Rapid quantitative analysis of microcystins in raw surface waters with MALDI MS utilizing easily synthesized internal standards

Article abstract of DOI:10.1016/j.toxicon.2013.12.007

The freshwater cyanotoxins, microcystins (MCs), pose a global public health threat as potent hepatotoxins in cyanobacterial blooms; their persistence in drinking and recreational water has been associated with potential chronic effects in addition to acute intoxications. Rapid and accurate detection of the over 80 structural congeners is challenged by the rigorous and time consuming clean up required to overcome interference found in raw water samples. MALDI-MS has shown promise for rapid quantification of individual congeners in raw water samples, with very low operative cost, but so far limited sensitivity and lack of available and versatile internal standards (ISs) has limited its use. Two easily synthesized S-hydroxyethyl-Cys(7)-MC-LR and -RR ISs were used to generate linear standard curves in a reflectron MALDI instrument, reproducible across several orders of magnitude for MC-LR, -RR and -YR. Minimum quantification limits in direct water samples with no clean up or concentration step involved were consistently below 7 μg/L, with recoveries from spiked samples between 80 and 119%. This method improves sensitivity by 30 fold over previous reports of quantitative MALDI-TOF applications to MCs and provides a salient option for rapid throughput analysis for multiple MC congeners in untreated raw surface water blooms as a means to identify source public health threats and target intervention strategies within a watershed. As demonstrated by analysis of a set of samples from Uruguay, utilizing the reaction of different MC congeners with alternate sulfhydryl compounds, the m/z of the IS can be customized to avoid overlap with interfering compounds in local surface water samples.

Full text of DOI:10.1016/j.toxicon.2013.12.007

Toxicon 78 (2014) 94–102
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Rapid quantitative analysis of microcystins in raw surface
waters with MALDI MS utilizing easily synthesized internal
standards
,
Amber F. Roegner a, Macarena Pírez Schirmer b c, Birgit Puschner a,
,
Beatriz Brena b, Gualberto Gonzalez-Sapienza c
*
a Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
b Cátedra de Bioquímica, Facultad de Química, Universidad de la República, Montevideo, Uruguay
c Cátedra Inmunología, Facultad de Química, Universidad de la República, Instituto de Higiene, Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 August 2013
Received in revised form 13 December 2013
Accepted 19 December 2013
Available online 31 December 2013
The freshwater cyanotoxins, microcystins (MCs), pose a global public health threat as
potent hepatotoxins in cyanobacterial blooms; their persistence in drinking and recrea-
tional water has been associated with potential chronic effects in addition to acute in-
toxications. Rapid and accurate detection of the over 80 structural congeners is challenged
by the rigorous and time consuming clean up required to overcome interference found in
raw water samples. MALDI-MS has shown promise for rapid quantification of individual
congeners in raw water samples, with very low operative cost, but so far limited sensitivity
and lack of available and versatile internal standards (ISs) has limited its use. Two easily
synthesized S-hydroxyethyl-Cys(7)-MC-LR and -RR ISs were used to generate linear stan-
dard curves in a reflectron MALDI instrument, reproducible across several orders of
magnitude for MC-LR, -RR and -YR. Minimum quantification limits in direct water samples
Keywords:
Microcystins
Cyanotoxins
Cyanobacterial blooms
Quantitative MALDI-TOF
Internal standards
with no clean up or concentration step involved were consistently below 7
mg/L, with
recoveries from spiked samples between 80 and 119%. This method improves sensitivity by
30 fold over previous reports of quantitative MALDI-TOF applications to MCs and provides
a salient option for rapid throughput analysis for multiple MC congeners in untreated raw
surface water blooms as a means to identify source public health threats and target
intervention strategies within a watershed. As demonstrated by analysis of a set of samples
from Uruguay, utilizing the reaction of different MC congeners with alternate sulfhydryl
compounds, the m/z of the IS can be customized to avoid overlap with interfering com-
pounds in local surface water samples.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
(MCs) are the most common hepatotoxins and tumour
promoters produced by freshwater cyanobacteria (de
Contamination of natural surface waters by toxic cya-
nobacteria is a growing problem worldwide, resulting in
serious animal and human health hazards. Microcystins
Figueiredo et al., 2004). They encompass a large family of
cyclic heptapeptide toxins, composed of uncommon amino
acids, including several D-amino acids and the character-
istic Adda group (3-amino-9-methoxy-2,6,8-trimethyl-10-
phenyldeca-4(E),6(E)-dienoic acid) (Fig. 1). Structural vari-
ation is observed in all amino acids, but major MC ana-
* Corresponding author. Cátedra Inmunología, Facultad de Química,
Universidad de la República, Instituto de Higiene, Av. Alfredo Narro 3051,
piso 2, Montevideo 11600, Uruguay. Tel.: þ598 24874334.
