Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk.

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha-ergocryptine, and ergocristine are extracted from wheat with methanol-0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 microg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovine and 20-400 ng/g for ergotamine, ergocornine, alpha-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of <5 and <20 ng/g, respectively, for each ergot alkaloid tested.

Determination of vitamin K1 in medical foods by liquid chromatography with postcolumn reduction and fluorometric detection.

A liquid chromatographic (LC) method is described for the determination of vitamin K1 in medical foods. The sample is enzymatically digested with lipase and alpha-amylase and extracted with 1% sodium bicarbonate solution-isopropanol (1 + 1). After C18 solid-phase extraction, vitamin K1 is separated by nonaqueous reversed-phase LC, converted to the hydroquinone by postcolumn zinc reduction, and quantitated by fluorescence detection. The limit of detection is 8 pg (3 sigma), and the limit of quantitation is 27 pg (10 sigma) on column. Linear response ranged from 0.1 to 1.0 ng vitamin K1 (r= 0.9999). The mean recovery (n = 38) for all spiking levels was 101.6 +/- 2.85%. Analysis of Standard Reference Material 1846, Infant Formula, gave a mean value of 0.95 +/- 0.088 mg vitamin K/kg (K or K1?) (n = 31) with a coefficient of variation of 9.26.

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.

Liquid chromatographic determination of zearalenone and zearalenol in animal feeds and grains, using fluorescence detection.

A liquid chromatographic (LC) method was developed for the determination of zearalenone and zearalenol in grains and mixed animal feeds. Samples are extracted with chloroform and purified by a base-acid liquid-liquid partition. Zearalenone and zearalenol are separated by reverse phase LC and determined by fluorescence detection, excitation wavelength 236 nm with a 418 nm cutoff filter. The method was applied to the determination of zearalenone and zearalenol in 395 survey samples of corn, oats, barley, sorghum, silage, and finished feeds. The limit of detection is 10 ng/g for both toxins. The range of naturally occurring toxins found was 10-4,000 ng/g. Average recoveries were 84% for zearlenone and 69% for zearalenol. Coefficients of variation were 24.6% for zearalenone and 30.8% for zearalenol for crop year 1980, and 28.3% for zearalenone and 22.0% for zearalenol for crop year 1981.

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.

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

A method is described for the determination of deoxynivalenol (DON) in wheat. The method involves sample extraction with chloroform-ethanol (8 + 2), column chromatographic cleanup on silica gel of small particle size, and derivatization with heptafluorobutyric acid anhydride using 4-dimethylaminopyridine as a catalyst. The derivative DON tris-heptafluorobutyrate is determined by gas chromatography using an electron capture detector. Recoveries of DON added to wheat at levels of 118-1184 ng/g averaged 88% with a coefficient of variation of 8.6%.

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%.

Determination of roquefortine in blue cheese and blue cheese dressing by high pressure liquid chromatography with ultraviolet and electrochemical detectors.

A method is described for the determination of roquefortine in blue cheese and blue cheese dressing. The method involves sample extraction with ethyl acetate, cleanup by liquid-liquid partition, and determination by high pressure liquid chromatography with ultraviolet and electrochemical detectors connected in series. Recoveries of roquefortine added to cheese at levels of from 16 to 320 ng/g averaged 74.9%. This method was applied to the analysis of 12 samples of blue cheese and 2 samples of blue cheese dressing, all of which were produced in the United States; roquefortine was found in all of the samples at average levels of 424 ng/g for the blue cheese and 45 ng/g for the blue cheese dressing.

Determination of zearalenone in corn by high pressure liquid chromatography and fluorescence detection.

A method is reported for determining zearalenone in corn at levels as low as 10 ng/g. Samples are extracted with chloroform-water and cleaned up by liquid-liquid chromatography, and the zearalenone is detected by a fluorescence detector after separation by reverse phase high pressure liquid chromatography (HPLC). Recoveries of zearalenone added to corn at levels from 10 to 200 ng/g averaged greater than 89%. In addition, a confirmation procedure is described which involves sequential HPLC analysis of the sample and a zearalenone standard, using 4 different excitation wavelengths and comparing fluorescence responses obtained. This method was successfully applied to the analysis of 11 samples of cornmeal; zearalenone was detected in 9 of the samples at levels from 11 to 69 ng/g.

High-pressure liquid chromatographic method for the determination of patulin in apple butter.

Patulin is extracted from apple butter samples with ethyl acetate and the extract is cleaned up on a silica gel column, using benzene-ethyl acetate (75+25) as the eluant. High-pressure liquid chromatography, using a 25 cm ZorbaxSil column, isooctane-ethyl ether-acetic acid (750+250+0.5) as the mobile solvent, and a 254 nm ultraviolet detector, is used for the determinative step. Under these conditions, patulin is eluted before 5-hydroxymethylfurfural, a component of apple butter which interferes with other liquid chromatographic and thin layer chromatographic methods. Recoveries of patulin added at levels of 34.6, 138.4, and 276.8 mug/kg ranged from 89.0 to 112.1%.