Hemoglobin adducts and mercapturic acid excretion of acrylamide and glycidamide in one study population

The aim of this study was to determine the relationship between the oxidative and reductive metabolic pathways of acrylamide (AA) in the nonsmoking general population. For the first time both the blood protein adducts and the urinary metabolites of AA and glycidamide (GA) were quantified in an especially designed study group with even distribution of age and gender. The hemoglobin adducts N-carbamoylethylvaline (AAVal) and N-( R, S)-2-hydroxy-2-carbamoylethylvaline (GAVal) were detected by GC-MS/MS in all blood samples with median levels of 30 and 34 pmol/g of globin, respectively. Concentrations ranged from 15 to 71 pmol/g of globin for AAVal and from 14 to 66 pmol/g of globin for GAVal. The ratio GAVal/AAVal was 0.4-2.7 (median = 1.1). The urinary metabolites were determined by LC-MS/MS. Of all urine samples examined 99% of N-acetyl- S-(2-carbamoylethyl)- l-cysteine (AAMA) levels and 73% of N-( R/ S)-acetyl- S-(2-carbamoyl-2-hydroxyethyl)- l-cysteine (GAMA) levels were above the LOD (1.5 microg/L). Concentrations ranged from 相似文献   

Development of a class-selective enzyme-linked immunosorbent assay for mercapturic acids in human urine

Epidemiological and toxicological studies often require the analysis of large numbers of samples for biological markers of exposure. The goal of this work was to develop a class-selective ELISA to detect groups of structurally closely related mercapturic acids with small nonpolar S-substituents. An assay was developed with strong recognition for mercapturates including S-benzylmercapturic acid (IC50 = 0.018 micromol/L), S-n-hexylmercapturic acid (IC50 = 0.021 micromol/L), S-phenylmercapturic acid (IC50 = 0.024 micromol/L), and S-cyclohexylmethylmercapturic acid (IC50 = 0.042 micromol/L). The same assay also showed weaker recognition for S-(1-hydroxynaphthal-2-yl)mercapturic acid and S-allylmercapturic acid (IC50 = 1.1 and 1.7 micromol/L, respectively). Subtle modifications to the hapten linker structure of the coating antigen proved to have a strong impact on the selectivity and the specificity of the assay. A slightly modified assay showed high recognition for S-benzylmercapturic acid (IC50 = 0.018 micromol/L) and weaker recognition for seven other mercapturic acids (IC50 = 0.021-10 micromol/L). Strong positive assay responses were detected in 12 urine samples obtained from persons with no known occupational exposure to exogenous electrophilic xenobiotics. Solid phase extraction and cross-reactivity indicated that the presumptive immunoreactive materials were similar in size and polarity to S-benzylmercapturic acid. The assay was more selective to mercapturic acids than the spectrophotometric thioether assay.     

Analysis of isothiocyanate mercapturic acids in urine: a biomarker for cruciferous vegetable intake

Cruciferous vegetables contain glucosinolates, which are degraded to isothiocyanates. These are easily absorbed, conjugated to glutathione, and excreted into the urine as their corresponding mercapturic acids. We have developed and validated a solid phase extraction-high-performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry method for the specific analysis of individual isothiocyanate mercapturic acids in urine. The range of reliable analysis was 1.0-310 microM in urine. Urine samples fortified with three different levels of isothiocyanate mercapturic acids were measured on six different days by three independent technicians. The relative standard deviation (RSD) of repeatability was 12, 6, and 3%; the RSD of reproducibility was 19, 14, and 8%, and spike recoveries were 103, 104, and 103%, respectively, for 1.04, 10.5, and 313 microM levels. In 24 h urine collected from two volunteers after they consumed broccoli and cauliflower, clearly sulforaphane mercapturic acid (133 micromol) and allyl isothiocyanate mercapturic acid (4.7 micromol) were found. This procedure demonstrates a reliable and efficient method to study the intake and mode of action of isothiocyanates in animal studies and clinical trials.     

