LaChrom Elite Application Note - uk.vwr-cmd.comuk.vwr-cmd.com/.../chrom/applications/Aflatoxins_with_Coring_Cell.pdf · LaChrom Elite® HPLC: Aflatoxins in foodstuff using Coring

LaChrom Elite® HPLC: Aflatoxins in foodstuff using Coring Cell (former Cobra Cell)

Food & Beverage Applications

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1. Introduction Aflatoxins B1, B2, G1 and G2 are the main toxins produced by Aspergillus flavus, A. parasiticus and A. nomius. These toxins are of relatively high molecular weight and contain one or more oxgenated alicyclic rings. They can contaminate food products when storage conditions are favorable to fungal growth. The main aflatoxin contaminations has been reported in maize, peanuts, brazil or pistachio nuts, copra and cottonseeds. Aflatoxins are carcinogenic, mutagenic, teratogenic and immunosuppressive to ost animal species. The Internal Agency for Research on Cancer (IARC) has classified all four aflatoxins as group of one carcinogens. Confirmation of the presence of aflatoxins in a sample by HPLC requires derivatisation of the aflatoxins B1 and G1 in order to enhance their natural fluorescence under UV light and make them more easily detected. Previously, the only options available for derivatising aflatoxins involved the use of either trifluoroacetic acid (TFA) or iodine. Both of these methods are reliable, however they do have some significant limitations which can be overcome by use of the Coring Cell. TFA method as pre-column derivatising technique requires a very accurate reaction performance and results in Aflatoxin derivatives, which do elute from HPLC very early and therefore can be easily covered by matrix peaks usually eluting at retention times similar to Aflatoxin B1 and G1. Iodine derivatisation as a post column technique does have other disadvantages like longer reaction time at higher temperatures (peak broadening), additional HPLC pump necessary and daily preparation of the corrosive iodine solution. The Coring Cell is an electrochemical cell which generates the derivatising agent, bromine, frompotassium bromide present in the mobile phase. The derivatisation of aflatoxins occurs rapidly(reaction time is approximately 4 seconds) at ambient temperature. Further advantages are: • no daily preparation of derivatising reagent necessary • no stability problems of mobile phase even after addition of potassium bromide and nitric acid • no handling with halogeneous solutions (i.e. iodine) • no additional pump for addition of derivatising reagent • no corrosion of pumps • low maintenance costs • high increase of Aflatoxins fluorescence activity • derivatisation in 4 seconds at ambient temperature, no heating block necessary • simple to install/uninstall • high reproducibility of results When such a system is combined with the AflaStar™ or AflaStar™ Fit immunoaffinity columns, asignificant amount of time can be saved since the set up of the HPLC system and the maintenanceof equipment is simpler. Optimising a system with such a combined approach maximises theadvantage of investing in an HPLC. This paper describes the clean-up of the aflatoxins B1, B2, G1 and G2 by means of an immunoaffinitycolumn (Aflastar column) and the following HPLC detection with an electrochemical cell (CoringCell). This HPLC method features a very high-sensitive fluorescence detection and is an officialGerman method for measuring compounds in food products (§35 LMBG methods).

Fluorescence spectrum of undeviratised Aflatoxins

LCE system with Coring cell

AF B2

AF G 2

AF B1

AF G 1

400 450 500 nm

Emmissionswellenlänge

VWR International GmbH, Scientific Instruments, Darmstadt 8-2005 1/15


Injection of 100 µl Aflatoxin-Standard: 0.2 ng/ml for B1 and G1 and 0.05 ng/ml for B2 and G2

2. Chromatograms

Injection of 100 µl Aflatoxin-Standard: 2 ng/ml for B1 and G1 and 0.5 ng/ml for B2 and G2


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Injection of 100 µl Aflatoxin-Standard: 10 pg/ml for B1 and G1 and 2.5 pg/ml for B2 and G2

Injection of 100 µl Aflatoxin-Standard: 20 pg/ml for B1 and G1 and 5 pg/ml for B2 and G2


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Injection of 100 µl musk seed extract (1:1 dilution with water to have an organic modifier concentration of 50 % methanol) after sample clean-up with AflaStar™ immunoaffinity column – this was a positive tested sample from a customer which could not be used for the production of liqueur.

