Application Note # LCMS-61 Identification and Quantitation of four Aflatoxins using the Bruker amaZon SL Ion Trap Mass Spectrometer
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,
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
Figure 1: Bruker amaZon SL ion trap mass spectrometer. Quantitative analysis of four aflatoxins
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 liquidliquid 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 Figure 2: Chemical structures, molecular weights (MW) and chemical formulae of the four aflatoxins under investigation.
Table 1: HPLC Gradient Time
%B
Flow Rate (µL/min)
0
30
200
2
30
200
7
75
12
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 Table 2: HPLC and MS/MS transition parameters Retention time
MS/MS Transition
Analyte
(min)
[qualifier] (m/z)
200
B1
9.75
313 → 285 [257]
75
200
B2
9.5
315 → 287 [259]
12.01
30
200
G1
9.25
329 → 311 [283]
14
30
200
G2
9.0
331 → 313 [285]
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.
Detection of quantifier and qualifier ion(s) MS/MS of m/z 329 311.0
Relative Intensity (arbitrary units)
Simultaneous Quantitation of Four Aflatoxins in Matrix 243.0
301.0
283.0 270.0
MS/MS of m/z 331 313.0
285.0 243.0 200
220
240
257.0
275.0
260
303.0 295.0
280
300
320
m/z
Figure 3: MS/MS spectra of aflatoxins G1 and G2. The blue diamond shows m/z of the parent ion. Extracted ion MS/MS chromatograms
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.
G2 G1
Relative Intensity (arbitrary units)
B2 B1
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
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.
11.5
Summary
Time [min]
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)
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.
Dynamic range of concentrations 3,E+08
B
2,E+08
Relative Intensity
3,E+08
Relative Intensity
3,E+08
A
2,E+08 2,E+08 y = 3E+06x + 2E+06 R² = 0,9973
1,E+08 5,E+07
2,E+08 1,E+08 5,E+07
0,E+00 0
20
40
60
80
100
y = 2E+06x + 2E+06 R² = 0,9969
0,E+00
120
0
20
Concentration (ppb) 4,E+08
2,E+08 2,E+08 1,E+08 y = 3E+06x + 6E+06 R² = 0,9945
2,E+07 2,E+07 1,E+07 y = 296498x + 311066 R² = 0,9968
0,E+00 20
40
60
Concentration (ppb)
100
3,E+07
5,E+06
0,E+00 0
80
D
3,E+07
3,E+08
Relative Intensity
Relative Intensity
4,E+07
5,E+07
60
Concentration (ppb)
C
3,E+08
40
80
100
0
20
40
60
Concentration (ppb)
80
100
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.
Table 3: Aflatoxin quantitation using the amaZon SL (n = 3)
Peanut
Corn Meal
Almond
Chili Powder
Leafy Greens
Analyte
Limit of detection1
Limit of Quantita-
Linear Calibration
(ppb)
tion 2 (ppb)
Range (ppb)
R2
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
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
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
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
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
S/N = 3 2 S/N = 10
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
Keywords
Instrumentation & Software
Foodstuffs. Official Journal of the European Union 2006, 49,
L364, 5–24.
Aflatoxin
amaZon SL
[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.
Ion Trap quantitation For research use only. Not for use in diagnostic procedures.
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Spiked Extract
