Gas-liquid chromatographic/mass spectrometric screening method for T-2 toxin in milk

A method for the analysis of T-2 toxin in milk is presented. Ethyl acetate extracts of milk samples which had been spiked with T-2 toxin were purified by thin layer chromatography and derivatized with N,O-bis(trimethylsilyl)acetamide to produce the T-2 toxin trimethylsilyl ether (T-2 toxin-TMS). N,O-bis(trimethylsilyl-d9)acetamide was used to make T-2 toxin d9-trimethylsilyl ether (T-2 toxin-d9 TMS) which was added to the derivatized milk extract as an internal standard. Samples were analyzed by combined gas-liquid chromatography/mass spectrometry using either electron impact ionization or chemical ionization mass spectrometry. In electron impact ionization analyses, simultaneous monitoring of the T-2 toxin-TMS fragment ion at m/z 436 and the T-2 toxin-d9TMS fragment ion at m/z 445 gave a T-2 toxin-TMS detectability estimated at 6 microgram/kg. In chemical ionization analyses, the T-2 toxin-TMS fragment ion at m/z 377 and the T-2 toxin-d9TMS fragment ion at m/z 386 were simultaneously monitored to give a T-2 toxin-TMS detectability estimated at 3 microgram/kg. Average recovery was 85% at 200 microgram/kg and 65% at 20 microgram/kg.     

Determination of zearalenone in cornflakes and other corn-based foods by thin layer chromatography, high pressure liquid chromatography, and gas-liquid chromatography/high resolution mass spectrometry

A simple cleanup procedure based on pH adjustments was used to obtain extracts of corn foods. The method gave good recoveries of zearalenone determined by thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). As little as 5 ng zearalenone was detected by TLC, using Fast Violet B Salt as the spray reagent; the lower limit of detection in cornflakes was about 20 microgram/kg. With HPLC on Spherisorb silica (5 micrometer) and detection by fluorescence at an excitation maximum of 310 nm as little as 5 microgram zearalenone/kg cornflakes could be determined. While the TLC method was also applicable to corn chips, cornmeal, popcorn, and frozen corn, an interference was observed in HPLC of the latter 3 products. This interference was separated from zearalenone by adding a second HPLC analytical column (Spherisorb ODS). Gas-liquid chromatography coupled with mass spectrometric single ion monitoring at high resolution, although of limited availability, was shown to be the most sensitive and selective method for determining zearalenone in corn foods. The natural occurrence of zearalenone in a sample of cornflakes (13-20 microgram/kg) was demonstrated by all 3 detection procedures.     

Alternaria mycotoxins in weathered wheat from China

This is the first report of the natural occurrence of Alternaria mycotoxins in Chinese wheat. Wheat kernels were significantly invaded by Alternaria species, mostly A. alternata, with an average infection frequency of 87.3%. A total of 22 samples of weathered wheat kernels from the 1998 crop, representing three locations in the suburbs of Beijing, China, were examined for the presence of Alternaria mycotoxins by high-performance liquid chromatography. Alternariol (AOH) was detected in 20 of 22 samples ranging between 116 and 731 microgram/kg (mean = 335 microgram/kg) and alternariol methyl ether (AME) at a mean level of 443 microgram/kg (range = 52-1426 microgram/kg) in 21 samples. The presence of tenuazonic acid (TA), a major Alternaria toxin in terms of quantity, was detected in all samples analyzed at an average concentration of 2419 microgram/kg with a maximum of 6432 microgram/kg. All samples were free from altertoxin I and altenuene. Samples with high levels of AOH and AME also contain a high level of TA. There was significant linear regression of correlations between the levels of AOH over AME (r = 0.850) and total benzopyrone derivatives (AOH + AME) over TA (r = 0.796).     

Liquid chromatographic multicomponent method for determination of residues of ipronidazole, ronidazole, and dimetridazole and some relevant metabolites in eggs, plasma, and feces and its use in depletion studies in laying hens

A simple, rapid liquid chromatographic (LC) method that uses UV/VIS detection has been developed for the determination in eggs of residues of the histomonostats dimetridazole (DMZ), ronidazole (RON), ipronidazole (IPR), and side-chain hydroxylated metabolites of DMZ and RON. Sample pretreatment includes an aqueous extraction, purification with an Extrelut cartridge, and acid partitioning with isooctane. An aliquot of the final aqueous extract is injected into a reverse-phase LC system; detection is performed at 313 nm. The limits of determination are in the 5-10 microgram/kg range. A UV/VIS spectrum can be obtained at the 10 microgram/kg level by using diode-array UV/VIS detection. Recoveries are between 80 and 98% with a coefficient of variation of about 5%. Some 20 samples can be analyzed per day. A side-chain hydroxylated metabolite of IPR can also be detected with this method, as demonstrated with samples from animal experiments. After a single oral dose of the drugs to laying hens, residues of the parent compound and/or the hydroxylated metabolites could be detected in eggs 5-8 days after dosing. Plasma distribution and excretion in feces were established both with and without deconjugation. DMZ and IPR were extensively metabolized to hydroxylated nitroimidazole metabolites; RON was excreted mainly as the parent compound.     

