FROM SAMPLING TO MEASUREMENT

MYCOTOXIN ANALYSIS

Mycotoxins
Mycotoxins are a diverse group of compounds comprised of hundreds of secondary metabolic products from various fungal species.

Awareness regarding the contamination of the food supply with Occur during growth, harvest, transportation, processing Broad range of complex matrixes can be contaminated Several mycotoxins show marked toxicity in humans
Therefore, the removal of contaminated products from the food chain is a primary means of eliminating human exposure. or storage mycotoxins is increasingly prevalent

The sensitive and accurate detection of very low levels of these compounds is critical to government efforts to identify contaminated product. Chromatographic methods such as GC and HPLC are most commonly used for analysis, usually preceded by a number of operations such as sampling, sample preparation, extraction and cleanup. In recent years, Sigma-Aldrich, has developed a comprehensive range of products and methods for the analysis of mycotoxins. This brochure contains our current product offering. Contact us if you need support with your application; we will be delighted to help.

MYCOTOXIN ANALYSIS WORKFLOW

Sigma-Aldrich offers valuable products for the entire mycotoxin analysis workflow.

Matrix Processing
Homogenization and Grinding

page 3

Isolation and Concentration

Derivatization
page 6 HPLC Reagents page 9 GC Reagents page 8 page 9 page 9

Chromatographic Separation

Quantification
page 10 Standards and CRMs page 13

page 3

SPE Cartridges and Accessories page 6 Solvents

HPLC and UHPLC page 11 GC and GC-MS page 10 Performance Accessories

page 13

Extraction page 4 Filtration and Evaporation

Labeled Standards page 13

page 5

page 15

LC-MS Grade Solvents

page 8

Mycotoxin Analysis

Matrix Processing
A range of well-developed techniques is available. The criteria for choosing a suitable method include available time and equipment, specificity and sensitivity. The workstation provides a unique and universal dispersing, stirring, homogenizing and grinding system, with hermetically sealable and disposable sample tubes.

HOMOGENIZATION AND GRINDING

Homogenization
Homogenization is an important first step because mycotoxins are formed by mold that occur either in isolated pockets of bulk materials, or in individual seeds or nuts. To ensure accurate test results, the sample should be representative of the lot from which is was obtained. Mycotoxins are often distributed in food commodities such as maize, ground nuts (peanuts), tree nuts and cottonseed.

Disperse, stir, homogenize and grind using a single drive unit Hermetically sealable disposable sample tubes eliminate Gamma-sterilized tubes Tubes (215 mL and 1550 mL ) with pierceable membrane covers Anti-locking function and chemical-resistant plastic
Protection and security for infectious sample materials, toxic substances, high-odor substances.
cross-contamination

Milling
The grinding of solid samples is essential to ensure precise analysis. Proper grinding leads to the homogeneity and desired fineness of the sample.

Cat. No.
Z722472 Z722464 Z722456 Z722375

Description
IKA ULTRA-TURRAX dispersers, T-10 Basic, for volumes of 0.5-100 mL, 230 V, 1/cs IKA ULTRA-TURRAX dispersers, T-25 Digital, for volumes of 1-2,000 mL, 230 V, 1/cs ULTRA-TURRAX Tube Drive Workstation IKA ULTRA-TURRAX disperser tubes, DT-20 dispersing tube with rotor-stator element, 25/CS

ULTRA-TURRAX Tube Drive Workstation

The workstation consists of an ULTRA-TURRAX Tube Drive: 2 x ST-20 (Z722383), 2 x DT-20 (Z722367), a removal hook for disengagement of the rotor-stator unit, 2 x BMT-20 G / S (Z722405 and Z722421) and a power supply.

The type of mill used depends on the properties of the matrix and the quantity of the sample. For example, brittle materials are ground with a beater, fibrous materials with a blade and hard materials are ground with a special metal cutter while small and large sample quantities are generally ground with a batch or inline mills, respectively.

A continuously operating grinder with a powerful drive, an easy to clean working surface made of stainless steel, and easily changeable heads.
Cat. No.
Z645176 Z645249 Z645257

Description
MF 10 basic Microfine grinder drive dringing heads not included with delivery MF 10.1 Cutting-grinding head (for crushing fibrous substances) MF 10.2 Impact grinding head (for crushing brittle, hard materials)

For more information, visit sigma-aldrich.com/analytical

EXTRACTION
Sample cleanup is the removal of substances from the matrix that may interfere with the detection of the analyte and can also be used for pre-concentration of the analyte by reducing the amount of solvent. Matrix components such as lipids, carbohydrates and peptides that are usually present in the raw extract make an additional purification step necessary prior to the ultimate separation and detection step.

Liquid-Solid Extraction (LSE)

This is a basic technique used in mycotoxin analyses, if the sample is available in a solid form, which is the case for cereals including maize and most other products. If solid samples are not available, freezedrying or dehydration is sometimes an option for making the sample easier to handle. It is the purpose of this purification step to dissolve the analyte quantitatively in the solvent, with as little additional compounds as possible in order to avoid interferences.
Soxhlet Extraction Apparatus and Thimbles

Liquid-Liquid Extraction (LLE)

Liquid-liquid partitioning is a well-known and well established cleanup technique. After the partitioning step, rotary evaporation is performed to reduce the amount of solvent and to pre-concentrate the analyte. The method is simple and easy-to-perform with standard laboratory equipment and often still forms part of official methods.

