Melissa Phillips1, Jennifer Ness2, Tomás López Seal4, Carolyn Q. Burdette1, Kai Zhang3

1National Institute of Standards & Technology, Gaithersburg, MD, USA and 2Charleston, SC, USA

3US Food and Drug Administration (FDA), College Park, MD USA

4Instituto Nacional de Tecnología Industrial (INTI), Buenos Aires, Argentina

Fungi of the genera Aspergillus, Fusarium, and Penicillium produce mycotoxins such as aflatoxins, fumonisins, deoxynivalenol, zearalenone, ochratoxin A, HT-2 toxin, and T-2 toxin onto foods

and feeds during harvests, storage, or onto finished products under warm or humid conditions. Although mycotoxin contamination can be minimized with the implementation of good

agricultural and manufacturing practices, dietary exposure to humans and animals is unavoidable as these toxins are resistant to degradation by current food-processing procedures. Due to

the numerous negative health impacts associated with the consumption of foodstuffs contaminated with mycotoxins, accurate determination is an international concern. Many commerciallyavailable certified RMs for mycotoxins mainly address a single mycotoxin or class of mycotoxins, requiring the use of multiple RMs for multi-target methods. In addition, none of these RMs

contain all of the FDA-regulated mycotoxins and mycotoxins of health significance. To address the increasing needs of laboratories moving toward LC-MS-based multimycotoxin analysis, the

U.S. National Institute of Standards and Technology (NIST) collaborated with the U.S. Food and Drug Administration (FDA) to produce a naturally incurred RM for multiple mycotoxins in corn.

Preliminary Screening by LC-MS/MS

Homogeneity Testing-Sample Size

Particle Size Analysis

Bottling

Homogeneity Testing

Value Assignment

Interlaboratory 

Comparison

Disclaimer: Certain commercial equipment, instruments, or materials are identified to specify the experimental procedure adequately. Such identification is not intended to imply 

recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Sample Preparation

Why a Multi-Mycotoxin Material?

Material Design and Preparation

Interlaboratory Comparison

Target particle size for SRM 1565

was ≤ 841 μm (#20 sieve; based on

guidance from AOAC).

Samples of blank (top), incurred

(center), and blended (bottom)

corn (≈ 0.5 g) were dispersed in

ethanol, allowed to disperse for

10 s before 10 individual measurements were recorded at an

obscuration of 10 % and 20 %

using a Malvern Mastersizer 3000.

Final packaged units of SRM 1565 were irradiated to an absorbed

dose of 11.9 kGy to 18.7 kGy to reduce the probability of microbial

growth and additional mycotoxin production.

Value Assignment by NIST and FDA/CFSAN

Assigned values for mycotoxins in SRM 1565 were determined by combination of the means from NIST ID-LC-MS/MS 

and FDA ID-LC-MS/MS measurements and the median from qualified laboratories from the interlaboratory comparison 

using a linear pool method (2). The uncertainty represents the SD of the aggregate probability distribution.

• A stratified random sampling scheme was used by

NIST and FDA to select 10 bottles of SRM 1565

(Incurred) and SRM 1565 (Blank) to prepare in

duplicates.

• A QC material with know levels of mycotoxins was

purchased from FAPAS to use as a control and was

prepared in duplicates.

• Recovery studies revealed automated shaking was

optimal for extraction and no significant increase in

extraction yield was observed from additional

extractions.

• NIST used a relative response factor approach to

calibration.

• Homogeneity was evaluated for each determined

mycotoxin as a function of packaging order, sample

preparation order, and chromatographic run order.

• FDA evaluated sample size variability between 1 g

and 5 g sample sizes.

Future Work

Corn cryomilled at 

NIST Cryogenic 

Reference Material 

Production Facility

Collected from 14 

different batches of 

naturally contaminated 

corn. 

Instrumentation: Agilent Series 1260 LC / Applied 

Biosystems SCIEX API 5000 LC-MS/MS system, 

electrospray ionization in positive ion mode.

MRM transitions and Compound MS Parameters:

Column: Phenomenex Kinetex XBC18 LC column 

(100 × 2.1 mm i.d., 2.6 μm particles) with a 10 ×

2.1 mm guard cartridge, 40 °C

Mobile Phase A: 10 mmol/L ammonium formate 

in water containing 0.1 % formic acid (v/v)

Mobile Phase B: 10 mmol/L ammonium formate 

in methanol containing 0.1 % formic acid (v/v)

Flow Rate: 0.3 mL/min

Sample chromatogram of 

mycotoxins in SRM 1565 by 

NIST ID-LC-MS/MS.

LC Gradient: Time (min) %B

0 5

2.0 40

10.0 100

11.5 100

12.0 5

20.0 5

Summary of ID-LC-MS/MS Measurements by NIST and

FDA/CFSAN

• All values obtained for the QC material using the ID-LC-MS/MS

method were within the value range assigned by the

manufacturer.

• NIST ID-LC-MS/MS measurement precision varied between

7.5 % and 41 % RSD in duplicate preparations of 10 samples of

incurred and blank SRM 1565.

• Based on FDA ID-LC-MS/MS measurement data (1), 1 g sample

size is recommended for SRM 1565 (Blank). Using 5 g aliquots

decreased measurement precision for deoxynivalenol (17 % to

35 % RSD) and fumonisin B1 (11 % to 23 % RSD). 5 g sample size

is recommended for the remaining mycotoxins.

• NIST did not provide data for fumonisin B2, fumonisin B3, total

fumonisins, and HT-2 toxin.

• FDA data for ochratoxin A was not used for value assignment.

• FDA did not provide data for aflatoxin G2 based on failed

identity criteria.