Association between consumption of cruciferous vegetables and condiments and excretion in urine of isothiocyanate mercapturic acids

A high intake of cruciferous vegetables is associated with a reduced risk of cancer and cardiovascular diseases. This protective effect has been linked to isothiocyanates, enzymatic hydrolysis products of glucosinolates. In this study, the metabolic fate of glucosinolates and isothiocyanates after ingestion of 19 different cruciferous vegetables was studied in three male subjects. After the consumption of 13 cruciferous vegetables (glucosinolate content, 0.01-0.94 mmol/kg) and six condiments (isothiocyanate content, 0.06-49.3 mmol/kg), eight different isothiocyanate mercapturic acids were determined in urine samples. Excretion levels after the consumption of raw vegetables and condiments were higher (bioavailability, 8.2-113%) as compared to cooked vegetables (bioavailability, 1.8-43%), but the excretion rate was similar (t1/2=2.1-3.9 h). Isothiocyanates in urine remain longer at a nonzero level after the consumption of glucosinolates from cooked vegetables, as compared to raw vegetables and condiments, and maximal levels in urine were reached about 4 h later. Isothiocyanate mercapturic acids can be used as a biomarker to reflect the active dose of isothiocyanates absorbed.     

Development of an enzyme-linked immunosorbent assay for the detection of dicamba

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.     

Quantification of uronic acids in tea polysaccharide conjugates and their antioxidant properties

A technique of high-performance liquid chromatography (HPLC) was described for the measurement of total uronic acids in tea polysaccharide conjugates. This method was applied to polysaccharide conjugate extracts obtained from green tea after most of the components that produce interference were removed. The preliminary extraction process was according to the procedure of isolation of polysaccharide conjugates. The uronic acid content of different polysaccharide conjugate fractions was quantified by HPLC on a Sugar-Pak I column with a 1.0 x 10(-)(4) mol x L(-)(1) calcium disodium ethylenediaminetetraacetic acid solution as the mobile phase and refractive index detection. The validation study showed high recoveries (>97.0%) and low coefficients of variance (<3.0%). The minimum detectable limit concentration of uronic acid was 10 microg x mL(-)(1). The analysis of a standard range of galacturonic acid concentrations (100-4000 microg x mL(-)(1)) yielded linear results. The use of the method on different polysaccharide conjugate fraction samples confirmed its effectiveness. With the high content of uronic acids in polysaccharide conjugates, the stronger reactive oxygen species scavenging activities were found.     

Phenolic antioxidants richly contained in corn bran are slightly bioavailable in rats

Phenolic acids (PAs) have been shown to be beneficial to human health and are found most abundantly in corn bran ( approximately 4%, w/w), one of the main dietary fibers. This study therefore evaluated the bioavailabilities of phenolic antioxidants ferulic acid (FA) and p-coumaric acid (PCA) in refined corn bran (RCB) by determining their recovery in the plasma, urine, and feces of rats fed a single meal of a RCB diet containing 5% RCB or adapted to the RCB diet for 10 days. In both studies, 0.4-0.5% of ingested FA and 1.2-2.3% of ingested PCA were recovered in rat urine. By contrast, approximately 81% of FA and approximately 64% of PCA ingested with the single meal were excreted through the rat feces within 3 days after the ingestion. On the other hand, after rats were fed the RCB diet, total FA (all forms of FA) was recovered in plasma at a concentration of 35.0 +/- 2.0 microg/L, total FA and total PCA were excreted through urine at levels of 155.4 +/- 5.8 and 50.9 +/- 6.6 microg/day, respectively. These parameters showed no significant change (P = 0.93, 0.09, and 0.66, respectively) after rats were fed the RCB diet continuously for up to 10 days. These results suggest that the PAs in RCB are bioavailable in rats. Their bioavailabilities, however, are relatively low compared with their high content in RCB and not improved by the adaptation for 10 days to the enriched RCB diet. Additionally, comparison with the results of other studies revealed that high contents of FA and, especially, diferulic acids in cereal bran, which act as cross-links between bran cell wall polysaccharides, may not improve but, rather, limit the bioavailabilities of PAs in vivo.     