Extraction Systems and Thimbles

Cat. No.
64815-U 64816-U 64817-U 64818 64819-U 64820-U 64821-U 64822-U 64823 64803-U 64804-U 64805-U 64806 64824 64825 64826 64840-U 64841-U 64842 64836-U 64837 64838

Description
Condenser size small Condenser size medium Condenser size large Extractor size small Extractor size medium Extractor size large Flat bottom flask size 125 mL Flat bottom flask size 250 mL Flat bottom flask size 300 mL Separatory funnel glass stopcock plug, size 250 mL Separatory funnel glass stopcock plug, size 2000 mL Separatory funnel PTFE stopcock, size 250 mL Separatory funnel PTFE stopcock, size 2000 mL Soxhlet extraction apparatus size small Soxhlet extraction apparatus size medium Soxhlet extraction apparatus size large Thimble cellulose, size 25 mm 80 mm Thimble cellulose, size 33 mm 94 mm Thimble cellulose, size 43 mm 123 mm Thimble glass, size 25 mm 85 mm Thimble glass, size 35 mm 90 mm Thimble glass, size 45 mm 130 mm

Extraction Glassware
Sigma-Aldrich has a wide range of extraction glassware, including:

Liquid-Liquid Extraction including extractor-concentrator kits, Accessories for Sample Concentration / Extraction
flasks and liquid-liquid extractor

Whatman Filter Papers

Mycotoxin Analysis

FILTRATION AND EVAPORATION

Filtration
After the extraction step, filtration could be necessary to remove solids or to eliminate particles before direct injection. Sigma-Aldrich offers a complete solution of membrane, syringe filters or special devices to meet your requirements. For HPLC and GC, to remove particles or recover the liquid part of the extraction, the use of pre-pleated (SV) membrane is recommended.

GD/X Syringe Filters for Hard-to-filter Samples that Require More Than One Filter
Whatman GD/X Syringe Filters are an excellent choice for filtering high-particulate or viscous solutions. They feature glass microfiber prefilters enabling you to filter more of your sample in less time.

Large selection of membranes (0.2 or 0.45 m) Increased flow rate Four layers of filtration media: Reduces blockage and need to
replace filter

Grade
Diameter 90 mm 110 mm 125 mm 150 mm 185 mm 240 mm 270 mm 320 mm 385 mm Whatman Cat. No. 1002-090 1002-110 1002-125 1002-150 1002-185 1002-240 1002-270 1002-320 1002-385

2
Sigma-Aldrich Cat.No. Z240214-1PAK Z240222-1PAK Z240230-1PAK Z240249-1PAK Z240257-1PAK Z240265-1PAK Z752304-1PAK Z240281-1PAK Z695165-1PAK

2SV (prepleated)
Whatman Cat. No. 1202-125 1202-150 1202-185 1202-240 1202-270 1202-320 1202-385 Sigma-Aldrich Cat.No. Z240303-1PAK Z240311-1PAK Z240338-1PAK Z240346-1PAK Z240354-1PAK Z240362-1PAK Z740330-100EA

One GD/X filter replaces 2 to 5 conventional filters.

Description
GD/X 25 mm 0.2 m Nylon PTFE PP CA RC GD/X 25 mm 0.45 m Nylon PES PVDF PTFE RC

150 per pack Cat. No.

Z277355-1PAK Z277401-1PAK Z277436-1PAK Z745375-150EA Z747599-150EA Z277363-1PAK Z286583-1PAK Z277398-1PAK Z277428-1PAK Z747610-150EA

1,500 per pack Cat. No.

Z745294-1500EA Z745332-1500EA Z747602-1500EA Z671185-1500EA Z745553-1500EA Z671193-1500EA Z671207-1500EA Z747629-1500EA

Whatman Mini-Uniprep for Direct Injection on LC-MS-MS

Avoid contamination Syringeless filter, all-in-one Replace the syringe, syringe

filter and vial Ideal for UHPLC

Available in clear or amber PP

or glass Wide range of membrane choices Compatible with most major autosamplers

Evaporation
For some applications, an evaporation step is necessary; either to have the components in a solvent compatible with the chromatographic system or just to concentrate the extract before the analysis. Various systems are available; one of the most well-known is the Rotavapor system from Bchi.

