Predicting aflatoxin and fumonisin in shelled corn lots using poor-quality grade components

A study was conducted to determine if aflatoxin and fumonisin are concentrated in the poor-quality grade components of shelled corn. Four 1.0 kg test samples were each taken from 23 lots of shelled corn marketed in North Carolina. Inspectors from the Federal Grain Inspection Service divided each test sample into 3 grade components: (1) damaged kernels (DM), (2) broken corn and foreign material (BCFM), and )3) whole kernels (WH). The aflatoxin and fumonisin concentration was measured in each component and a mass balance equation was used to calculate the total concentration of each mycotoxin in each test sample. Averaged across all test samples, the aflatoxin concentrations in the DM, BCFM, and WH components were 1300.3, 455.2, and 37.3 ppb, respectively. Averaged across all test samples, the fumonisin concentrations in the DM, BCFM, and WH components were 148.3, 51.3, and 1.8 ppm, respectively. The DM and BCFM components combined accounted for only 5.0% of the test sample mass, but accounted for 59.8 and 77.5% of the total aflatoxin and fumonisin mass in the test sample, respectively. Both aflatoxin mass (ng) and aflatoxin concentration (ng/g) in the combined DM and BCFM components had high correlations with aflatoxin concentration in the lot. The highest correlation occurred when aflatoxin mass (ng) in the combined DM and BCFM components was related to aflatoxin concentration in the lot (0.964). Similar results were obtained for fumonisin. This study indicated that measuring either aflatoxin or fumonisin in the combined DM and BCFM grade components could be used as a screening method to predict either aflatoxin or fumonisin in a bulk lot of shelled corn.     

Testing shelled corn for aflatoxin, Part III: evaluating the performance of aflatoxin sampling plans

The effects of changes in sample size and/or sample acceptance level on the performance of aflatoxin sampling plans for shelled corn were investigated. Six sampling plans were evaluated for a range of sample sizes and sample acceptance levels. For a given sample size, decreasing the sample acceptance level decreases the percentage of lots accepted while increasing the percentage of lots rejected at all aflatoxin concentrations, and decreases the average aflatoxin concentration in lots accepted and lots rejected. For a given sample size where the sample acceptance level decreases relative to a fixed regulatory guideline, the number of false positives increases and the number of false negatives decreases. For a given sample size where the sample acceptance level increases relative to a fixed regulatory guideline, the number of false positives decreases and the number of false negatives increases. For a given sample acceptance level, increasing the sample size increases the percentage of lots accepted at concentrations below the regulatory guideline while increasing the percentage of lots rejected at concentrations above the regulatory guideline, and decreases the average aflatoxin concentration in the lots accepted while increasing the average aflatoxin concentration in the rejected lots. For a given sample acceptance level that equals the regulatory guideline, increasing the sample size decreases misclassification of lots, both false positives and false negatives.     

Testing shelled corn for aflatoxin, Part II: modeling the observed distribution of aflatoxin test results

The suitability of several theoretical distributions to predict the observed distribution of aflatoxin test results in shelled corn was investigated. Fifteen positively skewed theoretical distributions were each fitted to 18 empirical distributions of aflatoxin test results for shelled corn. The compound gamma distribution was selected to model aflatoxin test results for shelled corn. The method of moments technique was chosen to estimate the parameters of the compound gamma distribution. Mathematical expressions were developed to calculate the parameters of the compound gamma distribution for any lot aflatoxin concentration and test procedure. Observed acceptance probabilities were compared to operating characteristic curves predicted from the compound gamma distribution, and all 18 observed acceptance probabilities were found to lie within a 95% confidence band. The parameters of compound gamma were used to calculate the fraction of aflatoxin-contaminated kernels in contaminated lots. At 20 ppb, it was estimated that about 6 in 10,000 kernels are contaminated.     

Sampling, sample preparation, and analytical variability associated with testing wheat for deoxynivalenol

The variability associated with testing wheat for deoxynivalenol (DON) was measured using a 0.454 kg sample, Romer mill, 25 g comminuted subsample, and the Romer Fluoroquant analytical method. The total variability was partitioned into sampling, sample preparation, and analytical variability components. Each variance component was a function of the DON concentration and equations were developed to predict each variance component using regression techniques. The effect of sample size, subsample size, and number of aliquots on reducing the variability of the DON test procedure was also determined. For the test procedure, the coefficient of variation (CV) associated with testing wheat at 5 ppm was 13.4%. The CVs associated with sampling, sample preparation, and analysis were 6.3, 10.0, and 6.3%, respectively. For the sample variation, a 0.454 kg sample was used; for the sample preparation variation, a Romer mill and a 25 g subsample were used; for the analytical variation, the Romer Fluoroquant method was used. The CVs associated with testing wheat are relatively small compared to the CV associated with testing other commodities for other mycotoxins, such as aflatoxin in peanuts. Even when the small sample size of 0.454 kg was used, the sampling variation was not the largest source of error as found in other mycotoxin test procedures.     

