Preparative method for isolating alpha-zearalenol and zearalenone using extracting disk.

A liquid chromatographic method is described for the determination of zearalenol and zearalenone in corn. Zearalenol and zearalenone are extracted from corn with methanol-water (1 + 1) and cleaned up using a solid-phase extraction (SPE) disk, separated on a reversed-phase analytical column, and detected with a fluorescence detector. The SPE disk concentrated and cleanly separated zearalenol and zearalenone from sample interferences. Standard calibration curves for zearalenol and zearalenone for the concentration range 25-500 ng/mL were linear. The small extract disk had a column capacity equivalent to 1 g extracted corn. Zearalenol and zearalenone were added at levels ranging from 10 to 2000 ng/g to a control sample that contained no detectable levels of zearalenol and zearalenone. Both toxins were recovered from spiked samples at 106.3 and 103.8%, with coefficients of variation of 7.6 and 13.0%, respectively. The method has an estimated reliable limit of detection and limit of quantitation around 10 and 40 ng/g for each toxin, respectively.

Robotic automated analysis of foods for aflatoxin.

Immunoaffinity column-based sample preparation procedures for determination of aflatoxins B1, B2, G1, and G2 in several food matrixes and aflatoxin M1 in milk have been automated by using flexible automation, or robotics. Components used to assemble the system were purchased commercially or developed and built in-house. A liquid-level sensor developed in-house to assist elution of the immunoaffinity column is described. After immunoaffinity column cleanup, aflatoxins are separated by reversed-phase liquid chromatography and determined by fluorescence without derivatization. Mean recoveries of aflatoxins B1, B2, and G1 added to corn and nuts at 9-36 ng/g total aflatoxins were > 85% (coefficient of variation [CV] = 16%). Recoveries of aflatoxin G2 averaged 50% (CV = 28%). Recoveries of aflatoxin M1 added to milk at 0.12-0.50 ng/mL averaged 78% (CV = 19%). The ability of the automated system to reproduce its results is demonstrated by the fact that the CV of replicate assays is generally better than 10%. Comparability between the automated procedure and the AOAC official method is demonstrated.

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%).

Use of ten gram samples of corn for determination of mycotoxins.

Data were gathered, during a study on the development of an automated system for the extraction, cleanup, and quantitation of mycotoxins in corn, to determine if it was scientifically sound to reduce the analytical sample size. Five, 10, and 25 g test portions were analyzed and statistically compared with 50 g test portions of the same composites for aflatoxin concentration variance. Statistical tests used to determine whether the 10 and 50 g sample sizes differed significantly showed a satisfactory observed variance ratio (Fobs) of 2.03 for computations of pooled standard deviations; paired t-test values of 0.952, 1.43, and 0.224 were computed for each of the 3 study samples. The results meet acceptable limits, since each sample's t-test result is less than the published value of the /t/, which is 1.6909 for the test conditions. The null hypothesis is retained since the sample sizes do not give significantly different values for the mean analyte concentration. The percent coefficients of variation (CVs) for all samples tested were within the expected range. In addition, the variance due to sample mixing was evaluated using radioisotope-labeled materials, yielding an acceptable CV of 22.2%. The variance due to the assay procedure was also evaluated and showed an aflatoxin B, recovery of 78.9% and a CV of 11.4%. Results support the original premise that a sufficiently ground and blended sample would produce an analyte variance for a 10 g sample that was statistically comparable with that for a 50 g sample.

Thin-layer chromatographic determination of sterigmatocystin in cheese: interlaboratory study.

A collaborative study of a method for the determination of sterigmatocystin in cheese was conducted by 10 laboratories. The study included control samples and samples spiked at levels of 5, 10, and 25 ppb, in coded blind pairs. Recoveries were 60.0, 90.7, and 59.3%, outliers excluded, for the respective levels. The mean reproducibilities, outliers excluded, were 81.97, 17.13, and 52.77%, respectively. Mean repeatabilities, outliers excluded, were 77.66, 17.13, and 46.40%, respectively. Results of this collaborative study indicate that the method, modified as described in this report, is applicable to the determination of sterigmatocystin in cheese at low levels (5-50 ppb) for the purpose of surveys. With regard to the difficulty with thin-layer chromatography in this study, it is recommended that a more satisfactory determinative step be developed. Recommendation for official first action status is deferred.

The cardiolipin antigen: chemistry and composition.

Cardiolipin, the primary lipid hapten in the antigen suspension used for the detection of antitreponemal antibodies in the sera of syphilitic patients, was successfully coupled to glucose oxidase, peroxidase, and some other enzymes using different crosslinking agents. These complexes were used to replace the pure uncomplexed cardiolipin for the preparation of the antigen suspension. When these suspensions were allowed to react with serum that contained anticardiolipin antibodies the activity of the enzyme was inhibited. In the absence of antibody, no enzyme inhibition was observed.

Gas chromatographic determination of deoxynivalenol in wheat with electron capture detection: collaborative study.

Ten laboratories participated in a collaborative study of a method for the determination of deoxynivalenol in wheat by gas chromatography with electron capture detection. Each laboratory analyzed 6 samples in duplicate. Each collaborator received samples spiked at the 100.3, 501.3, and 1002.6 ng/g levels; a control sample; and 2 naturally contaminated samples. The average recovery (outliers excluded) for the spiked samples was 92.2%. The mean repeatability and reproducibility, respectively, were 32.2 and 41.3% for the spiked samples and 30.9 and 47.6% for the naturally contaminated samples. The method was adopted official first action.

Liquid chromatographic determination of ergot alkaloids in wheat.

A method is described for the determination of individual ergot alkaloids in wheat. The sample is extracted with ethyl acetate-4% ammonium hydroxide (100 + 10), and the extract is cleaned up by liquid-liquid partition. The ergot alkaloids are resolved by liquid chromatography (LC), using a porous cross-linked polystyrene-divinylbenzene resin column and a mobile phase consisting of acetonitrile-0.05 M dibasic ammonium phosphate (55 + 45) buffered at pH 10.0. The ergot alkaloids ergonovine, ergonovinine, ergotamine, ergotaminine, alpha-ergocryptine, alpha-ergocryptinine, ergocristine, and ergocristinine are separated by LC and detected with a fluorescence detector. Recovery of ergot alkaloids added to wheat at levels of 16-760 ng/g averaged 85.6% with a coefficient of variation of 11.1%.

Thin layer chromatographic determination of sterigmatocystin in cheese.

A one-dimensional thin layer chromatographic method has been developed for determining sterigmatocystin in cheese. Cheese is extracted with acetonitrile-4% KCl (85 + 15). A simplified liquid-liquid partition cleanup is used, and the sample extract is passed through a cupric carbonate column for final purification. Sterigmatocystin is visualized by spraying the plate with aluminum chloride. The fluorescence of the spot is enhanced 10-fold by additional plate spraying with a silicone-ether mixture, enabling sterigmatocystin detection and quantitation at 2 and 5 micrograms/kg, respectively. Average recoveries were 88.3 and 86.4% at the 10 and 25 micrograms/kg levels, respectively.

Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection.

A sensitive, highly selective liquid chromatographic (LC) method is described which uses electrochemical (EC) reduction of the analyte in the determinative step. The method is capable of determining xanthomegnin in mixed animal feeds and grains at levels ranging from 15 to 1200 ng/g. The method can detect as little as 0.5 ng xanthomegnin injected on the LC column. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by silica gel column chromatography using a Sep-Pak silica gel cartridge. A novel feature of the method is that xanthomegnin is "backed off" the column by reversing the flow of the eluant through the column. LC is then used to separate xanthomegnin from other interfering substances. Xanthomegnin is detected by EC reduction at -0.16 V. Recoveries of xanthomegnin added to samples at levels ranging from 15 to 1200 ng/g averaged 79% with a coefficient of variation of 7.9%. Results also demonstrate that this LC system can separate the related metabolites viomellein and rubrosulphin from each other and from xanthomegnin and that the same EC detection system can be used to detect these metabolites.