An all-in-one filtration system can reduce costs and processing time by up to 35%. No additional consumables are required.
Cat. No. Description
Whatman Mini-UniPrep G2 standard septum Z759422 PTFE membrane, pore size 0.2 m, clear vial color Z759481 PTFE membrane, pore size 0.45 m, clear vial color Z759384 starter pack supplied with single vial compressor, PTFE membrane, pore size 0.2 m, amber vial color Z759430 starter pack supplied with single vial compressor, PTFE membrane, pore size 0.2 m, clear vial color Z759414 starter pack supplied with single vial compressor, Nylon membrane, pore size 0.2 m, clear vial color Z759503 starter pack supplied with single vial compressor, PTFE membrane, pore size 0.45 m, clear vial color Whatman Mini-UniPrep (PP housing) Z557889 nylon, 0.2 m, 100/pk or 1000/pk Z506621 nylon, 0.45 m, 100/pk or 1000/pk Z671274 amber, nylon, 0.2 m, 100/pk Z557897 PTFE, 0.2 m, 100/pk or 1000/pk Z506648 PTFE, 0.45 m, 100/pk or 1000/pk

Selection Guide of Available Rotavapors (240V)

Model R210
Diagonal 29/32 Glass Plastic-coated Vertical 29/32 Glass Plastic-coated Cold Trap 29/32 Glass Plastic-coated Cold Trap Reflux 29/32 Glass Plastic-coated

Basic (no Vac. Cont.)

Z563846 Z563633 Z563862 Z563676 Z563889 Z563706 Z565466 Z565199

Advanced (with V850)

Z563854 Z563641 Z563870 Z563684 Z563897 Z563714 Z565474 Z565202

Professional (with V855)

Z563668 Z563692 Z563722 Z565210

For more information, visit sigma-aldrich.com/analytical

Isolation / Concentration (SPE)

SUPEL TOX CARTRIDGES INCREASE THROUGHPUT TEN-FOLD
The need for a quick, simplistic sample cleanup approach prior to chromatographic mycotoxin analysis has brought about SPE cartridges that significantly decrease sample prep time, increase reproducibility and are more user friendly as compared to industry-standard immunoaffinity columns.

Purification in six minutes with a percent recovery > 85% and a RSD < 5% for Aflatoxins in peanut paste. Easy to handle and to store (no refrigeration).
Interference Removal Principle: Interference Removal Principle
Original sample (analytes and internal standard in a matrix) 1. Condition SPE cartridge 2. Apply sample to SPE cartridge

3. Apply elution solvent

Unlike the multiple step bind and elute strategy implemented when using immunoaffinity columns, the Supel Tox AflaZea, DON and Tricho SPE cartridges employ an interference removal strategy which saves time by eliminating wash steps prior to elution of aflatoxin and zearalenone, deoxynivalenol and tricothecenes (type A and B), respectively. Cartridges removing interferences associated with analysis of fumonisins (B1 and B2) and ochratoxin A are also available as a part of our Supel Tox product offering.

Unwanted matrix remains on cartridge. Cartridge discarded.

Features and Benefits

columns

Remove interferences associated with mycotoxin analysis Better reproducibility than the industry standard immunoaffinity Sample preparation time is up to ten times less than that of Straightforward, cost-effective and quick methodology requiring Improved shelf life over immunoaffinity columns due to the thermally
stable format. No refrigeration is required for shipping and storage. little additional method development (generic method) immunoaffinity columns (fewer steps)

4. Evaporate elution solvent

Purified analyte eluted

5. Reconstitute Purified and concentrated analytes and internal standard

Comparison of Supel Tox AflaZea SPE to Immunoaffinity for Aflatoxins B1, B2, G1 and G2 in Peanut Paste
Immunoaffinity
Sample Prep Time (post-extraction to pre-analysis) Ease of Use

Supel Tox AflaZea SPE Cartridge

60 minutes 8 samples/day (if processing 1 at a time) Large volumes of liquid Controlled drop rates Numerous complicated steps Additional buffer salts required Must be refrigerated, brought to room temp before use

6 minutes 80 samples/day (if processing 1 at a time) Small volumes of liquid Vacuum filtration used Steps few and not complicated No additional reagents required Column does not require special storage conditions

Mycotoxin Analysis

Sample Purification Strategy for Supel Tox SPE Cartridges

SPE Cartridge
Supel Tox AflaZea Supel Tox DON Supel Tox Tricho Supel Tox TrichoBind Supel Tox FumoniBind Supel Tox OchraBind

Analyte(s)
Aflatoxin B1, B2, G1 and G2, and Zearalenone Deoxynivalenol (DON) Trichothecenes (Type A and B) Trichothecenes (Type A and B) Fumonisins (B1 and B2) Ochratoxin A

Matrix
Grains, feeds, TMR samples, peanuts, peanut products and aqueous solutions Wheat, flour and corn Grains and complex matrices Grains, feeds and other complex matrices Grains and cereals Grain and feed samples

Purification Strategy
Interference removal Interference removal Interference removal Multifunctional bind and elute Multifunctional bind and elute Multifunctional bind and elute

Qty.
30 30 30 25 25 25

Cat. No.
55314-U 55316-U 55308-U 55307-U 55315-U 55318-U

Discover this new range of products, or obtain a complementary sample, visit

sigma-aldrich.com/supeltox

SPE ACCESSORIES
Visiprep DL (Disposable Liner) Vacuum Manifold eliminates the possibility of cross-contamination when processing a new sample on the same port.

