis was a solvent extraction of the grain sample. Similarly, aflatoxins have been detected in simple grain extracts at the low part-per-billion level (7). Three basic elements are common to all quantitative MS analyses; these are sensitivity, selectivity and time required for completion of the analysis. Tandem MS experiments offer the analyst a great deal of freedom in selecting from among many possible ways of performing a targeted analysis and allows the analyst to optimize any specific analysis with respect to these three elements. In developing an MS/MS targeted analysis the operator makes selective trade-offs between these three elements to obtain an acceptable blend of speed, sensitivity and selectivity appropriate to the problem at hand. In this paper, selected mycotoxin analysis problems from the authors' laboratory are used to illustrate available choices and how the variables stated above can affect each analysis.
2253
Analysisoftrichothecenemycotoxins
RESULTS
AND DISCUSSION
Studies several levels
of trichothecene
produced
approach
uses
sambucinum) well
W-induced
mutant
biosynthesize
numbers
fungal
liquid
cultures,
altered
of Fusarium
in its ability
conventional First,
culture
a rapid
were
mycelia.
medium
analyses grain
analysis. ethers
will
examined
MB1716.
instead,
in a
MB1716
is
accumulates
were
determined
MS protocols MS/MS
were
acetate,
by
examined.
analysis
work-up.
to one microliter MS/MS.
were
of one
Secondly,
the
and aliquots
of the
of the initial
Thirdly,
samples
cultures
of Fusarium
occurring
Conversion
culture
were measured
produced
simple
simplify
contaminated
mixture
by
almost
Thus,
cultures,
for each
specific
in the
from the fungal
requirements it is likely
which
cultures.
and secondary
and naturally
requirements,
grain
exclusively
analytical
it
higher
Furthermore,
in liquid
of primary
by filtration
cultures.
species
at much
the fungus.
array
are found
solid
will
for that
contaminated lead the
analysis.
TRICHOTHECENES
trichothecenes
GC results,
samples
of free hydroxyl
results
with
concentrated
different
choices
often
in naturally
infected
a complex
greatly
cultures, have
for best
generally
ethyl
and liquid
of liquid
OF DERIVATIZED
naturally
and,
with
encountered
produce
factors
to different
Most
were
3299 that has been
levels
sample
may be a relatively
analysis
of liquid
GC AND GC/MS
groups
solid
and are easily
These
samples
analyst
with
but trichothecenes
trichothecene
and,
by a direct
that trichothecenes
may still
media
tandem
without
are only partially
metabolites, culture
measured
extracted
are usually
the culture The fungus
in
methods
procedure.
In our studies
that
T-2 toxin
Three
equivalent
than
protocols
Trichothecene
by direct
levels
a
we levels
analytical
NRRL
filtrates
were
GC/MS/MS
samples
in which
snorotrichioides
extracts
be noted
created
in large
trichothecene
appropriate
as
of
to have
present
by strain
analyzed
should
levels
was conducted
analytical
(9).
were
of culture
acetate
ability
(DAS) production
GC and by GC/MS.
filtrates
filtrate
production,
the generation
approaches
for measuring
to select
to produce
(DAS)
trichothecenes
microliter
(Fusarium
has been in their
One
species.
studies.
diacetoxyscerpenol
ethyl
in order
MS- and MS/MS-based
strain
study
trichothecene
of trichothecene
trichothecene
methods
of diacetoxyscerpenol
a mutant
Pusarium
Both of these
A preliminary
encompass
to measure
uicaris
approach
are altered
(9). about
MS-based
for use in these
Several
that
information
several
of several
for Gibberella
A second
strains
of samples.
evaluated
in our laboratory methods
the inheritance
(8).
trichothecenes
need to obtain
rapid
cultures
system
to investigate traits
biosynthesis
require
in liquid a genetic
as other
study
which
approaches,
in compounds
have
should
groups
one or more
free hydroxyl
be derivatized
to trimethylsilyl
of suitable
stability
and
prior (TMS)
to
2254
R. D. PLATTNER et al.
chromatographic
derivatives a modern
with
are easily
Since
other
of the target
with
positive
the outlet
produces
the one-dimensional
Thus
GC/MS
superior
for MS, produces la shows
fairly
the EI mass
ion is observed
useful
higher
290; these
complicated
spectrum
mass
ions,
to a mass a mass
spectra
spectrum,
to deal with
are m/z combined,
378
GC detectors. ionization
of DAS.