Parallel synthesis: a new approach for developing analytical internal standards. Application to the analysis of patulin by gas chromatography-mass spectrometry

The polymer-assisted reaction of 4-(hydroxymethyl)furan-2(5H)-one (4HM2F) with 21 carboxylic acids using polystyrene-carbodiimide (PS-carbodiimide) yielded an ester library. Four of the esters, (5-oxo-2,5-dihydrofuran-3-yl)methyl acetate (IS-1), (5-oxo-2,5-dihydrofuran-3-yl)methyl butyrate (IS-2), (5-oxo-2,5-dihydrofuran-3-yl)methyl 2-methylpropanoate (IS-3), and (5-oxo-2,5-dihydrofuran-3-yl)methyl chloroacetate (IS-4), were tested as internal standards for the quantification of patulin in apple juice by gas chromatography-mass spectrometry in the selected ion monitoring mode (GC-MS-SIM). The developed method combines an AOAC official extractive step and a GC-MS-SIM analysis. Using a chromatographic column containing trifluoropropylmethylpolysiloxane as the stationary phase and IS-1 as the internal standard, it was possible to perform an accurate and precise quantification of underivatizated patulin in apple juice at concentrations down to 6 microg/L. A detection limit of 1 microg/L was established.     

Spelt (Triticum spelta L.) and winter wheat (Triticum aestivum L.) wholemeals have similar sterol profiles, as determined by quantitative liquid chromatography and mass spectrometry analysis

From a nutritional point of view, cereal lipids include valuable molecules, such as essential fatty acids, phytosterols, and fat-soluble vitamins. Spelt (Triticum spelta L.) is an alternative hulled bread cereal mostly grown in Belgium, where it is mainly intended for animal feed but should increasingly be used for human consumption. The present research focused on phytosterol quantification by LC/APCI-MS2 in saponified wholemeal extracts of 16 dehulled spelt and 5 winter wheat (Triticum aestivum L.) varieties grown in Belgium during 2001-2002 at the same location. Glycosylated sterols and free and formerly esterified sterols could be determined in saponified extracts. Results show that the mean phytosterol content is comparable in both cereals (whereas other lipids, such as oleic and linoleic acids, are increased in spelt wholemeal): spelt extract has, on average, 527.7 microg of free and esterified sterols g(-1) of wholemeal and 123.8 microg of glycosylated sterols g(-1) of wholemeal versus 528.5 and 112.6 microg x g(-1) in winter wheat (values not corrected for recoveries). This is the first report on the application and validation of an LC/MS2 method for the quantification of phytosterols in spelt and winter wheat.     

Fatty acid and carotenoid composition of gac (Momordica cochinchinensis Spreng) fruit

In this study, we analyzed fatty acid and carotenoid composition of fruit tissues, including seed (which are surrounded by a bright red, oily aril) of Momordica cochinchinensis Spreng, known as gac in Vietnam. Carotenoid content was analyzed by reversed-phase HPLC, using a C(30) column and a method separating cis- and trans-isomers of the major carotenoids in this fruit. Mean values obtained in aril tissues were 1342 microg trans-, 204 microg cis-, and 2227 microg total lycopene; 597 microg trans-, 39 microg cis-, and 718 microg total beta-carotene; and 107 microg alpha-carotene/g FW. Mesocarp contained 11 microg trans-, 5 microg cis-beta-carotene/g FW, trace amounts of alpha-carotene, and no lycopene. Gac aril contained 22% fatty acids by weight, composed of 32% oleic, 29% palmitic, and 28% linoleic acids. Seeds contained primarily stearic acid (60.5%), smaller amounts of linoleic (20%), oleic (9%), and palmitic (5-6%) acids, and trace amounts of arachidic, cis-vaccenic, linolenic, and palmitoleic, eicosa-11-enoic acids, and eicosa-13-enoic (in one fruit only) acids.     

Microbial response to bensulfuron-methyl treatment in soil.

