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MYCOTOXIN ANALYSISFROM SAMPLING TO MEASUREMENT

2 Mycotoxin Analysis

MycotoxinsMycotoxins 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 mycotoxins is increasingly prevalent

• Occur during growth, harvest, transportation, processing or storage

• 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.

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.

Derivatization

page 9

Isolation and Concentration

page 6

Matrix Processing

page 3

Chromatographic Separation

page 10

Quantification

page 13

HPLC Reagents

page 9

GC Reagents

page 9

SPE Cartridges and Accessories

page 6

Solvents

page 8

Homogenization and Grinding

page 3

Extraction

page 4

Filtration and Evaporation

page 5

HPLC and UHPLC

page 11

GC and GC-MS

page 10

Performance Accessories

page 15

LC-MS Grade Solvents

page 8

Standards and CRMs

page 13

Labeled Standards

page 13

MYCOTOXIN ANALYSIS WORKFLOWSigma-Aldrich offers valuable products for the entire mycotoxin analysis workflow.

3For more information, visit sigma-aldrich.com/analytical

Matrix ProcessingA range of well-developed techniques is available. The criteria for choosing a suitable method include available time and equipment, specificity and sensitivity.

HOMOGENIZATION AND GRINDING

HomogenizationHomogenization 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.

Cat. No. DescriptionZ722472 IKA ULTRA-TURRAX dispersers, T-10 Basic, for volumes of

0.5-100 mL, 230 V, 1/cs

Z722464 IKA ULTRA-TURRAX dispersers, T-25 Digital, for volumes of 1-2,000 mL, 230 V, 1/cs

Z722456 ULTRA-TURRAX Tube Drive Workstation

Z722375 IKA ULTRA-TURRAX disperser tubes, DT-20 dispersing tube with rotor-stator element, 25/CS

ULTRA-TURRAX Tube Drive WorkstationThe 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 workstation provides a unique and universal dispersing, stirring, homogenizing and grinding system, with hermetically sealable and disposable sample tubes.

• Disperse, stir, homogenize and grind using a single drive unit

• Hermetically sealable disposable sample tubes eliminate cross-contamination

• Gamma-sterilized tubes

• Tubes (2–15 mL and 15–50 mL ) with pierceable membrane covers

• Anti-locking function and chemical-resistant plastic

Protection and security for infectious sample materials, toxic substances, high-odor substances.

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

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. DescriptionZ645176 MF 10 basic Microfine grinder drive – dringing heads not

included with delivery

Z645249 MF 10.1 Cutting-grinding head (for crushing fibrous substances)

Z645257 MF 10.2 Impact grinding head (for crushing brittle, hard materials)

4 Mycotoxin Analysis

EXTRACTIONSample 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, freeze-drying 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.

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 GlasswareSigma-Aldrich has a wide range of extraction glassware, including:

• Liquid-Liquid Extraction – including extractor-concentrator kits, flasks and liquid-liquid extractor

• Accessories for Sample Concentration / Extraction

Whatman Filter Papers

Soxhlet Extraction Apparatus and Thimbles

Extraction Systems and ThimblesCat. No. Description64815-U Condenser size small

64816-U Condenser size medium

64817-U Condenser size large

64818 Extractor size small

64819-U Extractor size medium

64820-U Extractor size large

64821-U Flat bottom flask size 125 mL

64822-U Flat bottom flask size 250 mL

64823 Flat bottom flask size 300 mL

64803-U Separatory funnel glass stopcock plug, size 250 mL

64804-U Separatory funnel glass stopcock plug, size 2000 mL

64805-U Separatory funnel PTFE stopcock, size 250 mL

64806 Separatory funnel PTFE stopcock, size 2000 mL

64824 Soxhlet extraction apparatus size small

64825 Soxhlet extraction apparatus size medium

64826 Soxhlet extraction apparatus size large

64840-U Thimble cellulose, size 25 mm × 80 mm

64841-U Thimble cellulose, size 33 mm × 94 mm

64842 Thimble cellulose, size 43 mm × 123 mm

64836-U Thimble glass, size 25 mm × 85 mm

64837 Thimble glass, size 35 mm × 90 mm

64838 Thimble glass, size 45 mm × 130 mm

5For more information, visit sigma-aldrich.com/analytical

FILTRATION AND EVAPORATIONFiltrationAfter 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.

