Abstract

Aflatoxins are a type of mycotoxin that are toxic and extremely carcinogenic. Food for human consumption and feed for livestock are routinely tested for their presence. A sensitive analytical method for the determination and quantitation of four aflatoxins using the Bruker amaZon SL Ion Trap mass spectrometry system is presented here. Specifically, this method uses LC/MS/MS to quantify aflatoxins G1, G2, B1, and B2 in common food and feed matrices.

Introduction

Aflatoxins belong to a group of structurally similar secondary fungal metabolites produced primarly by Aspergillus flavus and A. parasiticus. Exposure to these compounds has been shown to cause cancer in humans and livestock. Aflatoxins have been classified as human liver carcinogens by the World Health Organization (WHO) and by the U.S. Environmental Protection Agency (USEPA). The United States Food and Drug Administration (FDA) has established action levels for aflatoxins present in food or feed to protect human and animal health down to 20 ppb [1]. The European Commission has set a maximum level for aflatoxin B1 of 8 ppb and the sum total of all four of these toxins of 15 ppb (μg/kg) in crops such as nuts,

Application Note # LCMS-61

Identification and Quantitation of four Aflatoxins using the Bruker amaZon SL Ion Trap Mass Spectrometer

groundnuts, grains, and dried fruits [2]. Routine quantitation of aflatoxins is therefore required to circumvent human and animal disease.

Ion trap mass spectrometry is a powerful analytical tool capable of measuring the mass to charge ratio (m/z) of ions. The analyte of interest can be isolated and fragmented once (MS/MS) or several times (MSn) to generate selective and rich molecular information. Here, the Bruker amaZonTM SL ion trap mass spectrometer (Figure 1) was coupled with High Performance Liquid Chromatography (HPLC) for quantitative analysis of four aflatoxins - B1, B2, G1 and G2 (Figure 2). By taking advantage of the versatility, high selectivity and sensitivity (MS/MS) of the amaZon SL, qualitative as well as quantitative analysis of a wide range of molecules is achieved simultaneously. The food sources used included peanuts, chili powder, corn meal, almonds and leafy greens representing the commonly affected crops of oilseeds, spices, cereals, nuts and salad and dried fruit.

Materials and Methods

Materials

Aflatoxin standards, G2, G1, B2 and B1 were obtained from Supelco (Bellafonte, PA). Ammonium acetate and formic acid (FA) were from Sigma (St. Louis, MO), while organic solvents were HPLC grade from Honeywell Burdick and

Jackson (Morristown, NJ). Samples of peanut, corn meal, chili powder, leafy greens and almonds were obtained from a local market and did not contain aflatoxins.

Sample Preparation

Liquid-liquid extractions were carried out as described by Sobolev [3]. Specifically, ground samples of peanut, corn meal, chili, and almonds (10 g) were mixed with 20 mL of MeOH/H2O (80/20, v/v) and thoroughly agitated using a vortex mixer. This liquid-liquid extract was then filtered through a 0.2 micron polypropylene filter. Standards were then prepared from a stock solution containing 1 µg/ml B1 and G1 and 0.3 µg/ml B2 and G2 in the filtered liquid-liquid extract from each food source. Matrix serial dilutions were performed to prepare standards down to 0.05 ppb. Leafy greens were prepared by the QuEChERs method as described by Anastassiades [4].

Chromatographic Conditions

Chromatographic separations were carried out using a Phenomenex Kinetex PFP column (2.1 x 150 mm, 2.6 μm, 100 Å) maintained at 40 oC on a Dionex Ultimate 3000 Rapid Separation LC system. The mobile phase consisted of (A) 5 mM ammonium acetate + 0.1 % FA and (B) 5 mM ammonium acetate + 0.1 % FA in MeOH. HPLC gradient conditions are shown in Table 1. 10 µL injections were made on column.

Ion Trap Parameters

The amaZon SL was equipped with the standard ESI ion source (Nebulizer pressure: 40 psi; Drying gas flow rate: 8 L/min; Drying gas temperature: 300 oC). MS/MS spectra were acquired in UltraScan (32,500 m/z / sec) mode between m/z 200-350, using positive ionization.

Results and Discussion

Optimization of MS and MS/MS parameters

Optimization of MS/MS parameters for each aflatoxin was carried out automatically using the Bruker Compass software by directly infusing 100 ppb stock aflatoxins in methanol (Figure 3, Table 2). Figure 3 shows an example of the MS/MS spectra of aflatoxins G1 and G2, respectively. The most intense fragment ion was used for quantitation, while the less intense fragment ions were used as qualifier

Quantitative analysis of four aflatoxins

Figure 1: Bruker amaZon SL ion trap mass spectrometer.

Figure 2: Chemical structures, molecular weights (MW) and chemical formulae of the four aflatoxins under investigation.

Time % B Flow Rate (µL/min)

0 30 200

2 30 200

7 75 200

12 75 200

12.01 30 200

14 30 200

Table 1: HPLC Gradient

Analyte

Retention time

(min)

MS/MS Transition

[qualifier] (m/z)

B1 9.75 313 → 285 [257]

B2 9.5 315 → 287 [259]

G1 9.25 329 → 311 [283]

G2 9.0 331 → 313 [285]

Table 2: HPLC and MS/MS transition parameters

ions. The ion trap allows for the detection of quantifier and qualifier ion(s) in a single MS/MS full scan rather than in separate scans.

