Multiple Mycotoxins Determination in Food by LC-MS/MS: An International Collaborative Study

An intercollaborative study was organized to evaluate the performance characteristics of a liquid chromatography tandem mass spectrometry procedure for the simultaneous determination of 12 mycotoxins in food, which were ochratoxin A, aflatoxins B1, B2, G1, G2, and M1, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. The method combined the simplicity of the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach with the efficiency of immunoaffinity column cleanup (the step used to enhance sensitivity and sample cleanup for some matrices only). Twenty-three entities were enrolled and were European reference laboratories for mycotoxin analysis, U.S. and European service laboratories, and Nestlé laboratories. Each participant analyzed 28 incurred and/or spiked blind samples composed of spices, nuts, milk powder, dried fruits, cereals, and baby food using the protocol given. Method performances were assessed according to ISO 5725-2. Relative standard deviations of repeatability and reproducibility and trueness values for each of the 115 mycotoxin/sample combinations ranged from 5% to 23%, 7% to 26%, and 85% to 129%, respectively, in line with requirements defined in EC 401/2006. The overall set of data gathered demonstrated that the method offered a unique platform to ensure compliance with EC 1881/2006 and EC 165/2013 regulations setting maximum limits for mycotoxins in food samples, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization.


Introduction
Mycotoxins are toxic fungal metabolites occurring in a wide range of foodstuffs such as cereals, nuts, spices, fruits, oil seeds, or coffee. Among several hundreds of mycotoxins identified so far, a few are of concern from a food safety perspective.
Trichothecenes are a group of mycotoxins produced particularly by Fusarium moulds. They are often grouped as groups A and B which are characterized by specific structural features. T-2 toxin (T-2) and HT-2 toxin (HT-2) are common type A representatives, while Deoxynivalenol (DON) is common type B representative. Trichothecenes initiate a wide range of effects in animal and human, such as reduced consumption of feed, skin irritation, diarrhea, multiple haemorrhages and immunosuppressive effects.
Ochratoxin A (OTA) is produced by several Aspergillus and Penicillium species in semitropical and temperate climates. OTA is a potent nephrotoxin and hepatoxin with teratogenic, mutagenic, carcinogenic and immunosuppressive effects even at trace levels.
Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin, which is frequently produced by Fusarium species. This mycotoxin exhibits striking estrogenic and anabolic properties in human and animal resulting in severe effects on the reproductive system. Fumonisins (FBs) are a group of structurally related toxic metabolites produced by Fusarium species. They occur at high incidence in corn and corn products all over the world. Fumonisin B1 (FB1) is the predominant metabolite, followed by Fumonisin B2 (FB2) and Fumonisin B3. FB1 is known to cause a range of species-specific toxic responses, including leukoencephalomalacia in horses, pulmonary oedema in swine, and hepatosis and nephrotoxicity in rodents. FB1 is carcinogenic for female mice and male rats.
Aflatoxins (AFLAs) are naturally related mycotoxins produced by numerous Aspergillus species. About 20 compounds have been described, but only Aflatoxin B1 (AFLA B1), Aflatoxin B2 (AFLA B2), Aflatoxin G1 (AFLA G1) and Aflatoxin G2 (AFLA G2) occurs in food commodities. AFLA B1 is the most toxic aflatoxin. When lactating cattle and other animals ingest aflatoxins in contaminated feed, toxic metabolites such as Aflatoxin M1 (AFLA M1) can be formed and transmitted to milk. This hydroxylated form is a potentially important contaminant in dairy products. The International Agency for Research on Cancer (IARC) has classified aflatoxins as carcinogenic to humans (Group 1).
To protect consumer health, maximum levels (MLs) for mycotoxins in foodstuffs have been established worldwide. Particularly, the European legislation has established MLs for AFLAs, OTA, ZEN, FBs and DON and recently indicative levels for T-2 and HT-2 toxins [1,2]. WARNING 1 -Suitable precaution and protection measures need to be taken when carrying out working steps with harmful chemicals. The European Union hazardous substances ordinance (EU) 1907/2006 [3], should be taken into account as well as appropriate National statements. WARNING 2 -The use of this document can involve hazardous materials, operations and equipment. This document does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
The range of concentrations covered for each mycotoxin is described hereafter and ensures quantification at or below EU regulatory limits [1,2]:  [4] with some modifications. The protocol involves an initial single phase extraction with water and acidified acetonitrile, followed by liquid-liquid partition by addition of magnesium sulphate and sodium chloride. The resulting acetonitrile supernatant obtained is then defatted with hexane. Depending on the mycotoxin/matrix combination and the sensitivity required for Aflatoxins and OTA, the sample extracts can then be submitted to two different clean-up procedures, named "QuEChERS" and "IAC": -"QuEChERS": Generic clean-up for all mycotoxins potentially present in cereals when an improved sensitivity for AFLAs and OTA is not required. An aliquot of the acetonitrile supernatant is evaporated to dryness and reconstituted in methanol-water prior injection onto the LC-MS/MS system.
-"IAC": Specific clean-up for AFLAs and OTA for sensitivity purpose in infant foods (e.g. infant cereals, infant formula) and "difficult" matrices (e.g. spices, dried fruits and nuts). An aliquot of the acetonitrile supernatant is first diluted in a phosphate buffered saline (PBS) solution and the whole extract is then applied onto an immunoaffinity column (IAC) containing antibodies specific to AFLAs and OTA. The IAC is washed with water and the toxins are eluted with methanol. The eluate is evaporated to dryness and reconstituted in methanol-water prior injection onto the LC-MS/MS system.
Positive identification of mycotoxins in the sample must fulfill the confirmation criteria defined in the SANTE/12089/2016 document for mass spectrometry analysis [5]. Quantification is performed by the isotopic dilution approach using 13 C-labeled mycotoxins as internal standards (ISs).

Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696, unless otherwise specified. Solvents shall be of quality for LC-MS analysis, unless otherwise specified. Commercially available solutions with equivalent properties to those listed may be used.

Unlabelled Working Standard Solutions -NOT NEEDED FOR THE COLLABORATIVE STUDY
The individual solutions are either prepared by dissolving neat (solid) standards in an appropriate solvent, or from individual stock solutions purchased as such. The mycotoxins covered in this standard dissolve well in acetonitrile, with the exception of fumonisins for which acetonitrile/water (50+50, v+v) is recommended for preparing individual stock solutions.
Prepare the unlabeled working standard solutions as described hereafter by combining the appropriate volumes of individual mycotoxin solutions, using the appropriate pipets (4.1) and the mentioned solvent.
These solutions are used to build the calibration curve (3.6).  Table 1.

Isotopically Labelled Working Standard Solutions -PROVIDED AS READY-TO-USE FOR THE COLLABORATIVE STUDY
Isotopically labelled mycotoxins are commercially available as certified standard solutions. Prepare the labelled working standard solutions as described hereafter by combining the appropriate volumes of individual labelled mycotoxin solutions, using the appropriate pipets (4.1) and the mentioned solvent.
These solutions are used to build the calibration curve (3.6) and to spike each test portions at the beginning of the sample preparation (6.2) for quantification purpose.  Table 2.

Standard Solutions for External Calibration Curve -PROVIDED AS READY-TO-USE FOR THE COLLABORATIVE STUDY
Note: The same batch of IS must be used for both making the calibration solutions and spiking the extracts as described in the extraction procedure (6.2). Any deviation from this may lead to wrong quantification.
Into nine separate 15-mL polypropylene tubes, prepare the standard solutions for calibration as described in Table 3. Concentration of each mycotoxin in each calibrant solution is given in Table 4. Transfer these solutions into glass vials and store them at -20 °C away from light for up to 3 months.  Note*: The calibration range can be extended for quantification of highly contaminated samples. Typically, CAL 7 and CAL 8 can be prepared as described in Table 3 to extend the range by a factor of 2 and a factor of 4, respectively.  C-T-2 & 13 C-HT-2  25  25  25  25  25  25  25  25  25  13 C-FBs  100  100  100  100  100  100  100  100  100  13 C-OTA  1  1  1  1  1  1  1  1  1

Extraction solvent: acetic acid 5 mL/L in acetonitrile
Into a 1-L volumetric flask, mix 500 mL of acetonitrile (3.1.4) and 5 mL of acetic acid (3.1.7). Complete to volume with acetonitrile and mix well. Store this solution at room temperature for no longer than 3 months.

Methanol-water (15+85, v+v)
Into a 100-mL volumetric flask, mix 15 mL of methanol (3.1.3) with 85 mL of water (3.1.1). Store this solution at room temperature for no longer than 3 months. Alternatively, a PBS solution with equivalent properties may be prepared from commercially available PBS material.
Alternatively, a ready-to-use MgSO4 -NaCl (4+1) salt mixture is an example of a suitable product commercially available. Store this solution at room temperature for no longer than 1 week.