Development and Value Assignment of an Incurred 

Multi-Mycotoxin Reference Material

Effects of Irradiation

References:

(1) Zhang, K., et al. (2017) J. Agric. Food Chem. 65, 7138–7152. doi:10.1021/acs.jafc.6b04872

(2) Koepke, A., Lafarge, T., Possolo, A., & Toman, B. (2017) Metrologia 54, S34–S62

(3) Phillips, M.M., et al. (2019) J. AOAC Int. 102, doi:https://doi.org/10.5740/jaoacint.19-0109

Acknowledgements: AJ Moors, J Trevillian, DL Ellisor (blending, packaging, particle size determination); KM 

Morehouse, L Cumberland (irradiation); CA Barber, BA Benner, JB Thomas, CQ Burdette, J Camara, SE Long, JA Murray, 

BJ Place, CA Rimmer, LJ Wood (HAMQAP); CQ Burdette (technical); H-K Liu (statistics); G Hahm (poster assembly)

Incurred 

Corn (14 kg)

Blank Corn 

(70 kg)

Corn collected from 

various markets in 

Maryland including 7 

brands and various types.

Estimated levels of mycotoxins in the blended incurred and blank corn

materials determined by FDA/CFSAN using ID-LC-MS/MS (1) after first

round of blending (left) and in final mixture for incurred corn (right).

Screened mass fraction values were used as target values and provided

to participating laboratories in interlaboratory comparison.

PCA scores plot of 1 g 

to 10 g sample sizes of 

SRM 1565 (Incurred) 

and QC material 

(FAPAS). 

Clustering of data 

points indicated that 

sample sizes of SRM 

1565 should be ≥ 3 g 

to ensure test portion 

homogeneity and 

reproducibility of 

analytical results.

Average particle size for SRM 1565 (Blank) was 588 μm

(SD = 17 μm, n = 10). For SRM 1565 (Incurred), the average

particle size was 676 μm (SD = 92 μm, n = 10).

Weigh Sample

1 g of Blank Corn

5 g of Incurred Corn

Extract mycotoxins by mixing for 30 min using Glas-Col Large Capacity 

Mixer with motor speed of 70 and maximum pulsation frequency

Add water 

5 mL to 1 g sample; 25 mL to 5 g sample

Hand-mix and equilibrate at room 

temperature for 15 min

Centrifuge for 10 min at 4200 × g

Filter supernatant through 13 mm × 0.2 μm PTFE syringe 

filter using 3 mL disposable syringe 

Add acetonitrile to achieve 

50:50 ACN:H2O (v/v)

Analyze by LC-MS/MS

Add 50:50 ACN:H2O (v/v)

5 mL to 1 g sample; 

25 mL to 5 g sample

Gravimetrically spike 

Internal Standard Mixture

Gravimetrically spike 

Internal Standard Mixture

• With the availability of reference standards

• Values for DON and ZON will be upgraded to certified 

with SI traceability

• Values will be assigned for fumonisin B2, fumonisin B3, 

HT-2 toxin, and T-2 toxin

• Development of other matrix-matched foods 

contaminated with mycotoxins

Results from Interlaboratory Comparison

• Data evaluated for quality based on the result provided for the

control sample from FAPAS. Data from poor results of the control was

excluded from the consensus data.

• Overall performance was good with between laboratory variabilities

were comparable and ranged from 16 % to 49% for most mycotoxins

in SRM 1565 (Incurred).

• Higher between-laboratory variability was observed for lower-level

mycotoxins in the materials: aflatoxin G2 (82 %) in SRM 1565

(Incurred), and deoxynivalenol (46 %) and fumonisin B1 (84 %) in

SRM 1565 (Blank).

• Consensus means were within the target ranges for all analytes in all

samples, except for total fumonisins in SRM 1565 (Blank). Low levels

of the fumonisins in the blank material may have resulted in

measurement challenges for some laboratories.

AFB1

DON

OTA

Results from interlaboratory comparison for Aflatoxin 

B1 (AFB1), Deoxynivalenol (DON), Ochratoxin A (OTA) 

in SRM 1565 Mycotoxins in Corn (Incurred).

• 38 participating laboratories for NIST Health Assessment Measurements

Quality Assurance Program (HAMQAP) Exercise 1.

• Participants were provided with blinded samples of SRM 1565 (Blank),

SRM 1565 (Incurred), and a QC material (FAPAS).

• Asked to prepare 3 samples and report 3 results for each mycotoxin or group

of mycotoxins in each sample, using their routine methods of analysis.

Blank Corn 

(70 kg)

14 g of contaminated corn 

and 28 kg of homogenized 

corn were mixed.

SRM 1565 

Myctoxins in 

Corn (Incurred)

Cryomilling

SRM 1565 

Mycotoxins in 

Corn (Blank)

Screening by LC-MS/MS

Particle Size Analysis

Incurred 

Corn (14 kg)Homogenized

Contains low natural levels of FB1 

and DON at 100x and 10x below 

the FDA lowest action levels (resp).

Contains mycotoxin 

concentrations near the FDA 

lowest action levels. 

Irradiation Blank Corn 

(28 kg)

Combined Results

Incurred Blank

600 units x 60 g 600 units x 60 g

MS Source Parameters:

Collision Gas (CAD) 10

Curtain Gas (CUR) 30

Ion Source Gas 1 (GS1) 50

Ion Source Gas (GS2) 50

IonSpray Voltage (IS) 5000

Temperature (TEM) 500

Entrance Potential 4

Analysis of mycotoxins by NIST ID-LC-MS/MS

Injection Volume: 3 μL

Mass fractions 

of mycotoxins in 

SRM 1565 were 

not affected 

significantly by 

exposure to 

γ-radiation at 10 

and 20 kGy

absorbed doses. 

SRM 1565 (Blank)

SRM 1565 (Incurred)