METHOD NUMBER: C-003.

01
POSTING DATE: 10/5/2017
POSTING EXPIRATION DATE: none
PROGRAM AREA: Mycotoxins
METHOD TITLE: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour
Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass
Spectrometry (LC-MS/MS)
VALIDATION STATUS: Level 3 Multilaboratory validation per the Guidelines for the
Validation of Chemical Methods for the FDA Foods Program 3rd Edition
AUTHOR(S): Kai Zhang, CFSAN/ORS/DBC/Bioanalytical Methods Branch
METHOD SUMMARY/SCOPE:
Analyte(s): Aflatoxin B1, B2, G1, G2; deoxynivalenol; fumonisin B1, B2, B3; HT-2
toxin, ochratoxin A, T-2 toxin, zearalenone
Matrices: Corn, peanut butter and wheat flour

REVISION HISTORY:

OTHER NOTES:
Title: Determination of Mycotoxins in Corn, Peanut Butter, and
Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid
Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Version: 1.0 (2017)

Author: Kai Zhang

CFSAN/ORS reviewers: Christine Parker, Gregory Noonan

Chemistry Research Coordination Group Concurrence: John Callahan, Chair, 10/5/2017

Table of Contents

2020.1 METHOD TITLE

2020.2 SCOPE OF APPLICATION

2020.3 PRINCIPLE

2020.4 REAGENTS

2020.5 STANDARDS

2020.6 PREPARATION OF SAMPLES OR TEST PORTIONS

2020.7 APPARATUS/INSTRUMENTATION

2020.8 METHOD

2020.9 CALCULATIONS

2020.10 VALIDATION INFORMATION/STATUS

2020.11 REFERENCES
2020.1 METHOD TITLE: Determination of mycotoxins in corn, peanut butter, and
wheat flour using stable isotope dilution assay (SIDA) and liquid chromatography-
tandem mass spectrometry (LC-MS/MS)

2020.2 SCOPE OF APPLICATION

This method describes a procedure for using stable isotope dilution assay (SIDA) and liquid
chromatography-tandem mass spectrometry (LC-MS/MS) to determine 12 mycotoxins of
regulatory and health significance. The 12 mycotoxins are aflatoxin B1, B2, G1, G2;
deoxynivalenol; fumonisin B1, B2, B3; HT-2 toxin, ochratoxin A, T-2 toxin and zearalenone.
The method has been validated in the following food matrices: corn, peanut butter and wheat
flour.

It is generally assumed that the more closely related a new food matrix is to a previously
validated matrix for a defined analyte, the greater the probability that the new matrix will behave
similarly. It is also usually the case that the regulatory chemical methods employed by FDA are
used to analyze a diversity of products representing a large spectrum of matrices. It becomes
unfeasible to carry out a matrix extension validation for each single matrix in order to expand the
scope of the method. A more reasonable approach to demonstrate the applicability of a method to
a set of product matrices is to validate the method for different “categories” of products. For
instance, a multi-residue pesticide method can be validated for “high-sugar”, “high-fat”, “high-
water”, “dry” and “high-protein” matrices. Food and Drug Administration (FDA) Guidelines for
the Validation of Chemical Methods for the FDA Foods Program Appendix 4 provides 1
guidance on commodity categories and gives examples of representative matrices in each
category.

This method should be used by analysts experienced in the use of SIDA and LC-MS/MS,
including but not limited to sample preparation, operation of LC-MS/MS, data analysis and
reporting results. Analysts also should be able to identify chromatographic and mass
spectrometric interferences in the course of sample analysis and take necessary actions following
validated procedures correct instrument/method performance issues. The method should be used
only by personnel thoroughly trained in the handling and analysis of samples for the
determination of mycotoxins in food and feed products.

2020.3 PRINCIPLE

Samples are prepared by fortifying with 13C uniformly labelled mycotoxins as internal standards
(IS), followed by extraction using 50% acetonitrile (50/50, v/v, acetonitrile/water), centrifugation
and filtration. The target mycotoxins are analyzed by LC-MS/MS and identified by retention
time alignment and product ion transition confirmation with calibration standards. The
identification criteria are detailed in FDA Guidelines for the Validation of Chemical Methods for
the FDA Foods Program and FDA Center for Veterinary Medicine. Guidance for Industry 118,
2003, specifically, all transitions must be present, with appropriate relative abundances. 1, 2
Quantitation was performed using solvent-only calibration standards. The concentration of each
target mycotoxin was determined using the peak area ratio of response of the mycotoxin to that
of the corresponding [13C]-IS, and calculating the concentration by preparing a calibration curve
using the peak area ratios of solvent-only calibration standards to that of the same [13C]-IS.