Mobile phase (B): formic acid (0.5 mL/L) in methanol
Into a 500-mL volumetric flask, add 250 mL of methanol Store this solution at room temperature for no longer than 1 week.
Store at room temperature for no longer than 3 months.

Apparatus
Standard laboratory apparatus may be used (graduated cylinders, glass funnels, beakers, pipette, etc.) and, in particular, the following.

Computer based control and data processing system 5 Sampling
Sampling is not part of the method specified in this document. The Commission Regulation (EC) No 401/2006 [6] can be used as a reference.
It is important that the laboratory receives a sample which is representative and has not been damaged or changed during transport or storage. Powdered samples must be homogenized before taking test portions. This could be achieved by transferring the whole sample into a container of capacity about twice that of the laboratory sample volume, and thoroughly mixing by repeatedly shaking and inverting the container. Alternatively, the powdered laboratory sample could be directly homogenised into its original container by means of a spoon before taking a test portion.

Cereals, infant cereals
Into a 50-mL polypropylene tube, weigh a test portion of 5.00 g of the laboratory sample to the nearest 0.05 g.

Milk powders, nuts, spices, dried fruits
Into a 50-mL polypropylene tube, weigh a test portion of 2.00 g of the laboratory sample to the nearest 0.02 g.

Spiking of 13 C-labelled mycotoxins used as internal standards
Note: Robustness of the method is not affected as long as the same mycotoxin standard solutions are used for both preparing calibration standards and spiking test portions.

Note:
The choice of the IS depends on the final purpose of the analysis.
Spike each test portion with 50 µL of individual and/or composite working standard solutions of 13 Clabeled mycotoxins used as internal standards (IS) as shown in Table 5.

Extraction
Add 10 mL of water (3.1.1) and shake vigorously by hand until the whole sample is completely dispersed in solution.
Add 10 mL of acidified acetonitrile (3.7.1), close the tubes and shake vigorously by hand for at least 5 s. Shake on a mechanical shaker for approximately 10 min at approximately 300 rpm.
Open the tubes and add 5.0 g ± 0.2 g of the magnesium sulphate-sodium chloride salt mixture (3.7.4) to the slurry. Close the tubes and immediately shake for a few seconds to avoid formation of lumps of magnesium sulphate. Shake the tube vigorously for about 1 min by hand or on a mechanical shaker.
Centrifuge the tubes at 4000 x g at room temperature for approximately 10 min.

General clean-up procedure: removal of co-extracted fat
Into a 15-mL polypropylene tube, transfer 5 mL of the supernatant acetonitrile phase (6.3) and add Centrifuge at 4000 x g for approximately 1 min to allow an efficient phase separation Discard the n-hexane (upper phase) by using a plastic Pasteur pipette (4.18).

Specific clean-up procedures ("QuEChERS" or "Immunoaffinity column (IAC)")
At this point of the method, the sample extract can be divided in two portions and submitted to two different clean-up protocols according to the general scheme presented in Annex A: -1 st option: all analytes are directed to the "QuEChERS" procedure (cereals-based samples).
-2 nd option: for AFLAs and OTA only, when extra sensitivity is needed (food intended for infants and young children) and for difficult matrices (e.g. spices, dried fruits and nuts), the sample extract is directed to the "IAC" procedure.

"QuEChERS" (generic clean-up for all mycotoxins)
Transfer a 1-mL aliquot of the defatted acetonitrile phase (lower phase, 6.4) into a new 15-mL polypropylene tube.
Evaporate the extract to dryness under a stream of nitrogen at about 40 °C.
Reconstitute the residue with 75 µL of methanol (3.1.3) and sonicate for about 1 min to re-suspend the residue. Add 425 µL of water (3.1.1) and mix for about 5 s using a vortex mixer. Transfer the resulting mixture in a 1.5-mL polypropylene microcentrifuge tube and centrifuge for about 10 min at 8'500 x g at room temperature.
Transfer the supernatant into a LC amber glass vial and proceed with LC-MS/MS analysis.

"IAC" (specific clean-up for OTA and AFLAs)
Transfer a 2-mL aliquot of the defatted acetonitrile phase (lower phase, 6.4) into a new 50-mL polypropylene tube. Dilute approximately to the 25-mL mark with the PBS solution (3.7.3.3) and mix well.
Allow the IAC to reach room temperature prior to use. Connect the IAC (4.19) to the vacuum manifold (4.15) and attach a SPE tube adapter with a reservoir with a minimum capacity of 25 mL at the top of the IAC.

