Sivagnanam et al.: Journal of aoaC international vol. 99, no.
4, 2016 895
SPECIAL GUEST EDITOR SECTION
Rapid Screening of Ergot Alkaloids in Sclerotia by
MALDI-TOF Mass Spectrometry
Kumaran Sivagnanam
Canadian Grain Commission, Grain Research Laboratory, 303 Main St, Winnipeg R3C 3G8, Manitoba, Canada
Emy KomatSu
University of Manitoba, Chemistry Department, 144 Dysart Rd, Winnipeg R3T 2N2, Manitoba, Canada
SuSan PatricK
Canadian Grain Commission, Grain Research Laboratory, 303 Main St, Winnipeg R3C 3G8, Manitoba, Canada
chriStoPh ramPitSch
Morden Research & Development Center, 101 Route 100, Morden R6M 1Y5, Manitoba, Canada
hélènE PErrEault
University of Manitoba, Chemistry Department, 144 Dysart Rd, Winnipeg R3T 2N2, Manitoba, Canada
tom gräfEnhan1
Canadian Grain Commission, Grain Research Laboratory, 303 Main St, Winnipeg R3C 3G8, Manitoba, Canada
Ergot is a common disease of wheat and other
cereal grains that is predominantly caused by
Claviceps purpurea in the field, often affecting
crop yield in addition to the environment. Infected
grain can be contaminated with dark sclerotia,
which contain fungal metabolites such as ergot
alkaloids. The occurrence of ergot alkaloids in cereal
grain is a major health concern for humans and
livestock. Effective and rapid screening of these
mycotoxins is crucial for producers, processors,
and consumers of cereal-based food and feed grain.
Established methods of ergot alkaloid screening
based on LC-MS or GC-MS require laborious
processes. A novel method using matrix-assisted
laser desorption ionization (MALDI)–time-of-flight
(TOF) MS was developed to identify four ergot
alkaloids. Using dihydroxybenzoic acid as the
matrix, ergosine, ergocornine, ergocryptine, and
ergocristine were readily detected in individual
sclerotia of C. purpurea. The accuracy of the
identified ergot alkaloids was further confirmed
by tandem MS analysis. MALDI-TOF MS is suitable
for high-throughput screening of ergot alkaloids
because it permits rapid and accurate identification,
simple sample preparation, and no derivatization or
chromatographic separation.
E
rgot alkaloids are well-known mycotoxins produced
by species belonging to the fungal genus Claviceps
on cereal grain, primarily by C. purpurea. These types
Guest edited as a special report on “Cutting-Edge Techniques for
Mycotoxin Analysis” by Mary Trucksess and Kai Zhang.
1
Corresponding author’s e-mail: tom.graefenhan@grainscanada.
gc.ca
This study was funded by Agriculture and Agri-Food Canada and
the AgriInnovation Program of Growing Forward 2 as a contribution
to “EmTOX: Research network on emerging mycotoxins of potential
importance for Canadian agriculture and regulation of domestic
commodities and international trade.”
DOI: 10.5740/jaoacint.16-0117
of fungal infections are characterized by the presence of
dark-colored wintering bodies called sclerotia or ergot bodies,
which replace sound kernels in cereal grains such as rye,
wheat, and barley. Sclerotia contribute most of the alkaloids
in a contaminated lot of wheat, showing large differences
in their total alkaloid content that may range from 0.01 to
0.5% (w/w; 1). Collecting sclerotia along with healthy grain
at harvest can lead to contamination of food and feed with
ergot alkaloids. Consumption of ergot-contaminated food
may cause convulsions, adrenergic cramps, vasoconstriction,
and limb necrosis in extreme cases (2). Ergot-contaminated
feed grain remains a major veterinary issue, with symptoms
including gangrene, miscarriage, oversensitivity, and/or
improper muscle movement coordination in cattle, sheep,
pigs, horses, and chicken (3).
In sclerotia, C. purpurea produces ergot alkaloids or
ergopeptines, such as ergosine, ergocornine, ergocryptine,
ergocristine, ergometrine, and ergotamine, which are
amide-like derivatives of lysergic acid (4). Even though the
European Food Safety Authority concluded that a consistent
relationship between the amount of sclerotia and total ergot
alkaloid concentration cannot be established due to highly
variable alkaloid concentrations, the European Commission
recommended monitoring these six ergot alkaloids in grains
and cereals that are intended for human or animal consumption
(1). Common methods for ergot alkaloid analyses follow a
distinct pathway of extraction procedures, such as solid-liquid
extraction and SPE, followed by separation using LC or GC,
and identification by MS (5). For high-throughput analysis by
grain handlers and end users, for instance, a simpler and more
rapid method for screening of ergot alkaloids is desirable.