2020.4 REAGENTS

(1) Acetonitrile, LC grade (Thermo Fisher Scientific, Part # 85188)

(2) Methanol, LC grade (Thermo Fisher Scientific, Part# A452SK-4)
(3) Water, LC grade (Thermo Fisher Scientific, Part# 85189)
(4) Formic acid, MS grade (Thermo Fisher Scientific, Part# 85178)
(5) Ammonium formate, MS grade (Thermo Fisher Scientific, Part# A11550)

NOTE: Reagents should be purchased from vendors that can provide certificates of analysis
(CoAs) to show traceability, purity, storage condition and duration.

2020.5 STANDARDS

(1) Aflatoxin B1, B2, G1, G2 (Romer Labs, Part# 002050)

(2) Deoxynivalenol (Romer Labs, Part# 002050)
(3) Fumonisin B1, B2, B3 (Romer Labs, Part# 002050)
(4) Ochratoxin A (Romer Labs, Part# 002050)
(5) HT-2 toxin ((Romer Labs, Part# 002050)
(6) T-2 toxin (Romer Labs, Part# 002050)
(7) Zearalenone (Romer Labs, Part# 002050)
(8) [13C17]-aflatoxin B1 (Romer Labs, Part # ILM010)
(9) [13C17]-aflatoxin B2 (Romer Labs, Part # ILM011)
(10) [13C17]-aflatoxin G1(Romer Labs, Part # ILM012)
(11) [13C17]-aflatoxin G2 (Romer Labs, Part # ILM013)
(12) [13C34]-fumonisin B1 (Romer Labs, Part# ILM003)
(13) [13C34]-fumonisin B2(Romer Labs, Part# ILM004)
(14) [13C34]-fumonisin B3(Romer Labs, Part# ILM005)
(15) [13C15]-deoxynivalenol (Romer Labs, Part# 002005)
(16) [13C20]-ochratoxin A (Romer Labs, Part# ILM007)
(17) [13C22]-HT-2 toxin (Romer Labs, Part# ILM 008)
(18) [13C24]-T-2 toxin (Romer Labs, Part# 002004)
(19) [13C18]-zearalenone (Romer Labs, Part# ILM009)

NOTE: Standards should be purchased from vendors that can provide CoAs to show traceability,
purity, storage condition and duration.

Standard preparation
Prepare or purchase the following three mycotoxin stock solutions A, B, and C (e.g., 5.0
mL/vial) from Romer Labs (Union, MO):
 (1) Solution A: Aflatoxin B1 (1.0 μg/mL), aflatoxin B2 (1.0 μg/mL), aflatoxin G1 (1.0
μg/mL), aflatoxin G2, (1.0 μg/mL) and ochratoxin A (1.0 μg/mL) in methanol
(2) Solution B: Fumonisin B1 (10 μg/mL), fumonisin B2 (10 μg/mL), and fumonisin B3
(10 μg/mL) in acetonitrile/water (50:50, v/v)
(3) Solution C: Deoxynivalenol (10 μg/mL), HT-2 toxin (10 μg/mL), T-2 toxin (10
μg/mL), and zearalenone (10 μg/mL) in acetonitrile.

The following [13C]-IS stock solutions can be purchased from Romer Labs:[13C17]-aflatoxin B1 +
B2 + G1 + G2, (0.5 μg/mL), [13C17]-aflatoxin M1 (0.5 μg/mL), [13C20]-ochratoxin A (10 μg/mL),
[13C7]-patulin (25 μg/mL), [13C34]-fumonisin B1 (25 μg/mL), [13C34]-fumonisin B2 (10 μg/mL),
[13C34]- fumonisin B3 (10 μg/mL), [13C15]-deoxynivalenol (25 μg/mL), [13C22]-HT-2 toxin (25
μg/mL), [13C24]-T-2 toxin (25 μg/mL), [13C18]-zearalenone (25 μg/mL). The working solution of
[13C]-IS can be prepared by mixing and diluting appropriate amounts of the stock solutions using
the extraction solution, acetonitrile/water (v/v, 50/50).