Features and Benefits

flow control to solvents

Patented screw-type valves within each SPE port for precise Glass basin will not dissolve, fog, or discolor when exposed Leg covers enable cover to rest on work surface after removed Various PP vessel racks for numerous type of glassware
To apply large volumes of sample, use our SPE tube adapters and reservoirs.
Empty SPE Barrel 10-100 cc Syringe

To avoid long decontamination of your vacuum manifolds, use our Visiprep DL.

from the manifold

Tube Adapter 57020-U

The liner consists of a PP female luer hub that attaches to the SPE and thin-walled PTFE tubing that is threaded through the SPE port. This ensures that all SPE port and valve surfaces coming in contact with the sample can be replaced following each extraction.

SPE Tube Adapters

Sample Solution Sample Solution

Cat. No.
57044 57265 57059 57020-U 57021

Description
DL (Disposable Liner), 12-port model (Supelco) DL (Disposable Liner), 24-port model (Supelco) Disposable Liners for Visiprep DL Manifolds (included with 57044 and 57265) - PTFE, pk of 100 SPE Tube Adapters for 1, 3, 6 mL tubes, pk of 12 Empty PP SPE Tube, 20 mL, pk of 12

For more information, visit sigma-aldrich.com/analytical

SOLVENTS FOR SPE, LLE AND ANALYSIS

Sigma-Aldrich offers a comprehensive range of high-quality solvents for dedicated analytical applications.
Each part of the workflow requires different grades of solvent. The table below provides the suggested minimum grade solvent that should be used within each stage of an application.

Common Extraction Solvents

Mycotoxins
Aflatoxins Type A Trichothecenes Type B Trichothecenes

Extraction Solvents
Acetonitrile/water Methanol/water Acetonitrile/water Methanol/water Acetonitrile/water Water/PEG Chloroform/methanol Etyl acetate Methanol Acetonitrile Chrloform and mixtures thereof Methanol Acetonitrile/water Water Water/tetrabutylammonium hydroxide (TBAH) Acetonitrile/water Methanol Methyl tert-butyl ether (MTBE) Chloroform Acetonitrile/water mixtures of toluene/HCl, MgCl2 Methanol/water (3:1) Acetonitrile/water (1:1) Ethyl acetate Acetone

Minimum grade suitable for:

SPE HPLC LC-MS GC and GC-MS applications
*PRA = Pesticide Residue Analysis

Zearalenone CHROMASOLV HPLC / CHROMASOLV Plus CHROMASOLV Plus / CHROMASOLV HPLC LC-MS CHROMASOLV Capillary GC / PRA Grade* Beauvericin Ochratoxin A Monililformin

Complete details and specifications for these solvents can be found at

sigma-aldrich.com/solvents

Grade Product Name

Acetone Acetonitrile Chloroform (w/ ~1% ethanol as stabilizer) Diethyl ether Ethanol Ethyl acetate Methanol tert-Butyl methyl ether Toluene acc. to FDA Water

PRA Cat. No.

34480 34481 25669 31671 2851 31063 34485 34498 34494 34478

LC-MS Cat. No.

34967 34972 34966 39253

LC-MS Ultra Cat. No.

14261 14262 14263

Fumonisins Patulin

To use the solvent selector tool, visit

sigma-aldrich.com/solvent_selector

Choose the right quality of solvent for accurate and reproducible results. Depending on your detection mode, use the corresponding solvent for the extraction step.

Mycotoxin Analysis

Derivatization
DERIVATIZATION FOR HPLC ANALYSIS
Derivatization is a suitable tool to make detection possible or to improve the detectability of an analyte. As a result, a modified analyte is produced emitting fluorescent light that is proportional to the amount of the analyte in the sample. When choosing an appropriate derivatization agent, a number of criteria should be considered:

DERIVATIZATION FOR GC ANALYSIS

When using GC-ECD detection, derivatization has to be performed. Most methods are based on trimethylsilylation or fluoroacylation.

A-trichothecenes are derivatized with heptafluorobutyrylimidazole Derivatization mixtures for B-trichothecenes include Tri-Sil TBT
(HFBI). and Sylon BTZ, which contain TMSI (40 5%), BSA (35 5%) and 13 C 13caused TMCS (25 5%). This is to avoid two peaks C 22 - by incomplete 24 10.003 derivatization. T- 2 HT-2 Toxin Toxin Generally, fumonisins are derivatized with ortho-phthalaldehyde (OPA).
9.541

Derivatization should be rapid and quantitative By products and excess reagents should not interfere with the The fluorophore must possess intense absorption bands The reagent must be stable The analyte must be reactive with the derivatization agent The derivatizing agent and the by-products should not be fluorescent
The sensitivity of HPLC determination of trichothecenes is limited to as many compounds show only minimal fluorescent or ultraviolet absorbing properties. Those with a conjugated C=O double bonding system (C9 and C10 and keto group at C8) can be detected, but derivatization is often applied as well, especially when detection is performed at ppb levels, because of interferences from impurities. formation of fluorescent light

SILYLATION USING MSTFA

Typically when using silylation, the excess of the silylating reagent had to be quenched by water followed by a re-extraction of the analyte. However, by using N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) containing 1% trimethylchlorosilane as a silylating reagent, one can eliminate the water-quenching and the re-extraction steps because the relatively low boiling point (131 C) allows for the use of MSTFA as a solvent for splitless injection GC.
9.50 10.00