Injection of 100 µl musk seed pure methanol extract (no dilution with water > chemical tailing) after sample clean-up with AflaStar™ immunoaffinity column – this was a positive tested sample from a customer which could not be used for the production of liqueur.


3. Coring Cell Membrane: Anion Capillary connections: Valco 1/16" (do not use metal nuts or tubes because these can damage the cell) Working pressure: max. 2 bar

Coring Cell Components 1 x Coring Cell 1 x Current Source + Connection Lead 1 x Electrode Connection Lead 1 x Spare Membrane

Platin electrode

Spacer

Membrane

Spacer

Stainless steel electrode

HPLC column Reaction coil

Fluorescense detector

Waste Black contact

Red contact

Figure 1: Diagram of the CoBrA Cell Connections

3.1. Important Notes Hazard: Aflatoxins are very hazardous substances. Suitable protective clothing, including rubbergloves, safety glasses and laboratory coats should be worn throughout the assay. • When not using the Coring cell always store it filled with water in order to keep the membrane

wet. • Never flush 100% v/v organic solvents through the Coring Cell as this can damage the

membrane. • Always ensure there is flow through the Coring Cell before plugging in the current source. • Never turn “off” the HPLC pump before having unplugged the Coring Cell current source first. • Always use HPLC grade solvents from a quality supplier, and maintain fresh supplies of

potassium bromide. • Never connect metal nuts or tubes directly with the Coring Cell. • Always use plastic tubing’s (Teflon, peek...) for connections. Always keep tube i.d’s the same.


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3.2 Installation of the Coring Cell a) Prepare one litre of mobile phase, methanol: acetonitrile: water (20:20:60 v/v), containing 119

mg of potassium bromide and 100 µl of 65% Nitric acid. Connect the reservoir of freshly prepared mobile phase to the HPLC, by immersing the tubing of the HPLC pump feed into the reservoir.

b) Disconnect all capillary tubing from the Coring Cell and connect the HPLC column with a plastic tubing to the Coring Cell inlet (marked “RP18”). Connect Coring Cell outlet (marked “FD-in”) with a plastic tubing to the detector inlet - this length of tubing critical, and the length is dependent upon flow rate and internal diameter (i.d.) of the tubing. Length calculation see equation and table 1.

c) Connect the detector outlet with a plastic tubing to the Coring Cell inlet (marked “FD-out”) and the Coring Cell outlet (marked “waste”) with a plastic tubing to waste. All connections are made to hand tightness (CAUTION : do not over tighten) and should be plastic not metal or stainless steel.

d) Switch on HPLC pump. e) Connect the Coring Cell current source to the cell, red lead to red terminal and black lead to

black terminal. Plug in current source and switch it on. One green and 2 red LED lights on the current source indicate the function as listed in the table:

LED OK Error

1 Error 2 Meaning Repair

on off off correct function -- on on off Cell not connected

No additives in mobile phase

Connect red lead to red terminal and black lead to black terminal Add KBr and HNO3 (see point 5, mobile phase)

on off on Short cut in circuit Internal defect current source

Contact Coring System Contact Coring System

f) After the HPLC has been allowed to stabilise (no more baseline drift, 20-30 minutes) the Coring

Cell is ready to be used. Table 1 Equation: 8.5 * Flow rate [ml/min] / I.D.² [mm]

0.4ml/min

0.5ml/min

0.6ml/min

0.8ml/min

1.0ml/min

0.20mm ∅ 84.9 cm 106.1 cm 127.3 cm 169.8 cm 212.3 cm 0.25mm ∅ 54.3 cm 67.9 cm 81.5 cm 108.6 cm 135.8 cm0.40mm ∅ 21.2 cm 26.5 cm 31.8 cm 42.4 cm 53.1 cm 0.50mm ∅ 13.6 cm 17.0 cm 20.4 cm 27.2 cm 34.0 cm 0.80mm ∅ 5.3 cm 6.6 cm 8.0 cm 10.6 cm 13.3 cm

Important: The Hitachi detectors have tubings with the following dimensions: Inlet 0.25 mm I.D. and Outlet 0.33 mm I.D. It is strongly recommended to use the same type of tubings for the reaction coil and the detector inlet (see the red values in Table 1). Example: When a flow rate of 1 ml/min is used the reaction coil should not be shorter than 135.8 cm to guarantee a reaction time of 4 sec.