logues occur due to variations in the
L-amino acids “X” and
“Z” (Rinehart et al., 1994). More than 80 MC congeners have
E-mail address: ggonzal@fq.edu.uy (G. Gonzalez-Sapienza).
0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.toxicon.2013.12.007

A.F. Roegner et al. / Toxicon 78 (2014) 94–102
95
Fig. 1. Structure and synthesis of thiol-modified microcystins. The general structure of microcystins is displayed at the left, denoting the characteristic ADDA b-
amino acid, and “X” and “Z” representing variable L-amino acids. Internal standards were prepared by the well-known reaction of sulfhydryl compounds with the
methylene group of the N methyldehydroalanine (Mdha) residue of MCs. The reaction occurred almost instantaneously under nitrogen gas.
been described to date, ranging in size between 909 and
1115 Da, and are produced by a broad range of cyanobac-
teria species in fresh and brackish surface water blooms
(Codd, 2000; Donohue et al., 2008). Potent inhibitors of
highly conserved protein phosphatase 1 and 2A (Runnegar
et al., 1995), microcystin toxicity impacts a broad range of
plant and animal species, including humans. In spite of
similar phosphatase inhibitory capacity, MCs differ greatly
in their acute toxicity from poorly toxic, LD50 > 1200 mg/kg,
to highly toxic variants such as MC-LR (LD50 ¼ 50 mg/kg)
(Rinehart et al., 1994). These differences appear to be
related to their toxicokinetics, since the mechanism of
cellular uptake occurs via specific organic anion trans-
porting polypeptides that mediate selective congener
transport (Fischer et al., 2010).
Enhanced by environmental perturbations and
increasing global water temperatures, cyanobacterial
blooms result from heavy nutrient loading, and their
increased occurrence has emerged as worldwide concern
(Codd, 2000; Pearson et al., 2010). While many cyano-
bacterial species are known producers of microcystins,
reasons for toxin production remain unknown and the
presence of a bloom does not guarantee the presence of
toxic compounds (Pearson et al., 2010). Moreover, the
production of individual MC congeners varies among
genera, species and strains, and toxin production can vary
considerably within hours (Wood et al., 2011). This vari-
ability presents a serious challenge to public health officials
and water treatment facility managers by requiring to
optimize sampling, minimize public health risk and target
interventions in the management of recreational or source
water bodies (Dodds et al., 2009). The WHO has established
chromatography (HPLC) coupled to UV or MS detection,
and reference standard HPLC/UV method (ISO
a
20179:2005) has been issued by the International Organi-
zation for Standardization. In normal practice and for reg-
ulatory purposes, the analysis of the MC content in drinking
water is restricted to the use of these reference methods. To
fulfill the sensitivity required by the WHO advisory level of
<1 mg/L of MC-LR, these chromatographic methods often
require large sample volumes, concentration and clean
steps, as well as the sequential analysis of samples
(Meriluoto et al., 2000). This processing makes the
methods comparatively less attractive for screening pur-
poses, particularly when large numbers of samples need to
be analyzed on a regular basis within a timely fashion. To
this end, other techniques, particularly immunochemical
methods, are currently used to study the ecology of cya-
nobacterial communities or monitor the evolution of these
communities in source or recreational waters (Pirez et al.,
2013; Qin et al., 2010; Znachor et al., 2006). ELISA is a
convenient option because it allows for the parallel analysis
of large sample loads in a fast and cost efficient manner.