Cat. No.
74382-10ML 74382-10X1ML 33031-U 33151 33030 79760-1G 79760-5G

Description
N-Heptafluorobutyrylimidazole (HFBI) N-Heptafluorobutyrylimidazole (HFBI) Sylon BTZ (1 x 25 mL) Sylon BTZ (144 x 0.1 mL) Sylon BTZ (20 x 1 mL) o-phthalaldehyde for fluorescence, 99.0% (HPLC) o-phthalaldehyde for fluorescence, 99.0% (HPLC)
13 C

HT-2 Toxin
9.541

22 -

10.003

24 T- 2 Toxin

13 C

HT-2
Toxin

T-2
Toxin

9.544 10.010

For more information or to request a Derivatization Reagents Brochure, visit

sigma-aldrich.com/derivatization
9.50 10.00 9.50 10.00

HT-2 T-2 MSTFA eliminates the water-quenching and re-extraction steps

Toxin Toxin

9.544 10.010

9.50

10.00

For more information, visit sigma-aldrich.com/analytical

Chromatographic Analysis
Coupling a suitable extraction with the right cleanup procedure, an optimized chromatographic separation and a selective derivatization can achieve an optimum level of specificity for reliable quantification. Depending on lab resources, mono-residues or multi-residues analysis can be performed. GC can be used with ECD, FID or MS detection or HPLC with fluorimetric, UV or MS-MS detection.

Figure 1. EI-Mass spectra of the TMS-derivatives of unlabelled and fully 13C isotope-labelled T-2 Toxin and HT-2 Toxin
Abundance 5500 5000 4500 4000 185 #962: HT-2 Toxin -TMS

HT - 2 Toxin

(TMS)
M=568
(not visible)

157

GAS CHROMATOGRAPHY
For GC analysis, a robust stationary phase with medium polarity such as a Poly (Diphenyl 35% / Dimethylsiloxane 65%) phase is required to withstand the effects of the silylating agent and temperature. A very good separation of T-2 toxin and HT toxin can be achieved with the application of a fast-temperature program. MS detection with selected-ion-monitoring (SIM) enables detection limits in the range of 25 ppb for HT-2 toxin and T-2 toxin, even in complex matrixes. As claimed by EU Guideline 96/23/EG, the identities of the toxins were confirmed not only by the retention time but also by the three SIM ions. A qualifier was measured for each internal standard to ensure peak purity.

3500 3000 2500 2000 105 1500 1000 500 221 122 245 303 377 204 275

(CH 3) 2 CH CH 2COOH - 102

Q1
347

T
466

Q2
478

139 406 320 437 495 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z--> Abundance 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 203 244 263 185 157 105 122

T-2 Toxin (TMS)

M=538
(not visible)

#963: T-2 Toxin -TMS

SPB-35 Capillary GC Columns

Cat. No.
24092 28568-U 24094 25331 25335

Description
L I.D. 30 m 0.25 mm, df 0.25 m L I.D. 60 m 0.25 mm, df 0.25 m L I.D. 30 m 0.32 mm, df 0.25 m L I.D. 30 m 0.53 mm, df 0.50 m L I.D. 30 m 0.53 mm, df 1.00 m

Q2
290

Q1
350

(CH 3) 2 CH CH 2COOH - 102

T
2(CH 3 CO) - 86
436

500

CH 3 COO 317 141 227 377 394 419 - 59 454 477 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

m/z--> Abundance

A New GC-MS Method for Mycotoxin Analysis Using 13 C-Labeled Mycotoxin Derivatives
Andreas Breidbach [1] and Wolfgang Brodacz [2,3,4] developed a new GC-MS method using fully 13C-isotope labeled analogues of T-2 toxin and HT-2 toxin that allows for the detection of these toxins at concentrations as low as 25 ppb. Isotopic dilution mass spectrometry (IDMS) takes advantage of the fact the chemical and physical properties of 13C isotope-labelled analogues are nearly identical to those of non-labeled analytes. This means that their behavior in the workup is essentially the same, but the labelled and non-labeled analogues can still be distinguished by mass spectrometry (Figure 1).

5000

#968: 13C22-HT-2 Toxin (voll 13C isotopenmarkiertes HT-2 Toxin) als TMS 198

4500

13 C

22

- HT - 2 Toxin
(TMS)
M=590
(not visible)

4000

3500 169 3000

(CH 3) 2 CH CH 2COOH - 107

2500

Q1
2000 113 130 147 216 288 367 1500

T
483

1000

500

234 305 393 500 410 331 349 439 456 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

257

m/z-->

Abundance 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 154 229 204 256 285 303 185 113 130
#967: 13C24-T-2 Toxin als TMS (voll 13C-isotopenmarkiertes T-2 Toxin)

Labeled mycotoxins allow IDMS

13 C

24

- T- 2 Toxin

(TMS)

Simplify sample handling Addition of the standard after sample extraction

Increase precision and accuracy

M=562
(not visible)