The concentration of each [13C]-IS in the working solution is as follows: [13C17]-aflatoxin B1, B2,
G1, and G2 (0.05 μg/mL), [13C17]-ochratoxin A (0.2 μg/mL), [13C34]-fumonisin B1 (2.0 μg/mL),
[13C34]-fumonisin B2 (2.0 μg/mL), [13C34]- fumonisin B3 (2.0 μg/mL), [13C15]-deoxynivalenol
(2.0 μg/mL), [13C22]-HT-2 toxin (2.0 μg/mL), [13C24]-T-2 toxin (2.0 μg/mL), and [13C18]-
zearalenone (2.0 μg/mL).

Solvent-only calibration standards are prepared from dilutions of stock solutions A, B, and C and
the corresponding [13C]-IS working solution, respectively. Multiple levels of solvent-only
calibration standards (0.5 mL for each level) can be prepared in acetonitrile/water (50/50, v/v)
and each standard is fortified with 10 μL of the [13C]-IS working solution. Concentration ranges
may be adjusted for quantitation. The fortification volume of the 13C-IS working solution may
be adjusted according to the sensitivity of LC-MS.

2020.6 PREPARATION OF SAMPLES OR TEST PORTIONS

2020.6.1 Water-slurry procedure used for food commodities (e.g., corn) that are
difficult to homogenize using dry milling.
(1) Weigh 25.0 ± 0.5g of a sample into a 40 or 100 mL disposable grinding chamber
(IKA, NC, USA) and add 25.0 ± 0.5 of water (HPLC grade).
(2) Blend the sample and water for 1.5 min at 25,000 rpm using an IKA Tube Mill.
(3) Weigh a test portion (2.00 ± 0.05 g) from the blended sample into a 15 mL
disposable screw-capped polypropylene centrifuge tube.
(4) Fortify the test portion with 100 μL of the [13C]-IS working solution, making the
concentrations of [13C]-IS in the sample as follows, [13C17]-aflatoxin B1 (0.005
μg/g), [13C17]-aflatoxin B2 (0.005 μg/g),[13C17]-aflatoxin G1 (0.005 μg/g), [13C17]-
aflatoxin G2, (0.005 μg/g) and [13C17]-ochratoxin A (0.02 μg/g), [13C34]-fumonisin
B1 (0.20 μg/g), [13C34]-fumonisin B2 (0.20 μg/g), and [13C34]- fumonisin B3 (0.20
μg/g), [13C15]-deoxynivalenol (0.20 μg/g), [13C22]-HT-2 toxin (0.20 μg/g), [13C24]-
T-2 toxin (0.20 μg/g), and [13C18]-zearalenone (0.20 μg/g). The concentrations of
 [13C]-IS fortified in each test portion were calculated based on mass of the sample
(1.0 g).
(5) Re-cap the tube and vortex it for 30 sec, followed by the addition of 4.0 mL of
extraction solvent (acetonitrile/water, 50/50, v/v) into the tube.
(6) Method blank (negative control QC) can be prepared using matrices of interest
containing non-detectable target mycotoxins following steps 1-5.
(7) Method spike (positive control QC) can be prepared by fortifying blank matrices
with target mycotoxins at pre-defined concentrations then following steps 1-5.
Alternatively, certified reference materials may be used as QC.
2020.6.2 For food commodities, such as infant rice cereal and peanut butter that are
generally considered homogenous, but can be homogenized using dry-milling.
(1) Weigh a test portion of homogenized sample (1.00 ± 0.05 g each) into a 15 mL
disposable screw-capped polypropylene centrifuge tube.
(2) Fortify the test portion with 100 μL of the [13C]-IS working solution, making the final
concentrations of [13C]-IS in the sample as follows, [13C17]-aflatoxin B1 (0.005 μg/g),
[13C17]-aflatoxin B2 (0.005 μg/g), [13C17]-aflatoxin G1 (0.005 μg/g), [13C17]-aflatoxin
G2, (0.005 μg/g) and [13C17]-ochratoxin A (0.02 μg/g), [13C34]-fumonisin B1 (0.20
μg/g), [13C34]-fumonisin B2 (0.20 μg/g), and [13C34]- fumonisin B3 (0.20 μg/g),
[13C15]-deoxynivalenol (0.20 μg/g), [13C22]-HT-2 toxin (0.20 μg/g), [13C24]-T-2 toxin
(0.20 μg/g), and [13C18]-zearalenone (0.10 μg/g)
(3) Re-cap the tube and vortex it for 30 sec, followed by the addition of 5.0 mL of extraction
solvent (acetonitrile/water, 50/50, v/v) into the tube.
2020.6.3 Sample extraction and cleanup
(1) Extract the samples prepared above for 30 min using a high-speed shaker with
pulsation (Glas-Col, Terre Haute, IN, USA) using a motor speed setting of 75
(1540−1560 rpm as measured by a DPM5 digital photo tachometer, Universal
Enterprises, Inc., Beaverton, OR, USA) and pulser frequency set at the middle mark of
the dial (∼30−35 pulsations/min), followed by subsequent centrifugation for 15 min at
4200g (ThermoElectro Corp., Milford, MA, USA).
(2) Filter the supernatant of samples through a 13 mm × 0.2 μm PTFE syringe filter (Pall
Life Sciences), using a 3 mL disposable syringe, directly into an amber autosampler vial
(National Scientific, Rockwood, TN, USA) for LC-MS/MS analysis.
(3) Alternatively, the supernatant (~2.0 mL) of samples can be transferred to an Amicon
Ultra-4 centrifugal filter with Ultracel-3 membrane (molecular weight cutoff value of 3
kDa) and centrifuged for 15 min at 4200g. The resulting filtrates are transferred into
autosampler vials for LC-MS/MS analysis.

