Collaborative study of a rapid method for determing aflatoxins in cottonseed products.

Eleven laboratories collaboratively studied a modification of the official final action AOAC method, 26.048-26.056, for determining aflatoxins in cottonseed products. An aflatoxin-negative meal, 6 contaminated meals, 4 contaminated meats (kernels) samples, and 2 ammonia-inactivated meals were used. Mean aflatoxin values, mug/kg, ranged from 6 to 223 (B1), 2 to 44 (B2), and 7 to 266 (total: B1 + B2). Only one laboratory reported a false-positive for the negative meal. The mean coefficients of variation for B1, B2, and total were 28, 56, and 29%, respectively, for meals; 35, 54, and 37%, respectively, for meats; and 35, 58 and 38%, respectively, for ammoniated meals. Statistical treatment of data from triplicate sets of meals and meats showed evidence for systematic error between laboratories. Since the modified method is considerably faster than the official method and yields precision estimates consistent with previous AOAC collaborative studies on determining aflatoxins, the method has been adopted as official first action.     

Rapid screening method for aflatoxins and zearalenone in corn.

The methanol-water extraction system used in AOAC Method II for aflatoxins extracts both the aflatoxins and zearalenone from corn. Using this methanol-water extraction system as a base, a rapid screening procedure has been developed for these mycotoxins. The methanol-water extract is defatted with hexane and the pigments are precipitated with copper carbonate. The aflatoxins and zearalenone are subsequently extracted into chloroform and are then detected by half-plate TLC. An elapsed time of about 1 hr is required to analyze 1 sample. The sensitivity of the method is about 2 mu-g/kg for aflatoxin B-1 and 100 mu-g/kg for zearalenone.     

A quantitative method for the determination of cyclopropenoid fatty acids in cottonseed, cottonseed meal, and cottonseed oil (Gossypium hirsutum) by high-performance liquid chromatography

Cyclopropenoid fatty acids (CPFAs), found in cottonseed, have been shown to have detrimental health effects to susceptible livestock. Previous quantitative analytical methods for the determination of CPFAs expressed these acids in terms of their relative abundance with respect to other fatty acids in the oil, necessitating the concurrent analysis of other fatty acids. The proposed analytical method describes the quantitation of three relevant CPFAs for cotton (malvalic acid, sterculic acid, and dihydrosterculic acid) in cottonseed in micrograms per gram fresh weight of sample. The method involves extraction of the oil, saponification, and derivatization of the free fatty acids with 2-bromoacetophenone to give the phenacyl esters. These esters are then separated by dual-column reverse-phase high-performance liquid chromatography and quantitated via external standards. This is the first method to include external calibration standards for CPFAs and, as such, is capable of direct quantification with no further data conversion required. CPFA data generated from the analysis of cottonseed, cottonseed meal, and cottonseed oil produced in the United States in 2002 are presented.     

Monoclonal antibodies for the analysis of gossypol in cottonseed products

Immunogens, prepared by conjugating either (+)-gossypol or (-)-gossypol to Limulus polyphemushemolymph protein, were used for immunization in the production of monoclonal antibodies. Hybridoma were evaluated for their relative affinity to racemic gossypol, (+)-gossypol, (-)-gossypol, gossypol analogues, and their lysine derivatives. The monoclonal antibody obtained showed higher affinity to gossypol and gossypol analogues as compared to their lysine derivative counterparts. An indirect competitive enzyme-linked immunosorbent assay (ELISA) with this antibody was used to measure gossypol in 15 cottonseed meal products; the results showed good correlation with results obtained using the AOCS (free gossypol) official method (R(2) = 0.89). The direct recognition of both free gossypol and bound gossypol using this antibody will be useful for rapid screening and quality control.     

Screening method for the detection of aflatoxins in mixed feeds and other agricultural commodities with subsequent confirmation and quantitative measurement of aflatoxins in positive samples.

The method described will detect total aflatoxins (B1, B2, G1, and G2) in mixed feeds, grains nuts, and fruit products in samples containing as little as 5-15 mug/kg. In addition, the presence of aflatoxins in the positive samples can be confirmed and the toxins can be quantitatively measured, using the same extract as that used for the screening method. In the screening method, aflatoxins are extracted with acetone-water (85+15), and interferences are removed by adding cupric carbonate and ferric chloride gel. The aflatoxins are extracted from the aqueous phase with chloroform and the chloroform extract is washed with a basic aqueous solution. A Velasco-type minicolumn is used to further purify the extract and capture the aflatoxins in a tight band. The screening method has been successfully applied to 24 different agricultural commodities. Quantitative thin layer chromatography was also performed with extracts of each of these commodities. An average recovery of 94% B1, 108% B2, 130% G1, and 103% G2 was obtained compared to the official final action AOAC method for cottonseed products, 26.048-26.056. Within-laboratory coefficients of variation of 10-15% were obtained for each of the aflatoxins and total aflatoxins in a sample of peanut meal naturally contaminated with 11 mug B1+3 mug B2+11 mug G1+5 mug G2/kg.     

Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC

A chemical cleanup procedure for low-level quantitative determination of aflatoxins in major economically important agricultural commodities using HPLC has been developed. Aflatoxins were extracted from a ground sample with MeOH/H2O (80:20, v/v), and after a cleanup step on a minicolumn packed with Florisil, aflatoxins were quantified by HPLC equipped with a C18 column, a photochemical reactor, and a fluorescence detector. Water/MeOH (63:37, v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5, 1.7, 5, and 1.7 ng/g were 89.5+/-2.2, 94.7+/-2.5, 90.4+/-1.0, and 98.2+/-1.1, respectively (mean+/-SD, %, n=3). Similar recoveries, precision, and accuracy were achieved for corn, brown and white rice, cottonseed, almonds, Brazil nuts, pistachios, walnuts, and hazelnuts. The quantitation limits for aflatoxins in peanuts were 50 pg/g for aflatoxin B1 and 17 pg/g for aflatoxin B2. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

Rapid quantitative method for simultaneous determination of benzoic acid, sorbic acid, and four parabens in meat and nonmeat products by liquid chromatography

A liquid chromatographic (LC) method for the simultaneous determinations of benzoic acid, sorbic acid, and methyl, ethyl, propyl, and butyl parabens (methyl, ethyl, propyl, and butyl-p-hydroxybenzoates) in meat and nonmeat products was developed. Benzoic acid, sorbic acid, and parabens were extracted from meat and nonmeat products with 70% ethanol. After filtration, extracts were analyzed by reverse phase liquid chromatography. Homogeneously ground samples of fresh sausage and hamburger were fortified with benzoic acid, sorbic acid, and each paraben at 5 different concentrations. Average recovery (after discarding outliers) for each preservative at all 5 levels was greater than 95% with a coefficient of variation less than 5%.     

Improved method for confirmation of identity of aflatoxins B1 and M1 in dairy products and animal tissue extracts

A method is described for confirming the identity of aflatoxins B1 and M1 in dairy products and liver extracts on a thin layer plate. Extracts and standards containing aflatoxins B1 and M1 are spotted on 10 x 10 cm plates, which are developed 2-dimensionally in mixtures of isopropanol-acetone-chloroform. After the first development, trifluoroacetic acid-hexane (1 + 4) is sprayed on that part of the plate containing the separated extract components and the underdeveloped standard spots of B1 and M1, and the plate is heated 6-8 min at 75 degrees C. Then the plate is developed in a second direction, and the reaction products of B1 and M1 with trifluoroacetic acid from the extract are compared with the same derivatives of the respective standards. The method has been used successfully on extracts of milk, cheese, and liver containing 0.1 ng B1 or M1/g and can be completed in 35-45 min.     

Rapid gas chromatographic method for determining ethyl carbamate in alcoholic beverages with thermal energy analyzer detection

A rapid column elution method has been developed for the determination of ethyl carbamate (EC) in alcoholic beverages. The beverage is mixed with Celite and packed in a column containing deactivated alumina capped with a layer of sodium sulfate. EC is then eluted with methylene chloride. The method, using a gas chromatograph-thermal energy analyzer with a nitrogen converter for detection and quantitation of EC, has been applied to a variety of alcoholic beverages. Recoveries +/- standard deviations of EC in wine and whisky fortified at the 20 and 133 micrograms/kg (ppb) levels averaged 87.3 +/- 5.3 and 88.7 +/- 3.6%, respectively. The method has a limit of detection of 1.5 ppb. Gas chromatography/mass spectrometry/mass spectrometry was used to confirm the identity and quantitation of EC in selected beverage extracts.     

Collaborative study of a screening method for the detection of aflatoxins in mixed feeds, other agricultural products, and foods.

A screening method for aflatoxins was collaboratively tested on 11 different agricultural and food products: white and yellow corn, peanuts, peanut butter, pistachio nuts, peanut meal, cottonseed meal, chicken, pig, and turkey starter rations, and dairy cattle feed. The method involves a rapid extraction and cleanup procedure followed by the detection of total aflatoxins (B1 + B2 + G1 + G2) as a fluorescent band on the Florisil layer of a Velasco-type minicolumn. The results of 32 collaborators from 10 different countries are presented. Samples containing 0, 5, 10, 15, 20, and 25 mug aflatoxins/kg were analyzed. Eighty-four per cent of the negative samples and 89% of the samples containing 10-25 mug total aflatoxins/kg were correctly identified. This method has been adopted as official first action for the detection of aflatoxins in corn, peanuts, peanut butter, peanut meal, cottonseed meal, mixed feeds, and pistachio nuts.     

Re-examination of chromogenic quantitative assays for determining flavonoid content

Flavonoids in plants have gained worldwide attention because of their benefits for human health. This study compared three analytical procedures commonly used for determining flavonoid content in plant samples in terms of chromogenic relationships and the reaction products of different flavonoid structures by means of using flavonoid standards with flavone, flavonol, flavanone, flavanol, and isoflavone and analytes such as phenolic acids commonly found in plant extracts. Procedure A produced a stable color reaction between 3-hydroxy-4-keto-flavonoids (flavonols) and 5-hydroxyflavones and was highly sensitive. Procedure B produced color reactions among most of the flavonoids, but the reaction products had different colors and faded over time. Procedure B also produced a color reaction with caffeic and chlorogenic acid. Procedure C was the most sensitive. It produced a color reaction and, like procedure A, could be used to quantify flavonols and 5-hydroxyflavones, but also showed color reaction toward caffeic and chlorogenic acid. On the basis of the results, the current three procedures are not satisfactory for determining all of the types of flavonoid. Two issues needed to be clarified before a promising determination of flavonoid content could be performed with chromogenic assays. The first is a survey of the literature to screen the possible predominant component of flavonoid in analytes. The other is guided by the predominant flavonoid; a promising calibration curve for flavonoid detection can be established on the basis of the selection of an appropriate method and a chemical standard with an equivalent dose response to the predominant flavonoid.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

Indirect spectrophotometric method for determining epicillin

A new UV spectrophotometric assay for determination of epicillin in capsules and plasma is reported. The method is based on the absorptivity of a degradation product obtained from the acidic hydrolysis of the antibiotic. The isolation, identification, individual capsule assays, composite assay, and recovery studies are described. Also, the proposed method is compared with a dc polarographic assay in plasma.