Aflatoxin detection limits

As regards the aflatoxin detection, UV spectrophotometry has been definitively abandoned, because of the lack of selectivity and the very poor analytical response, resulting in a very high detection limit. Since aflatoxins show fluorescence under UV light, spectrofluorimetric detection has been adopted in the last years. 

Plastic microdevices for high-throughput screening with MS detection were also prepared for detection of aflatoxins and barbiturates. These devices incorporated concentration techniques interfaced with electrospray ionization MS (ESI-MS) through capillaries [2], The microfluidic device for aflatoxin detection employed an affinity dialysis technique, in which a poly (vinylidene fluoride) (PVDF) membrane was incorporated in the microchip between two channels. Small molecules were dialyzed from the aflatoxin/antibody complexes, which were then analyzed by MS. A similar device was used for concentrating barbiturate/antibody complexes using an affinity ultrafiltration technique. A barbiturate solution was mixed with antibodies and then flowed into the device, where uncomplexed barbiturates were removed by filtration. The antibody complex was then dissociated and electrokinetically mobilized for MS analysis. In each case, the affinity preconcentration improved the sensitivity by at least one to two orders of magnitude over previously reported detection limits. 

SPR was developed for several mycotoxins. Different mycotoxins have been found simultaneously within a period of 25 min. with a detection limit for aflatoxin Bj, zearelenon, ochratoxin A, fnmonisin Bj and deoxinivalenol respectively - 0.2,0.01, 0.1, 50, 0.5 pg L (Zheng et al. 2005). 

The immunohistochemistry approach would be particularly informative in examining the distribution of methylation adducts amongst different cell types of a given tissue. In addition, if the methods can be adapted to fixed paraffin embedded tissues, then retrospective studies could be performed on samples already available. At present the detection limits for methylation- and aflatoxin-DNA adducts are of the order of 1 in 10 to 10 unmodified nucleotides (16, 33) and further studies are required to see if this level of sensitivity is sufficient for human studies. 

A last example of IMS methods applied to food quality is the determination of aflatoxins B1 and B2 on pistachio samples methanol extracts of samples were introduced into an IMS analyzer equipped with an IMS equipped with a nonradioactive, corona discharge ion source. The response to aflatoxins was acceptable for screening purposes, with a linear range of roughly 100, relative standard deviations 10% or lower, and a limit of detection of 0.25 ng for both aflatoxins. Ammonia could be used as a dopant, and detection limits were improved by a factor of 2.5. Authentic pistachio samples were analyzed without difficulties. 

As made evident in Table 2, immunoassays are also in routine use for analysis of mycotoxins, a group including the most dangerous and analytically elusive food-related toxins. In spite of the small structural differences within specific groups of mycotoxins (aflatoxins, ochratoxins, trichothecenes), assay specificity toward the other mycotoxin groups is always total. Determination of AEBi, toxin T-2, and ochratoxin A in a single extract from barley grains has been reported. Mycotoxins may be determined in crude extracts of various foodstuffs at concentration over 20 ng per kg. This detection limit can... 

The introduction of mass spectrometry and the subsequent coupling of liquid chromatography to this very efficient system of detection has resulted in the development of many LC-MS or LC-MS/MS methods for aflatoxin analysis. Because of the advantages of specificity and selectivity, chromatographic methods coupled to mass spectrometry continue to be developed they improve detection limits and are able to identify molecules by means of mass spectral fragmentation patterns. 

Two reviews [312, 1097] focus on the separation of aflatoxins B, B2, G, and G2. Often isocratic acetonitrile/water or acetonitrile/methanol/water mobile phases are used in conjunction with Cig columns (A = 360 nm). Normally acetonitrile levels of 25-40% are used. If methanol is used it is approximately at the 5-15% level and directly replaces an equivalent amount of acetonitrile. Most Importantly, TFA is almost invariably added at the 0.1-0.5% level. TFA forms a hemiacetal product with the aflatoxins that increases sensitivity by at least a factor of 3. Complete baseline resolution is usually achieved in <20 min. Fluorescence detection has been used, with 360 nm excitation and 440 nm emission wavelengths chosen. Detection limits of 1—lOpg were commonly reported. Postcolumn derivatization with iodine has also been used in conjunction with fluorescence detection to increase sensitivity. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

Despite the fact that biological and predominantly immunoenzymatic methods have received increasing attention during the last decade for the determination of aflatoxins, chemical and immunochemical assays have to be preferred for their characteristics, including a lower limit of detection and high specificity. 

