Synthesis of deuterated volatile lipid degradation products to be used as internal standards in isotope dilution assays. 1. Aldehydes.

The isotopically labeled compounds [5,6-(2)H(2)]hexanal (d-I), [2, 3-(2)H(2)]-(E)-2-nonenal (d-II), [3,4-(2)H(2)]-(E,E)-2,4-nonadienal (d-III), and [3,4-(2)H(2)]-(E,E)-2,4-decadienal (d-IV) were prepared in good yields using new or improved synthesis procedures. Labeling position, chemical purity, and isotopic distribution of the compounds were characterized by various MS and NMR techniques. These molecules are used as internal standards in quantification experiments based on isotope dilution assay. Synthesis of d-I, d-III, and d-IV has not yet been reported in the literature.     

Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays

[2H4]Folic acid was synthesized by deuterating p-aminobenzoic acid, which was then coupled to glutamic acid and 6-formylpterin. Using [2H4]folic acid as starting component enabled the preparation of labeled vitamers tetrahydrofolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and 10-formylfolate which were characterized by electrospray mass spectrometry and collision-induced dissociation. The mass spectrometric studies confirmed that the compounds could be used as internal standards in stable isotope dilution assays.     

Synthesis of deuterated gamma-lactones for use in stable isotope dilution assays

Two syntheses of deuterated gamma-lactones for use as internal standards in stable isotope dilution assays (SIDA) were developed. [2,2,3,3-2H4]-gamma-Octa-, -gamma-deca-, and -gamma-dodecalactones with >89% deuterium incorporation were prepared in 27, 17, and 19% overall yields, respectively, by the reduction of a doubly protected hydroxypropiolic acid with deuterium gas. [3,3,4-2H3]-gamma-Octa- and -gamma-dodecalactones were prepared in 6 and 23% yields with >92% deuterium incorporation by the free radical addition of 2-iodoacetamide to [1,1,2-2H3]-1-hexene and [1,1,2-2H3]-1-decene, respectively. Reaction yields were highly dependent upon the purity of the 1-alkene starting material. The deuterated gamma-lactones were evaluated as internal standards for SIDA.     

Synthesis of four carbon-13-labeled type a trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays

The first stable isotope dilution assay (SIDA) for the simultaneous quantitation of the most abundant type A trichothecenes in foods and feeds was developed. Synthesis of carbon-13-labeled T2-toxin, HT2-toxin, diacetoxyscirpenol, and monoacetoxyscirpenol was accomplished by [13C2]-acetylation of T2-triol and scirpentriol, respectively. Scirpentriol was prepared from diacetoxyscirpenol by complete alkaline hydrolysis and subsequently was converted to [13C6]-triacetoxyscirpentriol by peracetylation with [13C4]-acetic anhydride. The latter compound was selectively hydrolyzed using ammonium hydroxide to give [13C4]-diacetoxyscirpenol and [13C2]-monoacetoxyscirpenol in reasonable yields. Analogously, [13C6]-T2-triacetate was prepared from T2-triol and subjected to controlled hydrolysis to yield [13C4]-T2-toxin and [13C2]-HT2-toxin. All synthesized products were characterized by NMR and MS experiments. Using the prepared isotopically labeled standards, SIDAs were developed for the quantitation of type A trichothecenes in food and feeds. The mycotoxins were quantified by LC-single and tandem MS after cleanup on multifunctional columns. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery and good precision in interassay studies. Food samples were analyzed using the developed SIDA and showed substantial contamination of oat products with T2-toxin and HT2-toxin. Diacetoxyscirpenol was detected on potatoes, whereas monoacetoxyscirpenol was not present in the analyzed samples.     

Use of deuterated internal standards for quantitation of T-2 and HT-2 toxins in human blood by tandem mass spectrometry

Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero-HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.     

Can the extent of degradation of soil fungal mycelium during soil incubation be used to estimate ectomycorrhizal biomass in soil?