(M-60),
The three
m/z 350
only about
Figure
spectra
1.
Electron
ionization
of the TMS derivative
(a) and chemical of DAS.
(M-60,-28),
5% of the total
ionization
are
most
intensity.
Figure
from mode
NO molecular
of ions produced
fragments.
carry
each
rather
interference
for trichothecenes.
proportion
lower mass
interfere
spectrometer,
of conventional
of the TM.9 derivative
useful
fragments
three
can severely
(EI), the traditional
The greater
at m/z 438.
the less diagnostically
m/z
response
impact
of the
is a universal
from the GC near the elution
response,
to GC in its ability
Electron
background.
onto
column.
trichothecenes.
of the GC column
intensity
injected
capillary
by the complexity
components
of the various
when
silica
detector
may elute
a multidimensional
than
is
are affected
and these
levels
a fused
ionization
components
compound
identification
By coupling component
however,
with
may be analyzed
Trichothecene
detector.
at low nanogram
equipped
the flame
matrix
time
of trichothecenes
ionization
detected
of detection,
matrix.
detector,
sample
a flame
gas chromatograph
The limits sample
TMS ethers
properties.
by GC and detected
(b) mass
and ion
Analysisoftrichothecenemycotoxins
2255
For identification of unknown components, complete mass spectra are usually recorded. However, several approaches are possible for quantitative analyses. Full scans may be acquired and the total mass spectrum at the appropriate GC retention time can be compared with a standard spectrum for positive identifications. This approach provides the greatest certainty for positive identifications. Because the mass spectrometer is continually scanned across the entire mass range for this method, only a small portion of the total analysis time is actually spent measuring the most important mass-to-charge ratios (m/z 290, 350, and 378 for DAS); therefore, this method is relativly low in sensitivity. Many mass spectrometers can also operate in the selected ion mode (SIM). In the SIM mode, the mass spectrometer only measures signals at selected m/z values. The SIM technique improves sensitivity of ion abundance measurements (typically by a factor of 50 to 100) but the procedure also has its negative aspects. In SIM, instead of a complete mass spectrum for identity confirmation of the mycotoxin, only relative intensities of the selected ibns are available. It is debatable how many correct ion ratios are necessary to positively identify a component by GC/MS, but three ions are generally regarded as a sufficient number (10). Usable full-scan EI spectra of TMS derivatives of deoxynivalenol, DAS, neosolaniol and T-2 toxin require l-20 ng of the compound. In the SIM mode, DAS can be positively identified and quantitated in the 50-100 pg range. Liquid cultures of Fusarium sworotrichioides mutant MB1716 accumulate DAS in the medium. GC/MS analysis of an aliguot of derivatized ethyl
acetate
extract
equivalent
to 1 microliter
of culture
filtrate
gave
down to a few ng/ul. DAS was quantitated by using the response of three ions, m/z 378, 350, and 290 and a calibration curve prepared from injections of known amounts of pure DAS-TMS derivative. The use of SIM to improve sensitivity was not required. Typical coefficients of variation obtained with this procedure were on the order of 7-15% and DAS levels down to a few ng/pl could be routinely quantitated. Interference from the matrix increased considerably with extracts of solid cultures and was dependent on substrate. This increase in chemical noise resulted in poorer full-scan performance. Injection of an aliquot equivalent to 10 mg of solid culture using SIM (m/z 378, 350, and 290) provided routine quantitation of DAS down to the 10 rig/glevel. reasonable
full-scan
spectra
for DAS at levels
Chemical ionization (CI) is an alternate form of ionization available on many mass spectrometers. CI is a softer ionization mode that is accomplished by collision of sample molecules with a reagent gas ion; this procedure protonates many organic molecules. For many compounds, little or no fragmentation is observed. However, trichothecenes do produce varying amounts of fragmention in CI depending on their structures. Most fragments arise from loss of substituents as neutral molecules; but a substantial portion of the total ion current is normally carried by the protonated
molecule
the TMS derivative
(MH+). of DAS.