Grade 2 2SV (prepleated)

DiameterWhatman

Cat. No.Sigma-Aldrich

Cat.No.Whatman

Cat. No.Sigma-Aldrich

Cat.No.

90 mm 1002-090 Z240214-1PAK — —

110 mm 1002-110 Z240222-1PAK — —

125 mm 1002-125 Z240230-1PAK 1202-125 Z240303-1PAK150 mm 1002-150 Z240249-1PAK 1202-150 Z240311-1PAK185 mm 1002-185 Z240257-1PAK 1202-185 Z240338-1PAK240 mm 1002-240 Z240265-1PAK 1202-240 Z240346-1PAK270 mm 1002-270 Z752304-1PAK 1202-270 Z240354-1PAK320 mm 1002-320 Z240281-1PAK 1202-320 Z240362-1PAK385 mm 1002-385 Z695165-1PAK 1202-385 Z740330-100EA

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

An all-in-one filtration system can reduce costs and processing time by up to 35%. No additional consumables are required.

Cat. No. DescriptionWhatman Mini-UniPrep G2 standard septum Z759422 PTFE membrane, pore size 0.2 μm, clear vial color Z759481 PTFE membrane, pore size 0.45 μm, clear vial color Z759384 starter pack supplied with single vial compressor, PTFE

membrane, pore size 0.2 μm, amber vial color Z759430 starter pack supplied with single vial compressor, PTFE

membrane, pore size 0.2 μm, clear vial color Z759414 starter pack supplied with single vial compressor, Nylon

membrane, pore size 0.2 μm, clear vial color Z759503 starter pack supplied with single vial compressor, PTFE

membrane, pore size 0.45 μm, clear vial color Whatman Mini-UniPrep (PP housing)Z557889 nylon, 0.2 μm, 100/pk or 1000/pkZ506621 nylon, 0.45 μm, 100/pk or 1000/pkZ671274 amber, nylon, 0.2 μm, 100/pkZ557897 PTFE, 0.2 μm, 100/pk or 1000/pkZ506648 PTFE, 0.45 μm, 100/pk or 1000/pk

GD/X Syringe Filters – for Hard-to-filter Samples that Require More Than One FilterWhatman 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 µm)

• Increased flow rate

• Four layers of filtration media: Reduces blockage and need to replace filter

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

Description150 per pack Cat. No.

1,500 per pack Cat. No.

GD/X 25 mm – 0.2 µmNylon Z277355-1PAK Z745294-1500EAPTFE Z277401-1PAK Z745332-1500EAPP Z277436-1PAK —CA Z745375-150EA —RC Z747599-150EA Z747602-1500EAGD/X 25 mm – 0.45 µmNylon Z277363-1PAK Z671185-1500EAPES Z286583-1PAK Z745553-1500EAPVDF Z277398-1PAK Z671193-1500EAPTFE Z277428-1PAK Z671207-1500EARC Z747610-150EA Z747629-1500EA

EvaporationFor 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 Büchi.

Selection Guide of Available Rotavapors (240V)

Model R210Basic

(no Vac. Cont.)Advanced

(with V850)Professional (with V855)

Diagonal 29/32Glass Z563846 Z563854 —Plastic-coated Z563633 Z563641 Z563668Vertical 29/32Glass Z563862 Z563870 —Plastic-coated Z563676 Z563684 Z563692Cold Trap 29/32Glass Z563889 Z563897 —Plastic-coated Z563706 Z563714 Z563722Cold Trap Reflux 29/32Glass Z565466 Z565474 —Plastic-coated Z565199 Z565202 Z565210

6 Mycotoxin Analysis

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

Unwanted matrix remains on cartridge. Cartridge discarded.

4. Evaporateelution solvent

Purified analyte eluted

5. Reconstitute

Purified and concentratedanalytes and internal standard

SUPEL™ TOX CARTRIDGES – INCREASE THROUGHPUT TEN-FOLDThe 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.

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.

Features and Benefits• Remove interferences associated with mycotoxin analysis

• Better reproducibility than the industry standard immunoaffinity columns

• Sample preparation time is up to ten times less than that of immunoaffinity columns (fewer steps)

• Straightforward, cost-effective and quick methodology requiring little additional method development (generic method)

• Improved shelf life over immunoaffinity columns due to the thermally stable format. No refrigeration is required for shipping and storage.