2020.7 APPARATUS/INSTRUMENTATION
Disclaimer: The use of trade names in this method constitutes neither endorsement nor
recommendation by the U. S. Food and Drug Administration (FDA). Equivalent performance
may be achievable using apparatus and materials other than those cited here.
(1) Liquid chromatography-coupled with triple quadrupole mass spectrometry that can meet
instrument performance specifications defined Section 2017.13.
(2) HPLC guard and analytical columns –with a Phenomenex SecurityGuard ULTRA 10 mm ×
2.1 mm i.d. guard cartridge coupled with a Phenomenex Kinetex XB-C18 (100 mm × 2.1
mm i.d., 2.6 μm).
 (3) High-speed shaker with pulsation, Glas-Col.
(4) Centrifuge tubes—polypropylene conical tubes with caps, 15 mL.
(5) Vortex mixer
(6) Amicon Ultra-4 centrifugal filter with Ultracel-3 membrane (molecular weight cutoff value
of 3 kDa), Millipore.
(7) Plastic Syringes, disposable, general use and non-sterile, 5 mL, Luer-Loc tip.
(8) Syringe filters—used in filtering samples, disposable, 0.45 μm PTFE membrane with
polypropylene housing and Luer-Loc inlet.
(9) Analytical balance—precision of 0.01 g.
(10)Repeating pipettes and dispensers – 2 μL → 50 mL calibrated for organic and aqueous
liquids, i.e., Drummond Pipet Aid XP (Drummond Scientific Co) or equivalent for dispensing
10 and 20 mL liquid volumes, Gilson pipettes M10, M100, M1000 for dispensing 1-10 µL,
10-100 µL, 100 – 1000 µL range and appropriate CP10, CP100, and CP1000 pipet tips (or
equivalent brands)
(11)Benchtop Centrifuge (Thermo Scientific CR4i centrifuge or equivalent) for centrifuging 15
mL centrifuge tubes containing sample extracts.
(12)IKA Tube Mill and IKA Tube Mill 100.
(13)IKA disposable 40 mL and 100 mL grinding chambers.
(14)
(15)

2020.8 METHOD
The following instrument parameters are specific to two LC-MS/MS instruments listed below
used in the multi-laboratory validation of this method These parameters may change according to
the manufacturer, subtle variation in chemical composition of mobile phase, purity and type of
ESI source and collision gases used. Depending on LC columns and origin of mobile phases,
minor variations may occur.