However, commercially available ELISAs, detecting the side
chain ADDA common to all microcystins, show variable
cross reactivity and only detect total microcystins, missing
the information on individual congeners with variable de-
gree of toxicity (Msagati et al., 2006). In addition, most
ELISAs perform within a narrow linear range, about one
order, while environmental water and scum samples may
contain MC concentrations ranging from a few
mg/L to
hundreds of thousands. With an extended linear range,
short time of analysis (<30 s), high-throughput capacity
and minimal requirements for sample preparation, Matrix
Assisted Laser Desorption Ionization (MALDI) coupled with
time of flight (TOF) mass spectrometry (MS) offers a salient
option for comprehensive analysis of environmental water
samples (Pan et al., 2007; Pusch and Kostrzewa, 2005).
To date, MALDI-TOF MS mostly has been utilized qual-
itatively to identify MC variants in cyanobacterial colonies
and surface water blooms (Erhard et al.,1997; Ferranti et al.,
2011; Welker et al., 2002), and its use for MC quantification
is rather recent (Howard and Boyer, 2007; Puddick et al.,
2011). Quantitative detection by MALDI-TOF based on
peak response has poor reproducibility, mainly due to spot-
to-spot variability in crystal formation (Duncan et al., 2008;
Szajli et al., 2008). However, this can be overcome using an
a provisional guideline value of 1 mg/L for microcystin LR in
drinking water, but recent evidence in animal intoxications
suggests the prevalence and consequences of potentially
overlooked congeners (Dietrich and Hoeger, 2005;
Donohue et al., 2008). Similarly, a reference health alert
value of 20
mg/L has been suggested to minimize the
toxicity risk by accidental ingestion of recreational waters
(Chorus et al., 2000), and many countries have adopted
similar limits for bloom risk assessment ranging from 15 to
25
mg/L (Chorus, 2012).
The most widely used analytical methods for the
detection and quantification of MCs rely on chromato-
graphic separation techniques such as high pressure liquid

96
A.F. Roegner et al. / Toxicon 78 (2014) 94–102
internal standard (IS), and the best results are obtained
when this compound mimics the physicochemical prop-
erties of the analyte throughout all steps of the analytical
process (Szajli et al., 2008). The use of a stable isotope-
labeled form of the analyte (isotopomer) is an obvious
way to accomplish that goal, but requires special chemicals
and can be difficult to prepare and purify. In the initial
report of a quantitative MALDI-TOF application for MCs
(Howard and Boyer, 2007), three potential internal stan-
dards were assayed, and the best method detection limit
(MDL) for MC-LR was obtained with nodularin (a penta-
peptide cyanotoxin with similar basic ring structure to
microcystins), which outperformed a [15N]10-isotopomer
of microcystin-YR and the peptide hormone angiotensin I.
More recently, Puddick et al., in order to reduce the cost of
the analysis, used angiotensin I as internal standard in
combination with different sample preparation techniques,
but being a linear peptide, angiotensin as IS did not attain
the MDL values previously obtained with nodularin
(Puddick et al., 2011). This highlights the importance of
using internal standards that resemble the physicochem-
ical properties of the target compound.
With this in mind, we explored the use of derivatives of
MCs as internal standards for quantitative detection of MC
by MALDI-TOF MS. To this end, we utilized a well-known
and simple synthesis to prepare ISs by reaction of MCs
with thiol-containing compounds readily available in most
laboratories. To further improve the detection limit of the
method introduced by Howard and Boyer, we utilized
reflectron MALDI-TOF equipment that provides improved
resolution and better sensitivity. We demonstrate that
these ISs can be used for sensitive detection of MCs by
direct analysis of unprocessed surface water samples, with
an extended linear range and high recoveries. The ability to
identify and quantify common microcystins with minimal
processing of the raw water sample will dramatically
reduce time for screening of a broad number of samples.
mercaptoethylamine or b-mercaptoethanol were added
and allowed to react under nitrogen gas for several hours at
room temperature. One microliter of the reaction mix was
taken initially every 15 min and then hourly, and after
dilution in 100 ml of MilliQ water was analyzed by MALDI-
TOF for relative appearance and disappearance of peaks.
