Multi-mycotoxin analysis using dried blood spots and dried serum spots
Multi-mycotoxin analysis using dried blood spots and dried serum spots
Bernd Osteresch 0 1 2
Susana Viegas 0 1 2
Benedikt Cramer 0 1 2
Hans-Ulrich Humpf 0 1 2
0 Centro de Investigação em Saúde Pública, Escola Nacional de Saúde Pública, Universidade NOVA de Lisboa, Avenida Padre Cruz , 1600-560 Lisbon , Portugal
1 Environment and Health RG, Lisbon School of Health Technology, Polytechnic Institute of Lisbon , Av. D. João II, Lote 4.69.01, Parque das Nações, 1990-096 Lisbon , Portugal
2 Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster , Corrensstr. 45, 48149 Münster , Germany
In this study, a rapid multi-mycotoxin approach was developed for biomonitoring and quantification of 27 important mycotoxins and mycotoxin metabolites in human blood samples. HPLC-MS/MS detection was used for the analysis of dried serum spots (DSS) and dried blood spots (DBS). Detection of aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), trichothecenes (deoxynivalenol, DON; DON-3-glucoronic acid, DON-3-GlcA; T-2; HT-2; and HT-2-4-GlcA), fumonisin B1 (FB1), ochratoxins (OTA and its thermal degradation product 2'R-OTA; OTα; 10hydroxychratoxin A, 10-OH-OTA), citrinin (CIT and its urinary metabolite dihydrocitrinone, DH-CIT), zearalenone and zearalanone (ZEN, ZAN), altenuene (ALT), alternariols (AOH; alternariol monomethyl ether, AME), enniatins (EnA, EnA1, EnB, EnB1) and beauvericin (Bea) was validated for two matrices, serum (DSS), and whole blood (DBS). HPLC-MS/MS analysis showed signal suppression as well as signal enhancement due to matrix effects. However, for most analytes LOQs in the lower pg/mL range and excellent recovery rate were achieved using matrix-matched calibration. Besides validation of the method, the analyte stability in DBS and DSS was also investigated.
Biomonitoring; Dried blood spot; Dried serum spot; HPLC-MS/MS; Mass spectrometry; Mycotoxin
Mycotoxins are toxic secondary metabolites produced by
molds contaminating food during production, shipping,
processing, or storage . In order to protect consumers’ health
maximum levels for food and feed have been set by regulatory
authorities . Regulatory attempts are usually based on food
contamination and consumption data. However, this approach
does not take individual exposure due to personal, regional as
well as cultural divergences into account . Biomarker-based
approaches are more and more used to assess dietary exposure,
as they allow to analyze physiological samples like urine or
blood for each test person individually . Various sample
preparation and quantification techniques are described for
mycotoxins and their metabolites in several physiological samples
[5–9]. Main challenges in biomonitoring-based methods are
usually low analyte concentrations and matrix interferences
during analysis. Recently developed dilute-and-shoot
approaches for urine attempt to remove the majority of matrix
compounds by chromatographic separation and take advantage
of highly sensitive mass spectrometers [10, 11].
For blood analysis, dried blood spots (DBS) currently
undergo a comeback concerning medical or forensic issues
mainly due to improved detection limits and therefore
leading to new fields of application [12, 13]. For example, DBS
are suitable for extensive biomonitoring studies of
environmental contaminants in humans or animals . Particularly,
for animal studies the application of DBS is an effective
improvement concerning sample collection as only limited
blood volumes are often available for analysis due to the
low body weight of small animals . Advantages of
DBS compared to conventional blood collection are the
minimally invasive sampling, simple sample preparation, easier
storage, and shipping as well as the small volume required
. Samples can be collected on filter paper cards by heel,
ear, or finger pricking as well as by spotting a known blood
volume from ampoules after conventional vain puncture [17,
18]. Even if DBS are the standardized basis for medical
tests, dried serum spots (DSS) are an additional opportunity
for immunological tests and other blood counts. Likewise to
DBS, DSS take advantage of simplified storage and
shipment conditions [19, 20].