Estimation of measurement uncertainty for the determination of aflatoxin M1 in milk using immunoaffinity clean-up procedures

A measurement uncertainty estimated for aflatoxin M₁ determination in milk sample has been calculated using data generated from analytical method validation studies. The protocol adopted is described in detail in document LGC/VAM/1998/088. The uncertainty budget was based on precision, trueness and ruggedness data. The individual contributions are described in detail. The expanded uncertainty for aflatoxin M ₁ at a concentration of 20 ng L⁻¹ was estimated as 2.81 ng L⁻¹. This was calculated using a coverage factor of two which gives a level of confidence of approximately 95%. Presented at AOAC Europe / Eurachem Symposium March 2005, Brussels, Belgium     

Comparative study of different digestion procedures using supplementary analytical methods for multielement-screening of more than 50 elements in sediments of the river Elbe

The suitability of four different digestion procedures, i.e. i.) an aqua regia digestion according to DIN 38 414-S7, ii.) a pressure digestion using HNO₃/HF in PTFE-vessels, iii.) a HNO₃/HF + HCl-pressure digestion in PTFE-vessels and iv.) a HNO₃/HF + HCl-pressure digestion using microwave induction, has been evaluated with regard to the quantitative determination of about 50 elements in environmental samples. Three sediments of the river Elbe and two standard reference materials (MESS-1 and NIST 1645) have been employed. The analytical results from the dissolved samples, obtained using inductively coupled plasma mass- and optical emission spectrometry as well as total reflection X-ray fluorescence spectrometry, have been compared with those obtained by instrumental neutron activation analysis. Only digestion procedures using HNO₃/HF with a subsequent evaporation to dryness and dissolution in HCl have led to appropriate results for a wide range of elements (more than 50 elements in total). Because of its low contamination risk and its time saving, the microwave digestion is preferred. For this digestion procedure the accordance among the different instrumental methods used is high (better than 15% deviation) in general. A few elements (16) could be determined quantitatively only by a single method.     

Hierarchical classification designs for the estimation of different sources of variability in proficiency testing experiments

An approach to the estimation of possible different sources of variation found in proficiency testing experiments is described. Four errors namely, technique, analyst, laboratory and geographical location are considered and calculated by using a rational experimental design based on hierarchical classification. The treatment of the confidence of the design over different experimental arrangements is explored and visualised by calculating a function that depends only on the design and not on the experimental response. An illustrative example based on simulated data is used to show how the theory could be applied in practice.     

Selenium research in mammals using nuclear analytical methods and related techniques in conjunction with biochemical procedures

Due to the essential functions of selenium-containing enzymes and the relationships between changes in the selenium status and diseases, the determination of the element and its compounds is of great interest. Radiotracer studies with ⁷⁵Se have been valuable tools in selenium research. NAA and ICP-MS allow both total element and stable isotope measurements. ICP-MS in conjunction with chromatographic separation techniques and gel electrophoretic procedures coupled with scanning methods such as XRF, PIXE and laser ablation ICP-MS have been used in the determination of the selenium compounds. In this survey the application of these methods in selenium research is discussed with the help of examples on the regulation of the selenium metabolism and the detection and investigation of novel selenium-containing proteins. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Evaluation of purification procedures of DNA from maize-meal samples by exploiting different analytical techniques for the assessment of DNA quality

Two different approaches generally applied to achieve purification of DNA extracted from cells were compared: precipitation by organic solvents and enzymatic treatments. We investigated various experimental protocols reported in literature by evaluating DNA purity, integrity and yield. Reliability of analytical techniques normally employed to assess DNA purity and quantity was studied and comments and conclusions were suggested by comparing results obtained by different analytical techniques. Enzymatic treatments prove to be unable of increasing DNA purity while determining a significant degradation. In contrast, optimised conditions for solvent precipitation enabled a sharp increase of DNA purity to be obtained, associated with the maintenance of the initial DNA integrity. The application of the optimised protocol to maize-meal samples allowed us to achieve a good PCR amplification even with those samples which gave poor amplification by following the protocol recommended by the Italian legislation in force for GMO detection in food.     