After completion, the reaction mixture was diluted with
10% methanol, acidified to pH 3.0 with 5% glacial acetic acid
and purified using solid phase extraction (SPE C18, Phe-
nomenex, Torrance, CA). The methanol eluted (S-amino-
ethyl-/S-hydroxyethyl-Cys(7) microcystin) adduct was dried
under nitrogen gas to eliminate any residual sulfhydryl
compound, reconstituted in 200 ml of methanol, quantified
using HPLC, aliquoted into 50
further use.
m
l and stored at ꢀ20 ꢁC until
2.3. MALDI-TOF-MS
CHCA, which has been reported to deliver relatively
homogenous samples in crystal formation (Szajli et al.,
2008), was prepared as a saturated solution (>21 mg/ml)
in 50% (v/v) acetonitrile and 50% (v/v) 0.1% TFA MilliQ water.
The matrix was mixed with internal standard and sample
and permitted to air dry. A Microflex LRF MALDI-TOF
(Bruker Daltonics, Billerica, MS, USA) with a 337 nm nitro-
gen laser was operated in positive ion reflectron mode with
delayed extraction and optimized in the m/z range from
500 Da to2 kDa. Calibrations were performed with a peptide
calibration standard mix (Bruker Daltonics). The laser was
fired 100 times at each of ten locations for each sample well
on a 96 well plate for a cumulative 1000 shots per sample
well taken at 30% intensity. More specific details of sample
well preparation and quantification with internal standard
are provided in the Supplemental materials.
2.4. MALDI spot preparation and quantification with IS
Ten microliters of sample were thoroughly mixed with
2. Materials and methods
1
m
l of the appropriate concentration of internal standard
(30, 40, 300 or 400 g/L, as stated), followed by the addition
of 10 l of CHCA solution. Following thorough mixing, 2
of the solution was placed on the MALDI sample plate.
Unless otherwise specified, eight 2 l replicate spots were
m
2.1. Materials
m
ml
Microcystin-LR, and microcystin-RR were obtained from
m
GreenWater Laboratories (Palatka, FL); microcystin-YR,
b
-
aliquoted and allowed to air dry at room temperature. Ten
shots were taken at each spot on the 96 well stainless steel
plate with the laser firing 100 times at each shot for a cu-
mulative 1000 shots per well at 30% intensity. The data
point (intensity or area) was determined as an arithmetic
mean. Initially, ratios of both peak intensity and peak area
of microcystin congener divided by the equivalent intensity
and area of internal standard peak were determined for
each spot in order to account for spot-to-spot variation
across the plate and heterogeneity of crystal formation. The
stainless steel plate was thoroughly cleaned between ana-
lyses according to manufacturer instructions, and was
routinely checked to avoid any carry over contamination.
mercaptoethanol (99.9%), and trifluoroacetic acid (TFA)
were obtained from Sigma–Aldrich (St. Louis, MO). Alpha-
cyano-4-hydroxycinnamic acid (CHCA) and a standard
peptide calibration solution were obtained from Bruker
Daltonics (Denmark). HPLC grade acetonitrile and meth-
anol were obtained from Baker (Phillipsburg, NJ), along
with sodium borate and sodium chloride for the prepara-
tion of the borate buffer.
2.2. Preparation of internal standards
Two internal standards (IS-1 and IS-2) were prepared
from microcystin-LR (MC-LR) and microcystin-RR (MC-RR),
respectively, by reaction of the N methyldehydroalanine
(Mdha) group with sulfhydryl compounds, Fig. 1 (Smith
2.5. Calibration curves and detection limits
and Boyer, 2009). For each 0.1
m
M of MC dissolved in 50%
To study the linear range of the method, MC-LR, MC-RR
and MC-YR were dissolved in 100% methanol (1 mg/mL)
methanol/50% 100 M borate buffer, pH 10.5; 10
m
ml of
b-

A.F. Roegner et al. / Toxicon 78 (2014) 94–102
97
and further diluted in MilliQ water to twelve concentra-
tions ranging between 0 and 5000 g/L. These samples
were prepared for analysis as described (Supporting
information) using IS-1 and IS-2 at 300 g/L and 400 g/
L, respectively, in 100% methanol. Standard solutions were
prepared in MilliQ water for determination of a method
detection limit (MDL) or and in a reference cyanobacterial-
bloom water sample (ref-CB) for minimum quantification
limit (MQL). At least seven concentrations of the standards
performed equally well as IS but the hydroxyethyl conju-
gate (IS-1, MWaverage ¼ 1073.30 Da, MHþ ¼ 1073.7) showed
better reproducibility and thus was selected. A second in-
m
m
m
ternal standard, the b-mercaptoethanol derivative of MC-
RR (IS-2, MWaverage ¼ 1116.33 Da, MHþ ¼ 1116.6), was
prepared similarly, and the eventual fragmentation of both
IS was studied under different conditions of laser energy.
