Single Corn Kernel Aflatoxin B 1 Extraction and Analysis Method

Aflatoxins are highly carcinogenic compounds produced by the fungus Aspergillus flavus. Aspergillus flavus is a phytopathogenic fungus that commonly infects crops such as cotton, peanuts, and maize. The goal of this research was to design an effective method for sample preparation and analysis of aflatoxin B1 from Aspergillus flavus infected maize kernels. The method involves liquid extraction with a methanol/water solution. The samples were analyzed by liquid chromatography coupled to an electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in positive ion multiple reaction monitoring (MRM) mode. The spiked levels for the recovery experiment were 4 ppb and 20 ppb, respectively. Recoveries ranged between 72% and 113% (90.8% average), with Relative Standard Deviation below 15% (10.6% on average).


Introduction
Aspergillus flavus (A.flavus) is a fungus that commonly grows in the soil and is known to infect a variety of crops including cotton, peanuts, and corn.A. flavus can cause ear rot in maize, aflaroot in peanuts, and yellow spot disease in cotton [1]- [3].Environmental conditions such as high temperatures, high humidity, drought stress, and poor crop storage can cause A. flavus to start producing aflatoxins [4].Aflatoxins are secondary metabolites of the fungus and are extremely carcinogenic.The four major types of aflatoxins are aflatoxin B 1 , B 2 , G 1 , and G 2 .Aflatoxin B 1 and B 2 fluoresce blue under UV light while aflatoxin G 1 and G 2 fluoresce green in the presence of UV light.Aflatoxin B 1 can also be metabolized to aflatoxin M1 which would be found in the milk of lactating mammals.Aflatoxin B 1 is the most common and carcinogenic of these compounds as seen in Figure 1.Aflatoxin B 1 is converted in vivo to an aflatoxin B 1 -exo-8, 9-epoxide by the liver enzyme cytochrome p450 oxidase [5].The epoxide that is formed is a highly reactive electrophile and has an extremely high regiospecificity for the N7 position of the guanine residue in DNA [6].It specifically binds to the p53 encoding region to form a DNA adduct [7].This results in nonfunctioning p53 proteins which are important tumor suppressors in humans.
In 1988 the International Agency for Research on Cancer classified aflatoxin B 1 as a Class 1 human carcinogen [8].Therefore, the U.S. Food and Drug Administration (FDA) has restricted the amount of aflatoxin in food for human consumption to 20 parts per billion (ppb) in the United States.The European Commission (EC) limits aflatoxins in food for human consumption to 4 ppb in the European Union [9] [10].The Council of Agricultural Science and Technology has estimated that the US has an annual loss of almost a billion dollars due to crop damage from mycotoxins.An estimated $225 million of that are due to aflatoxin contamination in maize crops [11].Study of the maize genome has increased in recent years in hopes to discover genes that are involved with aflatoxin resistance in corn.The DNA from a single corn kernel can be used to analyze the gene expression that occurs in Aspergillus flavus inoculated maize.In order to compliment this procedure, aflatoxin extraction should be equally versatile.Aflatoxin extraction methods normally require several (10 -250) grams of ground maize which may not be feasible in determining aflatoxin accumulation for a smaller region of the A. flavus infected ear.A single kernel extraction method is needed in order to accurately track aflatoxin concentration and maize gene expression at specific A. flavus infected sites on the cob.

Reagents and Chemicals
All solvents and reagents were Optima LC/MS grade.Methanol, water, formic acid, and ammonium acetate were obtained from Fisher Scientific (Fair Lawn, NJ, USA).Purified aflatoxin B 1 and M 1 standards were acquired from Sigma Aldrich (St. Louis, MO, USA).

Sample Preparation
Maize kernels were flash frozen in liquid nitrogen.The kernels were ground into a fine powder with a mortar and pestle.200 mg of each ground sample was placed into a 1.5 mL micro-centrifuge tube.Aflatoxin free ground maize was spiked with aflatoxin B 1 to yield two levels of spiked samples (4 ppb and 20 ppb) of aflatoxin B 1 for recovery.These concentrations were chosen because they are the limit for aflatoxin in food for human consumption in the European Union and the United States, respectively.
A solution of 1 mL (70/30, v/v) methanol/water was added to each 1.5 mL microcentrifuge tube.The samples were mixed for 1 minute and then centrifuged for 5 minutes at 14,000 rpm.Polytetrafluoroethylene (PTFE) syringe filters (0.45 µm) were used to remove any large particles from the samples after centrifugation as shown in Figure 2. The liquid extracts were transferred to auto-sampler vials and analyzed using an Agilent 6460 LC/MS/MS Triple Quadrupole with electrospray ionization.Aflatoxin M 1 was added as an internal standard at a concentration of 10 ppb.The calibration curve was matrix-matched with extracted aflatoxin-free maize solution.

Experimental
The samples were analyzed using an Agilent 6460 LC/MS/MS Triple Quadrupole Mass Spectrometer with ESI, using an Agilent 1200 Series High Performance Liquid Chromatography (HPLC).The HPLC system consists of a binary pump (G1312B), infinity high performance degasser (G1379B), high performance autosampler (G1367E), thermostatted column compartment (G1316B), sampler thermostat (G1330B), and Mass Hunter data software.Aflatoxin B 1 and M 1 were optimized using the Agilent Optimization software.Aflatoxin M 1 was used as an internal standard (ISTD) at 10 ppb.The Agilent Optimization software produces the ideal fragmentor voltage and collision energy for each MRM transition of aflatoxin B 1 and M 1 as listed in Table 1.