3.3 Checking the performance of the Coring Cell the correct function of the Coring Cell can be checked by observing the baseline in the Preview mode.

1. Set up the instrument method according the application parameters 2. Install the Coring Cell correctly and do not power on the power supply 3. Start the Preview mode and wait 15-20 min until the baseline reaches a stable plateau 4. Power on the power supply of the Coring cell (red arrow of the picture below)

After powering on the power supply the baseline drops down after 10-20 sec. This is the sign that the Coring Cell is working properly and producing free bromine which absorbs some light and leads to this dropping of the baseline. In this case only the OK lamp at the power supply is on. If one or two of the error lamps are on please refer to chapter 3.2.


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3.4 Derivatisation reaction 3.5 Daily Cleaning Routine for the Coring Cell The HPLC system can be left running overnight at a reduced flow rate (.e.g. 0.1ml/min) and with thefluorescence detector and Coring Cell current source unplugged, if necessary. Alternatively, abetter practice to prolong the life of the system is to clean each day, as follows: 1. Switch off Coring Cell current source and fluorescent detector. 2. Switch off HPLC pump. 3. Disconnect Coring Cell from HPLC system, and reconnect HPLC column directly to

fluorescence detector. 4. For washing the HPLC column change the mobile phase to acetonitrile (100% v/v). 5. Switch on the HPLC pump and flush system for at least 30 minutes before switching "off"

system. 6. Meanwhile manually flush Coring Cell through with 5-10 ml distilled water using a syringe. Then

store Coring Cell with water inside the cell by closing off both ports on each housing with a tubebridge. This avoids a drying out of the membrane during longer storage periods.

3.6 Monitoring the Sensitivity of the Coring Cell It is necessary to regularly monitor the performance of the Coring Cell in order to detect anydeterioration in the membrane contained within. The sensitivity should be checked at the time ofinstallation and then weekly by comparing the peak areas of a known Aflatoxin standard solution(see our offer for standard solutions), from one week to another. Deterioration will occur over a period depending upon the frequency of use and the type of samplesanalysed, when the level of sensitivity becomes unacceptable the membrane should be replaced. Normally, it is found that even under extreme workloads the membrane need not be replaced for atleast 6 months.

+

O O

O

O

O O

H

H

O O

O

O

O O

H

H


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AFLATOXIN B1

O O

O

O

O O

H

H

O O

O

O

O O

H

H

Br2

+ Br2 Br

Br

Br

Br

AFLATOXIN G1



3.7 Maintenance: Changing the Membrane


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• Change the membrane when a decrease in sensitivity of Aflatoxin B1 and G1 is observed, and

the sensitivity of the 2’s stays the same. Additional membranes are available separately. • Using the integrated hexagon key crosswise remove the six locating screws in the top housing

over cross. • Carefully separate the top housing, and remove in turn the internal layers of the cell using

forceps. Make a note of the position and orientation of each layer as it is removed. Continueremoving the internal layers until the brown membrane is exposed.

• Remove the old membrane and replace it by a new membrane just having taken it out of thestorage bottle. Take care not to touch the middle of the membrane with hands or fingers. DONOT ALLOW THE MEMBRANE TO DRY-OUT. ADD DISTILLED WATER IFNECESSARY.

• Carefully replace the layers to be positioned on top of the membrane remembering their correctorder and orientation. Secure the layers in by re-fitting the six locating screws in the housingover cross. Tighten the screws just by hand, do not over tighten the screws. Maximum 2 Nm.

• Regularly monitor the sensitivity of the Coring Cell in order to detect any significantdeterioration of the membrane contained within.

RP-18 FD-in

Housing Spacer 1 Platin electrode Spacer 2 Membrane Spacer 2 Grid Spacer 2 Stainless Steel Electrode Spacer 1 Housing

FD-out Waste




3.8 Chromatogram without Derivatisation ("1") versus Coring-Cell derivatised Aflatoxins ("2") 4. AflaStar™ IMMUNOAFFINITY COLUMNS -

For the purification of AFLATOXIN B1, B2, G1, G2 in conjunction with HPLC

Absolutkonzentrationen AflatoxinG2..........14.25pgG1..........499.45pgB2..........72.5pgB1..........306.3pg

G2

G1

B2

B1

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4.1 Product Description AflaStar™ - Immunoaffinity Columns are reliable and user friendly designed for rapid and simplepurification of Aflatoxin B1, G1, B2, G2 from extracts of foodstuffs. AflaStar™ - ImmunoaffinityColumns contain monoclonal antibodies against Aflatoxin, which are covalently bound to gelparticles. Aflatoxins1 are metabolic products of the mould species Aspergillus flavus and Aspergillusparasiticus. Aflatoxins are highly carcinogenic.


