ANALYSIS OF MYCOTOXINS IN FOOD MATRICES - … Mycotoxins are compounds produced by fungi that grow...
Transcript of ANALYSIS OF MYCOTOXINS IN FOOD MATRICES - … Mycotoxins are compounds produced by fungi that grow...
IntroductionMycotoxins are compounds produced by fungi that grow on crops ranging from grains to fruits, vegetables, nuts, and spices. Mycotoxins can be harmful to humans and livestock through consumption of contaminated crops; therefore, mycotoxin levels are monitored in foods to minimize the risk of ingestion1. Regulatory agencies around the globe set Maximum Residue Limits (MRLs), which range from less than 10 ng/g (ppb) to more than 500 ppb to ensure harmful levels of mycotoxins do not enter the food supply. It is important to detect and accurately quantify mycotoxin contents at low levels across various food matrices, as each matrix composition poses different detection challenges.
This study demonstrates the accurate quantification of up to 12 regulated mycotoxin compounds in three commonly regulated foods using the Agilent Ultivo Triple Quadrupole LC/MS. (Figure 1).
ANALYSIS OF MYCOTOXINSIN FOOD MATRICES
Agilent Ultivo Triple Quadrupole LC/MS System
For more information, visit:www.agilent.com/chem/Ultivo
Figure 1. Agilent Ultivo Triple Quadrupole LC/MS integrated into the Agilent 1260 Infinity II Prime LC.
Agilent Ultivo Triple Quadrupole LC/MSUltivo is designed to address many of the challenges faced by labs performing environmental and food safety analyses. The innovative technologies housed within Ultivo allowed us to achieve a reduced overall footprint, while conserving the performance found in many larger MS systems.
Innovations such as VacShield, Cyclone Ion Guide, Vortex Collision Cell, and Hyperbolic Quads maximize quantitative performance in a small package, enhancing instrument reliability and robustness, resulting in greater uptime. Ultivo reduces user intervention for system maintenance, making it easy for the nonexpert MS user to operate and maintain.
Agilent MassHunter software simplifies data acquisition, method setup, data analysis, and reporting. This results in the fastest acquisition-to-reporting time possible, increasing lab productivity and confidence.
ExperimentalSample preparation
Corn, peanut, and black pepper were chosen as commonly regulated food crops of diverse matrix components for mycotoxins analysis. Twelve mycotoxins in corn and peanut matrices, and five mycotoxins in black pepper matrix were quantified using dynamic MRM (dMRM) in a 9-minute LC/Triple Quad method. Mycotoxin standards were post-spiked into matrix extracts for analysis.
A 5 g sample of corn and peanut, or 2 g of black pepper were extracted with 10 mL of ACN, 10 mL H2O, and EN Extraction Salts (5982-5650). Dispersive SPE (dSPE) for fruits and vegetables (5982-5058) was used on corn, and a universal dSPE kit (5982-0029) was used for black pepper. A novel modified lipid removal sorbent inflow through a cartridge format was used on each matrix as a final cleanup step. Spiked black pepper extracts were diluted 30:70 extract/water prior to analysis.
Results and DiscussionMycotoxin signal response
Excellent precision and sensitivity was attained for mycotoxins in various food matrices due to a combination of sample preparation techniques, LC separation, and the innovative technology designed into the Ultivo triple quadrupole mass spectrometer.
Instrument parameters
Table 1. LC and MS parameters.
Agilent 1290 Infinity II LC
Column Agilent Eclipse Plus C18, 3.0 × 150 mm, 1.8 µm
Column temperature 45 °C
Injection volume 2 µL (Corn and peanut extract); 10 µL (black pepper extract)
Mobile phase A) Water, 0.5 mM ammonium fluoride + 5 mM ammonium formate + 0.1 % formic acid B) MeOH, 0.5 mM ammonium fluoride + 5 mM ammonium formate + 0.1 % formic acid
Flow rate 0.450 mL/min
Gradient Time (min) %B 0 30 0.5 30 7.5 100 9.0 100 9.1 30
Agilent Ultivo
Drying gas temperature 250 °C
Drying gas flow 8 L/min
Sheath gas temperature 350 °C
Sheath gas flow 12 L/min
Nebulizer pressure 30 psi
Capillary voltage 3,300 V(+), 2,800 V(–)
Nozzle voltage 0 V(+), 0 V(–)
Cycle time 500 ms
×102
2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.80
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Coun
ts (%
)
Acquisition time (min)
Figure 2. Detection of mycotoxins at 1/10 MRL in corn matrix.
Mycotoxin maximum residue limits and sensitivity
Outstanding sensitivity was achieved, with the majority of the mycotoxin compounds in each matrix studied reaching a limit of quantitation (LOQ) at 1/20 the assigned MRL.
Table 2. Maximum residue limits for mycotoxins and specific matrices used in this study. EU reg No. 1881/2006 and 105/2010 used for reference. All assigned MRLs in this study are equal to or lower than SANTE MRLs.
