Lateral flow test
Rapid tests are packaged systems of the principal or key components of an analytical method used to determine the presence of a specific analyte(s) in a given matrix(es). Rapid test kits include directions for their use and are often self-contained, complete analytical systems; but they may require supporting supplies and equipment. Rapid tests can detect a specific analyte(s) in given matrix(es) in significantly less time than reference methods (source www.aoac.org).
For a rapid detection of mycotoxins the following technologies are widely accepted by the industry and recognized by the scientific community:
Enzyme linked immunosorbent assay (ELISA)
This is one of the most popular immunologically based methods used in test kits for the analysis of mycotoxins in foods and feeds. A commonly used approach is a competitive assay where a known amount of labeled (enzyme) toxin competes with any possible toxin in the sample for the specific antibodies attached to the reaction vessel. Any unbound toxin is then washed from the vessel. The quantification is dependent upon the amount of enzyme labeled toxin remaining in the vessel to react with the substrate for the enzyme. This reaction results in a colored product that can be measured optically.
The lateral flow test is a one-step lateral flow immunochromatographic concentration assay based on an inhibition immunoassay format. A typical immunochromatography test strip is composed of a sample pad, a conjugate pad, a membrane, an adsorbent pad and an adhesive backing. A sample extract is added onto the sample pad. Any mycotoxin present binds to the anti-mycotoxin antibody gold particle complex in the conjugate pad and they migrate together with the anti-2nd antibody gold particle complex along the membrane. The membrane contains a test zone and a control zone, onto which a mycotoxin-protein conjugate and a 2nd antibody are dried. The mycotoxin-protein conjugate in the test zone can capture any free anti-mycotoxin antibody gold particle complex, allowing color particles to concentrate and form a visible line. Hence, although a positive sample will result in no visible line in the test zone, the control zone will always be visible indicating the validity of the performed test.
The basis of fluorometry is the quantification of compounds by measuring their fluorescence using a fluorometer. In some cases the compounds may be innately fluorescent, and in others the compounds are rendered fluorescent through some chemical derivatization. The tests for compounds using fluorometry includes the extraction of the compound from the specific matrix, followed by a cleanup process using eitherimmunoaffinity columns or solid phase cleanup columns, and then a derivatization (if necessary) and measurement of the fluorescence.
A reference method is an internationally or nationally recognized standard analytical method applicable to the particular analyte being tested (source www.aoac.org).
The most common analytical reference methods for the detection of mycotoxins are the following:
Thin Layer Chromatography (TLC)
Thin layer chromatography is the separation of compounds in a mixture that are spotted near one end of a plate that is coated with a thin layer of an adsorbent matrix such as silica gel. Separation occurs when the end of the plate nearest the spotted mixture is placed in a solvent system in the bottom of a closed vessel and the solvent is allowed to migrate through the adsorbent matrix and moves toward the top of the plate. As this occurs the mixture of compounds separate based on their interactions between the solvent system (mobile phase) and the matrix (stationary phase). If the mycotoxin of interest is present in the mixture it will be identified by comparison with standard spots of this mycotoxin that are spotted on the plate in a similar manner as the mixture.
Gas Chromatography (GC)
Gas chromatography uses rather sophisticated equipment in which compounds are separated by a gas flowing through a heated glass column coated with a stationary nonvolatile liquid. Samples injected into the system are separated into the specific components on the column and the separated analytes coming off of the column are detected by a chemical or physical detection system.
Liquid chromatography- mass spectrometry (LC/MS)
High Performance Liquid Chromatography (HPLC)
HPLC is similar to TLC in principle but is a much more sophisticated system. A small portion of a sample to be analyzed is injected into a stream of solvent being pumped through a column of an adsorptive matrix. As the sample moves through the column the compounds are separated by basically the same principle as described for TLC. The sample components elute off from the column as separate entities, and the flow of solvent with the respective compounds passes through a detector to measure the response of the specific compound. The measurements are determined based on detector response which, are compared to selected concentrations of standard that were injected into the instrument as part of the analysis sequence.
HPLC can be coupled with a variety of detectors, e.g. spectrophotometric (UV-VIS) detectors, refractometers (RI), fluorescence detectors (FLD), electrochemical detectors, radioactivity detectors and mass spectrometers.
HPLC and LC/MS
The technology of liquid chromatography-mass spectrometry (LC/MS) or LC coupled to tandem mass spectrometry (LC/MS/MS) opens the perspective of efficient spectrometric assays for routine laboratory setting. This technique can be used for a wide range of potential analytes to be measured, with no limitations by molecular mass, a very straightforward sample preparation, no requirement for chemical derivatisation and has only limited maintenance needs. The main advantages include low detection limits, the ability to generate structural information, the requirement of minimal sample treatment and the possibility to cover a wide range of analytes differing in their polarities. Sometimes co-eluting matrix components influence the ionization efficiency of the analyte positively or negatively, impairing the repeatability and accuracy of the analytical method. Therefore a sample clean-up prior to liquid chromatography will be necessary. In order to overcome matrix effects stable isotope labeled internal standards are used.