Recently, a DBS method for the detection of ochratoxin A in
dried blood spots using HPLC-MS/MS has been published
including the optimization of various basic parameters such as
spotting volume and hematocrit which did not have a strong
influence [21, 22]. Thus, the application of DBS for mycotoxin
analysis showed positive findings in all samples for OTA as well
as 2’R-OTA in the blood of coffee drinkers . Here, we
present the further development of this method by the incorporation
of 27 relevant mycotoxins and metabolites and its application to
serum and blood samples. Compounds monitored were
aflatoxins (AFB1, AFB2, AFG1, AFG2, AFM1), Alternaria toxins
(ALT, AME, AOH), citrinin (CIT), and its metabolite
dihydrocitrinone (DH-CIT). Furthermore, trichothecenes as
deoxynivalenol (DON), deoxynivalenol-3-glucuronide
(DON3-GlcA), T-2-toxin (T-2), HT-2-toxin (HT-2),
HT-2-toxin4-glucoronide (HT-2-4-GlcA) have been incorporated. In
addition, the structurally related cyclic hexadepsipeptides beauvericin
(BEA) and enniatins (EnA, EnA1, EnB, EnB1) are included.
Lastly, fumonisin B1 (FB1), ochratoxin A, and its thermal
degradation product 2’R-ochratoxin A as well as ochratoxin α and
10-hydroxyochratoxin A (OTA, 2’R-OTA, OTα, 10-HOTA),
zearalenone (ZEN), and zearalanone (ZAN) are enclosed [1,
23–25]. Those mycotoxins and metabolites are frequently
determined in human fluids such as the urine, human breast milk, and
blood, or serum and plasma, respectively [26–29]. Their reliable
analysis in a multi-mycotoxin approach for physiological
samples is a challenging task but allows to determine the individual
mycotoxin exposure of humans and animals.
Mycotoxins should be handled with care due to their toxic
effects for humans. Blood samples underlie biological hazard
in principle. It is mandatory to autoclave all contaminated lab
material, e.g., ampoules and paper collection cards, which
encountered blood or serum.
Chemicals and reagents
Acetonitrile (ACN) and acetone were of LC gradient purity
(VWR, Darmstadt, Germany). Type 1 laboratory water was used
(Resistivity > 18 MΩ, TOC < 10 ppb; Purelab flex, Veolia Water
Technologies, Celle, Germany). Formic acid and acetic acid was
purchased from Grüssing (Filsum, Germany) with 99.5% purity.
Blood collection tubes were MonovetteR® EDTA KE/7.5 mL
and Monovette® Serum-Gel from Sarstedt (Nümbrecht,
Germany). Whatman 903 protein saver cards™ for sample
collection and preparation were acquired from Sigma-Aldrich
AFB1/2, AFG1/2, AFM1, BEA, CIT, EnA, EnA1, EnB,
EnB1, FB1, FB2, ZAN, and α-/β-zearalenol were from
Sigma-Aldrich. DH-CIT was purchased from AnalytiCon
Discovery (Potsdam, Germany). DON, FB1, 10-OH-OTA,
OTA, OTα, T-2, HT-2, and ZEN were isolated and purified
from fungal cultures [30–35]. 2’R-OTA was produced by
thermal isomerization of OTA . Glucuronic acid conjugates
(DON-3-GlcA and HT-2-4-GlcA) were synthesized
enzymatically using rat and pig liver microsomes . ALT, AME,
and AOH were isolated and purified according to a recently
published procedure . Tenuazonic acid was chemically
synthesized by Lohrey et al. . Stock solutions were
prepared at concentrations of 10 or 20 μg/mL in ACN or ACN/
H2O and stored at −20 °C in the dark until further use. Purity
of in-house produced standards was checked by HPLC-DAD/
ELSD (evaporative light scattering detector) and was ≥98%.
Structure verification was done via MS and NMR. Aflatoxins
B1/2 and G1/2 were applied as a combined solution at 2 μg/mL
for AFB1/AFG1 and 0.5 μg/mL for AFB2/AFG2. Two
working solutions were prepared at 20-fold concentration of the
highest calibration point at each day of measurement in
ACN/H2O (80:20, v/v) leading to concentrations of 1 μg/mL
for the majority of analytes. For AFB1/AFG1 the
concentrations in the working solution were 200 ng/mL and for AFB2/
AFG2 50 ng/mL.
Human blood and serum samples were provided by healthy
volunteers giving written consent as participants of
biomonitoring studies in Germany (Ethical committee of the
University Hospital Münster, Germany, file reference:
2014187-f-S). Of each sample 100 μL of blood was spotted four
times on filter paper cards.
Serum was prepared by whole blood collection in
Monovette® Serum-Gel tubes and centrifugation at 3000×g
for 10 min. Afterwards, the supernatant was used as serum
sample. For sample preparation, 100 μL of whole blood or
serum was pipetted on Whatman 903 protein saver cards™
and dried over night at room temperature to obtain DBS and
DSS. In order to assure no overlapping of the DSS, only three
of the five marked spotting positions were used when 100 μL
serum was applied to the cards, as serum spots show a larger
diameter compared do DBS due to lower viscosity. Next,
100 μL DBS or DSS were completely cut out leaving approx.