High performance liquid chromatography (HPLC) – Shimadzu Prominence equipped with a

binary pump (LC-20ADXR), autosampler (SIL-20ACXR) and column oven (CTO-20AC).

Mass spectrometer (MS) – Sciex 4000 and 6500 QTRAP with AnalystTM, version 1.6 or higher,
for instrument control software. LC-MS/MS data is processed using MultiQuanTM, version 2.0 or
higher (Sciex)

HPLC Binary Pump, autosampler and MS/MS, Positive Mode, parameters for FDA Mycotoxin
Analysis

HPLC:
Mobile phase A: Water (10 mM ammonium formate, 0.1% formic acid)
Mobile phase B: Methanol (10 mM ammonium formate, 0.1% formic acid)

Stop time 15 min

Pressure Limits ≥ 9000 Psi
Minimum Pressure 0 Psi
Maximum Pressure ≥ 9000 Psi
Timetable
Time Solvent B Flow
(min) (%) (µL/min)
0.0 5.0 300
2.0 40.0 300
10 100.0 300
11.5 100.0 300
12.0 5.0 300
15.0 5.0 300

Autosampler:
Injection volume: 3 μL
Rinsing volume: 200 μL
Needle stroke: 52 mm
Rinsing speed: 35 µL/s
Sampling speed: 5 µL/s
Rinse dip time: 5s
Controller temperature: 10 oC
Column temperature: 40 oC

Mass Spectrometry:

Sciex 4000
Collision gas: N2, medium
Curtain gas: N2, 30
IonSpray Voltage: 5000 Volts
Interface heater: ON
TurboIon source temperature: 500 °C
Gas 1: 50
Gas 2: 50
Q1 Resolution: Unit
Q3 Resolution: Unit
Scheduled MRM schemes for the target compounds are listed in the following table.

Sciex 6500
Collision gas: N2, medium
Curtain gas: N2, 30
IonSpray Voltage: 5500 Volts
Interface heater: ON
TurboIon source temperature: 600 °C
Gas 1: 60
Gas 2: 60
Q1 Resolution: Unit
Q3 Resolution: Unit
Table 8-1: Scheduled MRM parameters for the target mycotoxins.