Preliminary tests showed that dilutions of ISs of 30–300 mg/
L were suitable for quantitative analysis of MCs across a
broad range of concentrations. Using these concentrations,
a tradeoff value of 30% laser intensity was determined to
have maximal sensitivity at low concentrations of MCs,
while minimizing the fragmentation of the internal stan-
dards, Fig. 2.
were prepared in the 0–100-
mg/L range, using IS-1 and IS-2
at a working concentration of 30
m
g/L and 40 g/L,
m
respectively (Supporting information). The ref-CB water
sample consisted of a pool of representative water samples
from blooms with less than 0.25
mg/L of total MCs, as
determined by ELISA (Brena et al., 2006).
We then focused our study on the three most common
polar congeners found in cyanobacterial blooms, namely
MC-LR, MC-RR, and MC-YR, which produced peaks at 995.6
(m/z), 1038.6 (m/z), and 1045.6 (m/z), respectively. Fig. 3
shows their spectra at trace concentrations, in the pres-
2.6. Surface water sample preparation and spiking recoveries
Surface water samples collected from three water
bodies along the Rio Negro in northern Uruguay, kept
chilled under transport and frozen at ꢀ20 ꢁC until ready for
analysis. Following three cycles of freeze/thaw to fully
rupture the cyanobacterial cells, 1.0 mL of the water sample
was centrifuged at 10,000 ꢂ g for 5 min, followed by sy-
ence of 40 mg/L of IS-2. In all cases, there were peaks pro-
portional to the concentration of MC, no interference of IS
fragments were observed in the case of MC-LR and MC-YR,
despite the fact that a fragment, or a low concentration
contaminant of the IS, occurred at 1048.5 (m/z). In the case
of MC-RR a trace peak corresponding to this MC was
observed in the blank, presumably as a consequence of
marginal IS-2 fragmentation, but the signal was very weak
and did not interfere with the quantification of MC-RR.
Similar results with even less interference of contaminant
peaks were obtained when IS-1 was used (data not shown).
ringe filtration with a 0.22
mm filter (Millipore, MA). An
initial survey determination of the concentrations of MC-
LR, MC-RR, and MC-YR in the surface water samples were
made utilizing IS-2 with the method described in Section
2.4. Then nine samples covering a range of MC concentra-
tions were selected and spiked with 100 mg/L of MC-LR,
MC-RR and MC-YR, simultaneously, and percent recovery
was determined along with total concentrations. A bloom
showing extremely high MC concentration was spiked with
3.2. MDL and MQL with IS-1
1000
mg/L and diluted 10 fold before analysis. Calibration
IS-1 generated calibration curves (Supporting
curves were performed for each new analysis. MALDI well
spot preparation and laser operation were maintained as
for standard curves.
information, Fig. A.1–A.3) of standards with excellent
linearity in the 0–5000 m
g/L range and R2 values >0.99. As
previously described (Welker et al., 2002), the polar con-
geners generated strong [M þ H]þ signals likely because of
protonation of the basic arginine side chains. Not surpris-
ingly, MC-RR calibration curves generated a much greater
signal (approximately twice the slope) as compared with
MC-LR and MC-YR, confirming the role of protonation of
the basic arginine side chains; this increased signal prob-
ably explains the improve MDL observed for MC-RR as
compared with the two other congeners. Table 1 summa-
rizes calculated limit of detection and quantification for IS-
1 (Supporting information, Fig. A.1–A.3 and B.1-B.3) using
standard solutions prepared in MilliQ water.
2.7. Safety considerations
Acutely toxic to mammals, the LD50 (i.p.) for micro-
cystins range between 36 and 122
with an inhalation toxicity of 43
m
m
g/kg for mice and rats
g/kg. It is much less
bioavailable with an oral LD50 of 5 mg/kg2.3 Proper personal
protective equipment was worn (gloves, lab coat) when
handling any of the congener standards or handling any
bloom samples. Care was also taken to not aerosolize any
powder forms of standards or bloom materials.