Simultaneous Quantitation of Four Aflatoxins in Matrix

The PFP column provides chromatographic separation of the aflatoxins B1, B2, G1 and G2 with reproducible retention times in all matrices examined. Figure 4 shows the extracted ion MS/MS chromatograms (EIC) of aflatoxin G2 (3 ppb), G1 (10 ppb), B2 (3 ppb) and B1 (10 ppb). Various concentrations of aflatoxin standard solutions ranging from 0.015 ppb to 100 ppb were analyzed to evaluate the linearity of the calibration curves in each matrix. Figure 5 shows an example of the dynamic range of concentrations for which B1 can routinely be quantitated in different matrices.

Liquid-liquid extraction followed by filtration of peanut, almond and corn meal showed detection limits down to 0.05 ppb for B1 and G1 and 0.015 ppb B2 and G2 (S/N = 3, n = 3). The chili extract matrix and the QuEChERs extract of leafy greens showed detection limits down to 1 ppb. Table 3 shows the, limit of detection, linear dynamic range and R2 of spiked aflatoxin in the different matrices under the current experimental conditions.

Summary

The Bruker amaZon SL Ion Trap allows for the isolation of the desired precursor ions followed by fragmentation and quantitation on the most abundance MS/MS transition. This allows for reliable quantification at appropriate concentration limits in liquid-liquid extractions from common food and feed sources of aflatoxins.

Detection of quantifier and qualifier ion(s)

Extracted ion MS/MS chromatograms

243.0 257.0 275.0

285.0

295.0

303.0

313.0

200 220 240 260 280 300 320

m/z

243.0

270.0

283.0301.0

311.0

Relati

ve In

tens

ity

(arb

itra

ry u

nits

)

MS/MS of m/z 329

MS/MS of m/z 331

7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5

Time [min]

Relativ

e In

tens

ity(a

rbitr

ary

units

)

B2

B1

G2

G1

Figure 3: MS/MS spectra of aflatoxins G1 and G2. The blue diamond shows m/z of the parent ion.

Figure 4: Extracted ion chromatograph of aflatoxins G2 (green), G1 (grey), B2 (blue) and B1 (maroon) based on their MS/MS fragments (see table 2)

Dynamic range of concentrations

y = 2E+06x + 2E+06R² = 0,9969

0,E+00

5,E+07

1,E+08

2,E+08

2,E+08

3,E+08

0 20 40 60 80 100

Relati

ve In

tens

ity

Concentration (ppb)

B

y = 3E+06x + 6E+06R² = 0,9945

0,E+00

5,E+07

1,E+08

2,E+08

2,E+08

3,E+08

3,E+08

4,E+08

0 20 40 60 80 100

Relati

ve In

tens

ity

Concentration (ppb)

C

y = 296498x + 311066R² = 0,9968

0,E+00

5,E+06

1,E+07

2,E+07

2,E+07

3,E+07

3,E+07

4,E+07

0 20 40 60 80 100

Relati

ve In

tens

ity

Concentration (ppb)

D

y = 3E+06x + 2E+06R² = 0,9973

0,E+00

5,E+07

1,E+08

2,E+08

2,E+08

3,E+08

3,E+08

0 20 40 60 80 100 120

Relati

ve In

tens

ity

Concentration (ppb)

A

Figure 5: Linear calibration curves (ppb in solution) of aflatoxin B1 in (A) peanut, (B) almond, (C) corn meal and (D) chili matrix. In some cases, the error bars are within the data point.

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Keywords

Aflatoxin

Ion Trap

quantitation

Instrumentation & Software

amaZon SL

For research use only. Not for use in diagnostic procedures.

References

[1] Action Levels for Poisonous or Deleterious Substances in Human

Food and Animal Feed. Industry Activities Staff Booklet. U.S.

Food and Drug Administration, Washington, DC, 2000.

[2] Commission Regulation (EC) No 1881/2006 of 19 December

2006. Setting Maximum Levels for Certain Contaminants in

Foodstuffs. Official Journal of the European Union 2006, 49,

L364, 5–24.

[3] Sobolev, V.S. J. Ag. Food Chem. 55 (2007) 2136-2141.

[4] M. Anastassiades, S.J. Lehotay, D. Stajnbaher and F.J. Schenck,

J. Ass. Anal. Comm. Int. 86 (2003) 412.

Bruker Daltonik GmbH

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Bruker Daltonics Inc.

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www.bruker.com/ms

Spiked Extract Analyte

Limit of detection1

(ppb)

Limit of Quantita-

tion2 (ppb)

Linear Calibration

Range (ppb) R2

Peanut B1 0.05 0.1 0.1 – 100 0.9973

B2 0.03 0.15 0.15 – 30 0.9967

G1 0.05 0.1 0.1 – 100 0.9987

G2 0.015 0.03 0.03 – 30 0.9980

Corn Meal B1 0.05 0.1 0.1 – 100 0.9945

B2 0.015 0.03 0.03 – 30 0.9985

G1 0.5 1.0 1.0 – 100 0.9944

G2 0.3 1.5 1.5 – 30 0.9946

Almond B1 0.05 0.1 0.1 – 100 0.9969

B2 0.015 0.03 0.03 – 30 0.9920

G1 0.05 0.1 0.1 – 50 0.9986

G2 0.15 0.3 0.3 – 15 0.9975

Chili Powder B1 1 5 5 – 100 0.9968

B2 1.5 3 3 – 30 0.9987

G1 5 10 10 – 100 0.9948

G2 3 15 15 – 30 0.9926

Leafy Greens B1 0.05 0.1 0.1 – 100 0.9981

B2 0.15 0.3 0.3 – 30 0.9989

G1 0.5 1.0 1.0 – 100 0.9976

G2 0.03 0.15 0.15 – 30 0.9979

Table 3: Aflatoxin quantitation using the amaZon SL (n = 3)

1S/N = 32S/N = 10