Compensation of matrix effects and inaccuracies

(CH 3) 2 CH CH 2COOH - 107

T
365

Q1
2(CH 3 CO) - 90
455

347 331 393 411 439 499 473 0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

m/z-->

10

Mycotoxin Analysis

LIQUID CHROMATOGRAPHY
Analysis of Mycotoxins by LC-MS/MS
The sensitive and accurate detection of very low levels of mycotoxins is critical to identify contaminated products. LC-MS/MS is a popular analytical technique for this purpose. The combination of LC with MS/MS detection allows the quantification of multiple mycotoxins in the same run. Several different phase selectivities are required to achieve this separation. Using different phases can also decrease the analysis speed.
0.6 m

20.00 18.00 16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 0.00 0.50 H

Fused-Core 2.7 m Porous 3 m Fused-Core 5 m Porous 5 m

Column: 15 cm x 4.6 mm Mobile Phase: 60% Acetonitrile Temperature: 35 C Sample: 10 mL Toluene

0.5 m 5 m 2.7 m 1.7 m Solid Core 3.3 m Solid Core

1.00

1.50

2.00 2.50 Flow (mL/min)

3.00

3.50

4.00

4.50

12000 10000 8000 6000 4000 2000 0 0.00

2.7 m

5 m
Pressure (PSI)

Fused-Core 2.7 m Porous 3 m Fused-Core 5 m Porous 5 m

Column: 15 cm x 4.6 mm Mobile Phase: 60% Acetonitrile Temperature: 35 C Sample: 10 mL Toluene

The 5 m Fused-Core particle achieves much higher efficiency than fully porous 5 m particles at comparable pressures. It operates at the same efficiency levels or better than 3 m particles and is well suited for high performance with routine 400 bar instruments, while maintaining good analyte retention and loading capacity. These columns are used at a higher flow rate than the equivalent porous ones, without sacrificing efficiency, on your conventional HPLC system. The tables below demonstrate ways to accelerate systems and save solvent. However, to analyze multi-mycotoxins in the same run, one could face problems in the resolution of some on C18.

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

Flow (mL/min)

Current method
Pressure 4,000 psi Flow rate 1.0 mL/min Column length 5 cm 10 cm 15 cm 20 cm 25 cm
Back Pressure reaches the limits of conventional HPLC systems Back Pressure adapted to conventional HPLC systems

Efficiency (p/col)
5 m particles totally porous 4,500 9,000 13,500 18,000 max. 22,500 3 m particles totally porous 6,000 12,000 18,000 max. 24,000 1.8 m particles totally porous max. 12,000 Ascentis Express Fused-Core 5 m 7,000 15,300 max. 23,000

Analyze 15 mycotoxins in the same run in less than 10 minutes, decrease your solvent consumption by 3 without the need of a UHPLC system.

Particle Size
8 mL of solvent 5 m 5 m FC 2.7 m

Flow Rate
1.0 mL/min 1.0 mL/min 2.4 mL/min

Column Efficiency Retention Length (N) Time

25 cm 15 cm 10 cm 22,500 23,000 24,000 25.0 min 8.0 min 4.2 min 25 mL

For more information, visit sigma-aldrich.com/analytical

11

Improvements in resolution can also be achieved by changing the selectivity of the column stationary phase. Although we offer seven different chemistries within the Ascentis Express range, the F5 and RP-Amide are especially efficient for the analysis of mycotoxins because they provide both polar and ionic interactions. These two columns present orthogonality versus C18 and have been used to separate 15 mycotoxins in the same analytical run, with MS-MS detection.

Ascentis Express RP-Amide

1. 2. 3. 4. 5. 6. 7. 8. sample/matrix:  5 grams of cereal spiked with 15 mycotoxins; extract with 20 mL acetonitrile:1% formic acid in water (75:25); shake for 1 min; centrifuge; filter through a 0.45 m syringe filter column:  Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 2.7 m particles (53913-U) mobile phase:  (A) 1 mM ammonium acetate, 0.5% acetic acid in water; (B) 1 mM ammonium acetate, 0.5% acetic acid in methanol gradient: 0 min: 5% B; 0.5 min: 10% B; 12 min: 95% B; 15 min: 95% B flow rate: 400 L/min column temp.: 40 C detector: MS/MS, ESI(+) and ESI (-) injection: 2 L Nivalenol (NIV) Deoxynivalenol (DON) 15-Acetyldeoxynivalenol 3-Acetyldeoxynivalenol Aflatoxin G2 Aflatoxin G1 Aflatoxin B2 Aflatoxin B1 9. 10. 11. 12. 13. 14. 15. Atrazine-d5 Fumonisin B1 T-2 Toxin Fumonisin B2 Ochratoxin A Sterigmatocistin TFF

Selectivity differences between Ascentis Express chemistries: RP-Amide vs. C18

14 11

15

log k (RP-Amide)

14 11
10 9 6 Min 7 13 12

15

Increasing solute polarity

3,4

6 5 5

8 7

log k (C18)

Selectivity differences between Ascentis Express chemistries: C18 vs. F5

Chromatogram courtesy of Enio Belotti (Water & Life Lab s.r.l., Entratico (BG), Italy)

12
14 13

5 Ascentis Express F5

3,4

8 7 9

10

11 15

column:  Ascentis Express 7 4 5 F5, 10 cm x 2.1 6mm I.D., 2.7 m particles (53569-U) Min Peak IDs and all other conditions the same as Figure 1.