Validation study of immunoaffinity column chromatography coupled with solution fluorometry or HPLC for the detection of aflatoxin in peanuts and corn

Neogen Corp. has developed the Neocolumn for Aflatoxin DR for the detection of total aflatoxin by HPLC or solution fluorometry. The purpose of this study was to validate the method under the requirements of the AOAC Research Institute Performance Tested Methods (PTM) program. There are several AOAC Official Methods for detection of total aflatoxin in corn; they consist of rapid and analytical-based methods and two rapid methods (PTMs 030701 and 050901) that have been performance tested by the AOAC Research Institute. A widely used reference method, however, is AOAC Official Method 991.31, which uses immumoaffinity cleanup followed by HPLC or solution fluorometry and is referred to as the reference method in this document. In internal studies, the Neocolumn method coupled with solution fluorometry demonstrated a relative recovery from peanuts of 101.6% of the reference value, with a CV of 3.9% across all levels analyzed; when coupled with HPLC, the Neocolumn method demonstrated a relative recovery from peanuts of 103.0% of the reference value with a CV of 3.5% across all levels analyzed. The Neocolumn method coupled with solution fluorometry demonstrated a relative recovery from corn of 116.9% of the reference value with a CV of 6.1% across all levels analyzed; when coupled with HPLC, the Neocolumn method demonstrated a relative recovery from corn of 91.2% of the reference value, with a CV of 5.4% across all levels analyzed. Calculations were made by comparison with the mean result obtained by the HPLC reference method, which showed respective CV values of 3.9 and 2.0% for recoveries from peanuts and corn, respectively.     

Comparison of different cleanup procedures for oil crops based on the development of a trace analytical method for the determination of pyraclostrobin and epoxiconazole

The effects of different cleanup procedures in removing high‐molecular‐mass lipids and natural colorants from oil‐crop extracts, including dispersive solid‐phase extraction, low‐temperature precipitation and gel permeation chromatography, were studied. The pigment removal, lipid quantity, and matrix effects of the three cleanup methods were evaluated. Results indicated that the gel permeation chromatography method is the most effective way to compare the dispersive solid‐phase extraction and low‐temperature precipitation. Pyraclostrobin and epoxiconazole applied extensively in oil‐crop production were selected as typical pesticides to study and a trace analytical method was developed by gel permeation chromatography and ultra high performance liquid chromatography with tandem mass spectrometry. Average recoveries of the target pesticides at three levels (10, 50, and 100 μg/kg) were in the range of 74.7–96.8% with relative standard deviation values below 9.2%. The limits of detection did not exceed 0.46 μg/kg, whereas the limits of quantification were below 1.54 μg/kg and much lower than maximum residue limit in all matrices. This study may provide the essential data for optimizing the analytical method of pesticides in oil‐crop samples.     

Sampling and analytical variability associated with the determination of aflatoxins and ochratoxin A in bulk lots of powdered ginger marketed in 1-lb bags

Ginger has been used as a food, dietary supplement, and condiment for centuries. Mycotoxins such as the aflatoxins (AF) and ochratoxin A (OTA) have been reported in ginger roots in several studies. It is important to design effective sampling methods that will accurately and precisely predict the true mycotoxin level in a bulk lot. The objective of this study was to measure the sampling and analytical variability associated with the test procedure used to measure AF and OTA in a bulk lot of powdered ginger using a 5-g laboratory sample and HPLC analytical methods. Twelve 5-g laboratory samples were taken from each of two lots. Duplicate aliquots were removed from each 5-g laboratory sample/solvent blend, and each aliquot was simultaneously analyzed for AF and OTA by HPLC analytical methods. Using a balanced nested design, the total variance associated with the above AF and OTA test procedures was partitioned into sampling and analytical variance components for each lot. Averaged across both lots, the sampling and analytical variances accounted for 87% and 13% of the total variance, respectively, for AF and 97% and 3%, respectively, for OTA. The sampling and analytical coefficients of variation were 9.5% and 3.6%, respectively, for AF, and 16.6% and 2.9%, respectively, for OTA when using a single 5-g laboratory sample and HPLC analytical methods. Equations are derived to show the effect of increasing laboratory sample size and/or number of aliquots on reducing the variability of the test procedures used to estimate OTA and AF in powdered ginger.     

Quantitative approximation for the selectivity of analytical spectrophotometric procedures with systems which do not obey Beer's law

A selectivity index is proposed for defining the selectivity of a spectrophotometric procedure that is subject to interference by species which do not obey Beer's law in the system. The interactions between analyte and interferents which affect the absorbance of an analytical system are studied by means of a simple mathematical model. Theoretical expressions are derived which represent the selectivity as a function of the analyte or interfering species concentration. The treatment is illustrated by a study of the Zr(IV)-chloranilic acid system in presence of thorium as interferent.     

