Practical immunochemical method for determination of 3,5, 6-trichloro-2-pyridinol in human urine: applications and considerations for exposure assessment.

An analytical method is described for the quantitative determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in human urine. This is the primary analyte found in urine as a result of exposure to chlorpyrifos, chlorpyrifos-methyl, triclopyr, or 3,5,6-TCP. Conjugates of 3,5,6-TCP are released from urine by acid hydrolysis. The free 3,5,6-TCP is purified using C(18) solid-phase extraction, eluting the analyte with 1-chlorobutane. An aliquot of 1-chlorobutane is placed in a vial containing Trichloropyridinol Sample Diluent and evaporated, leaving the 3,5,6-TCP in the aqueous sample diluent. The samples are assayed using the Trichloropyridinol RaPID Assay immunoassay test kit. Final results are calculated using a standard curve constructed by linear regression after a ln/Logit data transformation is performed of the concentration and the absorbance readings, respectively. The calculated lower limit of quantitation for 3,5,6-TCP in fortified control urine samples is 2. 96 ng/mL (2.96 ppb). Residues of 3,5,6-TCP determined using both immunochemical and gas chromatography with mass spectrometric detection correlate well.

Determination of spinosad and its metabolites in food and environmental matrices. 3. Immunoassay methods.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and is effective on several classes of insects, especially Lepidopteran larvae. Spinosad is registered in many countries for use on a variety of crops, including cotton, corn, soybeans, fruits, and vegetables. Residue methods utilizing a magnetic particle-based immunoassay (IA) test kit have been developed and validated for determining spinosad in environmental and food matrices. These methods involve an extraction of the residues from the matrices with appropriate solvents. For some matrices, the sample extracts can be diluted and measured directly by IA without any cleanup. For other matrices, sample extracts are purified using liquid-liquid partitioning and/or solid phase extraction prior to measurement by IA. The methods determine the total residue of spinosad, which includes the active ingredients (spinosyns A and D) and several minor metabolites, including spinosyn B, spinosyn K, and N-demethylspinosyn D. The methods have validated limits of quantitation of 0.0001 microgram/mL in water, 0.05 microgram/g in sediment, and 0.010 microgram/g in crops, crop processed commodities, and animal tissues. This paper briefly summarizes the residue methodology and method validation data for spinosad in 34 food, feed, and environmental matrices.

Plant biomass partitioning and chemical defense: Response to defoliation and nitrate limitation.

Plant growth and allocation to root, shoot and carbon-based leaf chemical defense were measured in response to defoliation and nitrate limitation inHeterotheca subaxillaris. Field and greenhouse experiments demonstrated that, following defoliation, increased allocation to the shoot results in an equal root/shoot ratio between moderately defoliated (9% shoot mass removed) and non-defoliated plants. High defoliation (28% shoot mass or >25% leaf area removed) resulted in greater proportional shoot growth, reducing the root/shot ratio relative to moderate or non-defoliated plants. However, this latter effect was dependent on nutritional status. Despite the change in distribution of biomass, defoliation and nitrate limitation slowed the growth and development ofH. subaxillaris. Chronic defoliation decreased the growth of nitrate-rich plants more than that of nitrate-limited plants. The concentration of leaf mono- and sesqui-terpenes increased with nitrate-limitation and increasing defoliation. Nutrient stress resulting from reduced allocation to root growth with defoliation may explain the greater allocation to carbon-based leaf defenses, as well as the defoliation-related greater growth reduction of nitrate-rich plants.

Changes in leaf mono- and sesquiterpene metabolism with nitrate availability and leaf age inHeterotheca subaxillaris.

The concentration of leaf mono- and sesquiterpenes is greater in nitrate-limited than in nitrate-richHeterotheca subaxillaris plants and is highest in young leaves and declines with leaf age. To determine whether rates of volatile terpene synthesis and/or loss vary with nitrate availability and leaf age, incorporation of(14)C from photosynthetically fixed(14)CO2 and the subsequent loss of label was measured in plants grown under nitrate-limited and nitrate-rich conditions.(14)C incorporation into mono- and sesquiterpenes was greater in nitrate-limited than in nitrate-rich plants and was highest in young leaves and declined with leaf age. Incorporation continued for several days after exposure, while loss of label was slow until leaves were 4-6 weeks old. These results suggest that the higher leaf volatile terpene content observed under nitrate limitation apparently results from increased synthesis per leaf and accumulation of mono- and sesquiterpenes in immature leaves of nitratepoor plants. Furthermore, volatile terpene synthesis is highest in young leaves, declines with leaf age, and is very low in older leaves. Carbon used for synthesis of this pool may be derived from both current photosynthesis as well as carbon transported to young leaves from older leaves. These data are consistent with hypotheses that predict that greater levels of carbon-based chemical defenses occur in plants under nutrient limitation. The apparent low metabolic cost of maintenance (i.e., slow turnover) of the accumulated terpenoid pool would limit the energetic cost of volatile terpenes as a chemical defense.

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.

Inhibition of feeding by a generalist insect due to increased volatile leaf terpenes under nitrate-limiting conditions.

Nitrogen-limited plants ofHeterotheca subaxillaris accumulate greater quantities of leaf volatile terpenes than do nitrogen-rich plants. A series of feeding trials were performed to determine if such nitrate-limited plants are better defended against generalist-feeding insect herbivores. Soybean looper (Pseudoplusia includens) larvae were fed leaves fromH. subaxillaris rosettes grown under high and low nitrate supply regimes. Larval consumption, growth, and survival declined as the leaf volatile terpene content increased. Larval consumption and growth were enhanced by higher plant nitrate supply and with increasing leafage. The results suggest that the higher quantity of volatile terpenes in the leaves of nitrate-limited plants may better defend these leaves against generalist-feeding insects.

Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves.

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.