11 15

14
12

log k (C18)

3
Increasing solute polarity

4 5 6 Min

9 7 8 7 13 8

10

2 3 4 5

912

log k (F5)

Chromatogram courtesy of Enio Belotti (Water & Life Lab s.r.l., Entratico (BG), Italy)

4 5 6 Min

9 7 8 7 13 8

10

Ascentis Express Columns

Cat. No.
53823-U 53825-U 53913-U 53914-U 53569-U 53571-U

Description
C18, 10 cm x 2.1 mm I.D., 2.7 m C18, 15 cm x 2.1 mm I.D., 2.7 m RP-Amide, 10 cm x 2.1 mm I.D., 2.7 m RP-Amide, 15 cm x 2.1 mm I.D., 2.7 m F5, 10 cm x 2.1 mm I.D., 2.7 m F5, 15 cm x 2.1 mm I.D., 2.7 m

For additional details on the Ascentis Express HPLC column range or to request an application note, visit

sigma-aldrich.com/express

12

Mycotoxin Analysis

Standards
For a precise detection of regulated mycotoxins, Sigma-Aldrich offers a comprehensive range of standards, including single- and multicomponent standard solutions, 13C-isotope labelled standards and certified reference materials (CRMs).

SINGLE COMPONENTS STANDARD SOLUTIONS

For precise quality control of food and feed , available in various concentrations and different solvents.

MYCOTOXIN REFERENCE MATERIALS (NEATS)

For calibration of your analytical instruments and recovery experiments.

Cat. No.
34133 34029 46323-U 44647-U 46324-U 34034 34032 46325-U 34033 46326-U 34031 46319-U 35406 35407 46911 34124 34139 34142 32606 34136 34131 34037 46912 32411 34127 46914-U 34071 46916-U 34126

Component
15-Acetyldeoxynivalenol Aflatoxin B1 Aflatoxin B1 Aflatoxin B1 Aflatoxin B2 Aflatoxin B2 Aflatoxin G1 Aflatoxin G1 Aflatoxin G2 Aflatoxin G2 Aflatoxin M1 Aflatoxin M1 -Zearalanol -Zearalanol Deoxynivalenol Deoxynivalenol Fumonisin B1 Fumonisin B2 Fumonisin B3 HT-2 Toxin Nivalenol Ochratoxin A Ochratoxin A Ochratoxin B Patulin Patulin T-2 Toxin Zearalenone Zearalenone

Con. (g/g)
100 2 3 20 3 0.5 2 3 0.5 3 0.5 10 10 10 200 100 50 50 50 100 100 10 50 10 100 100 100 50 100

Solvent
ACN ACN Benz/ACN MeOH Benz/ACN ACN ACN Benz/ACN ACN Benz/ACN ACN ACN ACN ACN Et-Ac/MeOH ACN ACN/H2O ACN/H2O ACN/H2O ACN ACN ACN Benz/Ac Acid ACN ACN CHCl3 ACN ACN ACN

Cat. No.
32928 32754 32755 32756 32757

Component
15-Acetyldeoxynivalenol Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

Cat. No.
32943 32936 32937 33947 32939

Component
Deoxynivalenol Fumonisin B1 Ochratoxin A T-2 Toxin Zearalenone

ISOTOPICALLY LABELED INTERNAL STANDARD SOLUTION FOR LC-MS

For an accurate detection, as they show an optimal mass unit difference between the analyte and the standard, which prevents interference.

Cat. No.
32962 32764 32771 32772 34128 33621 32915 32916 33842 33416 33892 32758

Component
3-Acetyldeoxynivalenol 13C17 Aflatoxin B1-13C17 Aflatoxn B2-13C17 Aflatoxin G1-13C17 Deoxynivaleno-13C15 Fumonisin B1-13C34 Fumonisin B2-13C34 Fumonisin B3-13C34 HT-2 Toxin-13C22 Ochratoxin A-13C20 T-2 Toxin-13C24 Zearalenone-13C18

Con. (g/g)
25 0.5 0.5 0.5 25 25 10 10 25 10 25 25

Solvent
ACN ACN ACN ACN ACN ACN /H2O ACN /H2O ACN /H2O ACN ACN ACN ACN

Molecular structure of a fully 13C-isotope labeled Aflatoxin B1-13C17 (32764)

For more information, visit sigma-aldrich.com/analytical

13

CERTIFIED MATRIX REFERENCE MATERIALS AND MYCOTOXIN SOLUTIONS (CRMS) FROM IRMM
CRMs are produced with raw materials to more accurately resemble actual samples in their natural state. For performance control of mycotoxin.

MIXTURE STANDARDS SOLUTION FOR MULTI-ANALYTE DETECTION

For a detailed analysis of individual mycotoxins in multitoxin samples.

