Application
Note: 523
Detection of Mycotoxins in Corn Meal Extract
Using Automated Online Sample Preparation
with LC-MS/MS
Yang Shi, Catherine Lafontaine, François Espourteille
Thermo Fisher Scientific, Franklin, MA
Introduction
Key Words
s4RANSCEND4,8
s4URBO&LOW
4ECHNOLOGY
s4316ANTAGE
s&OOD3AFETY
Since the discovery of aflatoxin in 1960, mycotoxin
research has received considerable attention. Mycotoxins
are a group of naturally occurring toxic substances
produced by certain molds, which can contaminate food
and feed. The inhalation or absorption of mycotoxins
into the body may cause harm, including kidney or liver
damage, cancer, or even death in man or animals.1 From
a food safety perspective, the aflatoxins, ochratoxin A,
patulin, fumonisins, trichothecenes, and zearalenone are
the mycotoxins of major concern.
Many countries now monitor mycotoxin levels in food
and feed products. Liquid chromatography-tandem mass
spectrometry (LC-MS/MS) is currently a common analytical approach for the quantification of mycotoxin contamination.2 Sample preparation for LC-MS/MS analysis can
be time and labor intensive, often involving pH modification, solid phase or immunoaffinity column clean-up
extraction, multi-step extract clean-up, and pre-concentration.3 The strict regulation published by the European
Union in 1999 asking for lower detection limits and higher
method reliability presented a new analytical challenge.4
In this study we describe an easy, comprehensive,
LC-MS/MS method using a Thermo Scientific Transcend
TLX-1 system powered by Thermo Scientific TurboFlow
technology to analyze multiple mycotoxin residues in corn
meal extract. Figure 1 illustrates a typical Transcend™
TLX-1 system with the Thermo Scientific TSQ Vantage
triple stage quadrupole mass spectrometer.
Goal
Develop a rapid and sensitive automated, online sample
preparation LC-MS/MS method to detect and quantify
multiple mycotoxins in corn meal extract resulting in a
shorter assay time and increased throughput.
Experimental
4HEMATRIXSTANDARDCURVE
Five grams of corn meal purchased from a local grocery
store were extracted using 25 mL of 70% methanol in
water followed by 60 minutes of ultra-sonication. The
extract sat overnight at room temperature. The resulting
solution was then centrifuged at 6000 RPM for
20 minutes. The supernatant was used to prepare the
matrix calibrators and QC samples. Each milliliter of
supernatant corresponds to 0.2 g solid corn meal powder
as the unit of conversion.
Figure 1. Thermo Scientific Transcend TLX system with TSQ Vantage triple quadrupole mass spectrometer
The stock mix solution of analytes was prepared in
methanol. Table 1 lists selected reaction monitoring
(SRM) transitions and stock concentrations for individual
analytes. Eight mycotoxins were analyzed under positive
electrospray ionization (ESI) mode. The remaining three
compounds, deoxynivalenol (DON), nivalenol (NIV), and
3-acetyl-DON (3-AcDON), were analyzed under negative
electrospray ionization (ESI) mode.
,#-3-ETHODSUSINGNEGATIVE%3)MODE-ETHOD"
Table 1. Analytes list
#OMPOUNDS
3TOCK
0ARENT 0RIMARY 3ECONDARY CONCENTRATION
(m/z)
(m/z)
(m/z)
«GM,
4URBO&LOW-ETHOD0ARAMETER
Column:
Research column A 0.5 x 50 mm
Injection Volume:
10 µL
Solvent A:
water
Solvent B:
methanol
Solvent C:
0.1% ammonium hydroxide
Solvent C:
45:45:10 ACN: isopropanol: acetone (v:v:v)
(0,#-ETHOD0ARAMETERS
Aflatoxins B1
313
241
285
0.050
Aflatoxins B2
315
259
287
0.015
Aflatoxins G1
329
243
283
0.050
Aflatoxins G2
331
245
275
0.015
Zearalenone (ZEA)
319
187
185
10.000
Ochratoxin A (OTA)
404
239
221
1.000
-ASS3PECTROMETER0ARAMETERS
Fumonisins B1 (FB1)
722
334
352
2.500
MS:
Fumonisins B2 (FB2)
706
336
318
2.500
TSQ Vantage triple stage quadrupole mass
spectrometer
Deoxynivalenol (DON)
295
138
265
20.000
MS Ionization Source:
H-ESI
Nivalenol (NIV)
311
281
205
20.000
Spray Voltage:
4.5 kV
3-Acetyl-DON (3-AcDON)
337
307
173
20.000
Sheath Gas Pressure (N2):
50 arbitrary units
,#-3-ETHODSUSINGPOSITIVE%3)MODE-ETHOD!