Matrix-assisted laser desorption ionization (MALDI)–
time-of-flight (TOF) MS enables high-throughput analyses
using only small sample amounts within a few minutes. Thus,
the MALDI-TOF MS method has the potential for fast and
accurate detection of ergot alkaloids in sclerotia. Of interest,
MALDI methods for the identification of ergot alkaloids have
not been published so far, which constitutes the rationale for
this study. This work opens up the possibility for accurate
and rapid screening of ergot alkaloids in sclerotia using
MALDI-TOF MS.
896 Sivagnanam et al.: Journal of aoaC international vol. 99, no. 4, 2016
Experimental
Chemicals
Four ergot alkaloid toxin standards, i.e., ergosine (purity,
95.9%), ergocornine (purity, 97.8%), ergocryptine (purity,
97.6%), and ergocristine (purity, 98.7%), were purchased from
Romer Labs (St. Louis, MO). Standard stock solutions of all
four ergot alkaloids were prepared in acetonitrile and stored
at −20°C until use. All other chemicals and analytical grade
reagents were purchased from Sigma Aldrich (Oakville, ON,
Canada), unless otherwise mentioned.
Sample Preparation
Samples were extracted based on the method described
in Tittlemier et al. (6), with minor modifications. Individual
sclerotia were weighed and crushed in a piece of paper
with a hammer tool. The crushed sclerotia were transferred
into a 2 mL Eppendorf tube, and 1 mL extraction solvent
(acetonitrile–3.03 M ammonium carbonate buffer, 84 + 16, v/v)
was added. The samples were mixed on a vortex mixer for
5 min and then sonicated for 5 min at room temperature,
followed by brief centrifugation at 10 000 rpm for 2 min. The
supernatant was collected and spotted on a MALDI target plate
for MS analysis. A dried droplet technique was followed for
MALDI-TOF MS analysis, whereby 1 μL sample and 0.5 μL
2,5-dihydroxybenzoic acid (DHB) matrix solution (10 mg/mL
concentration prepared in acetonitrile–0.1% trifluoroacetic
acid, 70 + 30, v/v) were spotted to the MALDI target plate
and dried at room temperature. Standards were prepared as
1 mg/mL concentration in acetonitrile and spotted on a MALDI
target plate under the same conditions as the samples.
MALDI-TOF MS Analysis
MALDI-TOF MS analyses were performed on an
UltrafleXtreme MALDI-TOF/TOF system (Bruker Daltonics,
Leipzig, Germany) equipped with a 355 nm laser operating in
reflector mode. All spectra were acquired by summing 3500 laser
Figure 1. MALDI mass spectra of ergot alkaloids in (A–D) standards and (E–H) corresponding sclerotia samples.
shots for reflector ion mode, 1000 laser shots for the precursor
mass and 4000 laser shots for the fragments in lift mode. Laser
power was adjusted between 28 and 32% of its maximal intensity
using a 1000 Hz smartbeam laser. MS spectra were acquired in
the reflector positive ion mode within a mass range from 100 to
3500 Da. The ion source 1 voltage was 20 kV, and the ion source
2 voltage was maintained at 17.8 kV. The lens voltage was 7 kV,
and the pulse ion extraction time was 140 ns.
Tandem MS (MS/MS) spectra were acquired in the lift
positive ion mode within a mass range from 10 to 3500 Da. The
ion source 1 voltage was 7.5 kV, and the ion source 2 voltage
was maintained at 6.8 kV. The lens voltage was 3.5 kV, and the
pulse ion extraction time was 120 ns.
Results and Discussion
To achieve the goal of rapid screening of ergot alkaloids in
individual sclerotia, an appropriate MALDI-TOF MS method
was designed. Initially, MALDI matrix noise in the m/z range,
which covered the mass range of known ergot alkaloids
ions, was evaluated by acquiring a mass spectrum on a blank
spot with 1 μL DHB matrix. No ions from the matrix that
could interfere with the alkaloid detection were observed.