Figure
lb shows
The MH' at m/z
an isobutane
CI spectrum
of
439 and the fragment ion at
R. D. PLATTNER el al.
2256 m/z
379 account
ionization
mode,
for most
of the
chemical
noise
fragments
and,
responses
for CI and EI are approximately
portion mass
therefore,
of the total
fragments,
Overall
with
CI.
limits
equivalent With
solid
to or better culture
that
SIM for measurement
OF CULTURE
Tandem conduct
sample
MS has the potential
but
analysis
Figure
2.
mode
since
ion a greater
diagnostic
are somewhat EI, can easily
CI identification
can be accomplished
samples
analyses
times
Chemical spectrum
to greatly
High
higher better. be and
at levels
may rapidly
mass
of of
be inserted
of a minute
spectrum
and total
or so.
of DAS
(a) and its MS/MS (b).
387
217 749
325
per
into the mass
probe,
molecule
‘I/./
to
separation
the solids
367, the protonated
16w.8-
required
for analysis
from 20-30 minutes
are made using
ionization
times
method resolution
GC requires
are on the order
of m/z
reduce
a sensitive
is time.
by capillary
when
sample
but,
in the EI mode.
provides
its cost
In contrast,
spectrometer,
daughter
GC/MS
components
analysis.
than
samples,
fewer
Total
FILTRATES
analyses.
trichothecenes,
equivalent
in CI are the more
of DAS in the full-scan
MS/MS
problem.
for trichotheoenes
quantitation require
CI is a softer
also produces
a less severe
ions generated
performance,
achieved
is often
detection
Since
ion current. from the matrix
i
-
54144.
-
218%
2257
Analysisoftrichothecene mycotoxins
Trichothecenes MS/MS
produce
of MB1716 Figure
by direct
2a shows
daughter
acid
This
ion.
fragmentation
of water
spectrometer
and then
The
ionized.
collided
(1 mtorr
with
argon
analyzed
produces Figure
by 43.
a profile 3b shows
culture
filtrate
the selected same time
Figure
daughter
MS/MS
3.
of m/z
as a pure
profile
from
188.8 1
A full daughter
scan
with
while
full daughter
different.
filtrate
that
fractional measuring standards.
8) total
intensity
are
each second, is heated. of
The profile
3a elutes
at about
of
the
of DAS.
of m/z
367 from
of strain
daughter
at this
spectrum
1 microliter
MB1716.
A)
ion response.
spectrum
than
eluting
(figure
daughter
material
probe.
307 daughter
(figure
(figure
fragment
in this
component
367 in isobutane
DAS and was
4) is virtually
standard
of an unknown
from the solids of the m/z
time
from the pure
scan of later
less volatile
only
2304.
also has an ion at m/z
distillation the
daughters
of MB1716.
of daughters
obtained
a daughter
is the daughter
is fortuitously
is set to pass
from a 1 ul aliquot
in figure
sample
is placed
ions are then
of 367 as the probe obtained
M/z. 307 is not an intense
The latter
filter
1
identical the
filtrate
and the resulting
four day old culture
307 daughter.
from
into the mass
mass-selected
daughters
(m/z 307) shown
elution
filtrate
molecules
to the
43 from m/z 90 to 400, once
ion profile
and acetic
from consecutive
of culture
from a four day old culture
(temperature)
of culture response
of eluting
the total
MS
Intense
of DAS itself.
first mass
and -2OV),
Scanning
349),
similar
inserted
These
filtrate.
acid as neutral
is quite
to dryness,
ion of DAS.
mass
and acetic
a 1 ul aliquot
m/r. 367, the parent
(at m/z
in
cultures
2b is the tandem
(m/z 367).
are observed
in the CI spectrum
evaporated
of culture
DAS molecule
pattern
can be used in liquid
of DAS and figure
fragments
daughter
MS analysis,
vial,
of an aliquot
spectrum
from the loss of water
observed
For tandem in a probe
analysis
Additional
307).
of combinations
the parent
Mli+ ions in CI that
DAS can be determined
of the protonated
are observed
(at m/z
losses
MS/MS
the CI mass
spectrum
daughters
abundant
For example,
experiments.
CI.
separated
Zb),
5) is quite spectrum.
of the culture This
component
from DAS by
DAS may be guantitated signal
and comparing
by it to
R. D. PLATTNERet al.
2258
Figure
MS/MS daughter
4.
1W.B
spectrum
of scan 54 of analysis
shown
in fig. 3.
10912.