4URBO&LOW©-ETHOD0ARAMETERS
Column:
TurboFlow Cyclone-P 0.5 x 50 mm
Injection Volume:
10 µL
Solvent A:
10 mM ammonium acetate in water
Solvent B:
0.1% formic acid in acetonitrile (ACN)
Solvent C:
1:1:1 ACN: isopropanol: acetone (v:v:v) with
0.3% formic acid
Analytical Column:
Hypersil GOLD™ 2.1 x 50 mm, 1.9 µm
Solvent A:
0.1% formic acid in water
Solvent B:
0.1% formic acid in ACN
Auxiliary Gas Pressure (N2): 20 arbitrary units
Vaporizer Temperature:
250 °C
Capillary Temperature:
270 °C
Collision Gas Pressure:
1.5 mTorr
The LC method views from Thermo Scientific Aria
Operating Software are shown in Figures 2 and 3.
(0,#-ETHOD0ARAMETERS
Analytical Column:
Thermo Scientific Hypersil GOLD 2.1 x 100 mm,
1.9 µm
Solvent A:
0.1% formic acid in water
Solvent B:
0.1% formic acid in ACN
Figure 2. Method A view in Aria OS software
-ASS3PECTROMETER0ARAMETERS
MS:
TSQ Vantage™ triple stage quadrupole mass
spectrometer
MS Ionization Source:
Heated Electrospray Ionization (H-ESI)
Spray Voltage:
5 KV
Sheath Gas Pressure (N2):
50 arbitrary units
Auxiliary Gas Pressure (N2): 20 arbitrary units
Vaporizer Temperature:
209 °C
Capillary Temperature:
270 °C
Collision Gas Pressure:
1.5 mTorr
Figure 3. Method B view in Aria OS software
Results and Discussion
Figure 6 presents the linear fit calibration curves for
DON and NIV, indicating excellent linear fits over the
dynamic range. Table 3 summarizes detection, quantitation
limits, and standard curve linearity for three analytes analyzed in negative ion mode. For all analytes, the quantitation limits obtained using the present methodology comply
with the maximum levels in foods defined by European
Union.6 To the best of our knowledge, this is the first application of its type to detect these three compounds using an
automated online sample preparation technique coupled to
tandem mass spectrometry.
In addition, a lower limit of quantitation (LOQ) could
be achieved by increasing sample injection volume since
TurboFlow columns can handle larger injections (up to a
few hundred microliters) while regular HPLC or UHPLC
columns can not.
Figure 4 shows the comparison of chromatograms of eight
analytes at 1:100 dilutions in methanol and corn meal
extract, indicating excellent chromatographic separation
in both solvent standard and matrix. Matrix-matched
calibration standards showed linear response of two
orders of magnitude (r2 > 0.99) for six of them (Table 2).
Significant signal enhancement was observed for FB1 and
FB2 due to matrix-induced ionization variability, which
was previously reported by other researchers.5 In future
work, the isotope-labeled internal standard might be used
to compensate for the matrix interference.
Because DON, NIV, and 3-AcDON have a better
signal response under negative ionization mode, a separate
LC-MS/MS method was developed. Figure 5 shows the
chromatograms of DON, NIV, and 3-AcDON identified at
100 ng/mL fortified in the corn meal extract.