1 mm space between the cutting edge and the visually detected
border of the spot, followed by extraction with 1 mL
extraction mixture consisting of water/acetone/acetonitrile
(30:35:35, v/v/v) in 2 mL safe-lock tubes for 30 min under
sonication. After extraction, an aliquot of 800 μL was
transferred into a new 2-mL safe-lock tube and evaporated
to dryness at 50 °C under reduced pressure. The residues were
reconstituted with water/acetonitrile/acetic acid (95:5:0.1, v/v/
v) followed by centrifugation at 22,000×g for 10 min and
injection of 30 μL of the supernatant into the HPLC-MS/MS
system. Sample preparation was performed in duplicate for
the volunteer samples and in triplicate for each fortified
Analysis was carried out on a 1260 Infinity LC system
(Agilent, Waldbronn, Germany) coupled to a QTRAP
6500 mass spectrometer (SCIEX, Darmstadt, Germany).
During method development four different column
rials, Nucleodur C18 ISIS, Pyramid, Gravity, and Gravity
SB (all Macherey-Nagel, Düren, Germany) were
compared for retention behavior and peak shape (see
Electronic supplementary material). Finally, a Nucleodur
C18 Gravity SB column (3 μm, 2.0 × 100 mm) equipped
with a guard column (2.0 × 4.0 mm) of the same material
was used at a column temperature of 45 °C. Eluent A was
acetonitrile containing 2% acetic acid and eluent B was
water with 0.1% acetic acid. The binary gradient and flow
rate was set up as follows: 0 min, 3% A (750 μL/min);
3 min, 15% A (750 μL/min); 4.5 min, 55% A (750 μL/
min); 6.0 min, 55% A (850 μL/min); 8.0 min, 100% A
(850 μL/min); 10.0 min, 100% A (850 μL/min);
10.1 min, 3% A (750 μL/min); 11.5 min, 3% A
(750 μL/min). Electrospray ionization (ESI) was
performed in both polarities with +5500 or −4500 V in
scheduled multiple reaction monitoring (sMRM™)
detection mode. Latter increases the number of data points as
l es s t r a n s i t i o n s a r e mo n i t o r e d a t t h e s a m e t i m e .
Furthermore, longer dwell times are possible resulting in
decreased noise levels and better signal-to-noise ratios.
Analyst 1.6.2 software was used for data acquisition. In
order to avoid a high entry of unwanted polar compounds
into the mass spectrometer, a diverter valve was applied
between column and mass spectrometer allowing to
discard the first 2.6 min of each chromatographic run before
entering the ion source. ESI-source parameters were
optimized and set to a source temperature of 500 °C, curtain
gas to 40 psi, the collision activated dissociation gas to
Bhigh^, GS1 (nebulizer gas) to 45 psi and GS2 (heater
gas) to 50 psi. The two MRM transitions which showed
the best signal-to-noise ratios were monitored per analyte,
the one with the highest signal intensity was selected as
quantifier. Table 1 lists detailed HPLC-MS/MS
parameters, and Fig. 1 shows a reconstructed HPLC-MS/MS
chromatogram with the quantifier transition of each
271 [M − H]−
257 [M − H]−
784.4 [M + H]+
471 [M − H]−
704.4 [M + Na]+
668.4 [M + H]+
640.4 [M + H]+
662.4 [M + Na]+
654.4 [M + H]+
404.1 [M + H]+
489 [M + Na]+
319 [M − H]−
317 [M − H]−
Detailed scheduled multiple reaction monitoring (sMRM) parameters for all mycotoxins quantified
MS transitions are listed for quantifier on top and qualifier below for each analyte. Expected retention time (Exp. tR) is equivalent to programmed time for
sMRM algorithm and set against actual measured mean retention time (mean tR) followed by standard deviation (SD) of all analyzed calibration and
a Quantifier top, qualifier below
b Expected retention times, as they were programmed for scheduled MRM algorithm
c Analyte mean retention time ± SD [min]
d OTA and 2’R-OTA show identical fragmentation patterns but are chromatographically separated with OTA eluting before 2’R-OTA
Fig. 1 Reconstructed HPLC-MS/MS chromatogram of whole blood (DBS) recovery samples spiked with 27 analytes. Quantifier transition and
retention times are given in brackets for each signal
Calibration, recovery, and stability testing
Matrix-matched calibration was applied by adding standards to
dried blood spot or dried serum spot extract. As no blood
sample without detectable amounts of OTA and EnB was available,
a sample with low natural contamination of both compounds
was used to evaluate method parameters. OTA validation was
performed using the diastereomer 2’R-OTA as previously
described . The level of EnB in the matrix solution was
determined as cEnB, Matrix = 0.0361 ± 0.0011 ng/mL via standard
addition and taken into account for all further calculations.