Mycotoxin
European Union MRL for mycotoxins2,3 Assigned MRL level used for this study
Corn Peanut Black pepper Corn and peanut Black pepper
Aflatoxin B1 2 ppb 2 ppb 5 ppba 2 ppb 5 ppb
Aflatoxin B2 Sum of Aflatoxins: 4 ppb
Sum of Aflatoxins: 4 ppb
Sum of Aflatoxins: 10 ppb
2 ppb 5 ppb
Aflatoxin G1 2 ppb 5 ppb
Aflatoxin G2 2 ppb 5 ppb
Ochratoxin A 3 ppb n/a 15 ppbb 3 ppb 15 ppb
Fumonisin B1 500 ppb n/a n/a 500 ppb Not included
Fumonisin B2 Sum of Fumonisins: 1,000 ppb
n/a n/a 500 ppb Not included
Fumonisin B3 n/a n/a 500 ppb Not included
Deoxynivalenol 750 ppb n/a n/a 75 ppb Not included
Zearalenone 100 ppb n/a n/a 100 ppb Not included
T-2 Toxin n/a n/a n/a 100 ppb Not included
HT-2 Toxin n/a n/a n/a 500 ppb Not included
Corn Peanut Black pepper0
2
4
6
8
10
12
14
No. o
f myc
otox
ins
Matrix
1/5 MRL1/10 MRL1/20 MRL
1210
11
3
2
Figure 3. Quantitation limit for all mycotoxins studied in each matrix, defined as a fraction of the assigned MRL.
Precision and linearity of mycotoxins
Excellent linearities were obtained for all compounds in this study, with R2 values >0.99 over the calibration ranges used. Precision values obtained for mycotoxins in the matrices investigated rendered %RSD values of <10 % at the LOQs.
Corn Peanut Black pepper02468
101214
No. o
f myc
otox
ins
Matrix
RSD 5–10 %RSD 0–5 %3 3
99
2
3
Figure 4. The mycotoxins show excellent precision in all three matrices, with all RSD <10 % at LOQ.
×102
5.50
CornAflatoxin B1200 pptRSD% = 1.75Avg. S/N = 326
5.55 5.60 5.65
00.10.20.30.40.50.60.70.80.61.0
Coun
ts (%
)
Acquisition time (min)
×102
5.50
PeanutAflatoxin B1200 pptRSD% = 2.34Avg. S/N = 169
5.55 5.60 5.65
00.10.20.30.40.50.60.70.80.61.0
Coun
ts (%
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Acquisition time (min)
×102
5.50
Blackpepper
Aflatoxin B1200 pptRSD% = 5.65Avg. S/N = 53.6
5.55 5.60 5.65
00.10.20.30.40.50.60.70.80.61.0
Coun
ts (%
)
Acquisition time (min)
Figure 5. Excellent precision demonstrated for Aflatoxin B1 at 1/10 MRL (200 ppt or 500 ppt) in all matrices.
www.agilent.comInformation, descriptions and specifications in this
publication are subject to change without notice.
© Agilent Technologies, Inc. 2017 Published in USA, June 22, 2017
5991-8162ENRev. 1.0
Conclusions• The Agilent Ultivo Triple Quadrupole LC/MS is an exceptionally innovative new
mass spectrometer that can minimize laboratory workspace needs, as well as reduce maintenance challenges, creating a productive work environment for high-throughput laboratories.
• Ultivo is a small, yet powerful tool enabling the accurate and sensitive detection of commonly regulated mycotoxins in various food matrices well below established MRL levels.
• Agilent MassHunter software provides an easy-to-use, all-inclusive tool for managing and reporting LC/MS data.
×103
0 3 6 9 12 15 18 21 24 27 300
0.40.81.21.62.02.42.83.23.64.04.4
Resp
onse
Concentration (ng/mL)
Ochratoxin A0.5–30 ppbR2 = 0.999
×103
0 75 150 225 300 375 450 525 600 675 7500
0.30.60.91.21.51.82.12.42.73.03.3
Resp
onse
Concentration (ng/mL)
Deoxynivalenol0.375–750 ppbR2 = 0.999
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00.20.40.60.81.01.21.41.6
00.20.40.60.81.01.21.41.6
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Aflatoxin B10.1–20 ppbR2 = 0.999
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Resp
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Concentration (ng/mL)
Aflatoxin G20.1–20 ppbR2 = 0.999
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0 800 1,600 2,400 3,200 4,000 4,800
00.61.21.82.43.03.64.24.85.46.0
Resp
onse
Concentration (ng/mL)
HT-2 Toxin25–5,000 ppbR2 = 0.999
×104
0 75 150 225 300 375 450 525 600 675 750
00.81.62.43.24.04.85.66.4
Resp
onse
Concentration (ng/mL)
Zearalenone5–1,000 ppbR2 = 0.999
Figure 6. Exceptional linearity was demonstrated for all compounds, with R2 values ≥0.99 for all compounds, in all matrices. Pictured above, examples of linearity for 6 mycotoxins in corn matrix.
References1. Bennett, J. W.; Klich, M.
Mycotoxins. Clinical Microbiology Reviews 2003, 16(3), 497-516.
2. Commission Regulation (EC) No 1881/2006. Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union. L 364/5-24. 19 December 2006.
3. Commission Regulation (EU) No 105/2010. Amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. L 35/7-8. Official Journal of the European Union. 5 February 2010.