Improved HPLC-MS/MS method for determination of isoxaflutole (balance) and its metabolites in soils and forage plants

A robust multi-residue procedure is needed for the analysis of the pro-herbicide isoxaflutole and its degradates in soil and plant materials at environmentally relevant (<1 microg kg-1) levels. An analytical method using turbo-spray and heat-nebulizer high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the analysis of isoxaflutole (IXF) and its two metabolites, diketonitrile (DKN) and the benzoic acid metabolite (BA), at sub-microgram per kilogram levels in soil and plant samples. The average recoveries of the three compounds in spiked soil and plant samples ranged from 84 to 110% and 94 to 105%, respectively. The limits of quantification were validated at 0.06 microg kg-1 for soil and 0.3 microg kg-1 for plant samples. The limits of detection (LOD) for soil analysis were 0.01, 0.002, and 0.01 microg kg-1 for IXF, DKN, and BA, respectively. Corresponding LOD for the plant analysis method were 0.05, 0.01, and 0.05 microg kg-1. The developed method was validated using forage grass and soil samples collected from a field lysimeter study in which IXF was applied to each of four forage treatments. Forage plants and soils were sampled for analyses 25 days after IXF application to the soil. In soils, IXF was not detected in any treatment, and DKN was the predominant metabolite found. In forage plants, the concentrations of DKN and BA were 10-100-fold higher than that in soil samples, but IXF was not detected in any forage plants. The much higher proportion of BA to DKN in plant tissues (23-53%), as compared to soils (0-5%), suggested that these forages were capable of detoxifying DKN. The developed methods provided LODs at sub-microgram per kilogram levels to determine the fate of IXF and its metabolites in soils and forage plants, and they also represent considerable improvements in extraction recovery rates and detection sensitivity as compared to previous analytical methods for these compounds.     

Effect of dips in a 1-methylcyclopropene-generating solution on 'Harrow Sun' plums stored under different temperature regimes

The effect of postharvest dips in a 1-methylcyclopropene-generating solution of the formulation AFxRD-038 (Rohm & Haas) on plum fruit (Prunus salicina Lindell cv. 'Harrow Sun') quality and ripening during storage was determined. Fruit weight loss, tissue firmness, soluble solids content (SSC), titratable acidity (TA), ethylene production, respiration, and the activities of the cell wall modifying enzymes polygalacturonase (PG), 1,4-beta-D-glucanase/glucosidase (EGase), beta-galactosidase (beta-gal), and pectin methylesterase (PME) were measured. Fruit reddening, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity were also analyzed. The 1-MCP-treated fruit showed reduced ethylene production and respiration rate and delayed softening, which was associated with the reduction in the activity of PG, EGase, and beta-gal. The immersion in 1-MCP-generating solutions also decreased weight and acidity loss without modifying the fruit SSC. The immersion treatment was particularly effective in the fruit stored at 5 degrees C, keeping higher overall quality, maintaining lower levels of anthocyanins and PAL activity, and preventing flesh reddening. The present data show that beneficial effects in delaying plum fruit ripening and controlling chilling injury can be obtained by dipping the fruits in a solution of this novel 1-MCP-generating formulation.     

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.     

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis

Background

Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody – the serological marker of type 2 autoimmune hepatitis (AIH-2)- is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1) – either in association with anti-LKM1, or in isolation- and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients.

Methods

One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls.

Results

Six out of 18 (33%) anti-LKM^pos/HCV^pos patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM^neg/HCV^pos patients and 0/120 (0%) of the anti-LKM1^neg/HBV^pos patients (p < 0.001 for both comparisons). Anti-SLA antibodies were not present in any of the HCV (with or without anti-LKM1) or HBV-infected patients.

Conclusion

We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of anti-LKM1 negative patients with chronic hepatitis B and C infections. Based on these results we cannot find any justification for the application of anti-LC1 and anti-SLA tests in the routine laboratory testing of viral hepatitis-related autoantibody serology with the only potential exception being the anti-LC1 screening in anti-LKM1^pos/HCV^pos patients.

     

The Chinese grass carp, Ctenopharyngodon idella, its biology,introduction, control of aquatic macrophytes and breeding in the Sudan

The paper describes the distribution and biology of the grass carp following its introduction into Sudan. It provides aspects on the culture of the grass carp and its breeding under Sudanese conditions with emphasis on future research.     

VH1-44 gene usage defines a subset of canine B-cell lymphomas associated with better patient survival

The use of specific immunoglobulin heavy chain variable region (VH) genes has been associated with increased patient survival in human B-cell lymphomas (hBCL). Given the similarity of human and canine BCL (cBCL) in morphology and clinical treatment, we examined the choice of VH in cBCL and determined whether VH gene selection was a distinct feature associated with survival time in dogs. VH gene selection and mutational status in 52 cBCL, including 29 diffuse large B-cell lymphomas (cDLBCL, the most common subtype of cBCL), were analyzed by comparison with the 80 published canine germline VH gene sequences. We further examined the prognostic impact of the subgroups defined by these features on canine survival. We found that VH1-44 was preferentially expressed in the majority of the 52 cBCLs (60%) as well as in the majority of the cDLBCL subset (59%). VH1-44 gene expression was associated with a statistically better overall survival (p = 0.039) in cBCL patients, as well as in the cDLBCL subset of patients (p = 0.038). These findings suggest that VH gene selection in cBCL is not random and may therefore have functional implications for cBCL lymphomagenesis, in addition to being a useful prognostic biomarker.     

The establishment and impact of Pseudorasbora parva,an exotic fish species introduced into Lake Mikri Prespa (north-western Greece)

• 1. The topmouth gudgeon Pseudorasbora parva Schlegel, 1842, a south-east asian cyprinid, was introduced accidentally in the Danube Delta in Romania in the 1960s and has now achieved a pan-Danubian distribution. P. parva has been introduced into other countries, such as Greece, usually inadvertently included with other species imported for fish farming. In 1984–85, during a study of the fish populations of Lake Mikri Prespa (north-west Greece), several topmouth gudgeon were caught. Our aim was to study the changes in the population size of P. parva over nearly 10 years, to study its life history traits and to assess its potential impacts on native fish species.
• 2. The catch per unit effort (CPUE) in spring increased significantly between 1984 and 1992. We found evidence that P. parva has established a breeding population in the lake. P. parva combines many characteristics likely to favour a successful colonization (resistance to harsh climatic conditions, early sexual maturity, extended breeding season, broad dietary spectrum). Growth in Lake Prespa, where the oldest individuals captured were 3 years old, is very similar to that observed within its native range.
• 3. There is evidence for dietary overlap between P. parva and three endemic species: Paraphoxinus epiroticus prespensis, Cobitis meridionalis and Alburnoides bipunctatus ohridanus. No decline in the populations of these three species has yet been demonstrated. Other possible impacts are discussed. The successful colonization by P. parva was certainly favoured by the absence of a true piscivorous fish in the lake and by the isolation and high level of endemism of fish communities.
• 4. The conservation of the many endemic species in the lake should be a priority and the introduction of exotic species should be banned.

     

Metallothionein induction in cultured fibroblasts and liver of a marine flatfish,the turbot,Scophthalmus maximus

Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.Abbreviations used AMP adenosine monophosphate - CHSE chinook salmon embryo - DMSO dimethyl sulphoxide - HPLC high performance liquid chromatography - KB kenacid blue - MT metallothionein - Mr molecular weight (kDaltons) - NR neutral red - PEG polyethylene glycol - RTH rainbow trout hepatoma - SDS sodium dodecyl sulphate - SSC 0.15M NaCl, 0.015M sodium citrate - TF turbot fibroblast