Excretion profile of boldenone and its metabolites after oral administration to veal calves.

The residue profiles of boldenone (17beta-Bol), its epimer (17alpha-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17alpha-Bol decreased rapidly after end of treatment; detectable amounts of 17alpha-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17beta-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum.

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17beta-estradiol (17beta-E2) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17beta-E2 polyclonal antibodies (PAb), the competition was performed using 17beta-estradiol-alkaline phosphatase conjugate (17beta-E2-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum. According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCbeta), was determined. The CCbeta value resulted below the action limit (40 pg mL(-1)) fixed for 17beta-E2) Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%. Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.

Confirmatory analysis of residues of stanozolol and its major metabolite in bovine urine by liquid chromatography-tandem mass spectrometry.

A reliable method for the confirmation of the synthetic hormone stanozolol and its major metabolite, 16beta-hydroxystanozolol, in bovine urine by liquid chromatography coupled with tandem mass spectrometry has been developed. [2H3]Stanozolol was used as internal standard. Sample preparation involved enzymatic hydrolysis, liquid-liquid extraction and purification on an amino solid-phase extraction column. The analytes were ionized using atmospheric pressure chemical ionization with a heated nebulizer interface operating in the positive ion mode, where only the protonated molecules, [M+H]+, at m/z 329 and m/z 345, for stanozolol and 16beta-hydroxystanozolol, respectively, were generated. These served as precursor ions for collision-induced dissociation and three diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring liquid chromatography-tandem mass spectrometry. The accuracy ranged from 19.7 to 14.9% and from 18.9 to 13.2% for stanozolol and 16beta-hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16beta-hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16beta-hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods.

Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry.

Blue-green algae (cyanobacteria) in tablets and capsules, which are marketed as health food supplements, were investigated for the presence of neurotoxins related to anatoxin-a. These neurotoxins, which are nicotinic agonists, were investigated using isocratic micro-liquid chromatograph-tandem mass spectrometry (micro-LC-MS-MS). The investigated compounds were anatoxin-a and homoanatoxin-a, together with their degradation products, dihydroanatoxin-a, epoxyanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a which were synthesized from the parent toxins. The analytes were extracted with methanol followed by isocratic chromatography on a micro C18 reversed-phase column using acetonitrile-water, 50:50 (v/v), containing 20 mm acetic acid at 30 microl min(-1). The toxins were ionized in an ionspray (IS) interface operating in the positive ion mode, where the intact protonated molecules, [M + H]+, were generated at m/z 166, m/z 168, m/z 182, m/z 180, m/z 182 and m/z 196, for anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, homoanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a, respectively. These served as precursor ions for collision-induced-dissociation (CID) and diagnostic product ions for these anatoxins were identified to carry out toxin confirmation by selected reaction monitoring (SRM) LC-MS-MS analysis. Dihydrohomoanatoxin-a and a novel isomer of epoxyanatoxin-a were identified in blue-green algae tablets. This finding suggests that a potential human health hazard could be associated with the consumption of these food supplements.

Accelerated solvent extraction and liquid chromatography-tandem mass spectrometry quantitation of corticosteroid residues in bovine liver.

A new method for the rapid extraction and unequivocal confirmation of two highly potent fluorinated synthetic corticosteroids, dexamethasone and its beta-epimer betamethasone, in bovine liver was developed. Flumethasone was used as internal standard. An extraction procedure using an accelerated solvent extraction system was employed for the isolation of the analytes in liver samples. The procedure was highly automated, including defatting and extraction steps, sequentially carried out under 1.0 x 10(4) kPa in about 35 min. The extracts were then directly analysed by tandem mass spectrometry with on-line liquid chromatography. The analytes were ionised in a heated nebulizer interface operating in the negative ion mode where the molecular related ions [M-H-CH2O]- were generated for each analyte, at m/z 361 for betamethasone and dexamethasone and at m/z 379 for flumethasone. They served as precursor ions for collision-induced dissociation and three diagnostic product ions for the drugs were identified to carry out analyte confirmation by selected reaction monitoring. Assessment of recovery, specificity and precision for betamethasone, dexamethasone and flumethasone proved the method suitable for confirmatory purposes. The limit of quantification of betamethasone and dexamethasone in liver tissue was 1.0 microg/kg.

