Luca Palleschi

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.

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.

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.

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.

New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry.

A new approach using combined liquid chromatography-mass spectrometry (LC-MS) with ionspray ionization is proposed for the direct detection of known and new toxins in mussels and phytoplankton. A first stage reversed-phase, negative ion mode, selected ion monitoring (SIM) LC-MS analysis was performed in order to detect DSP toxins in the same chromatographic run with a total run time of 20 min. The toxins analysed included yessotoxin (YTX), okadaic acid (OA) and four of its analogues, dinophysistoxins (i.e. DTX-1, DTX-2, DTX-2B, DTX-2C), and pectenotoxins (PTXs), involving PTX-2, two PTX-2 secoacids (PTX-2SAs), PTX-2SA, 7-epi-PTX-2SA, and AC1, the three isomeric toxins structurally related to PTX-2 recently identified in Irish phytoplankton. Positive samples can, therefore, be analyzed through reversed-phase, positive ion mode SIM LC-MS, in order to perform complete chromatographic separations of the structurally related toxins within the OA and PTX groups. Detailed toxin profiles of a number of toxic phytoplankton and shellfish, from different marine areas, were easily obtained through the new approach. PTX-2SAs and AC1 were found in phytoplankton and shellfish from Ireland as well as in Italian shellfish. Moreover, for the first time there was evidence of the presence of PTX-2 in Irish phytoplankton. YTX was present in Italian shellfish. Four isomeric OA toxins were detected in samples from Ireland with OA, DTX-2 and DTX-2B present in shellfish, and OA, DTX-2 and DTX-2C in phytoplankton. In contrast, OA was the only toxin from this group to be detected in Italian mussels.

Improved ion chromatography-integrated pulsed amperometric detection method for the evaluation of biogenic amines in food of vegetable or animal origin and in fermented foods

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid-liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.

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.

Quantification of 17β-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.

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.

Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry

Abstract A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for confirmatory analysis of 17β-boldenone (17β-BOL), 17α-boldenone (17α-BOL) and androsta-1,4-diene-3,17-dione (ADD) in bovine urine was developed. [ 2 H 2 ]17β-Testosterone (17β-T-d 2 ) was used as the internal standard. Sample preparation involved enzymatic hydrolysis and purification on a C 18 solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C 18 HPLC column. LC–MS–MS detection was carried out with an atmospheric pressure chemical ionisation (APCI) source equipped with a heated nebulizer (HN) interface operating in the positive ion mode. For unambiguous hormone confirmation, three analyte precursor–product ion combinations were monitored during multiple-reaction monitoring (MRM) LC–MS–MS analysis. Overall recovery (%), repeatability (relative standard deviations, RSD, %) and within-laboratory reproducibility (RSD, %) ranged from 92.2 to 97.7%, from 6.50 to 2.94% and from 13.50 to 5.04%, respectively, for all analytes. The limit of quantification in bovine urine was 0.20 ng ml −1 for 17β-BOL and ADD and 0.50 ng ml −1 for 17α-BOL. The validated method was successfully applied for determination of 17β-BOL, 17α-BOL and ADD in a large number of bovine urine samples collected within the national Official Residue Control Program.