Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins

Three different methods were compared for the determination of total flatoxins in corn and peanuts naturally contaminated with aflatoxins and in corn, peanuts, cottonseed, peanut butter, and poultry feed spiked with aflatoxins B1, B2, and G1. The 3 methods were an enzyme-linked immunosorbent assay (ELISA) screening test; a monoclonal antibody-affinity column-solid-phase separation method; and the AOAC official thin-layer chromatography (TLC) methods for all except poultry feed, for which Shannon's TLC method for mixed feed was used. The ELISA test is designed to provide only positive results for total aflatoxins at greater than or equal to 20 ng/g or negative results at less than 20 ng/g. The affinity column separation is coupled with either bromination solution fluorometry to estimate total aflatoxins or liquid chromatography (LC) to quantitate individual aflatoxins. Fluorodensitometry was used to determine aflatoxins in commodities analyzed by the TLC methods. The LC and TLC results were in good agreement for all the analyses. The results for the affinity column using bromination solution fluorometry were similar except those for cottonseed, which were about 60% higher. The ELISA screening method correctly identified naturally contaminated corn and peanut positive samples. No false positives were found for controls. The correct response for spiked corn, raw peanuts, peanut butter, and cottonseed at greater than or equal to 20 ng aflatoxins/g was about 90%. The correct response for spiked poultry feed at greater than or equal to 20 ng aflatoxins/g was about 50%.     

Comparison of postcolumn derivatization-liquid chromatography with thin-layer chromatography for determination of aflatoxins in naturally contaminated corn

Quantitation of aflatoxins by liquid chromatography with postcolumn iodine derivatization (LC-PCD) and fluorescence detection was compared with quantitation by the AOAC CB method, 968.22. Thirty-seven naturally contaminated corn samples were ground and then divided. One portion was extracted, and the extract was cleaned up and analyzed by thin-layer chromatography according to the CB method. The second portion was extracted and cleaned up in a similar fashion, but quantitation was by the LC-PCD method. For aflatoxin B1 concentrations ranging from 0 to 150 ng/g, results obtained by the 2 methods were fitted to a linear equation with the LC-PCD results as the dependent variable. The correlation coefficient was 0.99, the intercept was near 0, and the slope was near 1. For aflatoxin B2, the correlation coefficient was 0.97, and the intercept was near 0. However, the slope of the equation relating LC-PCD concentration to TLC concentration was only 0.5. We believe that this lack of equivalence between the methods for determination of aflatoxin B2 is due to overestimation by the TLC method because the low levels present are near the TLC detection limit for B2.     

Liquid chromatographic determination of aflatoxins in feedstuffs containing citrus pulp

A liquid chromatographic (LC) method was developed for the determination of aflatoxins in feedstuffs containing citrus pulp. The feed-stuff sample is extracted with chloroform, followed by Sep-Pak Florisil cartridge cleanup and Sep-Pak C18 cartridge cleanup. The final eluate (water-acetone, 85 + 15, v/v) is submitted to reverse-phase liquid chromatography with water-methanol-acetonitrile (130 + 70 + 40, v/v/v) as mobile phase and postcolumn derivatization with iodine. Citrus components are removed from the extract efficiently. The limit of detection for aflatoxin B1 is less than 1 microgram/kg. Other aflatoxins can also be detected and measured. Recoveries of aflatoxins B1, B2, G1, and G2 for dairy rations spiked at 13, 5, 10, and 4 micrograms/kg were 87, 86, 81, and 82%, respectively. Corresponding coefficients of variation were 3.1, 3.6, 5.2, and 3.8%, respectively.     

High pressure liquid chromatographic determination of aflatoxins in corn.

A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.     

Beta-cyclodextrin post-column fluorescence enhancement of aflatoxins for reverse-phase liquid chromatographic determination in corn

beta-Cyclodextrin enhances the fluorescence of aflatoxins B1 and G1 in aqueous systems. This effect was utilized in developing a unique reverse-phase liquid chromatographic (LC) method for determination of aflatoxins B1, B2, G1, and G2 (B1 detection limit 1 ppb), without preparing derivatives of B1 and G1. The aflatoxins are dissolved in methanol or the mobile phase for injection onto the LC system. Using a mobile phase of methanol-beta-cyclodextrin (1 + 1), the aflatoxins are resolved on a C18 column. Fluorescence of the aflatoxins is enhanced by post-column introduction of an aqueous concentrated beta-cyclodextrin solution. All 4 aflatoxins elute within 10 min in the order G2, G1, B2, B1. Fluorescence responses for B1 and G1 standards were linear over the concentration range 0.5-10 ng, yielding correlation coefficients (r) of 0.9989 and 1.000, respectively. The average peak response ratio for G1:B1 for the mobile phase-enhancement solution described was 0.765 with a coefficient of variation (CV) of 0.98%. CVs were 6.2, 9.0, and 7.5% for multiple assays of aflatoxin B1 in 3 samples of naturally contaminated corn. For samples of corn spiked to a total B1 content of 8.3 ng/g, average B1 recovery was 90% (CV 11.7%).     

