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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© B
ruke
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05
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1, L
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1, #
276
642
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.
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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