Matrix-induced signal suppression and enhancement (SSE)
was assessed by calculating the ratio of the slopes of
matrixmatched and pure solvent calibration curve according to the
following equation (see Tables 2 and 3):
slopematrix‐matched calibration *100
slopepure solvent calibration
Values of 100% correspond to no impairment of analyte
detection by matrix while SSE > 100% describes a signal
enhancement due to a positive matrix effect and vice versa.
For every analyte, a calibration curve containing at least six
points in the working range (Tables 2 and 3) was generated by
dilution of the working solutions. Calibration solutions were
freshly prepared for every measurement day. Reproducibility
was determined by analyzing one real sample of the
previously mentioned German sample cohort, naturally containing
OTA, 2’R-OTA, and EnB, as quality control sample. The
determination of LOD and LOQ was based on a signal to noise
ratio (S/N) of 3 for LOD and S/N of 10 for LOQ. The
calculated LODs are given in Tables 2 and 3.
Recovery rates were determined by spotting blood and
serum samples spiked with low, medium, and high analyte
concentrations of the working range on DBS cards, followed
by standard sample preparation procedure as described above.
Three independent replicates for each concentration were
prepared, analyzed, and quantified via the respective
matrixmatched calibration for calculation of the recovery. Interday
performance was evaluated by analysis of fortified recovery
samples in both matrices on three different days. On the first
day, the samples were worked up in triplicate and on the other
2 days in duplicate for all spiking levels. Detailed data of
spiked concentrations, single and averaged results, and
relative standard deviations are shown in Tables 2 and 3.
Stability testing of DBS/DSS was done with fortified
samples which were stored at room temperature (T = 20 °C) and in
the dark, as these parameters are used for storage and shipping
of DBS when pharmaceutical and medical analysis are
applied. The samples were analyzed in duplicate for low,
medium, and high spiking levels after storage for 1-10 weeks.
Based on these values, mean relative degradation rates were
calculated by comparison to freshly prepared recovery
samples. Furthermore, two sets of spiked samples were prepared
up to 24 weeks and stored at 4 °C in a fridge as well as in a
freezer at −18 °C. The batches were kept in plastic boxes until
analysis. Humidity during storage in laboratory and fridge was
monitored by a hygro-/thermometer and was consistent at
60 ± 10%.
Results and discussion
In order to develop a multi-mycotoxin method based on dried
blood spots (DBS) and dried serum spots (DSS), HPLC-MS/
MS analysis of 27 mycotoxins and metabolites was performed
in positive and negative mode with electrospray ionization
and scheduled multiple reaction monitoring (sMRM™).
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Analytes have been selected based on their occurrence,
toxicological relevance, and availability [1, 23–29]. Polarity of the
compounds was not considered as an important criterion for
this study despite the fact that glucuronides are usually more
relevant biomarkers for urinary exposure assessment than for
blood-based human biomonitoring. However, kinetic studies
in humans or animals might require the detection of these
polar metabolites in order to investigate the metabolic activity
and biotransformation rates of individuals.
For sampling, the previously developed method  was
extended and optimized to cover a larger set of 27 analytes and
to include serum samples. A defined blood or serum volume
of 100 μL was spotted on standard filter paper cards, followed
by drying, cutting the entire spot out of the paper, and an
extraction with a mixture containing 35% acetonitrile, 35%
acetone, and 30% water. After the extraction, an aliquot of
the extract was evaporated to dryness, reconstituted, and
centrifuged, leading to a clear and colorless solution. During
optimization of the extraction procedure, it was observed that the
applied centrifugal force has a strong impact on the
subsequent HPLC-MS/MS analysis. Centrifugation at 22,000×g
was superior in matrix removal compared to the previously
applied 3000×g. For example, centrifugation at 22,000×g
yielded a higher signal intensity and a lower S/N ratio for
FB1 (see Fig. S1, Electronic Supplementary Material (ESM)).
Optimization of the HPLC-MS/MS conditions
Special attention was paid to column selection, solvent
additive and gradient to optimize peak shape, and signal response
for all compounds from this heterogeneous group of analytes.