Comparative study of three different methods for the determination of aflatoxins in tahini

Aflatoxins spiked at three different levels (6.5, 13.0, and 19.5 microg/kg) in tahini, a sesame butter, were analyzed by using three different methods: high-performance liquid chromatography (HPLC), fluorometry, and enzyme-linked immunosorbent assay (ELISA). An immunoaffinity column was used for cleanup and purification of extracts prior to detection by HPLC and fluorometry. All methods were statistically evaluated for accuracy, precision, and simple correlations. Additionally, 14 tahini samples randomly obtained from Turkish retail markets were analyzed using an immunoaffinity column cleanup procedure coupled with the HPLC detection method. The fluorometric determination method involving an immunoaffinity column cleanup step was found to be highly correlated with the HPLC method (r = 0.978). Both methods were found to be effective due to their high recoveries and low variance for the prediction of total aflatoxin contamination in tahini samples. The ELISA method, due to its high variation in replicates, was found to be applicable only as a screening method. The survey study demonstrated the need for control of aflatoxin contamination of foodstuffs involving sesame seeds as an ingredient.     

Comparison of four methods of digestion for the determination of total phosphorus content in hen droppings

Abstract

The objective of this study was to compare four methods of digesting for the determination of total phosphorus (P) content in hen droppings by colorimetric spectrometry. For this comparison, we used an homogeneous and oven‐dried sample of hen droppings and made 19 analyses with each method on this same sample. Method 1 was a dry and complete digestion with addition of calcium carbonate before ashing at 550°C for 12 h. Method 2 was a dry simplified digestion without the addition of calcium carbonate. Method 3 was a wet and complete digestion with sulfuric acid and successive addition of nitric acid and heating. Method 4 was a wet simplified digestion with sulfuric and nitric acids added at once and heating for 30 min. We obtained P levels of 1.93±0.049%, 1.93±0.047%, 1.96±0.079%, and 1.98±0.143% for the four methods, respectively. There was no statistical difference among those four methods, and therefore we recommend Method 2 since it requires the use of less concentrated acids, and in addition, the standard deviation is the lowest.     

Use of the Mycosep multifunctional cleanup column for liquid chromatographic determination of aflatoxins in agricultural products.

A liquid chromatographic (LC) technique has been developed that uses the Mycosep multifunctional cleanup (MFC) column. MFC columns provide a rapid 1-step extract purification. They are designed to retain particular groups of compounds that may create interferences in analytical methods. At the same time, MFC columns allow compounds of interest to pass through. In the method presented, test samples are extracted in a blender with acetonitrile-water (9 + 1). A portion of the extract is forced through an MFC column designed especially for analysis of numerous mycotoxins. Analytical interferences are retained, while aflatoxins pass through the column. Aflatoxins B1 and G1 are converted to their hemiacetals by heating a mixture of purified extract and water-trifluoroacetic acid-acetic acid (7 + 2 + 1) at 65 degrees C for 8.5 min. An aliquot of this mixture is analyzed by isocratic LC with acetonitrile-water mobile phase and fluorescence detection. A detection limit of less than 0.5 ng/g for aflatoxin B1 was obtained. Average recoveries greater than 95% total aflatoxins (B1, B2, G1, and G2) and coefficients of variation of less than 3% were obtained. The method was successfully applied to the following commodities: corn, almonds, pista-chios, walnuts, peanuts, Brazil nuts, milo, rice, cottonseed, corn meal, corn gluten meal, fig paste, and mixed feeds.     