Good correlation was observed between values gener-
ated by ratio of intensities (peak height) of microcystin
congener to internal standard and the corresponding ratio
of areas (peak area). Previously, it has been reported that
the peak height ratio is more suitable for MALDI quantifi-
cation because the integration of a selected baseline and
range can be highly variable, particularly when using a
linear, nonflectron mode (Duncan et al., 2008; Howard and
Boyer, 2007). Previous reports indicated little difference
between R2 values for MC standard solutions when using
non microcystin internal standards, but a higher coefficient
of variation (CV) for peak area ratios, a higher limit of
detection, and a smaller linearity of range (Howard and
3. Results and discussions
3.1. Preparation of internal standards
We initially optimized the conditions for the reaction of
b
-mercaptoethylamine and b-mercaptoethanol with the
Mdha group of MC-LR. The reaction proceeded very rapidly
at high pH as determined by MALDI analysis of time course
samples; prepared in borate buffer pH 10.5, all MC-LR
was converted to the S-aminoethyl- or S-hydroxyethyl-
Cys(7)-MC- LR derivative within seconds (data not shown).
Initial experiments suggested that both compounds

98
A.F. Roegner et al. / Toxicon 78 (2014) 94–102
Fig. 2. Mass Spectra of S-hydroxyethyl-Cys(7) microcystin LR (IS-1) and S-hydroxyethyl-Cys(7) microcystin RR (IS-2). Both standards (40
MALDI target as previously described (2 l per spot), and were analyzed at 30% of laser intensity.
mg/L) were spotted on the
m
Boyer, 2007). We did not observe a substantial difference in
limit of detection between peak area and peak height
minimum detection limits; however, the linear range ob-
tained using peak area appeared to decrease with
increasing microcystin standard solution indicated by the
divergence of the standard curves at higher concentrations,
so peak intensity was adopted for quantification.
water, it is worth noting that a concentration step was not
employed prior to the screening of samples. The method
has the potential to be adapted for screening of drinking
water with an additional concentration step. Regardless, a
major advantage is that it allows screening of a large
number of untreated surface water samples almost
simultaneously.
3.3. MDL and MQL with IS-2
3.4. Selection of IS
We expected similar results with respect to linearity of
range and minimum detection limit. Likely due to the
presence of two arginine side chains, IS-2 exhibited greater
ionization than IS-1, resulting in decreased slopes for the
calibration standard curves for all three microcystin con-
geners as compared with IS-1 (Supporting information,
Fig. C.1–C.3 and D.1–D.3). However, the limit of detection
The utility of any IS may be severely threatened by the
occurrence of common compounds of similar mass in the
sample, as demonstrated in the case of the bloom samples
taken from three water bodies in northern Uruguay along
the Rio Negro. Upon an initial screening without internal
standard, a number of the samples were determined to
contain an unknown peak at 1072.6 (m/z). Post source
decay (PSD) analysis of this peak did not show the
distinctive fragmentation products (ADDA fragment
[PhCH2CH(OCH3)]þ ¼ 135 (m/z), [Mdha-Ala þ H]þ ¼ 155
(m/z), [Glu-Mdha þ H]þ ¼ 213 (m/z), [ADDA-fragment-Glu-
Mdha]þ ¼ 375.0 (m/z)) of the MC core, ruling out that this
was a MC congener. Due to the close proximity to the peak
of IS-1 (1073.6 m/z) and the high signal intensity of the
1072.6 m/z compound IS-1 could not be used with our
particular set of samples. Nevertheless, IS-1 may prove
useful for surface water analysis in other regions of the
world, with different contaminants and should not be
discounted. This result actually prompted the synthesis of
IS-2 after an additional screening of up to 20 raw surface
water samples showed that no interfering compounds with
similar m/z value to the calculated mass of IS-2 (1116.6 m/z)
occurred in the samples. This case highlights the impor-
tance of a simple and flexible IS synthesis strategy that
makes it possible to design the m/z of the IS to maximally
avoid interference present in samples. In particular, blooms
was not affected, MDL ¼ 2.8, 1.4, and 4.5
mg/L, respectively
for MC-LR, MC-RR and MC-YR, Table 2, and were very close
to the values obtained with IS-1. These data suggest the
limit of detection operates independently of ionization of
internal standard. The use of lower concentrations of IS,
which produced peaks of similar size to those of the MC
standards, did not significantly affect the detection limit
(data not shown). To study the potential influence of the
sample matrix, this time MQL was quantified using MC
standards diluted in the ref-CB water sample. Under these
conditions, the calculated MQL were 6.5, 4.6, and 6.4 mg/L,
for MC-LR, MC-RR and MC-YR, respectively. Contributing to
the robustness of this method, sample matrix effects
appear to have minimal effect on MQLs. While the peak
height intensity and peak area curves appear to diverge a
bit, this discrepancy had minimal effect on quantification.