Besides Nucleodur C18 Gravity SB, columns of the same sizes
packed with ISIS, Pyramid and Gravity materials were
evaluated. Matrix compounds in the extracted blood spot solutions
strongly influence analyte separation while simultaneously
interfering mass spectrometric detection by falsely triggering the
measured transitions. Therefore, reducing the noise level
closely to the expected retention times of the substances was
mandatory. Another point was the baseline separation of OTA and
2’R-OTA within a short LC gradient as both isomers show the
same MRM transitions. Both aims were preferable achieved by
use of Gravity SB material when extracted matrix solutions
spiked with the analytes were injected. The selected column
material showed good retention properties for all analytes, in
particular for OTα and DH-CIT which usually elute near the
parent compounds OTA and CIT, respectively. Additionally,
previously used formic acid as eluent additive was replaced by
acetic acid. Despite being the weaker acid, chromatographic
separation was still excellent. The pH gradient of pH 3.5 to pH
2 caused by fortified concentrations of 0.1% acetic acid in
water and 2% acetic acid in acetonitrile, respectively, led to
improved peak shapes for DON as well as DH-CIT.
Moreover, the weaker protonation properties resulted in higher
ionization yields and therefore increased LODs. For instance,
the chromatograms in Fig. S2 (ESM) demonstrate the
influence of eluent additive on signal intensity and S/N ratio.
Figure 1 shows a reconstructed HPLC-MS/MS
chromatogram of spiked DBS recovery samples including all 27
compounds. All analytes and their phase-II-metabolites as well as
OTA and 2’R-OTA were baseline separated to ensure no
detection of artifacts. Chromatographic retention of the first eluting
mycotoxin DON was adjusted to about 3.1 min in order to
discar d unwan ted c om po unds by a diverter v al ve .
Furthermore, to achieve maximum sensitivity with short dwell
times, scheduled MRM was applied, reducing the detection
window for every MRM to 30 s around the expected retention time.
Table 1 lists all detailed parameters for MS/MS detection.
Moreover, programmed expected retention times are shown
and set against those actually determined of all analyzed
calibration and recovery standard solutions. During this analysis,
over a period of 12 weeks, retention time shifts below ±7 s were
observed, providing the possibility to apply scheduled MRM to
achieve maximum sensitivity with short dwell times. The
windows for every MRM were set at 30 s, except for the first and
last eluting compounds DON and DH-CIT as it was expected
that both would be the least accurately eluting. In fact, these
analytes had some of the highest standard deviations
concerning mean retention times as demonstrated in Table 1.
However, with a variation of about ±6 s for DH-CIT, a retention
time window of 30 s would have also been sufficiently broad.
Since matrix effects are a challenge in mass spectrometric
detection, the aim was to obtain a matrix effect at a maximum
of 10% of its intensity compared to neat eluent solutions in
order to decrease the limit of detection as low as possible.
Thus, an injection volume of 30 μL compromises on absolute
signal intensity as well as matrix suppression (Tables 2 and 3).
As no blank matrix solution was available, a blood sample with
a low natural concentration of OTA and EnB was used for
matrix-matched calibration. Figure 2 shows in sections A and
B the extracted ion traces for both analytes in whole blood and
serum matrix. The used matrix solution lacked the presence of
2’R-OTA, which was used for the determination of validation
parameters of OTA. Chromatograms C and D in Fig. 2 show
the ion traces of a human blood sample containing OTA,
2’ROTA, and EnB which was used as quality control sample.
The diastereomers OTA and 2’R-OTA are baseline
separated and show the same ratio of the signal transition intensities
in both processed whole blood and serum samples matrices.
Reproducibility was investigated by the analysis of control
Fig. 2 Extracted ion
chromatograms of OTA/2’R-OTA
(yellow transition m/z 404.1 →
239.0, blue transition m/z
404.1 → 102.0) and EnB (green
transition m/z 640.4 → 196.2,
orange transition m/z 662.4 →
336.3) in whole blood (a and c)
and serum (b and d). a, b Analyte
signals for the used matrix
solutions containing 0.291 ng/mL
OTA and 0.0349 ng/mL EnB. c, d
A real sample which was used as
quality control sample containing
0.283 ng/mL OTA, 0.193 ng/mL
2’R OTA, and 0.0366 ng/mL EnB
samples over a period of 7 days for whole blood and serum.
For this, a sample naturally contaminated with OTA,
2’ROTA, and EnB was chosen for performance control in order
to provide results most closely to available samples.
Therefore, quality control samples were freshly prepared and
analyzed on each measurement day and reproducibility was
accepted with 7.5% for OTA, 7.2% for 2’R-OTA, and 4.2%
for EnB, consequently.