Development of an analytical method for yam saponins using HPLC with pulsed amperometric detection at different column temperatures

Yam saponins (dioscin, gracillin, protodioscin, and protogracillin) were analyzed with three different C18 columns at incremental column temperatures from 15 to 45°C to investigate the effect of temperature on the retention and resolution of yam saponins. At low temperature, yam saponins showed decreased retention times and improved resolutions in the C18 columns. In the Kinetex C18 column at 15°C, the four saponins achieved baseline separation (Rs > 1.5) within 30 min. Pulsed amperometric detection was used to identify saponins with high sensitivity. The limits of detection and quantification of saponins were 0.11–0.31 and 0.33–0.95 ng, respectively. The correlation coefficients ranged 0.9986–1.0000. Intra‐ and inter‐day precisions were <4.2% of retention times and <9.5% of the calculated contents. Average recoveries ranged from 92.18 to 105.98%. Saponin contents in Dioscorea nipponica tubers and commercial yam foods were determined without sample purification or concentration. Among the ten commercial yam foods investigated, only three showed significant saponin contents.     

Study of the behaviour of the absorbent blanks in analytical procedures by using the H-Point standard additions method (HPSAM)

This paper studies the behaviour of reagent blank in different extractive-colorimetric procedures (determination of sympathomimetic amines with NQS reagent) by using the H-Point Standard Additions Method (HPSAM) in order to study and characterize the different possibilities that the blank can introduce in an analytical procedure. We define two kinds of blanks: the external blank (from reagent alone solutions data) and the internal blank (from extrapolation of reagent plus analyte solutions data). Comparison between both gives the information about the reproducibility of the behaviour of the reagent blank. A procedure to evaluate, and characterize, errors (if they exist) is described, and a guide for optimizing the measuring procedure is presented.     

Preservation and analytical procedures for the analysis of chloro-s-triazines and their chlorodegradate products in drinking waters using direct injection liquid chromatography tandem mass spectrometry

A direct injection, liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the analysis of the chloro-s-triazine herbicides and their degradates in finished drinking water. The target compounds in the method were selected based on their inclusion in a common mechanism group (CMG) because of their ability to induce a similar toxic effect through a common mechanism of toxicity. The target list includes the chloro-s-triazines (atrazine, simazine, cyanazine, and propazine) and their dealkylated degradates (desethylatrazine, desisopropylatrazine, and diaminochlorotriazine). Potential matrix effects are minimized by the use of individual isotopically enriched internal standards. Analyte stability in finished chlorinated drinking water samples is ensured through careful selection of proper dechlorinating and antimicrobial reagents and through buffering sample pH. In the absence of proper dechlorination, the target analytes were found to degrade over a short period of time, even under refrigerated storage conditions. The final method has adequate sensitivity to accurately detect all target analytes at or below 0.1 microg/L and displays sufficient precision and robustness to warrant publication as EPA Method 536.     

Assessment of suitability of the florisil and CB clean-up procedures for determining aflatoxin levels in sorghum by bidirectional HPTLC

Chromatographia - The Florisil clean-up procedure developed by Kamimura et al. for determining aflatoxin levels in cereals, oilseeds and cheese has been evaluated for its suitability for...     

Development of analytical procedures for the determination of hexavalent chromium in corrosion prevention coatings used in the automotive industry

The European directive 2000/53/EC limits the use of Cr(VI) in vehicle manufacturing. Although a maximum of 2 g of Cr(VI) was authorised per vehicle for corrosion prevention coatings of key components, since July 2007 its use has been prohibited except for some particular applications. Therefore, the objective of this work was to develop direct analytical procedures for Cr(VI) determination in the different steel coatings used for screws. Instead of working directly with screws, the optimisation of the procedures was carried out with metallic plates homogeneously coated to improve the data comparability. Extraction of Cr(VI) from the metallic parts was performed by sonication. Two extraction solutions were tested: a direct water extraction solution used in standard protocols and an ammonium/ammonia buffer solution at pH 8.9. The extracts were further analysed for Cr speciation by high-performance liquid chromatography (HPLC) inductively coupled plasma (ICP) atomic emission spectrometry or HPLC ICP mass spectrometry depending on the concentration level. When possible, the coatings were also directly analysed by solid speciation techniques (X-ray photoelectron spectroscopy, XPS, and X-ray absorption near-edge structure, XANES) for validation of the results. Very good results between the different analytical approaches were obtained for the sample of coating made up of a heated paint containing Zn, Al and Cr when using the extracting buffer solution at pH 8.9. After a repeated four-step extraction procedure on the same portion test, taking into account the depth of the surface layer reached, good agreement with XPS and XANES results was obtained. In contrast, for the coatings composed of an alkaline Zn layer where Cr(VI) and Cr(III) are deposited, only the extraction procedure using water allowed the detection of Cr(VI). To elucidate the Cr(VI) reduction during extraction at pH 8.9, the reactivity of Cr(VI) towards different species of Zn generally present in the coatings (metallic Zn and zinc oxide) was studied. The results showed that metallic Zn rapidly reduces Cr(VI), whereas this reaction is less evident in the presence of zinc oxide. Water was then retained for coatings containing metallic Zn.