The development of automated methods for the determination of trace concentrations of carbomate pesticides in water using solid sorbent pre-concentration methods and high performance liquid chromatography /

Several automated reversed-phase HPLC methods have been developed to determine trace concentrations of carbamate pesticides (which are of concern in Ontario environmental samples) in water by utilizing two solid sorbent extraction techniques. One of the methods is known as on-line pre-concentration'. This technique involves passing 100 milliliters of sample water through a 3 cm pre-column, packed with 5 micron ODS sorbent, at flow rates varying from 5-10 mUmin. By the use of a valve apparatus, the HPLC system is then switched to a gradient mobile phase program consisting of acetonitrile and water. The analytes, Propoxur, Carbofuran, Carbaryl, Propham, Captan, Chloropropham, Barban, and Butylate, which are pre-concentrated on the pre-column, are eluted and separated on a 25 cm C-8 analytical column and determined by UV absorption at 220 nm. The total analytical time is 60 minutes, and the pre-column can be used repeatedly for the analysis of as many as thirty samples. The method is highly sensitive as 100 percent of the analytes present in the sample can be injected into the HPLC. No breakthrough of any of the analytes was observed and the minimum detectable concentrations range from 10 to 480 ng/L. The developed method is totally automated for the analysis of one sample. When the above mobile phase is modified with a buffer solution, Aminocarb, Benomyl, and its degradation product, MBC, can also be detected along with the above pesticides with baseline resolution for all of the analytes. The method can also be easily modified to determine Benomyl and MBC both as solute and as particulate matter. By using a commercially available solid phase extraction cartridge, in lieu of a pre-column, for the extraction and concentration of analytes, a completely automated method has been developed with the aid of the Waters Millilab Workstation. Sample water is loaded at 10 mL/min through a cartridge and the concentrated analytes are eluted from the sorbent with acetonitrile. The resulting eluate is blown-down under nitrogen, made up to volume with water, and injected into the HPLC. The total analytical time is 90 minutes. Fifty percent of the analytes present in the sample can be injected into the HPLC, and recoveries for the above eight pesticides ranged from 84 to 93 percent. The minimum detectable concentrations range from 20 to 960 ng/L. The developed method is totally automated for the analysis of up to thirty consecutive samples. The method has proven to be applicable to both purer water samples as well as untreated lake water samples.

An HPLC method for simultaneous determination of residual benomyl and MBC on apple foliage without cleanup

A simple High Performance Liquid Chromatograph (HPLC) method has been developed to identify benamyl (methyl 1- (butylcarbamoyl)-2-benzimidazole carbamate) and MBC (methyl 2-benzimidazole carbamat~ residues on apple leaves without cleanup. Sample leaves are freeze dried in a Mason jar and residues are then extracted by tumbling them in chloroform containing 5,000 microgram per milliliter of n-propyl isocyanate (PIC) at 10 C. To the extract, n-butyl isocyanate (BIC) was added at 5,000 microgram per milliliter and 20 microliter of this mixture injected onto the HPLC system. Separation is accomplished by the use of a Brownlee LiChrosorb silica gel column with a guard column and' operated with a mixed mobile phase consisting of chloroform and hexane (4:1) saturated with water. MBC, a degradation product of benomyl is identified if present as methyl l-(npropyl carbamoyl)-2-benzimidazole carbamate (MBC-n-PIC). Both benomyl and MBC-n-PIC can be detected with aKUltraviolet (UV) detector (280nm) at a concentration as low as 0.2 microgram per milliliter in apple leaves. The fate of benomyl on apple foliage after spray application of benomyl (Ben late 50 per cent wettable powder) was investigated by the method thus described. Benomyl quickly dissipated during the first 3-7 days, but the dissipatio'n sltowed down thereafter. In contrast, the concentration of MBC in leaves gradually increased after repeated applications of Benlate.

