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%.     

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.     

Quantitative fluorodensitometric determination and survey of aflatoxins in nutmeg.

A study is presented for the quantitative fluorodensitometric analysis of aflatoxins in spices, in particular nutmeg (Semen myristicae). Samples were extracted with chloroform, followed by silica gel column cleanup according to the AOAC officail first action method, 26.019(a), and by 2-dimensional thin layer chromatography according to the antidiagonal technique. The method includes a confirmatory test for aflatoxins by hemiacetal formation. The concentrations of aflatoxins in samples were determined by measurement of the fluorescent intensities of the separated aflatoxin spots from sample and standards on the same chromato-plate with a reflectance flying-spot sensitometer. With such a technique, a coefficient of variation value of 5.22 plus or minus 1.24% (P = 99%) was calculated for a series of 5 standard B-1 spots and averaged for 13 TLC plates, demonstrating the precision of the chromatographic and densitometric procedures. An average recovery of 108.4 plus or minus 5.8% (P = 95%) was obtained for 11 spiked nutmeg extracts (5.0-20.0 mu-g B-1 added/kg), whereas an average recovery of 92.6 plus or minus 4.9 (P = 95%) was established for 13 spiked nutmeg samples (5.0-20.0 mu-g B-1 added/kg). The coefficient of variation of the complete analytical procedure for ground nutmeg was 8.80%. In a survey on the occurrence of aflatoxins in 40 commercial nutmeg samples (covering 12 different brands) in The Netherlands, aflatoxins were detected in 30 ground samples (32 ground samples analyzed) in concentrations ranging from 1.0 to 23.2 mu-g B-1/kg or 2.7 to 36.5 mu-g B-1 + B-2 + G-1 + G-2/kg, whereas no aflatoxins were present in whole nutmeg kernels (8 samples analyzed). The lowest level of detection was 1.0 mu-g B-1/kg. In addition, 50 commercial spices consisting of 19 different types of commodities other than nutmeg wer assayed for aflatoxins according to the same procedure. No aflatoxins were detected in these samples, with the exception of 1 sample of bay leaf which contained 5.1 mu-g B-1/kg.     

Some features of stelar tissues isolated from corn roots

In relation to radial ion transport to xylem vessels in higher plant roots, some features of stele, cortex, and root tip freshly isolated from 2-day-old corn roots grown under moistened or submerged condition were investigated. The contents of P, K, and Mg in primary roots decreased gradually from the apical to the basa1 region axially. The stele contained P, Na, Cu, Fe, and Mn in higher level than the cortex. The O₂ uptake rate of root tip was much higher and that of freshly isolated stele was as high as that of fresh cortex. In stele, more CN sensitive respiration was observed than in cortex. Although isolated stele absorbed tess ³²p than cortex and root tip, the incorporation of ³²p into organic compounds in stele was as much as in root tip. These results show that the freshly isolated stele has high metabolic activity and must play an inherent role in radial and selective ion transport in situ.     

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.     

Survey for aflatoxins and zearalenone in 1973 crop corn stored on farms and in country elevators.

A total of 315 marketable and 57 obviously damaged corn samples were collected at 116 different farms and country elevators located in the United States in countries selected from among those producing more than 1 million bushels of corn in 1972. The samples were analyzed for aflatoxins and zearalenone. The most striking correlations observed were between geographical area and mycotoxin contamination. Aflatoxin contamination was most frequently encountered in the Southeast-Appalachia areas with a 44% incidence of marketable corn with detectable aflatoxins. Zearalenone was most frequently encountered in the Corn Belt with 10% incidence in marketable corn from that region. When mycotoxin contamination was found in an establishment, most of the samples from that establishment were contaminated. There was no correlation between mycotoxin contamination and storage practices nor could the observed contamination of marketable corn be related to the contamination of the obviously damaged grain. These observations plus correlations with the geographic incidence and aflatoxin level distribution of published field contamination data suggest the possibility of a common contamination mode.     

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%).     