Isolation / Concentration (SPE)

Comparison of Supel Tox AflaZea SPE to Immunoaffinity for Aflatoxins B1, B2, G1 and G2 in Peanut Paste

Immunoaffinity Supel Tox AflaZea SPE Cartridge

Sample Prep Time (post-extraction to pre-analysis)

• 60 minutes• 8 samples/day (if processing 1 at a time)

• 6 minutes• 80 samples/day (if processing 1 at a time)

Ease of Use • Large volumes of liquid • Controlled drop rates• Numerous complicated steps• Additional buffer salts required• Must be refrigerated, brought to room temp before use

• Small volumes of liquid• Vacuum filtration used• Steps few and not complicated• No additional reagents required • Column does not require special storage conditions

7For more information, visit sigma-aldrich.com/analytical

Features and Benefits• Patented screw-type valves within each SPE port for precise

flow control

• Glass basin will not dissolve, fog, or discolor when exposed to solvents

• Leg covers enable cover to rest on work surface after removed from the manifold

• 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

Tube Adapter 57020-U

10-100 cc Syringe

Sample SolutionSample Solution

SPE Tube Adapters

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

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

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.

Cat. No. Description57044 DL (Disposable Liner), 12-port model (Supelco)

57265 DL (Disposable Liner), 24-port model (Supelco)

57059 Disposable Liners for Visiprep DL Manifolds (included with 57044 and 57265) - PTFE, pk of 100

57020-U SPE Tube Adapters for 1, 3, 6 mL tubes, pk of 12

57021 Empty PP SPE Tube, 20 mL, pk of 12

Sample Purification Strategy for Supel Tox SPE CartridgesSPE Cartridge Analyte(s) Matrix Purification Strategy Qty. Cat. No.Supel Tox AflaZea Aflatoxin B1, B2, G1 and G2,

and ZearalenoneGrains, feeds, TMR samples, peanuts, peanut products and aqueous solutions

Interference removal 30 55314-U

Supel Tox DON Deoxynivalenol (DON) Wheat, flour and corn Interference removal 30 55316-USupel Tox Tricho Trichothecenes (Type A and B) Grains and complex matrices Interference removal 30 55308-USupel Tox TrichoBind Trichothecenes (Type A and B) Grains, feeds and other complex

matrices Multifunctional bind and elute 25 55307-U

Supel Tox FumoniBind Fumonisins (B1 and B2) Grains and cereals Multifunctional bind and elute 25 55315-USupel Tox OchraBind Ochratoxin A Grain and feed samples Multifunctional bind and elute 25 55318-U

Discover this new range of products, or obtain a complementary sample, visit sigma-aldrich.com/supeltox

8 Mycotoxin Analysis

SOLVENTS FOR SPE, LLE AND ANALYSISSigma-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.

Minimum grade suitable for:SPE CHROMASOLV® HPLC / CHROMASOLV Plus

HPLC CHROMASOLV Plus / CHROMASOLV HPLC

LC-MS LC-MS CHROMASOLV

GC and GC-MS applications Capillary GC / PRA Grade*

*PRA = Pesticide Residue Analysis

Complete details and specifications for these solvents can be found at sigma-aldrich.com/solvents

Grade Product NamePRA

Cat. No. LC-MS

Cat. No. LC-MS Ultra

Cat. No. Acetone 34480 — —Acetonitrile 34481 34967 14261Chloroform (w/ ~1% ethanol as stabilizer)

25669 — —

Diethyl ether 31671 — —Ethanol 2851 — —Ethyl acetate 31063 34972 —Methanol 34485 34966 14262tert-Butyl methyl ether 34498 — —Toluene acc. to FDA 34494 — —Water 34478 39253 14263

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.