Flame absorption spectroscopic determination of cadmium, copper, iron, lead, and zinc in mussels

A rapid method is proposed for determination of Cd, Cu, Fe, Pb, and Zn in mussel samples. The elements are extracted with concentrated nitric acid in borosilicate glass tubes at 90 degrees C for 1 h, and determined by flame atomic absorption spectroscopy. Detection limits for a 300 mg sample corresponded to 0.3 microgram Cd/g, 0.7 microgram Cu/g, 33 microgram Fe/g, 0.7 microgram Pb/g, and 6 micrograms Zn/g. The coefficient of variation for 20 independent analyses was 7% for Cd, 7% for Cu, 6% for Fe, 14% for Pb, and 8% for Zn. Recoveries were 107 +/- 3% for Cd, 90 +/- 3% for Cu, 94 +/- 1% for Fe, 90 +/- 5% for Pb, and 96 +/- 3% for Zn. The accuracy of the method was determined by analyzing NBS Oyster Tissues.     

Capillary column gas chromatographic determination of ethyl carbamate in alcoholic beverages with confirmation by gas chromatography/mass spectrometry

A method is described for determining ethyl carbamate at low microgram/kg levels in several types of alcoholic beverages by capillary column gas chromatography with Hall electrolytic conductivity detection and confirmation by mass spectrometry. Samples are diluted to obtain a uniform concentration of ethanol (ca 10%) then saturated with NaCl and extracted with methylene chloride. Extracts are evaporated to a small volume and injected in ethyl acetate solution for chromatographic analysis. The method was evaluated by 5 laboratories, 4 employing the Hall detector and one using mass spectrometric detection. Overall between-laboratory mean percent recoveries were: wine, 85.3 +/- 21.0% coefficient of variation (CV) (spiking level 20-45 micrograms/kg); sherry, 83.8 +/- 16.1% CV (spiking level, 81-142 micrograms/kg); whiskey, 79.5 +/- 13.9% CV (spiking level 127-190 micrograms/kg); and brandy, 85.0 +/- 12.5% CV (spiking level 297-446 micrograms/kg). Mass spectrometric results agreed well with the Hall results for all commodities. Detection limits were about 5 micrograms/kg for the Hall detector and about 0.5 microgram/kg for mass spectrometric detection.     

Lead, fluoride, and other elements in bonemeal supplements

The Pb, Cd, F, Al, Cr, Cu, Fe, Mn, Mo, Ni, Ti, and Zn content of 20 commercial bonemeal supplements was determined. Samples were mineralized with nitric and perchloric acids prior to determination of all elements except F, for which a diffusion method was used. Pb and Cd were determined by differential pulse anodic stripping voltammetry, F was measured using an ion selective electrode, and all other elements were determined by inductively coupled argon plasma spectroscopy. The mean recoveries of Pb and F were 97 and 99%, respectively. The concentration range of Pb was 1.5-8.7 microgram/g. Cd was quantitated in only one sample at a level of 2.5 microgram/g; all other samples were estimated to contain less than 0.05 microgram Cd/g. The concentration of F ranged from 261 to 921 microgram/g.     

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.     

Disappearance of organochlorine residues from abdominal and egg fats of chickens, Ontario, Canada, 1969-1982

Abdominal fats were collected from 8-week old broilers slaughtered at provincially inspected abattoirs across Ontario between 1969 and 1982. Domestically produced hen eggs were collected from either egg grading stations or producers over the same period. Composite samples were analyzed for organochlorine insecticides and industrial chemicals. Between 1979 and 1982, organophosphorus insecticides were routinely included in these analyses. Sigma DDT and PCB residues declined rapidly between 1969 and 1982 in extractable lipids of both abdominal and egg fats, while dieldrin declined less markedly over the same period. Declines in residues followed first order logarithmic regression equations. Chlordane and heptachlor epoxide were rarely observed above the detection limit of 1 microgram/kg in egg fat; however, the incidence of these residues in abdominal fat increased after 1973 following the removal of aldrin, dieldrin, and heptachlor in 1969 and the subsequent increased use of chlordane for soil insect control until 1977. Lindane residues were rarely observed above the detection limit. In 1979, when the detection limit was reduced, both alpha-BHC and lindane were identified, but at levels below 1 microgram/kg. Endosulfan, methoxychlor, and fenthion were identified on one or 2 occasions over the 13-year period.     

