Aflatoxins, HPLC analysis

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

Figure 12.11—Comparison of UV andfluorescence detection after chromatographic separation. Aflatoxins, which are carcinogenic contaminants present in certain batches of grain cereals, are controlled by HPLC analysis. It can be seen that the peak intensities in UV detection vary with concentration whereas fluorescence detection is much more sensitive to aflatoxin G2 and B2. (Reproduced by permission of SUPELCO.)...

Figure 7.9. HPLC analysis of aflatoxins in a peanut extract sample using reversed-phase chromatography and fluorescence detection. Chromatogram courtesy of PerkinElmer, Inc.

However, HPTLC and HPLC techniques complanent each other the HPTLC for preliminary work to optimize LC separation conditions during the development of a method or it may also use as screening for the analysis of a large number of samples to limit the HPLC analysis only to positive samples. Liquid chromatographic methods for aflatoxin determination include both normal and reverse-phase separations, although current methods for aflatoxin analysis typically rely upon reverse-phase HPLC, with mixtures of methanol, water, and acetonitrile for mobile phases. 

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. 

Fig. 2 Postcolumn derivatization scheme for aflatoxin analysis 1, mobile phase 2, HPLC pump 3, injection valve 4, precolumn 5. analytical column 6, derivatizing agent solution 7, auxiliary HPLC pump 8, T-valve 9, oil or water bath 10, reaction coil 11, fluorescence detector 12, waste 13, chromatographic data handling system.

OG Roch, G Blunden, RD Coker, S Nawaz. The validation of a solid phase clean-up procedure for the analysis of aflatoxins in groundnut cake using HPLC. Anal Food Clin Methods. Section 93-98, 1994. 

Figure 11.12 Comparison, following a chromatographic separation, of UV and fluorescence detection. Aflatoxins, which are carcinogenic contaminants present in certain hatches of grain cereals, are the subject of analysis by HPLC. Detection by fluorescence is much more sensitive to Gj and B2 than with UV detection (reproduced courtesy of SUPELCO). Below left, schematic of the different components of a LC-detector based upon fluorescence. This detector is able to find rapidely, for each compound eluted, the best coupling of excitation/emission without interrupting the chromatography underway (reproduced courtesy of a document from Agilent Technologies).

The evaluation of a number of immunoassay diagnostic kits was undertaken to determine their usefulness in a regulatory analytical laboratory environment in the food, feed and pesticide areas. Four rapid enzyme immunoassay tests for the detection of aflatoxin residues at the 20 ppb level in animal feeds were compared to the official HPLC procedure. In the pesticide area, a commercial pentachlorophenol competitive inhibition assay for residues in water was investigated as to its applicability to poultry and pork liver matrices. In addition, an ELISA screening procedure for the herbicide fusilade was developed. Modifications were incorporated into the rapid immunoband 1-2 Test procedure for the detection of motile Salmonella in various food and animal feed products resulting in quicker analysis than the standard culture method. Also, a comparative evaluation of a Quik-Card Test for sulphamethazine drug residues in pork urine, liver and muscle tissue, is described. 

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. 

Food Analysis Preparative immunoaffinlty chromatography techniques are also applicable to the analysis of food samples for the presence of parent aflatoxins. To validate this method, naturally contaminated corn and peanut product samples were obtained and extracts made using methanol-water (60% 40%, vol/vol). An aliquot of extract was applied to the monoclonal antibody affinity column, aflatoxins eluted, and the product measured by reversed phase HPLC. In Figure... 

The analysis of mycotoxins, toxic substances produced by some fungi, is essential to guaranteeing the safety of foods such as cereals, nuts, oilseeds, and spices since the discovery of their significance to human health. The main mycotoxins, like aflatoxins and ochratoxin A, are determined using HPLC with octyl-or octadecyl-bonded phases and polar solvents such as methanol, acetonitrile, and water. For detection with the required sensitivity and selectivity, spectrofluori-metric detection is the most common method, with increasing use of mass spectrometric interfaces. 

Because of its higher separation power, higher sensitivity, and accuracy, and the possibility of automating the instrumental analysis, HPLC is now the most commonly used technique in analytical laboratories. HPLC using fluorescence detection has already become the most accepted chromatographic method for the determination of aflatoxins. For its specificity in the case of molecules that exhibit fluorescence. Commission Decision 2002/657/EC, concerning the performance of analytical methods, considers the HPLC technique coupled with fluorescence detector a suitable confirmatory method for aflatoxin identification. 

In this scene, HPLC is a paramount technique in the modern age of food analysis with hundreds of applications [3], some of which include the detection of aflatoxins, amino acid analysis, vitamins separation, profiling of various food components, analysis of colorants and their residues, or the determination of the sugars content. 

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