Cat. No.
46300-U 46303 46304-U 34036 33415 32926 34134

Component
Aflatoxin Mix, B1, G1, B2, G2 in Benzene/ACN Aflatoxin Mix, B1, G1, B2, G2 in MeOH Aflatoxin Mix, B1, G1, B2, G2 in MeOH Aflatoxin Mix 4, B1, G1 , B2, G2 in ACN Aflatoxin Mix 4, B1, G1, B2, G2 in ACN (20 g/mL each) Trichothecene Mix, 10 ug/mL each in acetonitrile (3-AcDON, DON, NIV, FusX, HT-2, T-2, DAS, ZON) B-Trichothecen Mix, 100 g/mL in acetonitrile (each of DON, NIV, 3-AcDON and 15-AcDON)

Package (mL)
5x1 1x5 5x1 2 2 1 2

Cat. No.
BCR375 ERMBE375 ERMBE376 ERMBC716 ERMBC717 BCR377 BCR401R BCR471

Description
Compound Feed (Aflatoxin blank) Compound Feedingstuff (Aflatoxins B1, B2, G1, G2, very low level) Compound Feedingstuff (Aflatoxins B1, B2, G1, high level) Maize (Zearalenone, very low level) Maize (Zearalenone, low level) Maize Flour (Deoxynivalenol, blank) Peanut Butter (Aflatoxins B1, B2, G1, G2, low level) Wheat (Ochratoxin A, blank)

The certified mycotoxin reference standard solutions provide an accurate determination of detection limits as well as validation of analytical methods. These standards are certified according to the ISO Guides 34 and 35. All mycotoxins are dissolved in acetonitrile and solutions are placed in 4 mL amber glass ampoules. We offer Aflatoxin M1 dissolved in chloroform (2.5 mL).

DRIED DOWN MYCOTOXIN STANDARDS (RMS)

Sigma-Aldrich offers some reference materials as dry, in small quantities, which are packaged in amber ampoules to ensure stability. The convenient reconstitution of these standards is described in the enclosed certificate of analysis.

Cat. No.
ERMAC057 ERMAC058 ERMAC059 ERMAC060 IRMM315 IRMM316 BCR423RM ERMAC699

Component
Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 4-Deoxynivalenol Nivalenol Aflatoxin M1 Zearalenone

Con. (g/g)
3.79 3.80 3.78 3.80 25.1 24.0 9.93 9.95

Cat. No.
35758 35762 37012 35878 35970 37025 35976 35977 37018

Component
Alternariol Alternariol-9-methyl ether Beauvericin (BEA) Citreoviridin A Meleagrin Retrorsine Stachybotrylactam Tentoxin Tenuazonic acid

Con. (g/g)
100 100 100 100 100 50 100 100 100

Package (mL)
0.1 0.1 0.1 0.1 0.1 0.05 0.1 0.1 0.1

If you cannot find the standard you are looking for, the concentration or the packaging format you need, contact your local office we have custom solutions to fit your needs.
Mycotoxin Standards

Single and Multi-Component Standard Solutions

13 C - Isotopically Labeled Standards

Matrix Certified Reference Materials Dried Down Standards

For a comprehensive list of Mycotoxins standards, visit

sigma-aldrich.com/mycotoxins

14

Mycotoxin Analysis

Accessories for GC or HPLC

GC INSTALLATION AND TROUBLESHOOTING AND PREVENTATIVE MAINTENANCE HPLC ACCESSORIES DESIGNED TO MAXIMIZE THE PERFORMANCE OF THE HPLC SYSTEM

Quality products are required for installation and troubleshooting tasks. To aid in accomplishing these tasks, the highest quality products are needed, including: column nuts, flow meters, tubing, fittings, valves, particle and oil filters, gas generators, leak detectors and pressure regulators.

To serve this rapidly growing area of High(er) Performance Liquid Chromatography, we have selected products from the most trusted names in the industry, making product selection easy. This selection of accessories for high speed and high sensitivity analytical applications, maximize the efficiency and reliability of the analysis while protecting the column investment.

GC Tips Periodic replacement of: Injection port items will minimize adsorption of analytes Purifiers will avoid their saturation and permit to maintain the continuous supply of chromatographic quality carrier gas
For more details on GC accessories or to request a free Maximize Performance guide, visit

HPLC Tips It is good laboratory practice to install the proper fittings, ferrules, and other accessories to ensure the analytical results show no peak broadening from extra column effects created by excessive volume or improper assembly.
For a complete listing or to request a brochure, visit

sigma-aldrich.com/gc

sigma-aldrich.com/hplc

Introducing SupelMIP SPE

Aids in the Extraction of Patulin from Food

SupelMIP solid phase extraction is based on molecularly imprinted polymer (MIP) technology. Each SupelMIP phase offers tailor-made selectivity for the extraction of trace analytes in complex matrixes. SupelMIP SPE Patulin is an SPE cartridge designed to be specific for the trace analysis of the mycotoxin patulin in apple matrices.

Benefits include:

Faster/simplified sample prep methods Robust and rapid methodology Improved sensitivity resulting in lower detection limits Better MS compatibility due to reduced ion suppression
To learn more, or to request a free sample pack, visit

sigma-aldrich.com/supelmip

For more information, visit sigma-aldrich.com/analytical

15

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