Common Extraction Solvents

Mycotoxins Extraction SolventsAflatoxins Acetonitrile/water

Methanol/water

Type A Trichothecenes Acetonitrile/waterMethanol/water

Type B Trichothecenes Acetonitrile/waterWater/PEGChloroform/methanol

Zearalenone Etyl acetateMethanolAcetonitrileChrloform and mixtures thereof

Monililformin MethanolAcetonitrile/waterWaterWater/tetrabutylammonium hydroxide (TBAH)

Beauvericin Acetonitrile/waterMethanol

Ochratoxin A Methyl tert-butyl ether (MTBE)ChloroformAcetonitrile/watermixtures of toluene/HCl, MgCl2

Fumonisins Methanol/water (3:1)Acetonitrile/water (1:1)

Patulin Ethyl acetateAcetone

9For more information, visit sigma-aldrich.com/analytical

Derivatization DERIVATIZATION FOR HPLC ANALYSISDerivatization 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 should be rapid and quantitative

• By products and excess reagents should not interfere with the formation of fluorescent light

• 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.

Cat. No. Description74382-10ML N-Heptafluorobutyrylimidazole (HFBI)

74382-10X1ML N-Heptafluorobutyrylimidazole (HFBI)

33031-U Sylon™ BTZ (1 x 25 mL)

33151 Sylon BTZ (144 x 0.1 mL)

33030 Sylon BTZ (20 x 1 mL)

79760-1G o-phthalaldehyde for fluorescence, ≥99.0% (HPLC)

79760-5G o-phthalaldehyde for fluorescence, ≥99.0% (HPLC)

For more information or to request a Derivatization Reagents Brochure, visit sigma-aldrich.com/derivatization

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

• A-trichothecenes are derivatized with heptafluorobutyrylimidazole (HFBI).

• Derivatization mixtures for B-trichothecenes include Tri-Sil® TBT and Sylon™ BTZ, which contain TMSI (40 ± 5%), BSA (35 ± 5%) and TMCS (25 ± 5%). This is to avoid two peaks caused by incomplete derivatization.

• Generally, fumonisins are derivatized with ortho-phthalaldehyde (OPA).

SILYLATION USING MSTFATypically 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.

13C24-T-2

Toxin

13C22-HT-2Toxin

HT-2Toxin

T-2Toxin

9.541

10.003

10.010

9.544

9.50

9.50 10.00

10.00

13C24-T-2

Toxin

13C22-HT-2Toxin

HT-2Toxin

T-2Toxin

9.541

10.003

10.010

9.544

9.50

9.50 10.00

10.00

MSTFA eliminates the water-quenching and re-extraction steps

10 Mycotoxin Analysis

Chromatographic AnalysisCoupling 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.

GAS CHROMATOGRAPHYFor 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 2–5 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.

SPB®-35 Capillary GC ColumnsCat. No. Description24092 L × I.D. 30 m × 0.25 mm, df 0.25 μm

28568-U L × I.D. 60 m × 0.25 mm, df 0.25 μm

24094 L × I.D. 30 m × 0.32 mm, df 0.25 μm

25331 L × I.D. 30 m × 0.53 mm, df 0.50 μm

25335 L × I.D. 30 m × 0.53 mm, df 1.00 μm

A New GC-MS Method for Mycotoxin Analysis Using 13C-Labeled Mycotoxin DerivativesAndreas 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 2–5 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).

• Labeled mycotoxins allow IDMS – Increase precision and accuracy

• Simplify sample handling

• Addition of the standard after sample extraction – Compensation of matrix effects and inaccuracies

Figure 1. EI-Mass spectra of the TMS-derivatives of unlabelled and fully 13C isotope-labelled T-2 Toxin and HT-2 Toxin

100 120 140 480 500

HT - 2 Toxin (TMS)

M=568

(CH 3) 2 CH CH 2COOH- 102

Q1

T

478

Q2

160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 4600

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

m/z-->

Abundance#962: HT-2 Toxin -TMS

185

157

347

204105

275 466122

245

221 377303

139 406320 437 495

(CH 3) 2 CH CH 2COOH- 102

(not visible)

260 280 300 380 400 440 460 480

350290

244

436263 2(CH 3 CO)

86

M=538

(CH 3) 2 CH CH 2COOH- 102

Q2Q1

T

CH3

100 120 140 160 180 200 220 240 320 340 360 4200

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

m/z-->

Abundance

#963: T-2 Toxin -TMS

122

105

185157

203

317141 227377

394 454 477419

T-2 Toxin (TMS)

2(CH 3 CO)