Rapid thin layer chromatographic determination of aflatoxin M1 in powdered milk

A method has been developed for the detection of aflatoxin M1 in milk. The toxin is extracted with chloroform, the extract is evaporated, and the residue is partitioned between carbon tetrachloride and an aqueous saline-methanol solution. The toxin is once again extracted with chloroform from the methanol solution and analyzed by thin layer chromatography. The limit of detection of M1 in powdered milk is 0.5 microgram/kg; recoveries of added M1 are about 83%. The limit of detection can be improved to 0.3 microgram/kg if the plate is sprayed with an aqueous solution of H2SO4 after development.     

Liquid chromatographic determination of chloramphenicol in calf tissues: studies of stability in muscle, kidney, and liver

A liquid chromatographic method has been developed for the measurement of chloramphenicol (CAP) in muscle, liver, and kidney. The mean recovery levels were 82.6, 75.3, and 79.2% in muscle, liver, and kidney, respectively. The method was repeatable and reproducible for CAP measurement in muscle, with a detection limit of 1 microgram/kg. Investigation of CAP stability in muscle, liver, and kidney showed that CAP stability in muscle was good at -20 degrees C; for spiked liver and kidney, degradation of CAP was observed, and the use of piperonyl butoxide (PB) for metabolism inhibition was recommended for recovery and linearity studies. However, PB was unnecessary for preservation of treated animal tissues if samples were cut into cubes and cooled at -20 degrees C, just after slaughter, pending analysis. With these limitations, CAP can be measured in liver and kidney.     

Liquid chromatographic determination of rotenone in fish, crayfish, mussels, and sediments

An analytical procedure is described for determining residues of rotenone in fish muscle, fish offal, crayfish, freshwater mussels, and bottom sediments. Tissue samples were extracted with ethyl ether and extracts were cleaned up by gel permeation chromatography and silica gel chromatography. Sediment samples were extracted with methanol, acidified, partitioned into hexane, and cleaned up on a silica gel column. Rotenone residues were quantitated by liquid chromatography, using ultraviolet (295 nm) detection. Recoveries from sediment samples fortified with rotenone at 0.3 microgram/g were 80.8%, whereas recoveries from tissue samples fortified with 0.1 microgram/g ranged from 87.7 to 96.8%. Samples fortified with 0.3 microgram/g and stored at -10 degrees C for 6 months before analysis had recoveries ranging from 83.2 to 90.5%. Limits of detection were 0.025 microgram/g for sediments and 0.005 microgram/g for tissue samples.     

Liquid chromatographic determination of chloramphenicol residues in meat: interlaboratory study

A liquid chromatographic (LC) method for the determination of chloramphenicol (CAP) residues in meat at the 10 microgram/kg level was tested in an interlaboratory study. The method used, based on aqueous extraction and sample cleanup with a cartridge containing Extrelut, was published earlier. A prestudy to familiarize collaborators with the method was performed before the actual interlaboratory precision study. The meat samples used in the precision study were prepared by diluting dosed chicken and pig muscle tissues with blank tissues from other species. Fourteen laboratories received 20 meat samples; 13 laboratories actually participated in the study. Two blank samples and 2 positive samples each of pig, calf, chicken, lamb, and cow meat were tested. The chloramphenicol concentrations in the positive samples ranged from 6.5 to 21 micrograms/kg. The overall mean reproducibility coefficient of variation was 17.9% after the results per laboratory were corrected for the mean recovery obtained within each sample series. The overall mean recovery was 55.1% with a coefficient of variation of 18.0% at the 10 micrograms/kg level. The limit of detection, based on chromatograms of blank samples, was estimated to be 1.5 micrograms/kg of chloramphenicol. No false positives or false negatives were observed in the concentration range tested; only 2 false positive results above the detection limit (1.7 and 6 micrograms/kg) on a total number of 60 blank analyses (3.3%) were observed.     