Comparison of three liquid chromatographic methods with FDA optimized Monier-Williams method for determination of total sulfite in foods

Three liquid chromatographic (LC) methods employing amperometric detection were compared with the collaboratively studied FDA optimized Monier-Williams distillation method for the determination of total sulfite in 5 food types. The foods included lemon juice, white wine, instant mashed potatoes, golden raisins, and onion flakes. Two of the LC methods (one employing headspace sampling and the other direct injection) used ion-exchange chromatography with a basic mobile phase (pH about 10.8) and a glassy carbon electrode; the third (employing direct injection) used ion-exclusion chromatography with an acidic mobile phase (pH about 2) and a platinum electrode. All 4 methods produced similar results for the wine, lemon juice, and raisins. Results were different for instant mashed potatoes and onion flakes. The headspace-LC method and direct ion-exclusion LC method, both of which employed an alkaline sample extraction, yielded significantly higher values for sulfite in instant potatoes than did the other 2 methods. A large interfering peak with both direct LC methods prevented quantitation of sulfite in the onion flakes. All methods can detect sulfite as low as about 1 microgram/g in 4 of 5 food types examined.     

Comparison of paired-ion liquid chromatographic method with AOAC fluorometric and microbiological methods for riboflavin determination in selected foods

A paired-ion liquid chromatographic (LC) technique coupled with fluorometric detection to determine riboflavin in various food matrices is described. Chromatograms of many foods showed 2 peaks of interest due to presence of riboflavin and flavin mononucleotide (FMN). Relatively high levels of FMN were found in raw beef, corned beef, chicken liver, and canned mushrooms. When riboflavin and FMN contents were summed, LC values were comparable to those obtained by the AOAC standard procedures. The LC technique was sensitive, rapid, and simple, yielding a mean standard deviation of 3.1% which was comparable to the AOAC fluorometric method (3.0%) and better than the AOAC microbiological assay (9.6%). Mean spike recoveries were 91.8% for LC compared to 90.5% and 89.6% for the AOAC fluorometric and microbiological methods, respectively.     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Comparison of an ELISA-based screening test with liquid chromatography for the determination of aflatoxins in corn.

An enzyme-linked immunosorbent assay (ELISA) screening test (CITE PROBE) was compared to liquid chromatography (LC) for the determination of aflatoxins in naturally contaminated corn samples. The CITE PROBE, with a positive/negative cutoff of 5 ng/g aflatoxin B1, was correct (based on LC results) on 47 of 51 samples. Two of the incorrect responses by the CITE PROBE were false positives on samples containing 4.4 ng/g and 4.1 ng/g aflatoxins by LC. Another incorrect response was a false negative on a sample containing 5.5 ng/g aflatoxins by LC. The fourth incorrect response was a false positive on a sample containing 1.9 ng/g aflatoxins by LC. On the basis of these results, the CITE PROBE was determined to be a reliable screening method for the detection of greater than or equal to 5 ng/g aflatoxins in corn.     

Comparison evaluation of liquid chromatographic and bioassay methods of analysis for determination of paralytic shellfish poisons in shellfish tissues

A liquid chromatographic (LC) method was compared with the AOAC mouse bioassay method (18.086-18.092) for determination of paralytic shellfish toxins in shellfish tissues. Shellfish samples were collected from Massachusetts coastal waters as part of a state surveillance program, and extracts of shellfish meat were analyzed for toxins by using both analytical methods. Overall correlation of the LC and bioassay methods is good (r = 0.943), but for samples with toxicities less than 100 micrograms saxitoxin/100 g shellfish meat, the correlation is significantly less (r = 0.531). Limits of detection are 10 micrograms saxitoxin/100 g shellfish meat and 40 micrograms saxitoxin/100 g shellfish meat for the LC and bioassay methods, respectively. Analytical capacity of the LC method is limited to 12 samples/person-day compared with 30 samples/person-day for the bioassay. Sampling capacity of the LC method could be increased by using a fluorescence detector with a wider response range, which would eliminate the need for dilution of concentrated samples.     

Survey of aflatoxins, ochratoxin A, zearalenone, and sterigmatocystin in some Brazilian foods by using multi-toxin thin-layer chromatographic method