Studies on oxamyl : analytical method development and investigation of fate in peach seedlings and corn seeds

A high performance liquid chromatographic method employing two columns connected in series and separated~y·a.switching valve has been developed for the analysis of the insecticide/ nematicide oxamyl (methyl-N' ,N'-dimethyl-N-[(methylcarbamoyl) oxy]-l-thiooxarnimidate) and two of its metabolites. A variation of this method involving two reverse phase columns was employed to monitor the persistence and translocation of oxamyl in treated peach seedlings. It was possible to simultaneously analyse for oxamyl and its corresponding oxime (methyl-N',N'-dimethyl-N-hydroxy-l-thiooxamimidate}, a major metabolite of oxamyl in plants, without prior cleanup of the samples. The method allowed detection of 0.058 pg oxamyl and 0.035 p.g oxime. On treated peach leaves oxamyl was found to dissipate rapidly during the first two-week period, followed by a period of slow decomposition. Movement of oxamyl or its oxime did not occur in detectable quantities to untreated leaves or to the root or soil. A second variation of the method which employed a size exclusion column as·the first column and a reverse phase column as the second was used to monitor the degradation of oxamyl in treated, planted corn seeds and was suitable for simultaneous analysis of oxamyl, its oxime and dimethylcyanoformamide (DMCF), a metabolite of oxamyl. The method allowed detection of 0.02 pg oxamyl, 0.02 p.g oxime and 0.005 pg DMCF. Oxamyl was found to persist for a period of 5 - 6 weeks, which is long enough to permit oxamyl seedtreatment to be considered as a potential means of protecting young corn plants from nematode attack. Decomposition was found to be more rapid in unsterilized soil than in sterililized soil. DMCF was found to have a nematostatic effect at high concentrations ( 2,OOOpprn), but at lower concentrations no effect on nematode mobility was observed. Oxamyl, on the other hand, was found to reduce the mobility of nematodes at concentrations down to 4 ppm.

Studies on the insecticide - nematicide-oxamyl and its quantitative determination by gas chromatographic method

Ox amyl , an insecticide/nematicide with the chemical name; methyl ~'. ~·-dimethyl-~-(methylcarbamoyl)oxy-l-thiooxamimidate, and its major degradation compound; oxime or oximino compound, methyl ~',~'-dimethyl-~-hydroxy-l-thiooxamimidate were studied in this work. NMR and mass spectrometry were utilized in the structural studies. An attempt was made to explain the fragmentation patterns of some major peaks in the mass spectra of oxamyl and oxime. A new gas chromatographic method for the detection and determination of submicrogram levels of intact oxamyl using a electron-capture detector was developed. The principle of this method is to produce a derivative which is highly sensitive to an electron-capture detector. The derivative described is dinitrophenyl methylamine( DNPMA ) • Experimental conditions such as pH , reaction temperature , reaction time, the amount of reagent ( Dinitrofluaro benzene) etc. were thoroughly investigated and optimized. This method was successfully applied to the determination of oxamyl residues in tobacco leaves and soil. Throughout this J9D:oject , thin layer chromatography was also used in the separation:and clean up of oxamyl and oxime samples.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

The identification, distribution and persistence of oxamyl and its degradation products in planted corn seed, seedling root and soil from oxamyl-treated corn seeds

A simple method was developed for treating corn seeds with oxamyl. It involved soaking the seeds to ensure oxamyl uptake, centrifugation to draw off excess solution, and drying under a stream of air to prevent the formation of fungus. The seeds were found to have an even distribution of oxamyl. Seeds remained fungus-free even 12 months after treatment. The highest nonphytotoxic treatment level was obtained by using a 4.00 mg/mL oxamyl solution. Extraction methods for the determination of oxamyl (methyl-N'N'-dimethyl-N-[(methylcarbamoyl)oxy]-l-thiooxamimidate), its oxime (methyl-N',N'-dimethyl-N-hydroxy-1-thiooxamimidate), and DMCF (N,N-dimethyl-1-cyanoformanade) in seed" root, and soil were developed. Seeds were processed by homogenizing, then shaking in methanol. Significantly more oxamyl was extracted from hydrated seeds as opposed to dry seeds. Soils were extracted by tumbling in methanol; recoveries range~ from 86 - 87% for oxamyl. Root was extracted to 93% efficiency for oxamyl by homogenizing the tissue in methanol. NucharAttaclay column cleanup afforded suitable extracts for analysis by RP-HPLC on a C18 column and UV detection at 254 nm. In the degradation study, oxamyl was found to dissipate from the seed down into the soil. It was also detected in the root. Oxime was detected in both the seed and soil, but not in the root. DMCF was detected in small amounts only in the seed.