Determination of carbadox-related residues in swine liver by gas chromatography/mass spectrometry with ion trap detection

The ion trap detector (ITD), in combination with a capillary gas chromatograph and under chemical ionization conditions, offers sufficient sensitivity to determine carbadox-related residues as the methyl ester derivative of quinoxaline-2-carboxylic acid at 3 micrograms/kg or higher in porcine liver. A tetradeuterated internal standard of QME effectively compensates for losses incurred during sample preparation. The method produced mean levels of 3.3 (+/- 0.5), 5.5 (+/- 0.8), and 10.1 (+/- 0.9) micrograms/kg for liver fortified at 3, 5, and 10 micrograms/kg. When applied to analysis of samples containing incurred residues of 14C-carbadox at the low microgram/kg level, results were comparable to those obtained by reverse isotope dilution analysis.     

Simultaneous determination of carbaryl, malathion, fenitrothion, and diazinon residues in sesame seeds (Sesamum indicum L.)

A method is described for the simultaneous determination of carbaryl (1-naphthyl methylcarbamate), malathion [diethyl (dimethoxythiophosphorylthio) succinate], fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate), and diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate) in sesame (Sesamum indicum L.) seeds. Sesame seeds were Soxhlet extracted with n-hexane, and the extract was subjected to a liquid-liquid partitioning and column cleanup to remove the oily coextractives prior to analysis by high performance liquid chromatography (HPLC). The mean percent recoveries (+/- standard deviations) from sesame seeds fortified with carbaryl (0.004 to 0.035 microgram/g), malathion (0.53 to 4.25 microgram/g), fenitrothion (0.22 to 1.78 microgram/g), and diazinon (0.54 to 4.35 microgram/g) were 83.3 +/- 5.7, 85.5 +/- 6.6, 85. 6 +/- 7.2, and 88.4 +/- 4.8, respectively. The method was used for the simultaneous analysis of carbaryl, malathion, fenitrothion, and diazinon residues in sesame seeds obtained from an Ethiopian field crop that had been treated with the pesticides during its growing period.     

Micellar electrokinetic capillary electrophoresis for rapid analysis of patulin in apple cider

A micellar electrokinetic capillary chromatography (MECC) mode was applied to a capillary electrophoresis (CE) method, which was developed for detection and quantitation of patulin in apple ciders. This method used a small sample amount (2 mL) and consumed minimal organic solvent compared to the most commonly used HPLC methods. The sample preparation procedure of the CE method was also simpler than other chromatographic techniques developed for patulin analysis. Patulin was detected with a photodiode array detector at 273 nm. The standard curve was linear (r(2) = 0.9984) from 75 microgram/L to 121 microgram/mL with patulin working solutions corresponding to 3.8 microgram/L to 6.1 microgram/mL patulin in the sample. The linearity was better in a narrower range of concentrations (r(2) = 0.9999) from 75 microgram/L to 24.1 microgram/mL. The limit of detection of the method was 3.8 microgram/L. Patulin recoveries at 4 levels in spiked samples (10-121 microgram/L) ranged from 95.2 to 105.4%. The recoveries were 96. 9% and 99.2% for 2 levels (22.3 and 223 microgram/L, respectively) of patulin in infected apple samples. This method represents a unique alternative method for rapid and sensitive analysis of patulin in apple ciders.     

Simultaneous extraction and fractionation and thin layer chromatographic determination of 14 mycotoxins in grains.

A simple, systematic analytical method for multiple mycotoxins was developed for detecting 14 mycotoxins; aflatoxins B1, B2, G1, and G2, sterigmatocystin, T-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon X, zearalenone, ochratoxin A, citrinin, luteoskyrin, and rugulosin. These mycotoxins were extracted with 20% H2SO4-4% KCl-acetonitrile (2 + 20 + 178), defatted with isooctane, and transferred to chloroform. The chloroform extract was cleaned up by silica gel column chromatography; the first 10 toxins were eluted with chloroform-methanol (97 + 3) and the remaining 4 toxins with benzene-acetone-acetic acid (75 + 20 + 5). Each fraction was analyzed by thin layer chromatography for the final determination. The method has been applied to polished rice, rough rice, corn, wheat, and peanuts as an analytical screening procedure. The detection limits in these commodities ranged from 10.00 to 800.0 microgram/kg, depending on the mycotoxin, but all limits were superior to those obtained for the individual mycotoxins by using other methods.     