MDL curves in MilliQ water yielded similar results (data not
shown). While these detection limits remain above the
provisional guideline provided by the WHO for drinking

Fig. 3. Mass spectra of low concentrations of MC-LR, MC-RR and MC-YR in presence of IS-2. The spectra were prepared with 10
ml of the relevant concentration of microcystin standard prepared in MilliQ water with 1 ml of
IS-2 (diluted to a final concentration of 40 g/L) and then thoroughly mixed with 10 l of CHCA matrix. The 2 l spots were allowed to dry and then shot at 30% intensity.
m
m
m

100
A.F. Roegner et al. / Toxicon 78 (2014) 94–102
Table 1
Determination of MDL and MQL for MC-LR, MC-RR, MC-YR standard solutions using both peak height and peak area ratios with IS-1.
Congener
MC-LR
Ratio parameter
Regression equation
R2
CVa
MDL (
m
g/L)b
MQL (m
g/L)c
Peak height
Peak area
Peak height
Peak area
Peak height
Peak area
y ¼ 0.0095x þ 0.04
y ¼ 0.0093x þ 0.06
y ¼ 0.0222x ꢀ 0.005
y ¼ 0.0214x ꢀ 0.01
y ¼ 0.0096x ꢀ 0.0001
y ¼ 0.0092x ꢀ 0.009
0.989
0.991
0.996
0.994
0.991
0.996
6.7
8.6
10.3
8.5
4.3
6.2
2.8
2.6
1.6
0.87
4.5
3.9
4.5
4.3
2.6
3.6
6.2
4.2
MC-RR
MC-YR
a
CV, coefficient of variation determined for 100
m
g/L of MCs.
MDL was determined based on standard solutions of 5 g/L for MC-LR and MC-RR and 10
MQL was determined with standard solutions prepared in MilliQ water.
b
c
m
m
g/L for MC-YR.
Table 2
Determination of MDL and MQL for MC-LR, MC-RR, MC-YR standard solutions using both peak height and peak area ratios with IS-2.
Congener
MC-LR
Ratio parameter
Regression equation
R2
CVa
MDL (
m
g/L)b
MQL (m
g/L)c
Peak height
Peak area
Peak height
Peak area
Peak height
Peak area
y ¼ 0.0033x ꢀ 0.017
y ¼ 0.0031x ꢀ 0.010
y ¼ 0.0124x ꢀ 0.057
y ¼ 0.0116x ꢀ 0.050
y ¼ 0.0025x ꢀ 0.013
y ¼ 0.0021x ꢀ 0.0087
0.984
0.991
0.991
0.992
0.992
0.985
18.5
25.3
6.2
3.1
7.7
2.8
1.8
1.4
2.3
4.5
3.6
6.5
4.4
4.6
4.3
6.4
4.5
MC-RR
MC-YR
9.8
a
CV, coefficient of variation determined for 100
m
g/L of MCs.
MDL was determined based on standard solutions of 5 g/L for MC-LR and MC-RR and 10
MQL was determined with standard solutions prepared in the reference water sample ref-CB.
b
c
m
mg/L for MC-YR; to study matrix effect.
in this region of the world have been shown to produce
uncommon MCs (Andrinolo et al., 2007; Neumann et al.,
2000), so we emphasize the importance of pre-screening
bloom samples for potential interfering peaks. In partic-
ular, the presence of [D-Asp3,ADMAdda5, Dhb7] MC-HtyR
(MW ¼ 1073) (Beattie et al., 1998) could interfere with IS-
1, and [L-MeLan7] MC-LR (MW ¼ 1115) (Namikoshi et al.,
1995) could interfere with IS-2, if present in the bloom
sample.
the same order than its original concentration in the sam-
ple, in order to fulfill our goal of developing a method that
could be used for direct screening of unprocessed raw
water samples. All spike recoveries were between 80 and
120%. Determined concentrations were highly reproducible
and values by peak height and peak area corresponded
closely throughout the spiking experiments. As mentioned,
for each spiking set, a calibration curve with three to four
data points was established with each microcystin
congener standard and IS-2. We did all initial experiments
using eight replicate well spots, but data analysis showed
that three replicates might be used without altering esti-
mated concentrations or recovery of spiked samples (data
not included).