Loading step:
Transfer the whole diluted extract (25 mL) into the IAC reservoir and pass it through the IAC by applying a gentle vacuum to get a flow rate of approximately 1-2 drops/s.

Washing Step:
Wash the column with 20 mL of water (3.1.1) at an approximate flow rate of 1-2 drops/s. Remove the reservoir and dry the column.

Elution
Step: Place a 15-mL polypropylene tube beneath the column and apply at first 800 µL of methanol Transfer the supernatant into a HPLC glass vial and proceed with LC-MS/MS analysis.

LC-MS/MS analysis 7.1 LC conditions
Using the column (4.20.6) and the mobile phases A and B (3.8.1 and 3.8.2) specified in this document, the following LC conditions are adapted for mycotoxin analysis:  Injection volume: 10 µL  Column Temperature: 50 °C  Flow rate: 400 µL/min  Gradient: as shown in Table 6

MS Conditions
The values given in Annex B.1.2 need to be checked and optimized for each instrument. It is recommended to optimize MS parameters by syringe-infusing each individual mycotoxin standard solution in electrospray ionisation (ESI) mode. At least two transition reactions per compound must be monitored for each mycotoxin and its relative internal standard. The most intense transition reaction may differ depending on the instrument and eluents used in LC (e.g. protonated molecules or sodium/ammonium adducts in ESI+, deprotonated molecules or acetate/formate adducts in ESI-).

Injection sequence
Start a batch of analysis by injecting a methanol-water (15+85) (3.7.2) solution to stabilize the LC-MS/MS system and to check for any contamination of the system.
Inject the calibration solutions from CAL 0 to CAL 6 (3.6) and carefully check that all toxins and their respective ISs are visible at the lowest calibration level (CAL 1). Inject a methanol-water (15+85) (3.7.2) solution to ensure that there is no carry over.
End the sequence by re-injecting at least one calibration solution (3.6) to ensure system stability. CAL 7 and CAL 8 can be injected at the end of the sequence. Rinse the column afterwards by injecting the methanol-water solution (3.7.2) at least twice.

Data Treatment
Process the data using the appropriate integration software. Peaks areas are used for subsequent calculations. Check peak area assignment and integration for the measured transition reaction and adjust if necessary.

Identification and confirmation
Mycotoxins are considered as positively identified in the sample when all the confirmation criteria defined in the SANTE/12089/2016 document [5] are fulfilled:  A signal is visible at least at two diagnostic transition reactions selected for each mycotoxin and each corresponding IS.  The retention time of the analyte in the sample extract should correspond to that of the average of the calibration standards measured in the same sequence with a tolerance of ±0.2 min.  The retention time of the analyte should correspond to that of its labelled internal standard with a tolerance of ±0.05 min.  The peak area ratio from the different transition reactions recorded for each analyte is ± 30%.

Calibration curve
Quantification is performed by the isotopic dilution approach using 13 C-labeled mycotoxins as internal standard (IS).
Draw the calibration curve (Area Ratio = [(Concentration Ratio) x Slope] + Intercept) by plotting peak area ratio of each mycotoxin and its IS (= y axis) against concentration ratio of each mycotoxin and its IS (= x axis) using calibration solutions from CAL 0 to CAL 6.
Calculate the slope and the intercept by linear regression and check that the calibration curve is linear:  Regression coefficient R 2 should be higher than 0.99.
 The deviation of the back-calculated concentration of the calibrants standards from the true concentration, using the calibration curve, should not be more than ±20%.

Note:
In order to improve the precision on the low calibration points, it is advisable to use a 1/x or 1/x 2 weighing factor for the calibration curve. Alternatively, ensure that the confidence interval around the intercept contains 0 and force the regression line through the origin (i.e. Intercept = 0).
Note: CAL 7 and CAL 8 (3.6) are only to be considered in case of highly contaminated sample whene mycotoxin levels are out of the calibration range.

Calculations
Calculate the mass fraction of each analyte, wa, in microgram per kilogram (µg/kg) in the sample using the following equation: = mass of the test portion, in g (either 2.0 g or 5.0 g) mis = mass of IS added to the test portion, in ng (see Table 7 below) * DP: declustering potential (V) / CE: collision energy (eV) / EP: entrance potential (V) / CXP: collision exit potential (V).