The National Institute of Health and the Italian Poison Centers Network: results of a collaborative study for the surveillance of exposures to chemicals.

Background: The Public Health Surveillance Systems are essential to improve and protect public health, as highlighted by the World Health Organization. According with this consideration, a systematic collaboration between the National Institute of Health and the Poison Centers of Northern, Central and Southern Italy was established. Its aim was to improve the national network for the surveillance of dangerous exposures to chemicals. The developed network provided harmonized data essential for evidence-based interventions and significantly ameliorated the data flow between the Poison Centers and the Central Health Institutions. Methods: The improvement of the system was obtained through several actions, such as the development of the "Online Surveillance Card" for the detection of sentinel events in real time and the harmonization of the data collection flow, including the product categorization according to the European Product Categorization System. Data analysis was carried out by Microsoft's IBM SPSS Statistics version 26, Access and Excel. Results: Important information was obtained, regarding also exposures to chemicals and their management in pediatric populations. The surveillance network was proved effective not only under "normal" conditions but also to promptly monitor changes during exceptional health emergencies, such as the COVID-19 pandemic. During the 2020 lockdown the surveillance system registered a significant increase in the frequency of exposures to disinfectants (p-value=0.002), an evidence that highlighted the need of tailored intervention. Conclusions: This Italian Project model proves to be reliable and suitable to be transferred to other European countries, in order to realize an European Poison Centers' Network, able to overcome unsolved health problems and to globally improve the "evidence-based" prevention of exposures to chemicals.

Insights into possibilities for grouping and read-across for nanomaterials in EU chemicals legislation.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

Quantification of natural and synthetic glucocorticoids in calf urine following different growth-promoting prednisolone treatments.

Over the last few years, low levels of prednisolone have been reported in several cattle urine samples by a number of laboratories within the EU at an average concentration of 2.0 ng mL(-1). The occurrence of prednisolone residues together with increased levels of hydrocortisone and cortisone in urine and tissue samples of untreated animals seems to demonstrate that traces of this steroid can be produced endogenously during stressful situations. Therefore, the endogenous origin of prednisolone makes difficult to correlate positive samples to a potential illicit treatment. An experimental study was developed to investigate the presence of natural and synthetic glucocorticoids and to evaluate levels of excreted prednisolone following growth-promoting treatments. Urine samples from calves undergone oral treatment with prednisolone, alone and in association with dexamethasone, were analyzed by a LC-MS/MS method, validated according to the Commission Decision 2002/657/EC. We also investigated if urinary free 6ß-hydroxyhydrocortisone/hydrocortisone ratio could be a reliable biomarker of illicit treatment with prednisolone and dexamethasone in calves. Our data revealed that urinary levels of prednisolone after both oral prednisolone treatments, never exceeded the value of 1.1 ng mL(-1). Similar prednisolone levels were found in urine samples of untreated calves. Moreover the presence of 6ß-hydroxyhydrocortisone below the CCα value made possible to estimate the 6ß-hydroxyhydrocortisone/hydrocortisone ratio only in a very limited number of samples. Obtained data suggest that further criteria have to be considered to allow correct decisions about the urinary presence of prednisolone during control activities.

Development of an enhanced histopathological approach to detect low-dose dexamethasone illicit treatment in veal calves.

Dexamethasone is one of a number of synthetic corticosteroids illegally used to promote growth in food-producing animals. Since these low-level drug cocktails evade detection by currently available chemical methods, simple biological indicators that can aid in laboratory analysis are needed. In an attempt to devise an accurate biological method that could detect illicit drug treatment in food-producing animals, we characterized microscopic morphologic alterations of the thymus in veal calves administered low-dose dexamethasone versus control animals. For this purpose, 122 male calves were farmed for 6 months in controlled condition: 81 animals were orally administered dexamethasone (0.4 mg day(-1)) for 20 days during the sixth month and the remaining 41 were kept as control. Urine samples were collected systematically during the treatment period, the suspension period and at the slaughterhouse. All animals were slaughtered 10 per day starting from 10 days after the last dexamethasone administration and the thymus was sampled for histological examination. The difference between the two animal groups was evaluated by means of a non-parametric test of hypothesis. No residues were detected in the urines collected since the third day after the last administration, whereas morphometric analysis of the thoracic thymus revealed a significant decrease in the cortex:medulla ratio in the treated animals (p<0.0005). We can conclude that this histological approach offers encouraging prospects as a screening method to overcome current limitations in controlling growth promoter abuse.