3.5. Spike recoveries for surface bloom samples
Table 3 shows the results and spike recoveries of sam-
ples taken during the blooming season from three reser-
voirs in northern Uruguay along the Rio Negro, utilizing
peak height ratios. The results and spike recoveries quan-
tified utilizing peak area ratio are available in Supporting
information, Table A. Fortification with congeners was
4. Conclusions
Previous work on the quantitative use of MALDI-TOF for
MC analysis showed that the best sensitivity was obtained
when nodularin was used as IS (Howard and Boyer, 2007;
Puddick et al., 2011). This is probably due to the high
performed simultaneously, using 100
mg/L of the respective
congener (except for wbB4 that was spiked with 1000
mg/
L). The concentration of the spiked MCs was chosen to be of
Table 3
Spike recoveries for surface water bloom samples calculated with peak height ratio using the internal standard IS-2.
Sample
MC-LR
MC-RR
MC-RR
Initial conc.
m
g/L
Recovery %
Initial conc.
m
g/L
Recovery %
Initial conc.
m
g/L
Recovery %
wbA 1
wbA 2
wbB 1
wbB 3
wbB 4
wbB 5
wbC 1
wbC 2
wbC I
307
157
235
76.3
12000
35.3
49.5
417
116
234
41.6
198
<MQL
1470
ND
<MQL
53.5
ND
119
439
55.7
347
45.0
1240
ND
<MQL
95.8
ND
107
80.2
97
109
79.0
108
108
94
93.9
94.5
84.5
84.4
84.2
81.1
80.4
86.2
87.7
80.0
82.1
99.8
86.2
90.3
93.5
89.9
27.4
110
All samples were spiked with 100
m
g/L of each congener except for sample wbB 4 4 that was spiked with 1000
m
g/L.

A.F. Roegner et al. / Toxicon 78 (2014) 94–102
101
similitude of nodularin with the common structure of MCs.
However, nodularin has a smaller amino acid ring, its size
(m/z 825.4) is close to interfering peaks of the MALDI ma-
trix; in addition, it is a cyanobacterial toxin and could
become target of the analysis. As an alternative, the reac-
tion of sulfhydryl groups with the Mdha moiety of com-
mercial or in-house purified MCs offers a simple and
versatile method to prepare ISs for MC quantification that
preserve the physicochemical characteristics of these
toxins. Additionally, the combination of different MC con-
geners paired with different sulfhydryl compounds allows
for adjustment of the m/z of the IS to the particular pattern
of common interfering compounds in the samples, as
demonstrated by the advantageous use of IS-2 versus IS-1
when a frequent interfering compound of 1072.6 m/z was
found in surface water blooms in Uruguay. The combina-
tion of ISs highly similar to the analyte with the high
sensitivity and resolution of the reflectron mode MALDI-
TOF allowed direct analysis of raw water samples with
increased sensitivity. Although still not sufficient for direct
screening of drinking water, the method fulfills our purpose
of developing a rapid screening tool for monitoring envi-
ronmentally relevant concentrations of MCs using small
volumes of untreated samples. Indeed, the MQL for un-
treated surface water obtained in this study was 6.5, 4.6
(Montevideo, Uruguay) and the USEPA STAR Graduate
Student Fellowship provided support for the first author.
Protocols
No human subjects or experimental animals were
involved with the research presented in this manuscript.
Conflict of interest
The authors have no conflicts of interest to report.
Appendix A. Supplementary data
Supplementary data related to this article can be found
at http://dx.doi.org/10.1016/j.toxicon.2013.12.007.
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Funding
This work was supported by NIH Fogarty International
Center Grant TW05718, CSIC 400/2008, and ANII FMV 7263.
The U.S. Fulbright Student Research Fellowship

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