Loss of pendimethalin in runoff and leaching from turfgrass land under simulated rainfall

A field study was undertaken to investigate runoff and leaching loss of the herbicide pendimethalin in turfgrass land of loamy sand soil. A series of plots constructed in a golf course fairway were surface-applied with pendimethalin SC formulation at the rate of 2. 25 or 4.50 kg a.i./ha and subjected to simulated rainfall at 2.0 cm/day for 10 consecutive days. Runoff losses of pendimethalin were the highest at the first rainfall and then gradually decreased with time. The first runoff event contained pendimethalin in its highest concentration, and in subsequent runoff samples the concentration decreased exponentially. The ranges of pendimethalin concentration were 80.9-18.2 and 177.4-48.6 microgram/L in the standard and double doses, respectively. Total losses by 20 cm of rainfall for 10 days reached 0.81 and 1.22% of the initial deposits at 2.25 and 4.50 kg a. i./ha, respectively. Pendimethalin concentration in the leachate collected at 30-cm soil depth was quite lower than that in the runoff, and the concentration rapidly decreased from 4.3-4.7 to 0. 2-0.4 microgram/L during the 10 days of rainfall treatment. Soil residue analysis at 45 and 90 days after pendimethalin treatment showed that more than 90% of the residue remained at the top 10 cm of soil depth. Low runoff and leaching confirmed that lateral and downward movement of the herbicide should be limited in turf soil. The half-life of pendimethalin under field conditions was 23-30 days and was not affected by application dose and rainfall treatment, but longer persistence was observed under laboratory conditions. Considering low runoff and leaching, as well as relatively short persistence in soil, it is concluded that little environmental carryover of pendimethalin would be expected in turfgrass land.     

Microbiological determination of penicillin G, ampicillin, and cloxacillin residues in milk

A fast cylinder plate microbiological method was developed for the quantitative determination of penicillin G, ampicillin, and cloxacillin in milk. Agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis were used and incubated at 64 degrees C for 4 1/2 hr. Standard curves were obtained for the following ranges of concentration of antibiotics: 0.004-0.064 IU penicillin G/mL, 0.0025-0.04 microgram ampicillin/mL, and 0.03-0.48 microgram cloxacillin/mL. The method is suitable for detecting penicillin residues in milk and for quantitative milk-out studies of the above antibiotics used in treatment of bovine mastitis.     

北方麦区土壤有效磷阈值及小麦产量、籽粒氮磷钾含量对监控施肥的响应

  【目的】  分析我国北方麦区不同土壤有效磷水平下,监控施肥后小麦籽粒产量与养分吸收利用变化,为保证减施磷肥后小麦的丰产、优质、绿色生产提供理论依据。  【方法】  于2018—2020年在我国北方麦区49个地点进行了田间试验。所有试验均设农户施肥(FF)、监控施肥(RF)和监控无磷(RF-P) 3个处理,监控施肥的磷(P₂O₅)肥用量较农户施肥平均减少60 kg/hm2,相当于减少了46%。在小麦成熟期调查了土壤不同磷素水平下,小麦产量、产量构成、籽粒氮磷钾含量,并计算了磷素养分吸收利用率;在小麦收获期,采样测定土壤有效氮磷钾含量。  【结果】  当土壤有效磷<15 mg/kg时,小麦产量最低,为5155 kg/hm2;当土壤有效磷在25～30 mg/kg时,产量达到最高,为7217 kg/hm2;有效磷过高并不能持续提高小麦产量,反而因穗数、千粒重低导致产量降低。土壤有效磷<15、15～20、20～25、25～30和>30 mg/kg时,监控施肥处理小麦产量与农户施肥处理相比差异虽然未达显著水平,但小麦的磷肥吸收效率与磷肥偏生产力平均分别为1.03和104.7 kg/kg,分别较农户处理显著提高了119.6%和112.2%,籽粒氮磷钾含量与农户施肥处理相比无显著差异。当土壤有效磷<15 mg/kg,或速效钾达171和200 mg/kg、有效磷为15～20和>30 mg/kg时,不施磷肥小麦显著减产;但土壤速效钾为147和158 mg/kg、有效磷在20～25和25～30 mg/kg时,不施磷肥不减产。土壤有效磷含量越高,小麦籽粒平均氮含量越低、磷含量越高,籽粒平均钾含量在有效磷为20～25 mg/kg时达到最高。  【结论】  在北方麦区,过高的土壤有效磷含量有降低小麦氮素营养的风险,适当降低磷肥用量在保证产量的同时,还可大幅提高磷肥的利用率。土壤有效磷维持在20～30 mg/kg时,减施或不施磷肥依然可以实现小麦高产,但若速效钾>170 mg/kg时不施磷肥小麦有减产风险。因此,应基于对小麦目标产量、籽粒养分含量和土壤有效磷钾的监控,确定合理的磷肥用量,实现北方麦区化肥减施,小麦稳产提质增效和绿色生产。