Boldenone, boldione, and milk replacers in the diet of veal calves: the effects of phytosterol content on the urinary excretion of boldenone metabolites.

Twenty-six veal calves were split into two groups and fed two milk replacers with a different content of phytosterols for 26 days; then, 14 calves (7 animals from each diet) were kept as controls and 12 calves (6 per diet) received daily, per os, a combination of 17beta-boldenone (17beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) for 38 days. The urinary elimination of 17 alpha-/17beta-boldenone conjugates (17 alpha/beta-Bol) and androsta-1,4-dien-3,17-dione (ADD) was followed by liquid chromatography-tandem mass spectrometry from all of the animals until slaughtering. In urine from treated animals, 17 alpha-Bol concentrations, despite a great variability, were greater than 17beta-Bol, both detected always as conjugates. At days 1, 2, and 3, the mean urine concentration of 17 alpha-Bol was higher than 12 ng/mL. A remarkable decrease was observed during the following days, but the 17 alpha-Bol concentration was still higher than the attention level of 2 ng/mL in 58% of the samples; the concentration of 17beta-Bol was around the action level of 1 ng/mL; two days after treatment withdrawal, no 17beta-Bol was detected in the urine. In urine from control animals, the 17 alpha-Bol concentration was strictly related to the phytosterol content of the diet, while, in urine from treated animals, the much higher 17 alpha-Bol levels were not modified by the production from diet precursors. The results confirmed that a 17 alpha-Bol level higher than 2 ng/mL should be considered as evidence of suspected illegal treatment and that the urinary excretion of 17beta-Bol is due to exogenous administration of 17beta-Bol. The discontinuous rate of elimination of both 17 alpha- and 17beta-Bol, despite the daily administration of 17beta-Bol plus ADD, indicates the necessity for further research to detect other urinary boldenone metabolites to strength surveillance strategy.

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.

Excretion profile of boldenone in urine of veal calves fed two different milk replacers.

The residue profiles of 17alpha-/17beta-boldenone conjugated (17alpha/beta-Bol) and ADD were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male veal calves fed two commercial milk replacers, with different content of cholesterol and phytosterols. The urine samples were collected within 4 h after feeding and further from all the animals. Detectable amounts of 17alpha-Bol conjugated were measured in urine collected from all calves, but the concentrations of 17alpha-Bol were higher in urine from calves receiving the milk replacer with the greater amount of phytosterols. During the whole experiment, 17beta-Bol and ADD were never detected in urine samples collected.

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.

Presence and metabolism of the anabolic steroid boldenone in various animal species: a review.

The review summarizes current knowledge on the possible illegal use of the anabolic steroid boldenone. The presence of' boldenone and metabolites in different animal species and the possibility of the occurrence of endogenous boldenone and metabolites is assessed, as are the methods of analysis used for detection. Different laboratories in the European Union have examined the occurrence of boldenone and its metabolites. The results were discussed at different meetings of a European Commission DG-SANCO Working Party) and summarized in an expert report. The situation of the different laboratories at this time is also covered herein. The overall conclusion of the Working Party was that there was a necessity for further research to distinguish between naturally occurring and illegally used boldenone forms. The confirmation of the presence of boldenone metabolites (free and conjugated forms) in certain matrices of animals is proposed as a marker for the illegal treatment with boldenone.

Analysis of erythromycin and tylosin in bovine muscle using disposable screen printed electrodes.

A disposable electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed using a screen printed electrode (SPE) system as a differential pulse voltammetry (DPV) transducer with mouse anti-erythromycin (and anti-tylosin) monoclonal antibodies (MAb) serving as molecular recognition elements. The immunochemical system makes use of the competition assay principle, and employs an erythromycin (or tylosin)-BSA conjugate as coating molecule. After competition between free and coated analyte for the antibodies, the activity of the alkaline phosphatase labelled antiglobulins was measured electrochemically using 1-naphthylphosphate as substrate. Using standard solutions of erythromycin and tylosin, the detection limit of the assay was 0.2 ng mL(-1) determined to be for erythromycin and 2.0 ng mL(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.0 ng mL(-1) for erythromycin and 3.0 ng mL(-1) for tylosin. The suitability of the assay for quantification of erythromycin and tylosin in bovine muscle was also studied. Spiked and real samples were analysed using the immunosensor system developed here. The ELISA showed precision values (relative standard deviation, RSD%) ranging from 4 to 9% for erythromycin and from 8 to 15% for tylosin; the accuracy (relative error, RE%) ranged from -11 to 6% and from -4 to 12% for erythromycin and tylosin, respectively. Results obtained on real samples were confirmed by micro-liquid chromatography coupled on line with tandem mass spectrometry (micro-LC-MS-MS), using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest.

Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection. Optimization and validation.

A high-performance liquid chromatography-diode array detection method (HPLC-DAD) combined with liquid chromatography-mass spectrometry was developed for the determination of enrofloxacin and its metabolite ciprofloxacin in goat milk. The HPLC-DAD method validation was compliant with the "DG SANCO 1805/2000" European regulation. The residues were extracted from milk with phosphate buffer, purified on a C18 Speedisk cartridge SPE (Baker) and then analysed using HPLC-DAD set at 277 nm. The decision limit (CCa) calculated by spiking samples at 100 microg/kg with both analytes, taking into account the maximum residue limit (MRL) of 100 microg/kg established by the European Union for the sum of enrofloxacin and its metabolite ciprofloxacin in milk, was 105.3 microg/kg for enrofloxacin and 105.5 microg/kg for ciprofloxacin. The detection capability (CCbeta) was 110.7 and 110.9 microg/kg for enrofloxacin and ciprofloxacin, respectively. The mean recoveries of the method, calculated by spiking samples at 50, 100 and 150 microg/kg were 84% for enrofloxacin and 88% for ciprofloxacin. The limit of quantification was 20 microg/kg for both analytes. The HPLC-DAD validated method was successfully applied for the first time in goats milk, and proved to be suitable for the sensitive and accurate quantification and confirmation analysis of enrofloxacin and ciprofloxacin for regulatory purposes.

Production of antibodies and development of highly sensitive formats of enzyme immunoassay for saxitoxin analysis.

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively. The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.

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.

Confirmatory method for macrolide residues in bovine tissues by micro-liquid chromatography-tandem mass spectrometry.

A new confirmatory method for three macrolides (tylosin, tilmicosin and erythromycin) in bovine muscle, liver and kidney by micro-LC-MS-MS using an atmospheric pressure ionisation source and an ionspray interface has been developed. Roxithromycin was used as internal standard. The molecular related ions, [M+2H]2+, at m/z 435 for tilmicosin, and [M+H]+, at m/z 734 and 916 for erythromycin and tylosin, respectively, were the precursor ions for collision-induced-dissociation and two diagnostic product ions for each macrolide were identified for the unambiguous confirmation by selected reaction monitoring LC-MS-MS. Precision values (relative standard deviations) were all below 14.9%, whereas the overall accuracy (relative error) ranged from -17.7 to -9.8% for tylosin, from -17.5 to -10.7% for tilmicosin and from -19.6 to -13.7% for erythromycin, in all the investigated bovine tissues. The limits of quantification were 30 (muscle) or 40 (liver, kidney) microg kg(-1), 20 (muscle) or 150 (liver, kidney) microg kg(-1), 50 (muscle, liver) or 80 (kidney) microg kg(-1), 20 (muscle, liver) or 50 (kidney) microg kg(-1) for tylosin, tilmicosin, erytromycin and roxithromycin, respectively.

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.

A new electrochemical enzyme-linked immunosorbent assay for the screening of macrolide antibiotic residues in bovine meat.

A new sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of two macrolides (erythromycin and tylosin) in bovine muscle was developed, using the mouse monoclonal antibodies anti-erythromycin and anti-tylosin. The competitive indirect assay was performed using an erythromycin (or tylosin)-BSA conjugate as a coating molecule; after competition between free and coated analytes for the antibodies, the activity of the horseradish peroxidase-labelled antiglobulins was measured electrochemically using 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. The detection limit of the assay was 0.4 ng ml(-1) for erythromycin and 4.0 ng ml(-1) for tylosin, while the sensitivity (25% inhibition concentration) was 1.4 ng ml(-1) for erythromycin and 13.0 ng ml(-1) for tylosin. The specificity of the assay was assessed by studying the cross-reactivity of various macrolides other than erythromycin and tylosin. The results indicate that the monoclonal antibodies anti-erythromycin and anti-tylosin can readily distinguish the target compound from other macrolides, with the exception of roxithromycin, a semisynthetic macrolide antibiotic derived from erythromycin. Fortified and real samples were analysed by the developed ELISA method and results confirmed by micro-LC-MS-MS using an atmospheric pressure ionisation (API) source and an ionspray (IS) interface. The latter provides unequivocal identification and quantification of the analytes at the level of interest. The ELISA assay showed precision (RSD) values ranging from 6.3 to 11.4% for erythromycin and from 7.5 to 12.6% for tylosin; the accuracy (relative error, RE) ranged from -16.0 to -9.8% and from -9.5 to 8.0% for erythromycin and tylosin, respectively. All results obtained demonstrate that the electrochemical ELISA is a suitable method for a sensitive, simple, rapid and reliable screening of the two macrolides in animal tissues.

Development of an electrochemical ELISA for the screening of 17 beta-estradiol and application to bovine serum.

A sensitive electrochemical enzyme-linked immunosorbent assay (ELISA) for the detection of 17 beta-estradiol (17 beta-E2) was developed. Optimisation of two ELISA competition assays, using monoclonal or polyclonal antibodies anti-17 beta-estradiol, coupled with the electrochemical detection was firstly performed. The activity of the label enzyme (horseradish peroxidase) was measured electrochemically using 3,3',5,5'-tetramethylbenzidine as substrate. The use of the polyclonal antibody resulted in a more sensitive assay and the detection limit of the assay was estimated to be 20 pg ml-1. The analytical performances of the method were compared to those obtained using a dissociation enhanced lanthanide fluorescence immunoassay (DELFIA). Although sample extraction is not usually required by DELFIA, both extracted and non extracted samples were assayed. The comparison between the two screening techniques revealed similar results for the extracted samples and showed a comparable precision (RSD%), ranging from 6.2 to 13.4 and from 6.7 to 14.3 for DELFIA and ELISA, respectively. The results obtained by these screening assays were confirmed by liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry which is currently used to confirm illegal hormone administration for regulatory purposes. The electrochemical enzyme immunoassay appears suitable as a screening tool for routine analysis of bovine serum estradiol and can be extended to other anabolic hormones using appropriate antibodies.

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC-APCI-MS-MS) for the quantitation of anabolic hormone residues (17beta-19-nortestosterone, 17beta-testosterone and progesterone) and their major metabolites (17alpha-19-nortestosterone and 17alpha-testosterone) in bovine serum and urine is reported. [2H2]17Beta-testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid-liquid extraction and purified by C18 solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]+, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC-MS-MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from -5.90 to -3.18% and from -6.40 to -2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.

Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry.

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.