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4.2 Intended Use AflaStar™ - Immunoaffinity Columns are intended for use in a wide range of commodities includingcottonseeds, cereals, maize, peanuts, walnuts, pistachio and spices. For this process, a representative sample2+3 of the commodity to be analysed is extracted with amixture of solvent. For a sample to be considered representative Romer Labs recommends the useof the Romer Analytical Sampling (RAS) mill, or Series II mill. The extract is filtered and the dilutedwith a buffer. The diluted extract can be run directly over the AflaStar™ - Immunoaffinity Columns, inwhich the Aflatoxins are bound and separated from the other substances of the extract. After a shortrinsing phase isolated Aflatoxins are eluted using a small volume Methanol. This eluate is used for determining the concentration of Aflatoxins with the HPLC by fluorescencedetection after derivatisation by the Coring Cell (or similar systems) in order to increasefluorescence activity of main Aflatoxins B1 and G1 by factors of 20 resp. 50. 4.3 Warnings and Precautions 1. Mycotoxins are highly toxic substances! Please take care about protective measures4! 2. Please decontaminate any equipment used with 4% solution of sodium hypochlorite. 3. Each AflaStar™ - Immunoaffinity Column contains sodium azide. 4. Do not use the AflaStar™ - Immunoaffinity Column after the expiration date indicated on the

label. 5. AflaStar™ - Immunoaffinity Columns are designed for single use only. 6. Do not freeze product. 4.4 Additional Equipment available but not provided Adapter Syringe barrel as a supply reservoir (20-50ml) Luer Lock valve Romer Analytical Sampling mill (RAS mill, 220 Volt) or Romer Series II Mill (220 Volt) Romer EVAP System (220 Volt) 4.5 Recommended solvents and buffers PBS-buffer (8g NaCl/L, 0.2 g KCl/L, 0.2 KH2PO4.1 H20/L, 2.9 g Na2HPO4 . 12 H20/L , pH 7,4) Extraction solution: 60:40 (v/v) acetonitrile/water or 60/40 (v/v) Methanol/water, Methanol, HPLC-grade All solvents and buffers should be at room temperature (+22-28°C). We recommend the use of our Biopure Aflatoxin standards.

O O

O

O

O

OCH3




4.6 Procedure 4.6.1 Extraction Grind a representative sample of the commodity. Various mixtures of solvents can be used for theextraction of Aflatoxins. However, a mixture of acetonitrile/water 60/40 (v/v) results in good recoveryrates. Also mixtures of Methanol/water 60/40 (v/v) give good results. For solid samples:

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e 1. Weight out 25g of sample into a 250 ml Erlenmeyer flask or a half-pint blender jar. 2. Add 100ml of extraction solution and stopper flask or seal blender jar. 3. Blend on high speed for 3 min. or in case of fine powder shake for 1 hour on a gyratory shaker 4. Using a funnel, filter extract into a sample jar through qualitative filter paper. Liquid samples can be diluted directly with PBS, without preceding extraction. Any alcohol content in the sample must be considered in the calculation of the dilution, or the sample can be vaporised if necessary. 4.6.2 Dilution of the Extracts Acetonitril extracts are diluted with PBS until the part of acetonitrile is not higher than 5 % (v/v). Methanol extracts are diluted with PBS until the part of Methanol is not higher than 20 % (v/v), e.g. 4 ml extract (equals 1 g sample) + 8ml PBS is adequate. Depending on the sensitivity required it is possible to carefully dry down an aliquot of the extract to a smaller volume (recommended equipment: Romer EVAP-System) and making it up to 10ml using PBS buffer. Please note the pH of the extract should be neutral. If necessary, neutralise with NaOH-solution. 4.6.3 Sample Application Column and sample extract must be at room temperature. The columns are ready to use and do not require rinsing before application of the diluted extract. Take off top cap and connect the AflaStar™ - Immunoaffinity Column with an adapter to a suitablesyringe barrel (luer connector), and fix construction using a clamp or similar. Fill in total volume of diluted extract into syringe barrel, take off bottom cap and let total volume passthrough the column under gravity (flow <= 2-3ml/min). The AflaStar™ - Immunoaffinity Column usuallydrops independently. Positive resp. negative pressure can be used as well to pass through thesample, but flow should not exceed 3ml/min. 4.6.4 Wash Step The AflaStar™ - Immunoaffinity Column is washed with 2 x 10 ml of distilled or deionised water. Thefirst portion of the wash is used to rinse the syringe barrel. Any remaining liquid is removed from the column through slight pressure from above or slight negative pressure from below. At the same time the column is not allowed to dry out. 4.6.5 Elution Remove syringe barrel from the AflaStar™ - Immunoaffinity Column and place a suitable vial underthe column for collecting the eluate. For the elution of bound Aflatoxins use only water free Methanol(HPLC-grade). Slowly elute AflaStar™ - Immunoaffinity Column in between 2-3 minutes with 1.5mlMethanol, which should be applied to the column in several small portions (for example 3 x 0.5ml).The Methanol should be left on the column for a short period of time before letting it run off. After the last portion is applied, the Methanol still remaining in the column is pressed out and added to the eluate. The eluate must be diluted at least 1:1 with water so that the organic modifier concentration is not higher than 50 %. If the eluate is not diluted you will get bad peak shape and double peaks because of chemical tailing (see lower chromatogram on page 4). In case of low-level contamination, the eluate can be carefully dried down (for example with the Romer EVAP-System) and re-constituted in a small portion of mobile phase.



5.1 HPLC Equipment


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VWR-Hitachi LaChrom Elite® (LCE) system Pump L-2130 equipped with low pressure gradient kit & built-in vacuum degasser &

standard static mixer unit Autosampler L-2200 Column Oven L-2300 with Peltier block

®Column Merck Purospher STAR RP-18e (5 µm) (4.6 x 150), art. 1.50358 ® STAR RP-18e (5 µm) (4.0 x 4.0 ), art. 1.50250 Precolumn Merck Purospher

Detector L-2480 fluorescence detector with standard cell Organizer with power supply for the other units

™ Acquisition CDS EZChrom Elite 3.1.6




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5.2 HPLC Conditions Detector settings (Fluorescence) Sampling Rate 400 ms, Time Constant = 2 sec PMT Voltage Medium Wavelengths EX 365 / EM 435 Column Purospher STAR RP-18e column, 150 x 4.6 mm, 5 µm Pre column Purospher STAR RP-18e guard column, 4 x 4 mm, 5 µm Temperature Oven 40 º C Flow Rate HPLC pump 1 mL/min

65%/L Eluent A: Water + 183.1 mg KBr/L + 154 µl HNO3Eluent B: Methanol Eluent C: Acetonitrile Isocratic: 65 % A / 17.5 % B / 17.5 % C (V/V/V), mixed by the pumps Derivatisation coil: PEEK coil, 1.38 m x 0.25 mm I.D (please refer to section 3.2) Injection volume 100 µl 6. Materials needed for the application • Purospher STAR RP-18e column, 150 x 4.6 mm, 5 µm (Merck , art. 1.50358.0001) • Purospher STAR RP-18e guard column, 4 x 4 mm, 5 µm (Merck , art. 1.50250.0001) • Manu-Cart NT cartridge holder for LiChroCart cartridges (Merck , art. 1.51486.0001) • Vici Jour one-piece finger tight fittings 6 pcs (jourm55020-1) • PEEK union kit (jour1061) • PEEK tubing OD 1/16” x ID 0.25 mm (Blue), 3 m (jour6001) • Teflon tubing for HPLC 1/16” x ID 0.33 mm, 10 m (jour4011) • Apparatus and filters (0.2 µm) to filter mobile phases • Pipettes and tips • Water LiChrosolv 2.5 L, (Merck , art. 1.15333.2500) • Acetonitrile gradient grade LiChroSolv 2.5 L, (Merck , art. 1.00030.2500) • Methanol gradient grade LiChroSolv 2.5 L, (Merck , art. 1.06007.2500) • Potassium bromide, (Merck , art. 1.04905.0500) • Nitric acid, HNO3 , (Merck , art. 1.00452.1000) • Coring Cell for aflatoxin applications, Coring art. 3200130 • Aflatoxin Mix 1 (2 µg/ml aflatoxin B1 & G1 each, 0.5 µg/ml aflatoxin B2 & G2 each , 5 ml in

acetonitrile, Coring art. 3009119) • Immuno affinity column (Coring AflaStar™, 10 tests: Coring art. 1001011, 25 tests: Coring

art. 1001006, 100 tests: Coring art. 1001009) or

• Immuno affinity column (Coring AflaStar™Fit, 25 tests: Coring art. 1001021)