A laboratory incubation study was conducted to evaluate the effect of bensulfuron-methyl treatment on soil microbial biomass and N-mineralization of a loamy sand soil. The herbicide was applied at 0 (control), 0.01 (field rate), 0.1, and 1.0 microg g(-1), and soil microbial biomass carbon (C(mb)), soil microbial biomass nitrogen (N(mb)), and N-mineralization rate (k) were measured at different times after herbicide treatment. Compared to the untreated soil, C(mb) and N(mb) decreased significantly (p < or = 0.05) within the first 7 days after herbicide treatment at 0.1 and 1.0 microg g(-1), and the impact was greater for N(mb) than for C(mb). Nitrogen mineralization was significantly suppressed during the first 5 days of incubation when the soil was treated with bensulfuron-methyl at 0.1 and 1.0 microg g(-1). The overall impact of bensulfuron-methyl to the soil microbial communities was closely related to the application rate in the range of 0.01-1.0 microg g(-1). This effect, however, was found to be transitory, and significant impact occurred only at high application rates.     

Chemiluminescence investigation of detection of rutin in medicine and human urine using controlled-reagent-release technology.

A novel continuous-flow sensor based on chemiluminescence (CL) detection was developed for the determination of rutin in pharmaceutical preparations and human urine by controlled-reagent-release technology. The analytical reagents involved in the CL reaction, including luminol and hexacyanoferrate(III), were both immobilized on an anion-exchange column in a flow-injection system. The CL signal produced by the reaction between luminol and hexacyanoferrate(III), which were eluted from the column through sodium phosphate injection, was decreased in the presence of rutin. CL intensity was inhibited by rutin; the decrement of CL intensity was linear over the logarithm of the rutin concentration range of 1.0-400 ng x mL(-1), and the detection limit was 0.35 ng x mL(-1) (3 sigma). The whole process, including sampling and washing, could be completed in 1.5 min with a relative standard deviation of <3.5%. The flow sensor showed remarkable stability and could be easily reused >450 time; the sensor proposed was applied successfully to the determination of rutin in pharmaceutical preparations and human urine.     

Metabolism of metamitron in goat following a single oral administration of a nontoxic dose level: a continued study

Disposition kinetic behavior and metabolism studies of metamitron and its metabolite in terms of the parent compound were carried out in black Bengal goats after a single oral administration of a nontoxic oral dose at 30 mg kg(-1) of body weight. Metamitron was detected in the blood sample at 5 min (2.23 +/- 0.04 microg mL(-1)), maximum at 1 h (3.43 +/- 0.02 microg mL(-1)) and minimum at 12 h (0.41 +/- 0.01 microg mL(-1)), after a single oral administration. Metabolite [3-methyl-6-phenyl-1,2,4-triazin-5(4H)-one] in terms of the parent compound was detected in the blood sample at 5 min (0.47 +/- 0.006 microg mL(-1)), maximum at 6 h (5.12 +/- 0.02 microg mL(-1)) and minimum at 96 h (1.06 +/- 0.016 microg mL(-1)), after a single oral administration. The t(1/2 K) and Cl(B) values of metamitron were 3.63 +/- 0.05 h and 1.36 +/- 0.016 L kg(-1) h(-1), respectively, whereas the t(1/2K)(m) and Cl(B)(m) values of the metabolite were 38.15 +/- 0.37 h and 0.091 +/- 0.001 L kg(-1) h(-1), respectively, which suggested long persistence of the metabolite in blood and tissues of goat. Metamitron was excreted through feces and urine for up to 48 and 72 h, whereas the metabolite was excreted for up to 168 and 144 h, respectively. Metabolite alone contributed to 96 and 67% of combined recovery percentage of metamitron and metabolite against the administered dose in feces and urine of goat, respectively. All of the goat tissues except lung, adrenal gland, ovary, testis, and mammary gland retained the metabolite residue for up to 6 days after administration.     