Molecular Molecular RT 6500 QTRAP 4000 QTRAP

Mycotoxins Adduct ion MRM transitions*
formula weight (min) DP (eV) CE (eV) CXP (eV) DP (eV) CE (eV) CXP (eV)
Aflatoxin B1 C17H12O6 312.1 6.1 [M+H] +
313.1→241.0/285.0 86/106 55/37 14/8 107/85 53/35 13/15
[ C17]-aflatoxin B1
13
C17H12O6
13
329.1 6.1 [M+H] +
330.1→255.2/301.0 86/106 55/37 14/8 107/85 53/35 13/15
Aflatoxin B2 C17 H14O6 314.1 5.8 [M+H] +
315.2→287.1/259.1 91/91 39/45 16/14 105/110 38/43 16/14
[ C17]-aflatoxin B2
13
C17 H14O6
13
331.1 5.8 [M+H] +
332.0→303.2/273.1 91/91 39/45 16/14 105/110 38/43 16/14
Aflatoxin G1 C17 H12O7 328.1 5.5 [M+H]+ 328.8→243.2/200.0 86/86 41/99 14/10 93/93 39/58 13/10
[ C17]-aflatoxin G1
13
C17 H12O7
13
345.1 5.5 [M+H] +
345.8→257.1/124.2 86/86 41/99 14/10 80/80 38/92 13.5/8
Aflatoxin G2 C17 H14O7 330.1 5.3 [M+H] +
331.2→313.0/245.0 111/111 36/49 18/20 88/100 36/42 18/13
[ C17]-aflatoxin G2
13
C17 H14O7
13
347.1 5.3 [M+H] +
348.0→330.0/259.0 111/111 36/49 18/20 88/100 36/42 18/13
Deoxynivalenol C15H20O6 296.1 3.6 [M+H] +
297.0→249.0/231.2 71/61 17/21 44/22 65/64 17/20 20/13
[ C15]-deoxynivalenol
13
C15H20O6
13
311.2 3.6 [M+H] +
312.0→263.0/345.2 71/61 17/21 44/22 65/64 17/20 20/13
Fumonisin B1 C34H59NO15 721.4 7.5 [M+H]+ 722.5→352.5/334.5 111/111 53/57 10/54 107/110 52/55 20/20
[ C34]-fumonisin B1
13
C34H59NO15
13
755.5 7.5 [M+H] +
756.4→374.5/356.4 111/111 53/57 10/54 107/110 52/55 20/20
Fumonisin B2 C34H59NO14 705.4 8.7 [M+H] +
706.3→336.3/318.3 106/106 55/59 10/20 115/105 51/54 20/18
[ C34] fumonisin B2
13
C34H59NO14
13
739.5 8.7 [M+H] +
740.3→358.4/340.5 106/106 55/59 10/20 125/92 51/55 20/26
Fumonisin B3 C34H59NO14 705.4 8.2 [M+H]+ 706.3→336.3/318.3 106/106 55/59 10/20 105/115 51/53 19/17
[ C34]- fumonisin B3
13
C34H59NO14
13
739.5 8.2 [M+H] +
740.5→358.4/340.4 106/106 55/59 10/20 105/115 51/53 19/18
Ochratoxin A C20H18ClNO6 403.1 7.6 [M+H] +
404.0→239.0/102.0 66/66 41/101 16/16 74/73 36/101 13/17
[ C20]-ochratoxin A
13
C20H18ClNO6
13
423.1 7.6 [M+H] +
424.1→250.1/110.1 66/66 41/101 16/16 74/73 36/101 13/17
HT-2 toxin C22H32O8 424.2 7.1 [M+NH4]+ 442.2→215.3/323..2 50/50 20/15 16/16 58/55 20/13 11/17
[ C22]-HT-2 toxin
13
C22H32O9
13
446.3 7.1 [M+NH4] +
464.2→229.2/340.2 50/50 20/15 16/16 58/55 20/13 11/17
T-2 toxin C24H34O9 466.2 7.7 [M+NH4] +
484.3→215.2/185.1 57/57 29/33 17/11 55/50 26/30 12/9
[ C24]-T-2 toxin
13
C24H34O9
13
490.3 7.7 [M+NH4] +
508.3→229.2/198.2 57/57 29/33 17/11 55/50 26/30 12/9
Zearalenone C18H22O5 318.1 8.2 [M+H] +
319.2→283.2/187.1 101/86 17/31 10/10 50/50 19/29 16/10
[13C18]-Zearalenone C18H22O5
13
336.2 8.2 [M+H]+ 337.2→138.2/124.0 71/81 79/87 10/10 45/35 70/77 10/21

9
2020.9 CALCULATIONS
Quantitation was based on linear least squares calibration of the relative response ratio of a
mycotoxin and its 13C-IS plotted versus mycotoxin concentration. In each sample, the
concentrations of mycotoxins were determined from the equation
C = 13C‐IS concentration ratio × (S − y‐intercept)/slope
Where C is the concentration in the sample, 13C-IS concentration ratio is the concentration of
13
C-IS in the sample/concentration of 13C-IS in each calibration standard, and S is the signal
(peak area) of native mycotoxin/signal (peak area) of 13C-IS in the sample (using the quantitation
ions of isotope and native mycotoxin). See Appendix IV for step-by-step calculation.