-

M=538(not visible)

(CH 3) 2 CH CH 2COOH- 102

Q2Q1

T

CH3 COO

- 59

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

216

500331

13 C 22 - HT - 2 Toxin(TMS)

M=590(not visible)

(CH 3) 2 CH CH 2COOH- 107

Q1

T

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

m/z-->

Abundance

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

198

169

367

113147 483130

288

257234 393305410

349 439 456

13 C 24 - T- 2 Toxin (TMS)#967: 13C24-T-2 Toxin als TMS (voll 13C-isotopenmarkiertes T-2 Toxin)

2(CH 3 CO)

M=562

(CH 3) 2 CH CH 2COOH- 107

Q1

T

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 4800

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000

9500

m/z-->

Abundance

130

113

185

365303

204455256

285154

229347

331 393 411 439 499473

- 90

(not visible)

(CH 3) 2 CH CH 2COOH- 107

11For more information, visit sigma-aldrich.com/analytical

LIQUID CHROMATOGRAPHY

Analysis of Mycotoxins by LC-MS/MSThe 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.

0.5 µm

1.7 µm2.7 µm Solid Core

0.6 µm

3.3 µm5 µm Solid Core

2.7 µm 5 µm

The 5 µm Fused-Core® particle achieves much higher efficiency than fully porous 5 µm particles at comparable pressures. It operates at the same efficiency levels or better than 3 μm 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.

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50Flow (mL/min)

H

Fused-Core 2.7 μmPorous 3 μmFused-Core 5 μmPorous 5 μm

0

2000

4000

6000

8000

10000

12000

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Flow (mL/min)

Pres

sure

(PSI

)

Fused-Core 2.7 μmPorous 3 μmFused-Core 5 μmPorous 5 μm

Column: 15 cm x 4.6 mmMobile Phase: 60% AcetonitrileTemperature: 35 °CSample: 10 mL Toluene

Column: 15 cm x 4.6 mmMobile Phase: 60% AcetonitrileTemperature: 35 °CSample: 10 mL Toluene

Particle Size

Flow Rate

Column Length

Efficiency (N)

Retention Time

5 µm 1.0 mL/min 25 cm 22,500 25.0 min5 µm FC 1.0 mL/min 15 cm 23,000 8.0 min

2.7 µm 2.4 mL/min 10 cm 24,000 4.2 min

8 mL of solvent

25 mL

Current method Efficiency (p/col)

Pressure Flow rateColumn

length5 µm particles totally porous

3 µm particles totally porous

1.8 µm particles totally porous

Ascentis Express Fused-Core 5 µm

4,000 psi 1.0 mL/min 5 cm 4,500 6,000 max. 12,000 7,000

10 cm 9,000 12,000 15,300

15 cm 13,500 18,000 max. 23,00020 cm 18,000 max. 24,00025 cm max. 22,500

Back Pressure reaches the limits of conventional HPLC systems

Back Pressure adapted to conventional HPLC systems

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.

12 Mycotoxin Analysis

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.

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

log k (C18)

log

k (R

P-A

mid

e)

Increasing solute polarity

Selectivity differences between Ascentis Express chemistries: C18 vs. F5

log k (F5)

log

k (C

18)

Increasing solute polarity

Ascentis Express ColumnsCat. No. Description53823-U C18, 10 cm x 2.1 mm I.D., 2.7 µm

53825-U C18, 15 cm x 2.1 mm I.D., 2.7 µm

53913-U RP-Amide, 10 cm x 2.1 mm I.D., 2.7 µm

53914-U RP-Amide, 15 cm x 2.1 mm I.D., 2.7 µm

53569-U F5, 10 cm x 2.1 mm I.D., 2.7 µm

53571-U F5, 15 cm x 2.1 mm I.D., 2.7 µm

Ascentis Express RP-Amide 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 µm syringe filter

column: Ascentis Express RP-Amide, 10 cm x 2.1 mm I.D., 2.7 µm 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 µL/min column temp.: 40 °C detector: MS/MS, ESI(+) and ESI (-) injection: 2 µL