The extent of degradation of the fungal biomass in forest soil during laboratory incubation was investigated as a measure of ectomycorrhizal (EM) biomass. The method simulates the disappearance of fungal mycelium after root trenching, where the EM fungi, deprived of its energy source (the tree), will start to die off. Incubating a forest humus soil at 25 °C resulted in a decrease in the relative proportion (mol%) of the phospholipid fatty acid 18:2ω6,9 (a fungal marker molecule) within 3-6 months, indicating that fungal biomass was disappearing. Incubation at 5 °C resulted in essentially no change in the amount of 18:2ω6,9. The measurement of ergosterol, another fungal marker molecule, gave similar results. Incubation of different forest soils (pine, spruce and spruce/oak), and assuming that the disappearance of fungal biomass during this period of time was entirely due to EM fungi, resulted in an estimation of EM biomass of between 47 and 84% of the total fungal biomass in these soils. The humus layer had more EM biomass than deeper mineral layers.     

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.     

Methods of analysis for toxic elements in food products. 2. Review of USSR standards on determinations of heavy metals and arsenic

Methods of analysis prescribed by USSR standards for Hg, Pb, Zn, As, Cd, Cu, Fe, and Sn in foods are described: for Hg--colorimetry of tetraiodide mercurate and atomic absorption spectroscopy (AAS); for Pb, Cd, Zn, and Cu--polarography; for Cu--colorimetry with sodium diethyldithiocarbamate and zinc dibenzyldithiocarbamate; for As--colorimetry with silver diethyldithiocarbamate; for Sn--colorimetry with quercetin; and for Fe--colorimetry with o-phenanthroline. All of the methods have the necessary metrological characteristics, including intralaboratory repeatability value (r), interlaboratory reproducibility value (R), minimum quantity of the element to be determined in the analytical test portion (MQSM), and the coefficients that account for mercury and arsenic losses during analysis. Establishing constant r- and R-values for the methods under consideration is expedient because (a) the methods suggested are used for safety purposes; and (b) the optimum amount of the element studied in the test sample is determined, to a certain degree, by the mass of the test portion.     

Development of a stable isotope dilution assay for the quantification of 5-methyl-(E)-2-hepten-4-one: application to hazelnut oils and hazelnuts.

A stable isotope dilution assay was developed for the quantitation of the hazelnut odorant 5-methyl-(E)-2-hepten-4-one by mass chromatography using synthesized [(2)H](2)-5-methyl-(E)-2-hepten-4-one as the internal standard. Application of the method on two batches of commercial hazelnut oils, processed from either roasted or unroasted nuts, revealed 6.4 microg 5-methyl-(E)-2-hepten-4-one per kg of unroasted oil whereas 315.8 microg per kg was determined in the roasted nut oil. The about 50-fold higher amount of 5-methyl-(E)-2-hepten-4-one in roasted hazelnut oil suggested the necessity of a thermal treatment to generate the flavor compound. Pan frying of raw hazelnuts (9 to 15 min) or boiling of the crushed nut material for 1 h in water led to an increase of 5-methyl-(E)-2-hepten-4-one by factors of 600 and 800, respectively, thereby corroborating that the major part of the nut flavorant is formed during heat treatment from a yet unknown precursor in hazelnuts.     

Improved detection of sugar addition to maple syrup using malic acid as internal standard and in 13C isotope ratio mass spectrometry (IRMS)

Stable carbon isotope ratio mass spectrometry (delta13C IRMS) was used to detect maple syrup adulteration by exogenous sugar addition (beet and cane sugar). Malic acid present in maple syrup is proposed as an isotopic internal standard to improve actual adulteration detection levels. A lead precipitation method has been modified to isolate quantitatively malic acid from maple syrup using preparative reversed-phase liquid chromatography. The stable carbon isotopic ratio of malic acid isolated from this procedure shows an excellent accuracy and repeatability of 0.01 and 0.1 per thousand respectively, confirming that the modified lead precipitation method is an isotopic fractionation-free process. A new approach is proposed to detect adulteration based on the correlation existing between the delta13Cmalic acid and the delta13Csugars-delta13Cmalic acid (r = 0.704). This technique has been tested on a set of 56 authentic maple syrup samples. Additionally, authentic samples were spiked with exogeneous sugars. The mean theoretical detection level was statistically lowered using this technique in comparison with the usual two-standard deviation approach, especially when maple syrup is adulterated with beet sugar : 24 +/- 12% of adulteration detection versus 48 +/- 20% (t-test, p = 7.3 x 10-15). The method was also applied to published data for pineapple juices and honey with the same improvement.     