The antiproliferative and differentiating effects of human leukemic U937 cells are mediated by cytokines from activated mononuclear cells by dietary mushrooms

Proliferation of human leukemic U937 cells was remarkably inhibited by conditioned medium (CM) of human peripheral blood mononuclear cells (MNC-CM) stimulated with cold-water extracts (CWE) (10-800 microg/mL of medium) of dietary mushrooms, Hypsizigus mamoreus (HM), Agrocybe aegerita (AA), Flammulina velutipes (FV), whereas insignificant results were observed when cells were cultured in the presence of CWE at the corresponding level. Water extracts from mushrooms were fractionated by Sephadex G-50 chromatography, and the pooled high molecular weight fraction (F1) (200 microg/mL) of HM (HM1) and AA (AA1) exhibited growth inhibitions >80% on U937 cells. Interestingly, the thus-cultured U937 cells showed high nitroblue tetrazolium (NBT) positive (>68%) and nonspecific esterase (NSE) positive (>47%) percentages, revealing the remarkable differentiation into monocytes/macrophages upon incubation with HM1- and AA1-stimulated MNC-CM. In addition, assays for the expressions of monocyte-associated antigens, CD11b, CD14, and CD68, also evidenced the remarkable differentiation of U937 cells into monocytes/macrophages by presenting high CD maker positive percentages (>50%). Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in CM of HM1-stimulated MNC for 1 day (MNC-CM-1) were 1350 and 1374 pg/mL, respectively, revealing the potent antitumor and differentiation-inducing activities of HM. Of note, MNC-CM-1 appeared to be more effective than day 5 MNC-CM (MNC-CM-5) in both antitumor and differentiation-inducing activities.     

Development of a solid-phase extraction method for phenoxy acids and bentazone in water and comparison to a liquid-liquid extraction method

A rapid solid-phase extraction (SPE) method was developed for the determination of bentazone and the phenoxy acids 2,4-D, dichlorprop, MCPA, and mecoprop in Norwegian environmental water samples. Cartridges with a high-capacity cross-linked polystyrene-based polymer were used for off-line preconcentration. The effects of elution solvent, elution volume, sample volume, sorbent mass, pH, and flow rate on the recoveries of the pesticides were investigated using HPLC. Average recovery of >90% was achieved with 500 mg sorbents using 2 mL of methanol with 5% NH3 as elution solvent. The recoveries were independent of sample pH in the tested range of pH 1-7. Using a sample volume of 200 mL, the limits of determination for the phenoxy acids and bentazone are 0.02 microg/L. Sample volumes up to 2000 mL at a flow rate of 60 mL/min could be handled without any loss of analytes, which makes it possible to lower the limits of determination. The SPE method was compared to a routinely used liquid-liquid extraction method. Three different water matrices spiked at 1.0 and 0.05 microg/L were extracted, and the quantification was performed by GC-MS. Both methods permitted the determination of phenoxy acids and bentazone in distilled water, creek water, and well water down to a level of 0.05 microg/L with recoveries >80% for 200 mL samples. Important advantages of the SPE method compared to the liquid-liquid extraction method were the short extraction times, lack of emulsions, use of disposable equipment, and reduced consumption of organic solvents.     

Some sulfur-containing metabolites of tri-n-butyltin chloride in male rats.

In an attempt to elucidate metabolic destination of TBTO, sulfur-containing metabolites were investigated in the urine. Tri-n-butyltin chloride (TBTC), tri-n-butyltin oxide (TBTO), and their in vitro metabolites in rat liver microsomal enzyme systems, di-n-butyl(3-hydroxybutyl)tin chloride (T3OH), di-n-butyl(3-oxobutyl)tin chloride (T3CO), dibutyltin dichloride (DBTC), and monobutyltin trichloride (MBTC), were intraperitoneally administered to rats. In particular, administration of T3OH and T3CO gave higher amounts of mercapturic acid derivatives, such as N-acetyl-S-(3-oxobutyl)-L-cysteine (3CO-MA) and N-acetyl-S-(3-hydroxybutyl)-L-cysteine (3OH-MA), than TBTC or TBTO. On the other hand, DBTC and MBTC did not yield measurable amounts of 3CO-MA and/or 3OH-MA. The appearance of organotin metabolites in urine indicates that T3OH, T3CO, and hypothesized secondary metabolites, such as n-butyl(3-hydroxybutyl)(3-oxobutyl)tin chloride, n-butyl(3-hydroxybutyl)(4-hydroxybutyl)tin chloride, etc., are subject to the action of glutathione S-transferase to give mercapturic acid derivatives. These sulfur-containing metabolites (3CO-MA and 3OH-MA) were also found in control rat urine.     