4 5 6 7 8Min

3,45

67

89

10

11

12

13

14 15

3 4 5 6 7 8 9Min

2

34

5

69

78

11 15 14

10

12

13

1. Nivalenol (NIV)2. Deoxynivalenol (DON)3. 15-Acetyldeoxynivalenol4. 3-Acetyldeoxynivalenol5. Aflatoxin G26. Aflatoxin G17. Aflatoxin B28. Aflatoxin B1

9. Atrazine-d5

10. Fumonisin B111. T-2 Toxin12. Fumonisin B213. Ochratoxin A14. Sterigmatocistin15. TFF

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

Ascentis Express F5 column: Ascentis Express F5, 10 cm x 2.1 mm I.D., 2.7 µm particles

(53569-U)

Peak IDs and all other conditions the same as Figure 1.

4 5 6 7 8Min

3,45

67

89

10

11

12

13

14 15

3 4 5 6 7 8 9Min

2

34

5

69

78

11 15 14

10

12

13Chromatogram courtesy of Enio Belotti (Water & Life Lab s.r.l., Entratico (BG), Italy)

For additional details on the Ascentis Express HPLC column range or to request an application note, visit sigma-aldrich.com/express

13For more information, visit sigma-aldrich.com/analytical

SINGLE COMPONENTS STANDARD SOLUTIONSFor precise quality control of food and feed , available in various concentrations and different solvents.

Cat. No. Component Con. (μg/g) Solvent34133 15-Acetyldeoxynivalenol 100 ACN

34029 Aflatoxin B1 2 ACN

46323-U Aflatoxin B1 3 Benz/ACN

44647-U Aflatoxin B1 20 MeOH

46324-U Aflatoxin B2 3 Benz/ACN

34034 Aflatoxin B2 0.5 ACN

34032 Aflatoxin G1 2 ACN

46325-U Aflatoxin G1 3 Benz/ACN

34033 Aflatoxin G2 0.5 ACN

46326-U Aflatoxin G2 3 Benz/ACN

34031 Aflatoxin M1 0.5 ACN

46319-U Aflatoxin M1 10 ACN

35406 α-Zearalanol 10 ACN

35407 β-Zearalanol 10 ACN

46911 Deoxynivalenol 200 Et-Ac/MeOH

34124 Deoxynivalenol 100 ACN

34139 Fumonisin B1 50 ACN/H2O

34142 Fumonisin B2 50 ACN/H2O

32606 Fumonisin B3 50 ACN/H2O

34136 HT-2 Toxin 100 ACN

34131 Nivalenol 100 ACN

34037 Ochratoxin A 10 ACN

46912 Ochratoxin A 50 Benz/Ac Acid

32411 Ochratoxin B 10 ACN

34127 Patulin 100 ACN

46914-U Patulin 100 CHCl334071 T-2 Toxin 100 ACN

46916-U Zearalenone 50 ACN

34126 Zearalenone 100 ACN

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

MYCOTOXIN REFERENCE MATERIALS (NEATS)For calibration of your analytical instruments and recovery experiments.

Cat. No. Component32928 15-Acetyldeoxynivalenol

32754 Aflatoxin B1

32755 Aflatoxin B2

32756 Aflatoxin G1

32757 Aflatoxin G2

Cat. No. Component32943 Deoxynivalenol

32936 Fumonisin B1

32937 Ochratoxin A

33947 T-2 Toxin

32939 Zearalenone

ISOTOPICALLY LABELED INTERNAL STANDARD SOLUTION FOR LC-MSFor an accurate detection, as they show an optimal mass unit difference between the analyte and the standard, which prevents interference.

Cat. No. Component Con. (μg/g) Solvent32962 3-Acetyldeoxynivalenol 13C17 25 ACN

32764 Aflatoxin B1-13C17 0.5 ACN

32771 Aflatoxn B2-13C17 0.5 ACN

32772 Aflatoxin G1-13C17 0.5 ACN

34128 Deoxynivaleno-13C15 25 ACN

33621 Fumonisin B1-13C34 25 ACN /H2O

32915 Fumonisin B2-13C34 10 ACN /H2O

32916 Fumonisin B3-13C34 10 ACN /H2O

33842 HT-2 Toxin-13C22 25 ACN

33416 Ochratoxin A-13C20 10 ACN

33892 T-2 Toxin-13C24 25 ACN

32758 Zearalenone-13C18 25 ACN

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

Standards

14 Mycotoxin Analysis

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

Cat. No. DescriptionBCR375 Compound Feed (Aflatoxin blank)

ERMBE375 Compound Feedingstuff (Aflatoxins B1, B2, G1, G2, very low level)

ERMBE376 Compound Feedingstuff (Aflatoxins B1, B2, G1, high level)

ERMBC716 Maize (Zearalenone, very low level)

ERMBC717 Maize (Zearalenone, low level)

BCR377 Maize Flour (Deoxynivalenol, blank)

BCR401R Peanut Butter (Aflatoxins B1, B2, G1, G2, low level)

BCR471 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).