Comparison of isolated cranberry (Vaccinium macrocarpon Ait.) proanthocyanidins to catechin and procyanidins A2 and B2 for use as standards in the 4-(dimethylamino)cinnamaldehyde assay

The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. However, this method suffers from issues of accuracy and precision in the analysis and comparison of PAC levels across a broad range of cranberry products. Current use of procyanidin A2 as a standard leads to an underestimation of PACs content in certain cranberry products, especially those containing higher molecular weight PACs. To begin to address the issue of accuracy, a method for the production of a cranberry PAC standard, derived from an extraction of cranberry (c-PAC) press cake, was developed and evaluated. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were 2.2 times higher than those determined by procyanidin A2. Increased accuracy is critical for estimating PAC content in relationship to research on authenticity, efficacy, and bioactivity, especially in designing clinical trials for determination of putative health benefits.     

Changes in nesting behavior and lipid content of a marine amphipod (Amphithoe valida) to the toxicity of a no. 2 fuel oil

Laboratory cultured amphipods, Amphithoe valida, were exposed to the water soluble fractions (WSF) of a No. 2 fuel oil for 6 days, and then transferred to clean sea water for one week. Survival and nesting behavior were observed daily, and the lipid contents were checked at the end of exposure and at the end of depuration. Survival of amphipods during exposure was high at all concentrations of WSF tested (0 to 25%). A delayed toxicity was observed; mortality was high in concentrations ? 15% WSF during depuration. The number of nests decreased with increasing concentration of WSF and length of exposure. Recovery of nest building activity in clean sea water was either lacking or small, indicating some damage to their nesting capability. The lipid contents of amphipods were close to the expected range (except at 25% WSF) following the exposure, but dropped to about 1 % following recovery compared with an expected value of 3.3%. Impairments of the chemosensory and locomotory systems may occur during exposure, which prevent amphipods from constructing nests for protection and food reserves, and eventually lead animals to use their stored energy of lipid for survival. Of the three biological parameters studied, nesting behavior and lipid content as a percentage of dry weight were better indicators of the sublethal stress of WSF than the response of survival; significant alterations of the former two parameters were found at the concentrations ? 5% WSF.     

Fate of airborne metal pollution in soils as related to agricultural management: 2. Assessing the role of biological activity in micro‐scale Zn and Pb distributions in A,B and C horizons

This work assesses relationships between characteristic aggregate microstructures related to biological activity in soils under different long‐term land use and the distribution and extractability of metal pollutants. We selected two neighbouring soils contaminated with comparable metal loads by past atmospheric deposition. Currently, these soils contain similar stocks, but different distributions of zinc (Zn) and lead (Pb) concentrations with depth. One century of continuous land use as permanent pasture (PP) and conventional arable (CA) land, has led to the development of two soils with different macro‐ and micro‐morphological characteristics. We studied distributions of organic matter, characteristic micro‐structures and earthworm‐worked soil by optical microscopy in thin sections from A, B and C horizons. Concentrations and amounts of total and EDTA‐extractable Zn and Pb were determined on bulk samples from soil horizons and on size‐fractions obtained by physical fractionation in water. Large amounts of Zn and Pb were found in 2–20‐µm fractions, ascribed to stable organo‐mineral micro‐aggregates influenced by root and microbial activity, present in both soils. Unimodal distribution patterns of Zn, Pb and organic C in size‐fractions were found in horizons of the CA soil. In contrast, bimodal patterns were observed in the PP soil, because large amounts of Zn and Pb were also demonstrated in stable larger micro‐aggregates (50–100‐µm fractions). Such differing distribution patterns characterized all those horizons markedly influenced by earthworm activity. Larger earthworm activity coincided with larger metal EDTA‐extractability, particularly of Pb. Hence, land use‐related biological activity leads to specific soil microstructures affecting metal distribution and extractability, both in surface and subsurface horizons.