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.

High levels of yessotoxin in mussels and presence of yessotoxin and homoyessotoxin in dinoflagellates of the Adriatic Sea.

Identification of YTX and homoYTX in natural phytoplankton populations containing significant amounts of Gonyaulax polyedra and determination of detailed toxin profiles of mussels (Mytilus galloprovincialis) periodically collected from two sites of the Northern Adriatic coast from February to October 1997 was performed by LC-FLD following derivatization with ADAM or DMEQ-TAD and LC-MS and LC-MS-MS. OA and YTX concentrations were recorded in the range 0.11-2.31 and 0.18-9.02 microg per g of hepatopancreas, respectively. HomoYTX was also detected both in phytoplankton and mussel samples.

Evaluation of musk contamination of freshwater fish in Italy by accelerated solvent extraction and gas chromatography with mass spectrometric detection.

Musk compounds play an important role as perfuming agents for household chemicals, detergents and cosmetics. It has been demonstrated that the oral absorption of these compounds in humans is significant in the case of contaminated fish. In this study we developed a new extraction procedure, using an accelerated solvent extraction system and a gas chromatography-mass spectrometry detection method, for the determination of 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta [g]-2-benzopyran, 7-acetyl-1,1,3,4,4,6-hexamethyltetralin, 4-acetyl-1,1-dimethyl-6-tert.-butylindan, 6-acetyl 1,2,3,3,5-hexamethylindan and 5-acetyl-1,1,2,6-tetramethyl-3-isopropylindan in freshwater fish samples, collected from several Italian rivers and one lake. 6,7-Dihydro-1,1,2,3,3-pentamethyl-4-(5H)-indanon was used as internal standard. The method provides a rapid and highly extraction procedure, and is sensitive in determining these musk compounds in freshwater fish samples. This is the first report on the contamination from musk compounds in freshwater fish collected in Italy.

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning.

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Quantification of 17 beta-estradiol residues in bovine serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A method for the quantification of the natural hormone 17 beta-estradiol (17 beta-E2) in bovine serum by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) was developed. Ethinylestradiol (EE2) was used as internal standard. Analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a polymeric reversed-phase (PLRP-S) LC column. They were ionized in a heated nebulizer (HN) interface operating in the negative ion mode, where only the intact deprotonated molecules, [M - H]-, were generated at m/z 271 and 295 for 17 beta-E2 and EE2, respectively. These served as precursor ions for collision-induced dissociation (CID) and diagnostic product ions were identified for the unambiguous hormone confirmation by selected reaction monitoring (SRM) LC-APCI-MS-MS. The method was validated on bovine serum and the limit of quantification (LOQ) was 30 pg ml-1 for 17 beta-E2. The inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) derived from the analyses of validation samples at three concentrations ranged from 1.76 to 3.76 and from -4.18 to -2.01%, respectively. This method is currently being successfully applied to measure the bovine serum concentration of 17 beta-E2 in order to discriminate between the physiological concentrations of 17 beta/E2 and the hormone levels resulting from illegal administration.

Biological activities of pseudomycin A, a lipodepsinonapeptide from Pseudomonas syringae MSU 16H.

Similarly to other Pseudomonas lipodepsinonapeptides, pseudomycin A inhibits proton extrusion from maize roots, promotes closure of stomata in Vicia faba, necrosis of tobacco leaves, haemolysis of human erythrocytes, affects H(+)-ATPase activity and proton translocation in plasma membrane vesicles, and stimulates succinate respiration in pea mitochondria. In general, the biological activities of pseudomycin A are lower than those of syringomycin-E, the prototype member of this family of bacterial metabolities. This difference might depend on the diverse number and distribution of charged residues in the peptide moiety of these compounds.