A previously published method for ochratoxin A was evaluated and proved appropriate for simultaneous determination of aflatoxins, ochratoxin A, sterigmatocystin, and zearalenone, with considerable savings in time and reagent costs. The detection limits were 2, 5, 15, and 55 micrograms/kg, respectively. The recoveries and coefficients of variation obtained with artificially contaminated samples were 91-101% and 0-16% for aflatoxin B1, 98-117% and 0-17% for sterigmatocystin, and 96-107% and 0-17% for zearalenone, respectively. The coefficients of variation for naturally contaminated samples (aflatoxins in rice and ochratoxin A in beans) ranged from 0 to 8%. The method was used to survey 296 samples that included 10 cultivars of dried beans, 8 types of corn products, 3 types of cassava flour, and both polished and parboiled rice between May 1985 and June 1986 in Campinas, Brazil. Only aflatoxin B1 (9 samples, 20-52 micrograms/kg), aflatoxin G1 (4 samples, 18-31 micrograms/kg), and ochratoxin A (5 samples, 32-160 micrograms/kg) were found. The average contamination percentage was 4.7%; beans showed the highest (6.6%) and rice showed the lowest (3.3%) incidence rates. Zearalenone and sterigmatocystin were not detected. Positive samples were confirmed by chemical derivatization, corroborated by development in 3 solvent systems.     

Comparison of two ELISA screening tests with liquid chromatography for determination of aflatoxins in raw peanuts

A study was conducted to evaluate the performance of 2 enzyme-linked immunosorbent assays (ELISA) for rapidly screening samples of peanuts for the presence of aflatoxin. The EZ-Screen Quick Card Test and the Afla-10 Cup Test were compared with liquid chromatography in duplicate analyses of common extracts of peanuts contaminated in the range of 0-70 ppb (ng/g). Each assay properly identified 95% of samples containing no detectable aflatoxin as negative and greater than 97% of samples containing greater than 10 ppb aflatoxin as positive. The card test, which had a 20 ppb detection threshold, identified as positive 32 of 34 samples in the 11-20 ppb range. This indicates that the card test might actually have a detection threshold closer to 10 ppb. Most of the errors associated with the assays occurred on samples containing less than 10 ppb aflatoxin. The cup and card tests identified 76 and 67% of the samples, respectively, as negative, in the range of 4-10 ppb. For samples either negative or contaminated above their detection thresholds for the assays, the methods are well suited for use as rapid screening tests.     

Comparison of some methods for determination of sulfate in soils

Abstract

Sulfate (SO₄ ^2‐) is present in soils as salts of various metals, and the different metals associated with sulfate may influence adsorption of SO₄ ^2‐by soils. The analytical method used for determination of SO₄ ^2‐could be affected by the type of metal associated with the SO₄ ^2‐. Four analytical methods based on different principles were evaluated for determination of SO₄ ^2‐in different metal salts and in soil extracts obtained with three extractants {0.1M lithium chloride (LiCl), 0.15% calcium chloride (CaCl₂), and 500 mg P/L as calcium phosphate [Ca(H₂PO₄)2]}. The analytical methods were: (i) a methylene blue (MB) colorimetric method after the reduction of SO₄ ^2‐to hyrogen sulfide (H₂S), (ii) an ion Chromatographie (IC) method, (iii) a turbidimetric (TD) method, and (iv) an indirect barium (Ba) atomic absorption spectrophotometric (SP) method. The recovery of SO₄ ^2‐associated with various mono‐, di‐, and tri‐valent metals was quantitative by the MB method. But, trivalent metals, such as aluminum (Al), indium (In), lanthanum (La), and scandium (IC), decreased the recovery of SO₄ ^2‐by the other three methods. The MB and IC methods gave similar values for SO₄ ^2‐in soils by using the three extractants. The TD and SP methods gave variable results and, in general, underestimated the amounts of SO₄ ^2‐in soils. Among the four methods, the MB and IC methods were the most accurate and precise.     

Comparison of four methods for digesting food samples for determination of trace levels of cadmium by flameless atomic absorption spectrophotometry

We compared 4 digestion procedures, namely, sulfuric-nitric acid in an open flask, nitric acid under pressure, sulfuric-nitric acid with refluxing, and nitric-hydrochloric-peroxide with refluxing, for the determination of cadmium by flameless atomic absorption spectrophotometry in 3 foodstuffs: rice, beef, and cream cheese. The foodstuffs were homogenized and divided into several batches for analysis. The results were evaluated using a 2-way cross analysis of variance. The study revealed that the digestion procedure was a highly significant factor (P less than 10(-4] in the analysis of the 3 foods; whereas the nature of the foodstuffs was not significant for rice and meat and only slightly significant (P less than 10(-2] for cream cheese. When the foodstuffs were spiked with a known amount of cadmium, we observed a loss of the metal when the sulfuric-nitric acid procedure in an open flask and the nitric-hydrochloric-peroxide digestion procedure were used. Taken together, the results of the present study indicate that the choice of the reagents used for digestion of foodstuffs is a crucial factor for cadmium determination by flameless atomic absorption spectrophotometry.