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 <10 to >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 and sensitive 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 in results.

ExperimentalSample preparation

Corn, peanut, and black pepper were chosen as commonly regulated food crops of diverse matrix components for mycotoxins. 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 spiked into matrix extracts for analysis.

A 5 g sample of corn flour, 5 g of peanuts, or 2 g of black pepper were extracted with 10 mL of ACN, 10 mL H2O, and EN Extraction Salts (5982-5650). Dispersive SPE for fruits and vegetables (5982-5058) was used on corn, and a universal dispersive SPE 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 Corn: 2 µL, Peanut: 10 µL, Black pepper: 10 µL

Mobile phase A) Water, 0.5 mM NH4F + 5 mM NH4 formate + 0.1 % formic acid B) MeOH, 0.5 mM NH4F + 5 mM NH4 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 1/10th 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 1/20th the assigned MRL.

Table 2. Maximum residue limits for mycotoxins 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

EU MRL for mycotoxins2,3 Assigned MRL level used

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 >0.999 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. Calibration curves for PFOS, PFOA, N-EtFOSAA, and PFBS.

×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 (%

)

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 1/10th 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, May 30, 2017

5991-8162EN

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

×104

0 2 4 6 8 10 12 14 16 18 20

00.20.40.60.81.01.21.41.6

00.20.40.60.81.01.21.41.6

Resp

onse

Concentration (ng/mL)

Aflatoxin B10.1–20 ppbR2 = 0.999

×104

0 2 4 6 8 10 12 14 16 18 20

Resp

onse

Concentration (ng/mL)

Aflatoxin G20.1–20 ppbR2 = 0.999

×104

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.