In vitro anti-inflammatory and anti-proliferative activity of sulfolipids from the red alga Porphyridium cruentum

A sulfoglycolipidic fraction (SF) isolated from the red microalga Porphyridium cruentum was analyzed for fatty acid composition and assayed for ability to inhibit, in vitro, the generation of superoxide anion in primed leucocytes and the proliferation of a panel of human cancer cell-lines. Results demonstrated that SF contained large amounts of palmitic acid (26.1%), arachidonic acid (C20: 4 omega-6, 36.8%), and eicopentaenoic (C20:5 omega-3, 16.6%) acids, and noticeable amounts of 16:1n-9 fatty acid (10.5%). It strongly inhibited both the production of superoxide anion generated by peritoneal leukocytes primed with phorbol myristate acetate (IC(50): 29.5 microg/mL), and the growth of human colon adenocarcinoma DLD-1 and to a lesser extent of human breast adenocarcinoma MCF-7, human prostate adenocarcinoma PC-3, and human malignant melanoma M4 Beu cell-lines, and therefore might have a chemopreventive or chemotherapeutic potential, or both. It was found markedly more cytotoxic than sulfoquinovosyldiacylglycerols from plant used as a standard (STD), due to a stronger ability to inhibit DNA alpha-polymerase (IC(50): 378 microg/mL, vs 1784 microg/mL for STD). After a 48-h continuous treatment, IC(50) values for growth inhibition were in the range of 20-46 microg/mL instead of 94 to >250 microg/mL for STD, and those for inhibition of metabolic activity were in the range of 34-87 microg/mL instead of >250 microg/mL for STD. The higher anti-proliferative effect was observed on colon adenocarcinoma DLD-1 cells, and the weaker effect was observed on breast adenocarcinoma MCF-7.     

Quantitation of ochratoxin A in South African wines

The natural occurrence of the carcinogenic mycotoxin ochratoxin A (OTA) in wines sold in local retail outlets in South Africa and Italy was investigated by HPLC analysis with fluorescence detection following cleanup by immunoaffinity column. All 24 local South African wines tested (15 white and 9 red) were found to contain detectable levels (>0.01 microg/L) of OTA, with a mean of 0.16 microg/L in the white wines and a mean of 0.24 microg/L in the red wines. Results were subsequently confirmed by LC-MS analysis using positive ion electrospray ionization with collision-induced dissociation of the protonated molecular ion [M + H](+) at m/z 404 and selected reaction monitoring of the resultant product ions [M + H - H(2)O - CO](+) at m/z 358 and [M + H - H(2)O](+) at m/z 386. Comparison with the fluorescence method gave a significant correlation (r = 0.87; p < 0.01). Although OTA contamination was present in all of the South African samples analyzed, levels were well below the suggested European Union limit of 0.5 microg/kg. The highest level found in a locally purchased wine was 0.39 microg/L in a blend of local and imported Spanish red wine. Of the eight Italian wines analyzed, only two red wines were contaminated above the suggested maximum level.     

Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin

Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine (hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-l-phenylalanine (hapten 5), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis) between antibody and coating antigen showed a high sensitivity for the target analyte. The IC50 was 1.3, 2.1, and 2.2 microg/L for the trans-target analyte and 0.4, 2.3, and 2.8 microg/L for the cis-target analyte. The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite (DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total cis/trans-DCCA-glycine in human urine samples.     

Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens

Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 microg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 microg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 microg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. Keywords: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry.