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.     

Thin layer chromatographic determination of deoxynivalenol in processed grain products

The thin layer chromatographic (TLC) method of Trucksess et al. (J. Assoc. Off. Anal. Chem. (1984) 67, 40-43) was modified for the determination of deoxynivalenol (DON) in high-sugar breakfast cereals, corn syrup, and beer. Celite was added to the substrate before extraction with acetonitrile-water (84 + 16). After filtration through an alumina-charcoal-Celite (0.5 + 0.7 + 0.3) column, the filtrate was evaporated to dryness and redissolved in water, which was passed through an octylsilyl reverse phase column. DON was eluted with anhydrous ethyl ether. The residue remaining after the eluate was evaporated to dryness was dissolved in CHCl3-acetonitrile (4 + 1) and chromatographed on AlCl3-impregnated silica gel TLC plates. The blue fluorescent DON spot was quantitated fluorodensitometrically after the TLC plate was heated at 120 degrees C for 7 min. Recoveries of DON added to breakfast cereals at 100, 200, and 400 ng/g levels and to syrup and beer at 50, 100, and 200 ng/g levels averaged 86%. The limit of determination in these products was about 50 ng/g.     

Thin layer chromatographic determination of aflatoxin B1 in eggs: collaborative study

The thin layer chromatographic method of Trucksess et al. for aflatoxin B1 in eggs was collaboratively studied. Each collaborator analyzed 3 known practice samples and 9 unknown samples containing added aflatoxin B1 at 0, 0.05, 0.10, and 0.30 ng/g. For 9 collaborators, recoveries for the 3 positive levels were: 0--0.13 ng/g (average 98%, coefficient of variation (C.V.) 83%), 0.05--0.18 ng/g (average 102%, C.V. 36%, and 0.11--0.42 ng/g (average 93%, C.V. 31%), respectively. The method has been adopted as official first action.     

Thin layer chromatographic method for the detection of uric acid: collaborative study

A collaborative study has been completed on an improved method for the detection and confirmation of uric acid from bird and insect excreta. The proposed method involves the lithium carbonate solubilization of the suspect excreta material, followed by butanol-methanol-water-acetic acid thin layer chromatography, and trisodium phosphate-phosphotungstic acid color development. The collaborative tests resulted in 100% detection of uric acid standard at the 50 ng level and 75% detection at the 20-25 ng level. No false positives were reported during tests of compounds similar to uric acid. The proposed method has been adopted official first action; the present official final action method, 44.161, will be retained for screening purposes.     

Gas chromatographic determination of deoxynivalenol in wheat

Modifications to a published method are described for the determination of deoxynivalenol (DON) in wheat by gas chromatography with electron capture quantitation of the heptafluorobutyrate derivative. In the modified method, DON is extracted by shaking the sample with methanol-water on a wrist-action shaker, followed by filtration through rapid flow paper. One concentration step is eliminated, and a hexane wash is incorporated to remove toluene from the silica gel column. Recoveries of DON from wheat samples spiked at 0.1, 0.5, and 1.0 ppm ranged from 77.3 to 86.3% and averaged 81.5%.     

Liquid chromatographic method for determination of azinphos-methyl in formulated products: collaborative study

A liquid chromatographic method for determination of azinphosmethyl (Guthion) in formulated products has been developed. Samples are dissolved in acetonitrile and analyzed by reverse-phase chromatography using n-butyrophenone as an internal standard. The method was subjected to a collaborative study involving 15 participating laboratories. Each collaborator was furnished with reference standard, internal standard, and blind duplicate samples of Guthion 50% wettable powder (50WP), 3 flowable (3F), and emulsifiable concentrate (2L and 2S) formulations. Collaborators were instructed to evaluate the method by peak height measurements only. Relative standard deviations for reproducibility (RSDR) were 1.11, 6.28, 2.47, and 1.17% for the 50WP, 3F, 2L, and 2S formulations, respectively. The method has been approved interim official first action for determination of azinphos-methyl in the 50WP, 2L, and 2S formulations.     

Thin layer chromatographic determination of citrinin.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.     

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.     

Headspace gas chromatographic method for determination of ethanol in canned salmon: collaborative study

Six laboratories collaboratively studied a headspace gas chromatographic method for determination of ethanol in the aqueous phase of canned salmon. Ethanol is determined by a headspace sampling technique with tert-butanol as the internal standard, using a gas chromatograph equipped with a Super Q column and a flame ionization detector. With outliers excluded, the mean recoveries from samples spiked with 25.1 and 78.4 ppm ethanol were 112 and 110%, respectively. For the 4 sample pairs quantitated, repeatability coefficients of variation ranged from 1.42 to 4.25% and reproducibility coefficients of variation from 2.55 to 8.09%, with 3 of the 4 reported values less than 5%. The method has been adopted official first action.     

Improved spray reagent for thin layer chromatographic method for detecting uric acid: collaborative study

A collaborative study was conducted to validate the substitution of an improved single spray in the official AOAC thin layer chromatographic method for identifying uric acid (UA) from bird and insect excreta. The proposed reagent, which is a dilute aqueous solution of ferric chloride and potassium ferricyanide, requires neither a heating step nor a pH indicator. Its preparation time, specificity, and sensitivity to low levels (5-50 ng) of UA were compared with those of the official sprays. The improved spray took 1/5 as long to prepare as the official sprays. Neither the proposed spray nor the official sprays gave false positive reactions with compounds similar to UA. For bird and insect excreta samples, at the 95% confidence limits, the false negative rate was between 0 and 9.7% for the proposed spray and between 0.7 and 18.7% for the official sprays. Sensitivity results showed that the proportion positive for the proposed spray was significantly higher (P less than 0.05) than for the official sprays at the 15 ng UA level. The proposed changes have been adopted official first action.     

IUPAC gas chromatographic method for determination of fatty acid composition: collaborative study.

An international collaborative study of IUPAC methods II.D.19 and II.D.25 for preparation and GLC analysis of fatty acid methyl esters was begun in 1976. The IUPAC methodology, applicable to animal and vegetable oils and fats and fatty acids from all sources, contains special instructions for preparation and analysis of methyl esters of fatty acids containing 4 or more carbon atoms (analysis of milk fat). Twenty-three collaborators participated in the analysis of 5 known mixtures, 4 vegetable oils, 1 fish oil, and 2 butterfats. Several blind duplicate samples were included. The experimental data were subjected to statistical analysis to examine intra- and interlaboratory variation. Reproducibility and accuracy data for the higher fatty acid (14:0-22:1) mixtures and fish and vegetable oils were satisfactory and were in good agreement with results from an AOCS Smalley Committee check sample program involving analysis of the same samples. Typical coefficients of variation (%) at various concentrations were 15 (2% level), 8.5 (5% level), 7 (10% level), and 3 (50% level). Low recoveries and poor reproducibility were characteristics of results obtained for butyric acid in the butterfat and related known mixtures. A coefficient of variation of about 19% was found for analysis of butyric acid in butterfat, vs coefficients of variation in the range of 4-13% for similar levels of other components in butterfat and other samples. The IUPAC methodology for GLC analysis of fats and oils other than milk fats has been adopted by the AOAC as official first action to replace the current GLC method, 28.063-28.067.     

Liquid chromatographic method for determination of furazolidone in premixes and complete feeds: collaborative study

A liquid chromatographic (LC) method for determining furazolidone in finished feeds and premixes was collaboratively studied. Finished feed sample is extracted with acetone-water (93 + 7) on a Goldfisch apparatus, extracting solvent is removed, and the residual material is dissolved in warm DMF. A solution of tetraethylammonium bromide is added, the fat layer is removed, and the sample is clarified by filtration and injected onto a reverse phase LC system with detection at 365 nm. Premixes, extracted by shaking with DMF and diluted so that the final furazolidone concentration is about 55 micrograms/mL, are chromatographed and detected the same as finished feed samples, using a mobile phase of acetonitrile-2% acetic acid (20 + 80). Ten commercial feed samples were preweighed and supplied to 14 collaborators. The 5 matched pairs were chosen to represent the following allowed levels: 0.0055, 0.022, 0.033, 2.2, and 22%. Two familiarization samples at the 0.0055 and 11% levels were also supplied. Instructions called for a single analysis of each sample. Two results were eliminated by the Dixon test. The coefficients of variation, following treatment by the ranking test, ranged from 2.0 at the 22% level to 6.5 at the 0.0055% level. Calculated F-values are not significant (P greater than 0.01) except for the 0.0055% level samples extracted overnight. This method has been adopted official first action.     

Thin layer chromatographic determination of aflatoxin in corn dust

Methods adopted by the AOAC and the American Association of Cereal Chemists for determining aflatoxin in corn were modified, and techniques were developed for application to samples of less than 1 to 10 g instead of the specified 50 g samples. Analysis included chloroform extraction of dust samples or dust collected from glass fiber filters, purification of extracts on a silica gel column of appropriate size, and measurement of aflatoxin by either 1- or 2-dimensional thin layer chromatography (TLC). The solvent for 1-dimensional TLC was chloroform-acetone-water (91 + 9 + 1). Solvents for 2-dimensional TLC were, first direction, ether-methanol-water (95 + 4 + 1, lined tank) and second direction, chloroform-acetone-water (91 + 9 + 1, unlined tank), or first direction, chloroform-acetone-water (91 + 9 + 1, unlined tank) and second direction, toluene-ethyl acetate-formic acid (60 + 30 + 10, unlined tank). When samples weighed less than or equal to 0.1 g, the entire concentrated extract was applied to the TLC plate. About 0.5-1.0 ng aflatoxin B1 could be detected on the plate, making the limit of detection about 9 ng/g for 0.1 g samples.     

Liquid chromatographic method for multiresidue determination of benzimidazoles in beef liver and muscle: collaborative study

A liquid chromatographic method for determination of thiabendazole, 5-hydroxythiabendazole, oxfendazole, mebendazole (MBZ), and fenbendazole (FBZ) in cattle liver and muscle was collaboratively studied in 7 laboratories in 1986. For blind fortified samples containing 800 ppb FBZ, average recovery and relative standard deviations for repeatability and reproducibility (RSDr and RSDR) based on results from 6 of the participating laboratories were 83%, 12.7%, and 14.0%, respectively. Recoveries of FBZ from incurred liver samples were more variable. Recoveries of MBZ from livers fortified at the 100 ppb level were encouraging; however, the drug levels were too low in the incurred samples used for MBZ studies. Except for FBZ and MBZ in liver, the study data were not satisfactory. The method has been adopted official first action by AOAC for determination of 800-1600 ppb fenbendazole in liver. The analysis should be repeated using a smaller sample size when initial analyses show levels greater than 1600 ppb FBZ.     

Liquid chromatographic determination of flucytosine in capsules: collaborative study

A liquid chromatographic method for the determination of flucytosine in capsules was collaboratively studied by 7 laboratories. The method uses a C18 reverse phase column, water-methanol-acetic acid mobile phase containing 1-octanesulfonic acid sodium salt, p-aminobenzoic acid as internal standard, and photometric detection at 285 nm. The mean recovery value (+/- SD) of flucytosine from a synthetic formulation representing capsules was 99.2 +/- 1.72% (CV = 1.73%). Composited samples of 250 and 500 mg commercial capsules gave assay values of (mean +/- SD) 103.17 +/- 2.21 and 99.29 +/- 1.29% of declared, respectively. CV values were 2.15 and 1.30%. Reproducibility and repeatability CVs were 2.19 and 1.50%, respectively, for the 250 mg capsules, and 1.34 and 0.63%, respectively, for the 500 mg capsules. The method has been adopted official first action.     

Liquid chromatographic determination of colchicine in pharmaceuticals: collaborative study

A liquid chromatographic (LC) method for the determination of colchicine in pharmaceutical dosage forms and the bulk drug was evaluated in an interlaboratory study which included 13 participating laboratories. The method involves extraction (or dissolution) of the active ingredient with methanol-water (1 + 1), followed by filtration of the extract and reverse phase LC using an octylsilane bonded phase column and UV detection at 254 nm. The mobile phase consists of a methanol-phosphate buffer mixture (pH = 5.5). Three commercial tablet formulations (0.5-0.6 mg colchicine/tablet), 1 synthetic injectable preparation (0.510 mg colchicine/mL), and 1 bulk drug sample were assayed in duplicate by the proposed method. The reproducibility and repeatability standard deviations based on nonpooled results for each sample ranged from 0.0062 mg/mL to 0.0147 mg/tablet and from 0.0037 mg/mL to 0.0127 mg/tablet, respectively; the corresponding coefficients of variation ranged from 1.21 to 2.54% and from 0.73 to 2.19%, respectively. The mean recovery from the synthetic injectable formulation was 100.0%. The method has been adopted official first action.     

Liquid chromatographic determination of diazepam in tablets: collaborative study

A stability indicating liquid chromatographic method for the determination of diazepam in tablets was collaboratively studied by 6 laboratories. The method uses a C18 reverse phase column, a methanol-water mobile phase, p-tolualdehyde as the internal standard, and photometric detection at 254 nm. The collaborators were supplied with a synthetic tablet powder and 3 commercial tablet samples. The mean recovery of diazepam from the synthetic tablet powder was 100.2%. For all samples analyzed, the coefficient of variation was less than 1.5%. The method has been adopted official first action.