RT: 4.50 - 10.00
RT: 4.50 - 10.00
SM: 13G
NL: 5.78E5
TIC F: + p ESI SRM ms2 313.109
[241.138-241.148, 285.172-285.182]
MS ICIS 1_100_std_B_002
RT: 7.12
AA: 15634265
100
SM: 13G
50
NL: 5.22E5
TIC F: + p ESI SRM ms2 313.109
[241.138-241.148, 285.172-285.182] MS
ICIS 1_100_std_B_corn_002
RT: 7.12
AA: 11351226
100
50
NL: 9.61E4
TIC F: + p ESI SRM ms2 315.123
[259.150-259.160, 287.194-287.204]
MS ICIS 1_100_std_B_002
RT: 6.95
AA: 2970358
100
RT: 8.54
AA: 420771
50
50
NL: 3.35E5
TIC F: + p ESI SRM ms2 329.000
[242.995-243.005, 282.995-283.005]
MS ICIS 1_100_std_B_002
RT: 6.91
AA: 10343546
100
NL: 1.25E5
TIC F: + p ESI SRM ms2 315.123
[259.150-259.160, 287.194-287.204] MS
ICIS 1_100_std_B_corn_002
RT: 6.91
AA: 4028649
100
RT: 8.54
AA: 313113
NL: 3.60E5
TIC F: + p ESI SRM ms2 329.000
[242.995-243.005, 282.995-283.005] MS
ICIS 1_100_std_B_corn_002
RT: 6.91
AA: 8571712
100
50
50
NL: 3.88E5
TIC F: + p ESI SRM ms2 331.132
[245.127-245.137, 274.995-275.005]
MS ICIS 1_100_std_B_002
RT: 8.55
AA: 8499060
100
50
50
RT: 6.74
AA: 880570
RT: 8.66
AA: 7959175
100
50
RT: 8.24
AA: 24390610
100
RT: 8.55
AA: 5872986
100
NL: 4.19E5
TIC F: + p ESI SRM ms2 319.160
[115.089-115.099, 185.067-185.077,
187.136-187.146, 283.188-283.198]
MS ICIS 1_100_std_B_002
100
NL: 1.15E6
TIC F: + p ESI SRM ms2 404.151
[220.912-220.922, 239.079-239.089]
MS ICIS 1_100_std_B_002
100
RT: 6.69
AA: 781042
RT: 8.71
AA: 11177335
50
50
NL: 3.19E5
TIC F: + p ESI SRM ms2 331.132
[245.127-245.137, 274.995-275.005] MS
ICIS 1_100_std_B_corn_002
RT: 6.45
AA: 4886413
NL: 5.88E5
TIC F: + p ESI SRM ms2 319.160
[115.089-115.099, 185.067-185.077,
187.136-187.146, 283.188-283.198] MS
ICIS 1_100_std_B_corn_002
NL: 2.04E6
TIC F: + p ESI SRM ms2 404.151
[220.912-220.922, 239.079-239.089] MS
ICIS 1_100_std_B_corn_002
RT: 8.29
AA: 40293551
50
NL: 5.70E5
TIC F: + p ESI SRM ms2 706.140
[318.387-318.397, 336.336-336.346]
MS ICIS 1_100_std_B_002
RT: 7.54
AA: 14738742
100
50
50
RT: 8.87
AA: 3101293
NL: 3.22E5
TIC F: + p ESI SRM ms2 722.116
[334.341-334.351, 352.377-352.387]
MS ICIS 1_100_std_B_002
RT: 6.79
AA: 7724904
100
NL: 2.92E6
TIC F: + p ESI SRM ms2 706.140
[318.387-318.397, 336.336-336.346] MS
ICIS 1_100_std_B_corn_002
RT: 7.49
AA: 63767620
100
50
NL: 2.42E6
TIC F: + p ESI SRM ms2 722.116
[334.341-334.351, 352.377-352.387] MS
ICIS 1_100_std_B_corn_002
RT: 6.79
AA: 49577376
100
50
RT: 8.65
AA: 1441806
6
5
7
Time (min)
8
9
10
5
6
7
Time (min)
8
9
10
Figure 4. Comparison of chromatograms of 8 SRM analytes in methanol and corn flour extract
(1:100 dilution of stock mixture)
NL: 1.25E5
m/z= 137.63-138.63 F: - p ESI SRM ms2
295.140 [138.123-138.133,
247.161-247.171, 265.169-265.179] MS
ICIS 100ng_ml_002
RT: 3.71
AA: 916274
SN: 15540RMS
100
80
DON
60
40
20
3.43
4.18
0
NL: 1.89E5
m/z= 280.67-281.67 F: - p ESI SRM ms2
311.148 [119.108-119.118,
183.072-183.082, 191.115-191.125,
197.078-197.088, 203.087-203.097,
281.170-281.180] MS ICIS 100ng_ml_002
NIV
Relative Abundance
100
80
RT: 3.51
AA: 1020801
SN: 618RMS
60
4.00
40
20
0
NL: 1.70E5
m/z= 306.63-307.63 F: - p ESI SRM ms2
337.148 [173.126-173.136,
217.155-217.165, 307.125-307.135] MS