Liquid chromatographic determination of sulfamoyldapsone in swine tissues

A liquid chromatographic (LC) method is described for the quantitative determination of sulfamoyldapsone (2-sulfamoyl-4,4'-diaminodiphenyl sulfone) in swine muscle, liver, kidney, and fat. Sulfamoyldapsone was extracted from tissues with acetonitrile saturated with n-hexane. The extract was washed with n-hexane saturated with acetonitrile, concentrated, and cleaned up by alumina column chromatography. Sulfamoyldapsone was separated on an ODS column by using acetonitrile-methanol-water (6 + 18 + 76) and was detected at 292 nm. Overall average recovery of sulfamoyldapsone added to tissues at levels of 0.1 and 0.5 microgram/g was 93.3% +/- 6.0. Detection limit was 0.02 microgram/g in these tissues.     

Comparison of isothiocyanate yield from wasabi rhizome tissues grown in soil or water

The isothiocyanate (ITC) yield of wasabi, the Japanese horseradish (Wasabi japonica), was measured on its release from glucosinolates in the rhizomes of plants grown in two traditional ways. Mature plants of 18 months old were harvested from two different commercial farms located in the South Island of New Zealand. At one farm, the plants were grown in raised soil beds, while the plants at the other farm were grown in gravel irrigated by river water. Following harvest, the rhizomes from each growth medium were divided into five size groups based on the weight and length of the rhizomes. The different sized rhizomes were also subdivided into proximal, medial, and distal portions of the rhizomes and each portion was further subdivided into epidermis plus cortex, and vascular plus pith. The individual and total ITC contents of each portion (proximal, medial, and distal) of the rhizomes were measured using dichloromethane extraction followed by the GC-FPD. The total ITC content of the rhizomes grown in soil increased (13 times) linearly from 6 to 114 g of rhizome weight, while the mean ITC content of the water-grown wasabi increased (10 times) nonlinearly for similar sized rhizomes. Water-grown rhizomes in the weight range from 18 to 45 g gave significantly (P = 0.030) higher total ITC (1-2 times) than similarly sized soil-grown rhizomes. Analysis of the tissues showed that the total and the individual ITCs were found in significantly higher levels (73 and 64%, respectively) in the skin and cortex tissue compared to the vascular and pith tissues. Analysis of the ITC content of the different locations of the wasabi rhizome showed that the distal portion of the rhizome contained significantly higher levels of both total and individual ITCs compared to the medial and proximal portions of the rhizome.     

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.     

Limited survey of residual tetracyclines in tissues collected from diseased animals in Aichi Prefecture, Japan.

Tissues were collected to survey the actual conditions of tetracycline antibiotics (TCs) residues in slaughtered animals that did not pass inspection at slaughterhouses in Aichi Prefecture, Japan, because of the presence of disease symptoms. Tissues were analyzed by liquid chromatography. Among 271 samples, 49 (18.1%) were positive for oxytetracycline (OTC), 5 (1.8%) for chlortetracycline (CTC), and 5 (1.8%) for doxycycline (DC), respectively. One sample (cattle kidney) was positive for both OTC and DC. However, tetracycline was not detected in any samples. Percentage frequencies of TCs residues were 29.1% (37/127) and 15.2% (22/144) for cattle and hogs, respectively. Kidney samples showed higher incidence of TCs residues and 1.5-7 times higher residual concentrations than liver and miscellaneous samples.     

Iron efficiency of different corn hybrids grown in nutrient solution culture

Growing Fe-efficient genotype(s) could be considered as a preferred genetic approach to tackle the widespread constraint of Fe-deficiency-/lime-induced chlorosis in crop grown on alkaline soil. This study aimed to investigate morphological and physiological traits linked to expression of Fe deficiency among four corn (Zea mays) including sweet (Z. mays sacchrata cvs. H403 and H404) and grain (Z. mays indentata cvs. H500 and H700) hybrids grown in nutrient solution using two Fe concentrations (5 and 50 µM Fe-ethylenediaminetetraacetic acid (Fe-EDTA)). Significant variation was found among studied hybrids in their tolerance to Fe-deficiency stress. Sweet corn hybrids were more sensitive to Fe deficiency as compared with grain corn hybrids and greater reduction was observed in their shoot dry matter at the 5 µM Fe-EDTA treatment. The greatest decrease in plant height, leaf area, and root and shoot dry matter weight under Fe-deficiency condition was found for H403 hybrid. No significant correlation was found between shoot and root Fe concentration with crop tolerance to Fe deficiency. Furthermore, different response of corn hybrids to Fe deficiency is an important factor, which has to be considered in Fe fertilizer recommendation as well as breeding programs.     