MALDI-TOF MS was then performed on all four ergot
alkaloid standards: ergosine (548.286 Da), ergocornine
(562.302 Da), ergocryptine (576.318 Da), and ergocristine
(610.302 Da). No analyte ions could be detected when a spectrum
was acquired without the addition of MALDI matrix. However,
efficient ionization was achieved by spiking the MALDI spot
with 1 μL of DHB matrix, and the analyte ions were successfully
detected. MALDI-TOF MS spectra of ergot alkaloid standards
showed the profile containing the monoisotopic ions m/z
+
548.306 [M + H] for ergosine, m/z 562.319 [M + H] for
+
+
ergocornine, m/z 576.310 [M + H] for ergocryptine, and m/z
+
610.313 [M + H] for ergocristine (Figure 1A–D). After the
MALDI-MS parameters were optimized using the standards, the
same method was followed for the detection of ergot alkaloids
in samples. Several samples of sclerotia were analyzed, and the
four alkaloid toxins were detected successfully (Figure 1E–H).
The mass accuracy was found to be in the range of ±50 ppm for
standards and ±150 ppm for samples.
 Sivagnanam et al.: Journal of aoaC international vol. 99, no. 4, 2016 897

Figure 2. MALDI tandem mass spectra of ergot alkaloids in (A, C) standards and (B, D) corresponding sclerotia samples.

The use of MS/MS as the main identification tool for small
molecules is largely described as a reliable approach (7).
Therefore, MS/MS analyses were performed for the [M + H]+
species of the ergot alkaloid standards and sclerotia samples.
The resulting fragmentation patterns were compared between
the standard and sample to further confirm confidence of
the identification of detected ergot alkaloids. The MS/MS
fragmentation patterns of ergosine and ergocornine standards
are shown in Figure 2A and C, respectively, and the
MS/MS fragmentation patterns of ergocryptine and ergocristine
standards are shown in Figure 3A and C, respectively. The
MS/MS fragmentation patterns of the detected ergot alkaloids
from samples (Figure 2B and D and Figure 3B and D) were
compared and found to be in agreement with their respective
standards.
The fragmentation patterns of all four ergot alkaloids
shown in this study using the MALDI-TOF MS/MS method
and their fragmentation patterns determined by electrospray
ionization (ESI)-MS/MS in the literature (8, 9) were found
to be similar with respect to the generation of common
product ions. The principal product ions m/z 530 in ergosine,
m/z 544 in ergocornine, m/z 558 in ergocryptine, and m/z
592 in ergocristine were generated from a consistent loss
of water (minus 18 m/z) and correlate well with previous
studies (8). Furthermore, the most abundant characteristic
product ions detected, i.e., m/z 223 and m/z 268, were
considered the quantifier ion and qualifier ion, respectively,
for the positive identification of ergot alkaloids (9). Both of
these product ions were detected in our standards and in our
samples, confirming the confident identification of all four
ergot alkaloids. In this study, we detected ergot alkaloids in
sclerotia as proof of concept to explore the application of the
MALDI-TOF MS technique in screening for ergot alkaloid
contamination. Certain other contaminants (e.g., ergotamine)
that are part of the ergot family of alkaloids were not included
in this study due to the unavailability of a Drug Enforcement

Figure 3. MALDI-tandem mass spectra of ergot alkaloids in (A, C) standards and (B, D) corresponding sclerotia samples.
898 Sivagnanam et al.: Journal of aoaC international vol. 99, no. 4, 2016
Administration license at our research facility. However,
efficient and rapid screening of the four major ergot alkaloids,
i.e., ergosine, ergocornine, ergocryptine and ergocristine, can
be accomplished by the MALDI-TOF MS method.
Conclusions
Commonly used methods of ergot alkaloid analysis require
either chemical derivatization, followed by GC-MS analysis,
or LC separation (with typical run times of around 45 min;
10, 11), followed by ESI-MS and MS/MS analysis. The detection
method for ergot alkaloids developed in this study benefits from
the superior features of MALDI-TOF MS analysis, such as speed,
simplicity, and ease of automation for high-throughput analysis,
with enhanced selectivity obtained with high-accuracy m/z
measurements. In conclusion, a novel method was successfully
developed for the rapid screening of ergot alkaloids in sclerotia by
MALDI-TOF MS. This method also appears to be applicable to
the detection and high-throughput screening of other mycotoxins
in contaminated grain.
Acknowledgments
Our special thanks are extended to Sheryl Tittlemier for
allowing us to use the laboratory, and to Dainna Drul for helping
with the sample preparation.
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