Validation experiments were carried out by analyzing
linearity, matrix effects, recovery, limit of detection (LOD), and limit
of quantification (LOQ) for the serum (Table 2) and whole
blood (Table 3) as matrices. Determination of LOD and
LOQ was made via linear regression of mean signal to noise
ratios of the four lowest matrix calibration standards of each
compound. Matrix effect (SSE) calculation was done by
comparison of signal intensities in aqueous and matrix solutions
by means of the slopes of both calibration curves (see
Experimental section for details). Furthermore, the
chromatograms indicate no distinct difference between the
two matrices concerning the noise levels around the signals
Best limits of detection were achieved for the enniatins at
about 0.001-0.006 ng/mL in both matrices. Next, the
aflatoxins, beauvericin, CIT, 10-OH-OTA, OTα, and
OTA/2’ROTA showed good LODs with calculated values up to
0.015 ng/mL. Poorest responses have been determined for
HT-2 and DON-3-GlcA with about 1.3–1.4 ng/mL for both
analytes. Regression coefficients (R2) of 0.9999 to 0.9784 were
accomplished by linear regression analysis. Maximum
calibration concentrations were set at 50 ng/mL as higher blood
contaminations were not anticipated. The LOQ values obtained
with this new method indicate that the DBS-based sample
preparation technique in combination with modern mass
spectrometers is able to reach LOQ values comparable to those of
previously published analyte-specific methods. For example,
current OTA detection methods based on liquid-liquid extraction
of the plasma, urine, or human breast milk lead to similar LOQs
of 0.03 ng/mL . Moreover, quantification of CIT can be
carried out by immunoaffinity column cleanup resulting in a
LOQ of 0.15 ng/mL for blood plasma samples . Analysis of
beauvericin and enniatins in pig plasma using HPLC-MS/MS
after solid phase extraction reached LOQs of 0.1–0.2 ng/mL
which are about one decade higher than the values achieved
here (Table 3). Similar method parameters were acquired when
human plasma samples were analyzed .
Recovery rates were determined by spiking blood and
serum samples with low, medium, and high analyte
concentrations (see Tables 2 and 3 for spiking levels). As shown in
Tables 2 and 3, average recovery rates of 80 to 120% were
achieved for 24 out of 27 mycotoxins in the serum and whole
blood. FB1 and DON reached lower recovery rates of 61 ± 6.0
and 77 ± 10.3%, whereas DON-3-GlcA showed a slightly
higher recovery rate of 130 ± 5.1% in serum matrix. In whole
blood, the lowest recovery rate was obtained for AFG1 with
81 ± 5.6% and the highest with 132 ± 6.2% for HT-2-4-GlcA.
For DON-3-GlcA, the determined mean recovery in DBS
was 249%, which is out of the range for reliable quantitative
analysis. The high polarity of this compound as well as the
low slope of the linear calibration curve due to strong matrix
effects are possible explanations. Carry-over effects through
the injection system could be excluded as injections of blank
solutions after the highest standards were always negative for
all compounds. Besides DON-3-GLcA, also DON-15-GlcA is
reported as main metabolite of DON in humans [28, 40–43].
Both compounds coelute on most chromatographic systems
and show mostly identical fragmentation behavior. As only
DON-3-GlcA was available, this was used as reference and
an accidental detection of coeluting DON-15-GlcA was
Matrix effects due to signal suppression or enhancement
are a major concern and a well-known phenomenon in mass
spectrometry. Consequently, its consideration is
recommended for the establishment of new analytical methods. For
example, matrix suppression down to 40% signal intensity for
CIT can be determined in urine samples even after
liquidliquid extraction followed by solid phase extraction .
Therefore, high signal suppression due to only little matrix
removal was expected and observed for most of the analytes
in both matrices, as it is often described for multi-analyte
biomonitoring methods [11, 45].
Among all compounds AOH undergoes the strongest
matrix effect in whole blood resulting in a signal intensity of only
14% compared to the standard solution in neat solvent. The
lowest signal intensity in the serum was observed for
alternariol monomethyl ether which was only 13% compared
to the standard solution. OTA and 2’R-OTA show in both
matrices only minor alteration concerning signal intensity
compared to blank solutions. In contrast, FB1, CIT, and the
enniatins undergo strong signal enhancement, which has
particularly for EnB already been reported . Enniatins stand
out with positive matrix effects of up to 939% for EnA in
processed serum matrix. Interestingly, beauvericin shows a
decent signal suppression when detected in whole blood and
an increased signal intensity in serum matrix (SSE of 56 and
Although the levels of SSEs are severe for some analytes,
the matrix effects are highly reproducible as can be seen by
their relative standard deviations (RSD). For example,
calibration curves for EnA and AME are shown in Fig. S3 (ESM).