Cat. No. Component Con. (μg/g)ERMAC057 Aflatoxin B1 3.79

ERMAC058 Aflatoxin B2 3.80

ERMAC059 Aflatoxin G1 3.78

ERMAC060 Aflatoxin G2 3.80

IRMM315 4-Deoxynivalenol 25.1

IRMM316 Nivalenol 24.0

BCR423RM Aflatoxin M1 9.93

ERMAC699 Zearalenone 9.95

MIXTURE STANDARDS SOLUTION FOR MULTI-ANALYTE DETECTIONFor a detailed analysis of individual mycotoxins in multitoxin samples.

Cat. No. Component Package (mL)46300-U Aflatoxin Mix, B1, G1, B2, G2 in Benzene/ACN 5 x 1

46303 Aflatoxin Mix, B1, G1, B2, G2 in MeOH 1 x 5

46304-U Aflatoxin Mix, B1, G1, B2, G2 in MeOH 5 x 1

34036 Aflatoxin Mix 4, B1, G1 , B2, G2 in ACN 2

33415 Aflatoxin Mix 4, B1, G1, B2, G2 in ACN (20 μg/mL each)

2

32926 Trichothecene Mix, 10 ug/mL each in acetonitrile (3-AcDON, DON, NIV, FusX, HT-2, T-2, DAS, ZON)

1

34134 B-Trichothecen Mix, 100 μg/mL in acetonitrile (each of DON, NIV, 3-AcDON and 15-AcDON)

2

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. Component Con. (μg/g) Package (mL)35758 Alternariol 100 0.1

35762 Alternariol-9-methyl ether 100 0.1

37012 Beauvericin (BEA) 100 0.1

35878 Citreoviridin A 100 0.1

35970 Meleagrin 100 0.1

37025 Retrorsine 50 0.05

35976 Stachybotrylactam 100 0.1

35977 Tentoxin 100 0.1

37018 Tenuazonic acid 100 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

13C - Isotopically LabeledStandards

Matrix Certified Reference Materials

Dried Down Standards

For a comprehensive list of Mycotoxins standards, visit sigma-aldrich.com/mycotoxins

15For more information, visit sigma-aldrich.com/analytical

HPLC – ACCESSORIES DESIGNED TO MAXIMIZE THE PERFORMANCE OF THE HPLC SYSTEM

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.

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/hplc

Accessories for GC or HPLCGC – INSTALLATION AND TROUBLESHOOTING AND PREVENTATIVE MAINTENANCE

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.

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 sigma-aldrich.com/gc

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

Introducing SupelMIP® SPE Aids in the Extraction of Patulin from Food

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©2013 Sigma-Aldrich Co. LLC. All rights reserved. SAFC, SIGMA-ALDRICH and SUPELCO are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries. Ascentis, Equity, Omegawax, SLB, SPB and SupelMIP are registered trademarks of Sigma-Aldrich Co. LLC. CHROMASOLV is a registered trademark of Sigma-Aldrich Laborchemikalien GmbH. Nukol, SAC, Solutions within, SP, Sup-Herb, Supel, Supel-Q, SUPELCOWAX, Sylon and Visiprep are trademarks of Sigma-Aldrich Co. LLC. Fused-Core is a registered trademark of Advanced Materials Technology, Inc. IKA and ULTRA-TURRAX are registered trademarks of IKA-Werke GmbH & Co. KG. Mini-Uniprep and Whatman are registered trademarks of GE Healthcare. Tri-Sil is a registered trademark of Thermo Fisher Scientific Inc. Supelco brand products are sold by affiliated Sigma-Aldrich distributors. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.