If necessary, the method LOQ of a target mycotoxin may be determined following the protocol
for determining a method detection limit (MDL) and subsequently an LOQ (LOQ = 3.33 ×
MDL) detailed in 40 CFR Part 136 App B.3 Briefly, the procedures include:
(1) Estimate an MDL (ng/g in matrix). This estimate can be based on previous work with
the method, response from low end calibration standards, or limited spiking studies.
(2) Fortify the matrix at between one and five times the Estimated MDL. In all cases a
minimum of 8 replicates should be processed and analyzed, but the fortification will
vary slightly depending on the matrix. For dry matrices that use the water slurry
method, we recommend separately fortifying each 25 g sample prior to the addition of
water. For more homogeneous matrices, it is appropriate to separately fortify each 1 g
sample or to fortify a larger sample, homogenize and then subsample 8 aliquots. For
liquid samples, fortifying a larger volume, mixing and then subsampling 8 aliquots is
appropriate.
(3) Process and analyze the samples and corresponding blanks using the method protocol.
(4) Determine the variance and standard deviation to calculate the MDL and LOQ using
the results from the replicate analyses. In addition to the determination of these
parameters, the quantitative results used in the calculations should meet the
identification and confirmation criteria described in FDA Guidelines for the
Validation of Chemical Methods for the FDA Foods Program; specifically, all
transitions must be present, with appropriate relative abundances, at the MDL.
(5) Report estimated LOD and LOQ. Report quantitative results only when quality
control criteria for a batch have been satisfactorily met. Report results for identified
and quantitated mycotoxins ≥LOQ as the final concentrations (ng/g or ppb) in
samples. Report results that are ≥LOD and <LOQ as “<LOQ”, indicating mycotoxin
is present at a trace level that is below the limit of quantification. Report results that
are <LOD as “not detected”. Due to variability between laboratories and
instrumentation, values for LOD and LOQ should be determined in each instrument
system at each laboratory.

2020.10 VALIDATION INFORMATION/STATUS

The method specified in this document was validated to level 3 under FDA Guidelines for the
Validation of Chemical Methods for the FDA Foods Program. 1 In addition to three certified
reference materials, the participating laboratories -analyzed corn, peanut butter, and wheat flour
fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. The majority
of recoveries ranged between 80 ─ 120% with relative standard derivations (RSDs) <20%.

10
Greater than 90% of the average recoveries of the participating laboratories were in the range of
90-110%, with repeatability RSDr (within laboratory) < 10% and reproducibility RSDR (among
laboratory) < 15%. All Z scores of the results of certified reference materials were between -2
and 2. More details regarding the method validation study can be found in Zhang et al., 2017.4

2020.11 REFERENCES
1. FDA Guidelines for the Validation of Chemical Methods for the FDA Foods
Program;http://www.fda.gov/downloads/ScienceResearch/FieldScience/UCM298730.pdf
(accessed May 1, 2017).

2. FDA Center for Veterinary Medicine. Guidance for Industry 118, 2003. Mass Spectrometry
for Confirmation of the Identity of Animal Drug Residues;
http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Guidan
ceforIndustry/UCM052658.pdf (accessed May 1, 2017)

3. 40 CFR Appendix B to Part 136 - Definition and Procedure for the Determination of the
Method Detection Limit-Revision 1.11 https://www.gpo.gov/fdsys/granule/CFR-2011-
title40-vol23/CFR-2011-title40-vol23-part136-appB/content-detail.html (accessed May 1,
2017)

4. Zhang, K., Schaab, M.R., Southwood, G., Tor, E.R., Aston, L.S., Song, W., Eitzer, B.,
Majumdar, S., Lapainis, T., Mai, H., Tran, K., El-Demerdash, A., Vega, V., Cai, Y., Wong,
J.W., Krynitsky, A.J., Begley, T.H. (2017) A Collaborative Study: Determination of
Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay
(SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). J. Agric.
Food Chem. 2017, 65, 7138-7152.

11
Appendix I. Example of procedures used to prepare 5 mL of working solution of 13C-IS

1. Mix 100 mL HPLC or Millipore-grade water and 100 mL of HPLC-grade acetonitrile in a 250 mL flask.
2. Manually shake the flask for 2 min.
3. Pipette appropriate amount of each 13C-IS from the corresponding stock solution vials into a 5 mL volumetric flask (as shown in the table below)
4. Bring the volume to the mark (5.0 mL) by adding prepared extraction solution (v/v, 50/50, acetonitrile/water)
5. Transfer prepared working solution to a 15 mL amber glass vial and stored at -20oC in

Stock solution Working solution

Pipetted volume of stock solution Working solution
13C-IS Conc. volume
(µL) conc. (µg/mL)
(µg/mL) (µL)
[13C]-aflatoxin B1+B2+G1+G2 0.5 500 5000 0.05
[13C]-deoxynivalenol 25 400 5000 2
[13C]-fumonisin B1 25 400 5000 2
[13C]-fumonisin B2 10 1000 5000 2
[13C]-fumonisin B3 10 1000 5000 2
[13C]-HT-2 toxin 25 400 5000 2
[13C]-ochratoxin A 10 100 5000 0.2
[13C]-T-2 toxin 25 400 5000 2
[13C]-zearalenone 25 400 5000 2