Stereochemical composition of clenbuterol residues in edible tissues of swine

A gas chromatography-mass spectrometry method was developed to measure the stereochemical residues of clenbuterol derivatives in edible tissues of swine. Clenbuterol present in tissue extracts was derivatized with phosgene to form clenbuterol oxazolidin-3-one, which was then separated into component enantiomers using a dimethyl beta-cyclodextrin capillary gas chromatographic column. Purified clenbuterol stereoisomers, isolated using published liquid chromatographic techniques, were used to determine stereoisomer elution order, stereoisomer racemization potential, and accuracy of the method. The stereochemical composition of clenbuterol could be measured at tissue concentrations of <2 ppb using the method. The dextrorotatory stereoisomer was the predominant clenbuterol stereoisomer present in edible tissues of hogs slaughtered after withdrawal periods of 0, 3, and 7 days, with a (+)/(-) isomer ratio of about 3:1. The prevalence of the dextrorotatory stereoisomer in edible tissues of hogs at all withdrawal periods suggests that stereoselective processes are occurring during the absorption, distribution, metabolism, and (or) excretion of clenbuterol. The effect of clenbuterol dose on its stereochemical composition in edible tissues is unknown but will be an area of further investigation.     

Gas chromatographic determination of incurred dimetridazole residues in swine tissues

A gas chromatographic method for determination of 2-hydroxymethyl-1-methyl-5-nitroimidazole (DMZOH), the hydroxy metabolite of dimetridazole, in swime muscle has been developed. The method uses cleanup steps similar to those of an earlier polarographic method. The present method is capable of quantitating levels as low as 2 ppb and detecting less than 1 ppb. Recoveries from 30 control tissues spiked at 1, 2, or 4 ppb averaged 80.4%. Performance of the method in incurred tissue was documented and limited data on the depletion of the metabolite in muscle were generated. The muscle of swine given 150 ppm dimetridazole in feed for 14 days contained less than 1 ppb DMZOH at 12 h withdrawal time.     

Determination of olaquindox residues in swine tissues by liquid chromatography

A liquid chromatographic (LC) method is described for determination of olaquindox residues in swine tissues. The drug is extracted from tissues with acetonitrile, and the extract is evaporated to dryness. This residue is cleaned up by alumina column chromatography. LC analysis is carried out on a Nucleosil C18 column, and olaquindox is quantitated by ultraviolet detection at 350 nm. The average recoveries of olaquindox added to tissues at levels of 0.2, 0.1, and 0.05 ppm were 74.0, 68.6, and 66.3%, respectively. The detection limit was 2 ng for olaquindox standard and 0.02 ppm in tissues.     

Residue depletion of cefquinome in swine tissues after intramuscular administration

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed for the detection of cefquinome (CEQ) residues in swine tissues. The limit of detection (LOD) of the method was 5 ng g(-1) for muscle and 10 ng g(-1) for fat, liver, and kidney. Mean recoveries of CEQ in all fortified samples at a concentration range of 20-500 ng g(-1) were 80.5-86.0% with coefficient of variation (CV) below 10.3%. Residue depletion study of CEQ in swine was conducted after five intramuscular injections at a dose of 2 mg kg(-1) of body weight with 24 h intervals. CEQ residue concentrations were detected in muscle, fat, liver, and kidney using the HPLC-UV method at 265 nm. The highest CEQ concentration was measured in kidney tissue during the study period, indicating that kidney was the target tissue for CEQ. CEQ concentrations in all examined tissues were below the accepted maximum residue limit (MRL) recommended by the Committee for Veterinary Medical Products of European Medical Evaluation Agency (EMEA) at 3 days post-treatment.     

Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin

Aflatoxicol (AFL) and aflatoxins B1 and M1 were found in tissues (kidney, liver, and muscle) of feeder pigs given an estimated LD50 oral dose of B1 (1.0 mg/kg body weight) provided as a rice culture of Aspergillus flavus and of market-weight pigs fed a naturally contaminated feed, containing aflatoxin B1 at a level of 400 ng/g from corn, for 14 days. The residues in all tissues decreased with time after treatment in both groups, with no detectable residues (approximate detection limits, ng/g, B1 0.03, M1 0.05, AFL 0.01) in pig tissues from the feeding experiment 24 h after withdrawal of aflatoxin-contaminated feed. B1 and M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the dominant aflatoxin. The level of AFL, when detected, was about 10% of the B1 level.