Of the 15 spices marketed in India and screened by Saxena and Mehrotra (1989) for the mycotoxins, aflatoxin, rubratoxin, ochra-toxin A, citrinin, zearalenone and sterigmato-cystin, samples of coriander and fennel were found to contain the largest number of positive samples and mycotoxins. Other spices like cinnamon, clove, yellow mustard and Indian mustard did not contain detectable amounts of the mycotoxins tested. Aflatoxins are the most common contaminants in the majority of samples, levels being higher than the prescribed limit for human consumption. 

The surveillance programs discussed above contain few data on aflatoxin Ml in cheese. In general, aflatoxin Mi was not detectable or occurred at concentrations lower than the current legal limits (0.2-0.25 jug/kg) in a few countries for aflatoxin Mi in cheese (van Egmond, 1989). 

If a test compound significantly increases the number of observed colonies relative to a control for spontaneous reversion, which always occurs to some extent, that compound must have caused mutation that resulted in reversion. Each type of strain, of which five are normally used for the full test and two for more limited versions, detects a specific type of mutation TA98 detects frameshift mutations while TAIOO picks up base pair substitutions. Since the mutagenic abilities of compounds often depend on metabolic activation, as it does for aflatoxin Bl, the test can be run in the presence of S9 liver fraction from rats whose CYPs have been induced by the polychlorinated biphenyl Aroclor 1254. An increase in reversion only in the presence of S9 shows that the mutagen must be a metabolite. 

Aflatest. This immunoaffinity column also met the lower detection requirement for the USFDA at a permissible limit of 20 ppb for aflatoxin. Qualitative results can be obtained using a florisil tip, however, the florisil tip was found difficult to interpret. Evaluation showed the quantitative analysis to be as accurate as other HPLC procedures, but much more rapid. Using the Aflatest for sample extract purification, and HPLC with post column derivatization using iodine, a limit of quantitation of 2.3 ppb for B and G is easily achieved. Sensitivity to B2 and G- is lower due to less specificity of the Aflatest antibodies to tnese toxins. 

Fluorescence spectroscopy plays an important function in modern food analysis as can be seen from its wide use in the determination of numerous food components, contaminants, additives, and adulterants. This technique has made available very sensitive and selective methods that satisfy the requirements of food analysis, which are usually very complex, taking into account the large number of species to be determined, frequently at very low concentrations, and the wide variety of foodstuffs available. Initially, the use of fluorescence spectroscopy in food analysis was limited to the determination of species with intrinsic fluorescence (e.g., vitamins, aflatoxins, and some polycyclic aromatic hydrocarbons (PAHs)), but now it is widely applied to nonfluorescent species, using several physicochemical means such as chemical or photochemical derivatization reactions. Numerous techniques involve fluorescence detection in liquid chromatography (LC), frequently using pre- or postcolumn derivatization. In addition to conventional fluorime-try, which is commonly chosen for this purpose, other fluorimetric techniques such as laser-induced... 

The usefulness of immunochemical techniques for the fluorimetric determination of mycotoxins has been widely demonstrated. In addition to the use of immunoaffinity column cleanup followed by LC with fluorescent detection, several FIAs have been described for the determination of aflatoxins, deoxynivalenol, and fumonisins using fluorescence polarization immunoassay or time-resolved fluor-oimmunoassay. Also, although the application of flu-oroimmunosensors to food analysis has been relatively limited, there are some examples of the use of this approach to the determination of some mycotoxins such as aflatoxin Bi and fumonisin B2. 

TLC, together with analytical features (plate material, elution, detection/visualization, limit of detection). Of particular interest are the following products aflatoxins Bl, B2, Gl, and G2, zearalenon, ochratoxin, and fiimo-nisins B1 and B2 in maize. Recently, high-performance TLC (HPTLC) techniques have also been applied to forage samples commonly infected with an endophyte considered as symbiotic but responsible for disorders in animals. These mycotoxins include lolitrems and ergot alkaloids. 

Atmospheric pressure chemical ionization (APCI) is the ionization source that provides lower chemical noise and, subsequently, lower quantification limit than electrospray ionization (ESI) which is more robust. The use of mass spectrometric methods can be expected to increase, particularly as they become easier to use and the costs of instrumentation continue to fall. Despite the enormous progress in analytical technologies, methods based on HPLC with fluorescence detection are the most used today for aflatoxins instrumental analysis, because of the large diffusion of this configuration in routine laboratories. 

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