For the calculations of RSD, five sets of matrix-matched
calibration and neat solvent calibration curves were used
(Tables 2 and 3). As a result, only slight differences between
both matrices were observed for most analytes. Endogenous
blood compounds are assumed to be co-extracted and to
interfere mass spectrometric detection. Due to the lack of blood
cells, we expected a lower matrix influence of serum
compared to whole blood; however, the results do not support this.
Erythrocytes appear to be intact but are distorted from
dehydration in DBS samples . However, it can be supposed
that cells are destroyed during the extraction procedure by
applying organic solvents as well as sonication. In fact, not
all analytes show comparable SSE in both extracted matrices
besides beauvericin. Nevertheless, determined recovery rates
are comparable for both matrices. In order to study any
influence of different matrix samples on DBS analysis, five
randomly chosen blood matrices from the study cohort (n = 50)
were fortified with the highest spiking level in triplicate. The
results are shown in ESM Table S1 and no significant
differences regarding the average recovery rate compared to the
prior obtained results were observed (P > 0.05). In summary,
the developed method achieves reliable validation parameters
for mycotoxin detection in DSS and DBS including a broad
range of analyte polarities. The developed method
accomplishes a stable and robust quantification performance by use
of matrix-matched calibration without the need of isotopically
labelled standards, which are not commercially available for
most of the mycotoxin metabolites as internal standards.
A crucial point in DBS and DSS analysis is the stability of the
analytes on the filter cards. Especially as DBS cards are
usually handled and shipped at room temperature, a certain
ageing of the sample material can be expected. Thus, the next step
in method validation was stability testing of the analytes in
DBS and DSS matrices. On the basis of fortified recovery
samples, relative recovery rates were investigated by storing
the DSS and DBS cards in the dark at room temperature (T =
20 °C), at 4 °C as well as −18 °C.
The stability of all 27 compounds was analyzed and the
results after storage for 1, 5, and 10 weeks at room temperature
and after storage for 24 weeks at 4 and −18 °C are summarized
in Table 4 for whole blood (the results for serum can be found
in Table S2 (ESM) as no clear difference between both DBS
and DSS matrix was observed when samples were stored at 4
or −18 °C). Detailed relative recovery rates for storage at
Table 4 Average relative analyte concentration ± RSD [%] of mycotoxins when stored as dried blood spots for 1, 5, and 10 weeks at room temperature
and 24 weeks at 4 and −18 °C in the dark
Freshly prepared recovery solutions were set at 100% and corresponding recovery rate after storage are depicted. Color gradient emphasizes the level of
nearly no degradation (≥95%, green) towards the highest (≤ 50%, red) with color steps of 5%
20 °C for 1–10 weeks are shown in Figs. S4 and S5 (ESM).
Besides OTA/2’R-OTA, all other compounds showed a
timedependent degradation at room temperature. Nearly all mean
recovery rates decrease already within the first week after
sample collection (Table 4). The enniatins,
HT-2/HT-2-4GlcA as well as 10-OH-OTA and OTα show a moderate
reduction over the 10-week period resulting in remaining toxin
concentrations of 37–75% after 10 weeks. The other
compounds undergo a relatively fast degradation under these
typical storage conditions to only 4–37% of the original
concentration. This trend can be observed for both matrices, DBS as
well as DSS (for the DSS results see Table S2, ESM).
However, storage for shorter periods like 1 week at room
temperature, a time frame sufficient for typical sample
shipping, still provides recoveries of 61% for DH-CIT, 63% for
AFG1, and for all other compounds at least 74% in serum. In
whole blood, after 1 week only DH-CIT (45%), AOH (64%),
and ALT (65%) show higher degradation, while all other
mycotoxins or metabolites show at least a recovery of 75% of the
Although room temperature is the typical condition for
shipping and storage of DBS in clinical studies, the relative
recovery rates of fortified DBS and DSS were investigated
when the samples were stored at 4 and −18 °C up to 24 weeks.
As representative example, stability curves for AFB1 under all
storage conditions are shown in Fig. 3. The summarized
results in Table 4 clearly demonstrate that most analytes are
relatively stable when stored at 4 or −18 °C. After 24 weeks
of storage, all recovery rates were still above 76 and 88% at 4
and −18 °C, respectively. During all studies, humidity was
held at a constant level of 60 ± 10%, which corresponds to
typical laboratory conditions.
Thus, we recommend to store the dried samples under light
exclusion in sealed containers at −18 °C to minimize potential
degradation of the mycotoxins as discussed above. To our
knowledge, this is the first evaluation of mycotoxin stability
in dried physiological samples such as DBS and DSS. The
results clearly show that sample storage under optimal
conditions is safe but can become an issue when temperature above
4 °C is reached.