HPLC Method Conditions
An Agilent 6460 LC/MS Triple Quadrupole Mass Spectrometer equipped with electrospray ionization (ESI) was used to analyze the aflatoxin concentration of the samples.The autosampler temperature was set to 4˚C.The HPLC used a Zorbax Eclipse Plus-C18 Narrow Bore 2.1 × 50 mm, 5 µm column with a temperature of 50˚C.The mobile phase consisted of 5 mM ammonium acetate with 0.1% formic acid in HPLC-grade water and 5 mM ammonium acetate with 0.1% formic acid in methanol.The flow rate of the mobile phase during the analysis was consistently 0.6 mL/min.The mobile phase gradient was transitioned from 95% water to 100% methanol during the six minutes of the analysis time.Then for the final three minutes of the run, the mobile phase reversed from 100% methanol back to 95% water.The total run time of the method was nine minutes which includes a six minute analysis time and an additional three minutes for the system to get back to equilibrium.The calibration curve was matrix-matched in order to reduce matrix effects.The six point calibration curve consisted of the concentrations 1 ppb, 5 ppb, 10 ppb, 25 ppb, 50 ppb, and 100 ppb as shown in Figure 3.

MS Method Conditions
The parameters for the mass spectrometer were the following.The Agilent 6460 Triple Quadrupole Mass Spectrometer (MS/MS) system coupled to an electrospray analyzed the samples while in positive mode.The drying gas temperature was 325˚C while the gas flow was set to 10 liters per minute.The nebulizer gas pressure was set to 50 psi and the capillary voltage was 4000V.The Sheath Gas Flow had an output of 11 liters per minute and the sheath gas temperature reached temperatures of 350˚C.The delta electron multiplier voltage (EMV) was     Counts vs. Acquisition Time (min)

Results and Discussion
Table 2 demonstrates the average percent recoveries and relative standard deviation (RSD) values obtained from the spiked corn samples after performing the single maize kernel aflatoxin extraction.The analysis was performed in replicates of five at each of the two levels.The average percent recoveries for the 4 ppb and the 20 ppb aflatoxin B 1 spiked maize were 90.83% with a RSD of 4.11% and 90.72% with a RSD value of 14.45% respectively.Calibration standards were matrix matched with a range from 1 ppb to 100 ppb with a linear correlation (R 2 ) of 0.996 as shown in Figure 3.In The limit of detection (LOD) and limit of quantification (LOQ) were estimated from the concentration of aflatoxin B1 required to give a signal to noise ratio of 3:1 and 10:1 respectively.The LOD was determined to be 0.344 ppb and the LOQ was calculated to be 1.042 ppb.The internal standard, aflatoxin M 1 , injected at 10 ppb accounts for possible instrument variation.Aflatoxin M 1 was chosen to be the internal standard because it has a structure similar to aflatoxin B 1 but is not produced by A. flavus.Aflatoxin M 1 is also less expensive and more readily available commercially than the radiolabeled form of aflatoxin B 1 .Methanol was the preferred solvent for aflatoxin extraction due to its compatibility with the ELISA testing and the cost effectiveness over other solvents like acetone and acetonitrile [12] [13].This simplified aflatoxin extraction method is analogous to other extractions methods in terms of aflatoxin recovery without the need for solid phase extraction or clean up columns [14].The single maize kernel extraction method was needed in order to more accurately determine the changes in aflatoxin production, gene transcription, and protein production between inoculated and control maize kernels.

Conclusion
This process demonstrates a fast, simple, and effective analytical method for determining aflatoxin concentrations in a single maize kernel using an Agilent 6460 Triple Quadruple Mass Spectrometer.The detection levels for aflatoxin B 1 were below both the limit set by the FDA in the United States and the limit set by the EC in the European Union.The recovery percentages for aflatoxin B 1 were 90.83% for 4 ppb and 90.72% for 20 ppb with a satisfactory average RSD less than 15%.The single kernel extraction method will be a useful technique in determining how aflatoxin producing Aspergillus flavus affects infected maize.
Analyze on the LC/MS Samples were filtered with 0.45 μm PTFE syringe filter Centrifuge for 5 minutes at 14000 rpm Vortex samples for 1 minute Add 1 mL methanol/water (70/30;v/v) to the tube Weigh 200 mg of ground corn in 1.5 mL microcentrifuge tube 800V and the dwell time lasted for 200 msec.The precursor ion for aflatoxin B 1 was 313.1 m/z and 329.1 m/z for aflatoxin M 1 .While in multiple reaction monitoring (MRM) mode, the mass spectrometer was set to look for the daughter ions after the precursor ion entered the collision cell.The transitions for aflatoxin B 1 included 313.1 > 285.1 m/z with a collision energy of 20kEV, 313.1 > 269.1 m/z with a collision energy of 25 kEV, and 313.1 > 241.1 m/z with a collision energy of 35 kEV.The fragmentor value was 166 for aflatoxin B 1 and for aflatoxin M 1 was 131.The cell accelerator (7) values were the same for both aflatoxin transitions.The two transitions used for identifying aflatoxin M 1 are 329.1 > 273.1 m/z and 329.1 > 229.1 m/z.The retention time of aflatoxin M 1 was 3.0 minutes and 3.4 minutes for aflatoxin B 1 as shown in Figure 4. Agilent MassHunter Quantitative Analysis Workstation Software v. B.04.0.225.19 was used to analyze the quantitative data obtained from the samples and the calibration curve.

Figure 3 .
Figure 3. Matrix matched calibration curve for aflatoxin B1 from concentration of 1.0 ppb to 100 ppb.

Figure 4 .
Figure 4. Chromatogram of aflatoxin B 1 standard at 20 ppb with aflatoxin M 1 as the internal standard at 10 ppb.

Table 1 .
MRM transitions for aflatoxin B1 and the internal standard, aflatoxin M1.

Table 2 .
Average percent recoveries and RSD values obtained from spiked corn samples.