High pressure liquid chromatographic determination of xanthomegnin in grains and animal feeds.

A high pressure liquid chromatographic (HPLC) method is described for the determination of xanthomegnin in grains and mixed animal feeds at levels ranging from 150 to 1200 ng/g. This is equivalent to actual amounts of xanthomegnin injected on the HPLC system at from 15 to 120 ng/injection. Xanthomegnin is extracted with chloroform and 0.1M phosphoric acid. An aliquot of the crude extract is purified by column chromatography using a commercially available silica gel cartridge. Xanthomegnin is then separated from the remaining interferences by HPLC with a reverse phase C-8 column, and subsequently determined by absorbance detection at 405 nm. Elapsed time for the method from initial extraction to final HPLC determination is approximately 1 h. Recoveries of xanthomegnin added to grains and animal feeds at levels from 150 to 1200 ng/g averaged 82% with a coefficient of variation of 10.2%.

Fluorometric and colorimetric enzymic determination of triglycerides (triacylglycerols) in serum.

We describe two fully enzymic methods, fluorometric and colorimetric, for determination of triglycerides (triacylglycerols) in serum. Samples are incubated with microbial lipase for 10 min, and the glycerol released from the triglycerides is oxidized by NAD+ in the presence of glycerol dehydrogenase. In the fluorometric method, the resulting NADH is in turn oxidized by resazurin as catalyzed by diaphorase to form resorufin, a highly fluorescent compound. In the colorimetric method, the NADH is oxidized by coupling with a tetrazolium salt/diaphorase system to form formazan, a highly colored compound. Calibration curves, constructed by plotting change in fluorescence or absorbance vs concentration of triglycerides, were linear up to 6 and 5 g of triglycerides per liter of serum for the fluorometric and colorimetric methods, respectively. The assays require only 5 and 15 microL of serum for fluorometry and colorimetry, respectively. The CV was 0.59% for the fluorometric method, 0.91% for the colorimetric procedure. The time for analysis for either method is less than 15 min. The results correlate well with those obtained by the Dow Diagnostic Kit method, a colorimetric method in which glycerol kinase and glycerol-1-phosphate dehydrogenase form NADH from ATP and NAD+ in the presence of glycerol and glycerol 1-phosphate.

Fluorometric enzymatic determination of serum creatinine on the surface of silicone-rubber pads.

An enzymatic semi-solid surface fluorometric method is described for the determination of serum creatinine on silicone-rubber pads. In this method, the glutamate dehydrogenase, alpha-ketoglutarate, ADP and NADH are mixed, then 30 microliters of diluted serum is added. After the free ammonium ion in serum is consumed, creatininase is added to initiate the assay. The rate of disappearance of NADH fluorescence at 460 nm (excitation wavelength 340 nm) is monitored and is proportional to the serum creatinine concentration. The whole assay uses only 0.41 I.U. creatininase, and takes less than 5 min. The calibration curve is linear up to 82 mg creatinine per liter. The proposed method offers a rapid, simple and inexpensive means for creatinine assay. The results obtained correlate well with the modified Jaffe method applied on Technicon SMA 12/60, with a correlation coefficient of 0.998. The recovery averages 99.3%.

An immobilized hexokinase enzyme stirrer for a simple and economical assay of plasma glucose.

We have adapted the hexokinase glucose procedure to an immobilized enzyme stirrer for the determination of glucose concentrations in human blood plasma. The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity.

Determination of plasma glucose with use of a stirrer containing immobilized glucose dehydrogenase.

A stirrer containing immobilized glucose dehydrogenase has been successfully used for determining glucose in plasma. The device is usable for at least two months and for about 500 assays. The reaction was measured kinetically and linearity was observed to 4 g of glucose per liter. Tested with aqueous glucose and with deproteinized plasma, within-day and day-to-day precision were good. Interference and method-comparison (hexokinase method) were examined. The performance of this system makes the technique useful and attractive for routine use in small-volume clinical laboratories.