Fig. 3 Relative recovery rates of AFB1 in fortified DBS and DSS; storage at room temperature (20 °C) for 1-10 weeks and at 4 °C as well as −18 °C for
Biomonitoring of blood samples
The developed multi-mycotoxin method was applied for the
analysis of blood samples of a German sample cohort which
have already been analyzed in our first DBS-study for OTA
and 2’R-OTA . For multi-mycotoxin analysis of this sample
cohort, DBS (n = 50) were prepared in duplicate and quantified
according to the matrix-matched calibration parameters. Besides
OTA and EnB, no other mycotoxins or metabolites were
detectable. Although DON and especially its phase-II-metabolite are
often detectable in human urine samples from Germany , it
seems that DON is rapidly converted and excreted as it is not
detectable in DBS samples. Figure 2 shows typical HPLC-MS/
MS chromatograms as the quality control is one sample out of
the German sample cohort.
Enniatin B was detectable in all samples with a mean value
of 0.0367 ± 0.0179 ng/mL and a median of 0.0330 ng/mL.
The lowest and highest concentrations were with 0.0144–
0.1071 ng/mL in the calibration range. Intake of EnB by food
is supposed to be the main reason of exposure. However, risk
assessment is currently not possible due to the lack of data for
dietary exposure as well as toxicity, so that no TDI is
established yet . Considering the low concentrations of
EnB in the analyzed DBS, this study contributes results for
the presence of EnB in human blood samples as it has already
been reported in urine and breast milk samples [11, 25].
Previously quantified OTA and 2’R-OTA  were
reanalyzed by this multi-mycotoxin method. OTA was found
in all samples as well as 2’R-OTA in the same samples as
before. However, some samples (n = 9 out of 34) showed
signals for 2’R-OTA between LOD and LOQ due to slightly
lower sensitivity of this multi-mycotoxin method. Therefore,
all positive findings were confirmed. Figure S6 (ESM)
demonstrates the correlation between the analyte amounts
calculated by both methods. The arithmetic mean of OTA (n = 50)
by  with cOTA = 0.211 ± 0.064 ng/mL is matching to
cOTA = 0.207 ± 0.063 ng/mL. Likewise, the average values
for 2’R-OTA are c2’R-OTA = 0.112 ± 0.092 ng/mL (n = 34)
and c2’R-OTA = 0.143 ± 0.100 ng/mL (n = 25), respectively.
Latter value is certainly higher because the amounts of nine
samples which show signals > LOD are not considered.
Calculated regression coefficients of R2 = 0.9043 (OTA) and
R2 = 0.9512 (2’R-OTA) support the comparability of both
sample preparations (Fig. S6, ESM). Thus, the results clearly
demonstrate the comparability of both methods for the
detection and quantification of OTA and 2’R-OTA in DBS.
A DBS- and DSS-based sample preparation technique for the
detection of various mycotoxins and metabolites was
established and validated. The developed multi-mycotoxin
method allows the simultaneous detection and quantification
of 27 analytes by HPLC-MS/MS in dried whole blood (DBS)
and serum spots (DSS). On the basis of previous DBS studies
[21, 22], the improved method has the same advantages such
as simplified storage and shipment conditions as well as
reduced use of chemicals and materials compared to other
The developed DSS and DBS-based method was evaluated
for linearity, limit of detection, and quantification, robustness,
recovery, and stability of fortified samples. It could be shown
that DSS and DBS are a suitable sampling technique for the
detection of multi-mycotoxin exposure due to adequate
validation parameters for most of the incorporated mycotoxins
and metabolites. Complications arise from stability issues
which have to be taken into account and for this reason storage
of samples at −18 °C is recommended. The developed method
was applied to DBS samples of a German cohort, showing
that besides OTA , all samples (n = 50) were positive for
EnB with mean levels of 0.0367 ng/mL. All in all, a novel
application for the use of DSS and DBS for multi-mycotoxin
exposure studies was developed and validated, proofing the
effective performance characteristics needed for biomarker or
biomonitoring approaches. As some mycotoxins or
metabolites are mainly detectable in urine (e.g., DON-3-GlcA) [10,
11] and others such as OTA, 2’R-OTA, EnB mainly in the
blood (Fig. 2), the analysis of both matrices is recommended
to evaluate the human and animal exposure to mycotoxins.
Besides dilute-and-shoot approaches used for urine samples,
the use of DBS and DSS provides an optimal extension of
human and animal biomonitoring due to the non-invasive
sample collection and easy sample preparation.
Compliance with ethical standards
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