7. Discussion: The handling and working with the Coring Cell is really comfortable and user-friendly compared to the classical post-column derivatisation with iodine. The aflatoxins can be detected at very low detection limits using the LCE HPLC system with L-2480 fluorescence detector and the Coring cell. The limit of detection (LOD) is about 2.5 pg/ml aflatoxin B2 and G2 (see page 3). For the sample preparation of real samples it is possible to be more sensitive when the AflaStar™Fit columns are used instead of the normal AflaStar™columns. The AflaStar™Fit column has a cartridge format of 1 ml whereas the AflaStar™column has 3 ml. The reduced cartridge format allows the customer to elute the aflatoxins only with 500 µl methanol or acetonitrile instead of 1500 µl. Therefore the sensitivity for measuring real natural samples using the AflaStar™Fit columns is increased by factor 3. The eluate has to be diluted at least 1:1 with water to have a final content of organic modifier not more than 50 %. Without this dilution step the chromatogram shows Chemical Tailing which becomes more serious the bigger the injection volume is (see the chromatograms on page 4). Also the standards should have organic modifier concentrations less than 50 %.

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8. Bibliography

• Castegnaro, M. et al.: "Laboratory Decontamination and Destruction of aflatoxins B1, B2, G1 and G2 in Laboratory Wastes" (ISBN 92 8 321137 5)z; WHO/IARC Scientific Publications No. 37 (1989).

• CAST-report, Mycotoxins Risks in Plant, Animal, and Human Systems, Jan. 2003 • USDA/ GIPSA, Aflatoxin handbook, Chapter 3, sample preparation

http://www.usda.gov/gipsa/reference-library/handbooks/aflatoxin/aflatoxin-ch03.pdf • EC-Directive 98/53/EC, 16.July 1998, EC-directive 2002/27/EC of 13th of March 2002,

laying down the sampling methods and methods for analysis for the official control of the levels for certain contaminants in foodstuff http://vm.cfsan.fda.gov/~acrobat/iuafl2.pdf

• Tauchmann, F., et al; Alimenta 11, 85 (1972): Schutzmaßnahmen beim Arbeiten mit Mykotoxinen (protective measures)

• EMAN, European Mycotoxin Awareness Network, Fact sheets on analytical methods, Fact sheet 1, Ochratoxin A, Extraction http:/www.Lfra.co.uk

9. References & Endnotes

®LaChrom , LaChrom Elite® and are registered trademarks of VWR International, AflaStar™ andAflaStar™Fit are trademarks of Coring System, EZChrom Elite™ is a trademark of Scientific SoftwareInc. Authors Volker Eckert, VWR International GmbH, Darmstadt, Germany (Mail: [email protected]) Klaus Lobeck, Coring Systems, Gernsheim, Germany (Mail: [email protected])

Coring System Diagnostix GmbH VWR International GmbH Magdalenenstrasse 21 Scientific InstrumentsD-64579 Gernsheim Hilpertstrasse 20 AGermany D-64295 DarmstadtTel.: +49 6258 9317-0 GermanyFax: +49 6258 3031 or 3050 Fax: +49-6151-3972101E-Mail: [email protected] [email protected] Homepage: http://www.coring.de Homepage: http://www.vwr.com


http://www.usda.gov/gipsa/reference-library/handbooks/aflatoxin/aflatoxin-ch03.pdf

http://vm.cfsan.fda.gov/%7Eacrobat/iuafl2.pdf

mailto:[email protected]




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LaChrom Elite Application Note - uk.vwr-cmd.comuk.vwr-cmd.com/.../chrom/applications/Aflatoxins_with_Coring_Cell.pdf · LaChrom Elite® HPLC: Aflatoxins in foodstuff using Coring