1
1 Figure
MS/MS
5.
daughter
spectrum
se.0 187 4
OF CULTURE
Ethyl of the
acetate
later
extracted
that
FILTRATE
into ethyl
obtained
EXTRACTS
of culture
component
because
acetate.
This
Response by probe
the parent
ions generated
MS/MS
required
about
experiments
obtaining specific
(SRM), because
of the parent daughter
50-100
by measuring
full daughter
monitoring
selected
scans.
is analogous both
a parent
(MB+, m/z
ng of DAS.
and a daughter
spectra
with
daughters
were
sensitive
to detect
good
obtained
at m/z
signal-to-noise
DAS in liquid
incubation
(i.e. when
toxin
ul aliquot
of culture
filtrate
cultures
accumulation extract.
daughter
can be improved ions rather
called
selected MS:
reaction
ion are defined.
This method
With
SRM
and 229;
ratios was
after
had just begun)
in
than
it is more
349,307,289,247,
of MB1716
than
is not
only
Full-scan
and the proper
at the 10 ng level.
lower
367) represented
daughter
This procedure,
most
simplifies
was
in MS/MS
for DAS.
to SIM in conventional
(m/z 367) and daughters
step
procedure
Sensitivity
selected
removes
is not efficiently
ion transmission
in the source only
effectively
purification
this MS/MS
MS, both because
100% and because 10% of the
filtrates
this contaminant
simple
for DAS using
about
spectra
in fig. 3
I
extraction
eluting
the analysis.
shown
20s
IWE-
MS/MS
of scan 70 of analysis
of all
sufficiently
24 hours
using
only a l-2
Analysis oftrichothecene mycotoxins
2259
GC/MS/MS OF DERIVATIZED CULTURE FILTRATE EXTRACTS MS/MS analysis of culture filtrate extracts using the solids probe is simple and rapid enough to be useful for analysis of large numbers of samples but analysis of samples using the solids probe is fairly tedious. The operator must place each sample vial on the probe, mount it, evacuate the inlet, open a valve, slide the probe into the source, heat the sample, cool the probe, slide it out and close the valve for each sample (one analysis requires about two minutes). A sample inlet system that requires fewer physical manipulations would greatly simplify this procedure. One alternative is use of the GC as the sample inlet system for MS/MS. Since MS/MS is being used to detect DAS, complete separation on the column is not an important factor for identification and, therefore, short columns and higher temperatures can be used to introduce samples into the mass spectrometer source rapidly. DAS could be introduced via the GC into the mass spectrometer for MS/MS without derivatization, however, reproducability problems and losses were encountered in preliminary experiments with underivatized T-Z toxin and neosolaneol. Therefore, TMS derivatives were chosen for MS/MS experiments using the GC inlet in order to avoid problems. Figure 6 shows the CI spectrum of DAS (6a) and the daughter spectrum of the MH+ (6b). Figure 6.
MS/MS daughter spectrum of DAS.
the TMS derivative
1ea.B
1
of m/z 439, the protonated
molecule
of
R. D. PLATTNER et al.
2260 As
in
arises closely MS/l@
the daughter
from a neutral parallels
the parent, sample.
of underivatized
the CI spectrum.
experiments
Acceptable
spectrum
loss of acetic
was slightly
m/z 439, carried
acid
Response
better
than
a higher
full scan daughter
were
daughter
typical
ratios
signals
(m/2439)
for three ratio
daughter;
this
lowered
but at the expense
Figure
7.
injections
MS/MS
to measure
the ,detection
on 20-50
379,319,289,
ionization. ng of
and 229 gave the Figure
of the protonated
was greater
than
The signal
30/l.
SRM
only the m/z 439 parent limit
about
an order
7 shows
DAS molecule
of 25 ng and 250 ng.
for the 25 ng injection
may be modified
of the total
obtained
in the
DA.9 because
of a few nanograms.
379 daughter
replicate
for the TMS derivative
at m/z
injections
for the m/z
to noise conditions
from
daughter
spectrum
for underivatized
percentage
spectra
SRM of m/z 439 and daughters
proper
DAS, the major
and the daughter
to m/z
379
of magnitude,
of selectivity.
response
for six injections
for the m/z 379 daughter
of DAS
of the parent
m/z 439
(3x 25ng and 3x 250nq).
lee.
I I_
373
v
6i0
Data presented in cultures analyze
above
was
of the mutant
several
different
for the detection
MB1716.
NRRL
3299,
derived,
high
levels
and neosolaniol. trichothecenes protonated neosolaniol fragment
than
scan,
of these
detection
MB1716.
MS/MS
three
limits
provided
trichothecenes
and precisions
was
levels
for these
selecting
In chemical
of DAS
three
each
ionization
of the MS,
and as the TMS derivatives)
Therefore,
the protonated
MB1716
as lower
by alternatively
(underivatized
DAS.
as well
to
Fusarium
from which
3299 may be analyzed
for MS/MS.
DAS,
may also be used
simultaneously. strain
the yield
and thus the response
None-the-less,
analysis
analysis
of one trichothecene,
MS/MS
of T-2 toxin,
as parents
10 fold less when
scan. with
more
the parent
of NRRL
in a single
and T-2 toxin
much
for the MS/MS about
Cultures
molecules
However,
trichothecenes
SDgrotrichioides, produces
m0e
6:40
for these
molecule a useable
method
are
for the MS/MS
for simultaneous
cultures
to those
both
molecules
trichothecenes,
is selected
in liquid similar
of protonated
of NRRL
obtained
3299
for DAS in
2261
Analysisoftrichothecenemycotoxins
STRENGTHS
AND
MS/MS
LIMITATIONS
can be a more
limitations
must
Daughter
for the target
mycotoxin.
fragment
is not a common
structural
relatively
large
amount
establish
responds
the same way
are most
selected
when
of other
are quantitated
of ionization
used
is introduced
that the parent
or internal
of this
selecting
an ion
scan to insure and that
trichothecenes
by MS/MS
ideal.
for the target matrix
must be added
it
or
it must be
are far from
sample
standards
spectra
be specific
into the ion source
ion selected
in the presence
must
trichothecene
related
in
full daughter
to the daughter
daughter
other
to be valid
when
for quantitation
to the target
compounds
of matrix
standard,
be shown
be exercised
that
from components
reliable
for a MS/MS
fragment
the conditions
one must
the reference
must
is unique
When
components. that
procedure
molecule
that the parent
remembered
may arise
Care must
than the protonated
matrix
filter
several
of the possibility
they are identical
ion(s)
however,
because
mass
and when
are recorded,
to GC/MS,
First,
Identifications
analyzed.
of standards.
other
alternative
a MS/MS
compound,
CC/MS
VS.
in mind.
in the first
than the target the matrix
rapid
be kept
the ion selected
spectra
OF MS/MS
A
and
component
as it did in for reliable
quantitation.
MS/MS culture
experiments
spectrometer
and the time
Analysis
of aliquots
analysis
of culture
provide
reliable
results
agreed
(typically
analysis
with
internal
quantitative
data,
methods
using
is not necessary.
were
precision. screening
to external
direct found to
standards.
MS/MS of
was slightly
GC/MS.
Under
poorer
these
MS/MS
for accurate
of an internal
The external
was
Coefficient
standard8 with
not necessary
type method
by either
extracts,
of analysis.
external
but the addition
for a rapid
acetate
than was observed
standards
improve
in the mass
full GC separation
data compared
analyses
for DAS in liquid
to l-5 ~1 of cultures,
other
20-30%)
method
from the matrix
or of ethyl
quantitative
conditions,
suitable
consuming
filtrates
well
about
significantly
a rapid
equivalent
for MS/MS
variation
provide
DAS can be separated
samples.
standard
standard
requiring
could
procedure
only
is
l-2 minutes
per
analysis.
Mutants
were
inhibition mutant
selected
enzyme-linked
cultures
from NRF&
in which
there
T-2 toxin
biosynthesis
(9).
production
by growing
small
CIEIA.