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Appendix II. Example of solvent-only calibration standards

Mycotoxin/13C-IS Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 Level 8 Level 9 Level 10
Aflatoxin B1 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 25 50
[ C]-aflatoxin B1
13
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Aflatoxin B2 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 25 50

[ C]-aflatoxin B2
13
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Aflatoxin G1 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 25 50

[ C]-aflatoxin G2
13
1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Aflatoxin G2 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 25 50

[13C]-aflatoxin G2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Ochratoxin A 0.05 0.1 0.25 0.5 1.0 2.5 5.0 10 25 50

[ C]-ochratoxin A
13
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0

Deoxynivalenol 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[ C]-deoxynivalenol
13
40 40 40 40 40 40 40 40 40 40

Fumonisin B1 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[ C]-fumonisin B1
13
40 40 40 40 40 40 40 40 40 40

Fumonisin B2 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[13C]-fumonisin B2 40 40 40 40 40 40 40 40 40 40

Fumonisin B3 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[ C]-fumonisin B3
13
40 40 40 40 40 40 40 40 40 40

HT-2 Toxin 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[ C]-HT-2 Toxin
13
40 40 40 40 40 40 40 40 40 40

13
 T-2 Toxin 0.5 1.0 2.5 5.0 10 25 50 100 250 500
[13C]-T-2 Toxin 40 40 40 40 40 40 40 40 40 40

Zearalenone 0.5 1.0 2.5 5.0 10 25 50 100 250 500

[ C]-zearalenone
13
40 40 40 40 40 40 40 40 40 40

14
Appendix III: Total ion chromatogram and extracted ion chromatograms of target mycotoxins and 13C-mycotoxins generetaed using
conditions listed in Section 2017.8

15
Appendix IV. Example for calculation
Step 1: establish calibration curve for a target mycotoxin (e.g., AFM1)

AFM1 13C-IS AFM1/13C-IS

Conc.
(ng/mL) Area Area Area ratio
0.01 8.08E+03 3.71E+05 2.18E-02
0.05 3.59E+04 3.68E+05 9.78E-02
0.1 7.45E+04 3.74E+05 1.99E-01
0.25 1.82E+05 3.76E+05 4.85E-01
1 7.62E+05 3.68E+05 2.07E+00
2.5 1.87E+06 3.63E+05 5.14E+00
5 3.70E+06 3.63E+05 1.02E+01
25 1.86E+07 3.80E+05 4.88E+01
50 3.63E+07 3.46E+05 1.05E+02
100 7.16E+07 3.57E+05 2.01E+02
Sample 1 3.77E+05 3.62E+05 1.04E+00
Sample 2 7.43E+06 3.74E+05 1.99E+01

Step 2

Calibration curve: y = 2.0205x + 0.1058

Use x =R*(y-b)/m to calculate the concentration of a detected mycotoxin:
. Where x is the calculated concentration, R is the relative concentration ratio of the [13C]-IS
fortified in the sample and calibration standards, which can be determined based on sample
preparation, y is the relative peak area ratio of the mycotoxin and the [13C]-IS in the sample, b is
the intercept on the Y axis, and m is the slope. In this case, R =5, m = 2.0205 and b = 0.1058

Step 3
Concentrations (ng/mL) of AFM1 in the two sample:

Sample 1 5*(A-0.1058)/2.205 = 2.12 (ng/g)

Sample 2 5*(B-0.1058)/2.205 = 44.80 (ng/g)

16
Appendix V. Sources of certified reference materials used for method verification

1. National Institute of Standards and Technology

• SRM 2387 (aflatoxins in peanut butter)

2. European Commission Joint Research Institute, Institute for Reference materials and
Measurements
• BCR-385R (aflatoxins in peanut butter)
• BCR-401 (aflatoxins in peanut butter)
• ERM-BC600 (fusarium mycotoxins in wheat flour)

3. FAPAS
• TET017RM (mycotoxins in maize flour)

17