AF

Silica gel-based immunoaffinity columns for Aflatoxin analysis

Users Manual

Introduction The rapid quantitative determination of aflatoxin B1, B2, G1 and G2 in dilute and/or difficult matrices is now feasible using the VENTURE AF immunoaffinity column. By immobilising anti-aflatoxin on wide porous silica, the VENTURE AF column selectively binds these aflatoxins. The surface of the silica has been treated with a proprietary process, ICEtechTM (Inert Coating Enhancement Technology), to create a passivated surface that prevents non-specific interactions. The wide porous silica has a narrow pore size distribution and a large internal surface area and exhibits the following advantages: Direct coupling with HPLC Efficient isolation, concentration and purification of aflatoxin B1, B2, G1 and G2 High selectivity towards aflatoxin B1, B2, G1 and G2 Quantitative recovery of aflatoxin B1, B2, G1 and G2 Long column lifetime No non-specific surface interactions High ligand stability Rapid analysis of aflatoxins

Since the silica particles are non-compressible, the immunoaffinity column can be used in combination with RP-HPLC at high flow rates for the quantitative analysis of aflatoxin. The particles do not swell or shrink in different solutions. Typical properties Composition Particle size support Ligand Capacity pH range Pressure Delivery conditions Storage Quality assurance Each batch of VENTURE AF columns is tested to determine the binding capacity with standard solutions of aflatoxin. The results are reported in the certificate of analysis that is included with each column. Wide porous spherical silica gel 15 - 20 m anti-human aflatoxin 0.5 nMol of total aflatoxin pH 2 to 8 maximum operating pressure 3000 psi (200 bar) PBS buffer, pH 7.0 +4 to +8 C in PBS buffer, pH 7.0

Typical performance characteristics Repeatability Dynamic range Sensitivity Accuracy 5 - 10 %, depending on the aflatoxin and the content analysed linear within the capacity of the column LOD 2 4 pg (0.2 0.4 g/kg) depending on the aflatoxin analysed quantitative recovery of aflatoxin B1, B2, G1 and G2 within the capacity of the column

Column preparation The columns are shipped in PBS buffer containing 0.02% sodium azide. Before using the column, it should be equilibrated with at least 5 to 10 columns volumes of binding buffer. Sample preparation Sample extracts should be filtered (0.22 or 0.45 m) or centrifuged and exchanged with binding buffer prior to column loading. The content of organic solvent should be equal or less than 10%v/v. If the aflatoxin concentration is high (> 100 g/l) dilution with binding buffer is required. When protease contamination is suspected, a protease inhibitor should be added to the sample prior to loading on the column. Protease can digest anti-aflatoxin and negatively effect column performance.

Chromatographic conditions for the quantitative analysis of aflatoxins For the quantitative analysis of aflatoxin B1, B2, G1 and G2, a HPLC apparatus, equipped with a 500 l sample loop, a programmable two-position six-way valve, a programmable low- or high-pressure (at least three-channel) gradient pump system, a Kobra cell and an fluorescence detector, is required.

Before loading the sample:

1. Program your HPLC according to the requirements mentioned below. 2. All buffers should be degassed and filtered (0.22 or 0.45 m). 3. Fill the pump tubing with the appropriate buffer solutions before connecting the column to the system to prevent air getting into the column(s). 4. Remove the end-cap of the column(s) and connect to the system. 5. Equilibrate the column(s) with at least 5 column volumes of binding buffer or until no signal is detected in the effluent;

6. Apply sample using the sample loop and analyse the aflatoxin content according to the chromatographic conditions mentioned below. Conditions Column 1 Column 2 Detection Binding buffer Elution buffer Mobile phase VENTURE AF (cat# AFVE1520405) anti-aflatoxin immunoaffinity column GENESIS C18 (cat# FM25960E), 4 m, 4.6 x 250mm Fluorescence detection (excitation at 369 nm/ emission at 422 nm) 0.10 M phosphate + 0.15 M NaCl, pH 7.0 20%v/v acetonitril in water 600 ml methanol + 80 ml acetonitril + 200 l of concentrated nitric acid + 50 mg of Potassium bromide and adjusted to 1000 ml with water Flow rate Column (ml/min) 1 0.5 On-line 0.5 1 1 1 On-line On-line Off-line On-line Column 2 Off-line Off-line On-line On-line On-line %Binding buffer 100 100 0 0 100 %Elution buffer 0 0 100 50 0 % Mobile phase 0 0 0 50 0

Program Time (min) 0-5 (inject) 5.1-10 (wash) 10.1-14.5 (elute) 14.6-40 (analyse) 40.1-50.1 (equilibrate) Storage

Columns should be stored at a temperature of +2 to +8 C. DO NOT FREEZE the columns. Columns that have been used should be cleaned with 10 column volumes of binding buffer containing 0.02% sodium azide. Always make sure that the column has sealed endcaps in place to prevent column drying. After storage, wash the column with at least 5 - 10 column volumes of binding buffer before use.

Safety information Read the safety datasheets of all chemicals involved. Make sure to wear the proper protective wear. The VENTURE AF immunoaffinity columns are for research purposes only. Column size The VENTURE AF anti-aflatoxin immunoaffinity column is available in the following dimension: Diameter (mm) 4.6 Length (mm) AFVE1520405 Catalog number 50

Standard column material is PEEK. For other column dimensions or materials please contact Grace Vydac. Ordering information To order the VENTURE AF immunoaffinity column, please contact your local Grace Vydac distributor or contact Grace Vydac direct: By phone (EU) +49 6241 403 90 759 (US and Other) 1-800-247-0924 or 760-244-6107 by Email experts@gracevydac.com or on the web www.gracevydac.com

VENTURE and ICEtech are trademarks of Grace Vydac, a wholly owned subsidiary of W.R. Grace & Co.-Conn. (C) Copyright 2004 W.R. Grace & Co.-Conn.