The MS/MS
trichothecenes, tandem have
procedure
other
MS procedure
altered
3299,
immunoassay were
than T-2,
biosynthesis
to detect
for altered
are not detected for which
and using
of measuring
by CIEIA.
screen
of
T-Z toxin
plates
as it is capable
alternative
of trichothecenes
a competitive
in the level
in microtiter
flexible
that
is a potential
changes
were screened
cultures
is more
because
was available
significant
Cultures liquid
in part,
(CIEIA)
Our rapid
for mutants
which
no antibodies
are
available. Isomeric present identical
compounds,
problems
such as monoacetoxyscerpenol
for MS/MS.
molecular
weights
The three
possible
MAS
(MAS) isomers isomers
and all show the same daughter
can
all have fragments
on
2262
R. D. PLATTNER et al.
CID of the protonated intensities separated, these
prior
compounds
Design for sample measure these
when'the
compounds are made
alternative
spectra
Therefore,
in order
to ionization,
introduction,
choices
Their
ions.
occur
of a quantitative
target
flexible
molecule.
of the daughter
MS/MS
analysis
carefully
to GC/MS
in relative must be
measure
levels
of
choices
of conditions
sample.
involves
and mass
at predetermined
only
isomers
to reliably
in the same
ionization,
differ these
analysis
levels
and correctly,
for the analysis
to detect
in a specific MS/MS
and
matrix.
can be a rapid
If
and
of trichothecenes.
EXPERIMENTAL
MS and MS/MS procedures. All MS and MS/MS experiments were performed on a Finnigan TSQ-46 triple quadrupole mass spectrometer. In chemical ionization, isobutane was the reagent gas at a pressure of 0.25 torr. Source temperature was maintained at 1OOC and electron energy was 70 eV. Argon was the target gas in the Q collision cell for CID experiments. The pressure in the collision ccl 3 was approximately 1 mtorr as measured by the Bastings gauge. The collision energy was set at either -12 or -20v. Samples were introduced either via the direct insertion probe, which was heated by the ballistic ramp heater or via the gas chromatograph. For GC/MS/MS, either a 3 foot x 2mm packed column coupled to the mass spectrometer via a glass jet separator, or a six foot DB-1 fused silica capillary directly coupled to the source, was used to inlet the samples. Samples were injected, splitless, onto the column at 250 C. Cultures, media and culture conditions. Cultures were maintained on V-8 juice agar (11) slants at 4C and as conidial suspensions in 10 to 15% LiqFid cultures were inoculated at a starting density glycerol at -70C. of 0.5 x lo5 to 1 x 10 conidia per ml of media [YEPD-5G (9)] with conidia washed from strains grown on V8 agar plates for 7 to 10 days. Liquid cultures were grown in Erlenmeyer flasks in a volume of medium equal to one-half the volume of the flasks for 6 to 7 days at 28 C on a At selected intervals, aliquots rotary shaker operated at 180 to 200 rpm. of the cultures were removed and filtered. Filtrates were stored at -20C or extracted with ethyl acetate (equal volumes, 2 times, until analyzed, combined) and stored until analyzed. For GC analyses, portions of ethyl acetate extracts were cleaned up using a charcoal column procedure (8). The clean-up procedure was omitted for GC/MS analyses. For preparation of TMS-derivatives a fraction of the sample equivalent to a l-ml portion of the original sample was evaporated to dryness at 80C under nitrogen, reacted with 100 ul of trimethyl silylating reagent (Tri-Sil/TBT; Pierce Chemical Co., Rockford, 111) for 1 hour at 8OC, and brought to 1 ml by addition of 900 pl of hexane. References John Wiley & Sons: McLafferty, F. W., Ed. "Tandem Mass Spectrometry"; New York, 1983. D. D., Mass Swectrom. Rev,_, 1983, 2, 2. Yost, R. A. and Fetterolf, l-45. G. L., Chem. Ena. News, 1981 (Nov. 30). 3. Cooks, R. G. and Glish, 40-52. F. W., Scinece, 1981, 214, 280-287. 4. McLafferty, 5. Johnson, J. V. and Yost, R. A., Anal. Chem, 1985, 57, 758-768a. Plattner, R. D. and Bennett, G. A., J. Assoc. Off. Anal. Chem., 6. 1983, 66, 1470-1477. R. D., J. Assoc. 7. Plattner, R. D., Bennett, G. A., and Stubblefield, Off. Anal. Chem., 1984, 67, 734-0. 1987, 77, Desjardins, A. E., and Beremand, M. N., Phvtowatholoay, 8. 678-683. Awolied and Environ. Microbial. 1987, 53, 1855-9. Beremand, M. N., 9. Mass Spectrometry, Aeyden, London, 10. Millard, 8. J., QuantitatG 1977. 1974, p. 691-703., University of 11. Stevens, R. B., Mycology Guidebook, Washington Press, Seattle, Washington. 1.