ICIS 100ng_ml_002
RT: 3.97
AA: 1142785
SN: 4961RMS
100
80
3-AcDON
60
40
20
0
3.0
3.61
3.14
3.2
3.4
3.6
Time (min)
3.8
4.0
4.2
Figure 5 Selected chromatograms of DON, NIV, and 3-AcDON detected at 100 ng/mL fortified
in the corn meal extract
Table 2. Limit quantitation (LOQ) and standard
curve linearity (r2) for analytes detected in
positive ion mode
#OMPOUNDS
,/1
NGG
0.50
0.9956
G1
0.50
0.9910
OTA
5.00
0.9937
ZEA
50.00
0.9955
FB1
12.50
0.9984
FB2
12.50
0.9965
Scientific maintains
,/1
NGG
Deoxynivalenol (DON)
25.00
0.9934
tative organizations
Nivalenol (NIV)
25.00
0.9933
throughout the world.
3-Acetyl-DON (3-AcDON)
25.00
0.9925
Deoxynivalenol
Y = 71516.1+10580*X R^2 = 0.9934 W: 1/X
a network of represen-
R2
Nivalenol
Y = 219026+8122.82*X R^2 = 0.9933 W: 1/X
12000000
9500000
11500000
NIV
9000000
11000000
DON
10500000
10000000
8500000
8000000
9500000
7500000
9000000
7000000
8500000
8000000
6500000
7500000
6000000
7000000
5500000
6500000
Area
Area
offices, Thermo Fisher
#OMPOUNDS
R2
B1
In addition to these
Table 3. LOQ and standard curve linearity for
analytes detected in negative ion mode
6000000
5500000
5000000
4500000
5000000
4000000
4500000
3500000
4000000
3000000
3500000
3000000
2500000
2500000
2000000
2000000
1500000
1500000
1000000
1000000
500000
500000
0
0
0
100
200
300
400
500
600
700
800
900
1000
0
1100
100
200
300
400
500
600
700
800
900
1000
1100
ng/ml
ng/ml
Figure 6. Calibration curves for DON and NIV
Conclusion
Developing a rapid and sensitive quantitative method is
always a major goal for mycotoxins analysis.7 Two quick,
automated online sample preparation LC-MS/MS methods
have been developed that are sensitive enough to detect
mycotoxins in corn meal extract. By eliminating manual
sample preparation, the reliability of this methodology was
improved significantly. The sample throughput could be
improved by multiplexing the two methods on different
LC channels using a Transcend TLX-2 (or TLX-4) system.
Future work will focus on the application of this methodology on various food matrices and references.
References
1
Pitt, J.I. What are mycotoxins? Australian Mycotoxin Newsletter 1996, 7(4),
1.
2
Spanjer, M.C., Rensen, P.M., Scholten, J.M. LC-MS/MS multi-method for
mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs. Food Addit. Contam. Part A
Chem. Anal. Control Expo. Risk Assess 2008, 25, 472-89.
3
Shephard, G.S., Determination of mycotixins in human foods, Chem. Soc.
Rev., 2008, 37, 2468-77.
4
Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting
maximum levels for certain contaminants in foodstuffs. http://eur-lex.
europa.eu/, accessed on Apr. 17, 2011.
5
Li, W., Herrman, T.J., Dai, S. Y., Rapid Determination of Fumonisins in
Corn-Based Products by Liquid Chromatography/Tandem Mass Spectrometry, J. AOAC Int., 2010, 93, 1472-81.
6
http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2006R18
81:20100701:EN:PDF. Accessed on Mar. 15, 2011.
7
Rahmani, A., Jinap, S., Soleimany, F., Quantitative and qualitative analysis
of mycotoxins, Compr. Rev. Food Sci. Food Safety, 2009, 8, 202-251.
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