SANIST: optimization of a technology for compound identification based on the European Union directive with applications in forensic, pharmaceutical and food analyses.

Electrospray Ionization and collision induced dissociation tandem mass spectrometry are usually employed to obtain compound identification through a mass spectra match. Different algorithms have been developed for this purpose (for example the nist match algorithm). These approaches compare the tandem mass spectra of the unknown analyte with the tandem mass spectra spectra of known compounds inserted in a database. The compounds are usually identified on the basis of spectral match value associated with a probability of recognition. However, this approach is not usually applied to multiple reaction monitoring transition spectra achieved by means of triple quadrupole apparatus, mainly due to the lack of a transition spectra database. The Surface Activated Chemical Ionization-Electrospray-NIST Bayesian model database search (SANIST) platform has been recently developed for new potential metabolite biomarker discovery, to confirm their identity and to use them for clinical and diagnostic applications. Here, we present an improved version of the SANIST platform that extends its application to forensic, pharmaceutical, and food analysis studies, where the compound identification rules are strict. The European Union (EU) has set directives for compound identification (EU directive 2002/657/EC). We have applied the SANIST method to identification of 11-nor-9-carboxytetrahydro-cannabinol in urine samples (an example of a forensic application), circulating levels of the immunosuppressive drug tacrolimus in blood (an example of a pharmaceutical application) and glyphosate in fruit juice (an example of a food analysis application) that meet the EU directive requirements. Copyright © 2016 John Wiley & Sons, Ltd.

Screening and confirmatory method for multiclass determination of 62 antibiotics in meat.

A multiclass method for screening and confirmatory analysis of antimicrobial residues in muscle has been developed and validated, according to Commission Decision 2002/657/EC. Sixty-two antibiotics belonging to ten different drug families (amphenicols, beta-lactams, diamino-pyrimidine, lincosamides, macrolides, pleuromutilins, quinolones, rifamycins, sulfonamides and tetracyclines) have been included in the method. After the addition of an aqueous solution of EDTA, the minced muscle was extracted with acetonitrile/water mixture and, later, with pure acetonitrile. The extract was evaporated and redissolved in ammonium acetate buffer prior to LC injection. Instrumental determination was performed by liquid chromatography coupled to hybrid high resolution mass analyser (LC-HRMS/MS) operating in positive electrospray ionization mode. Chromatographic separation was optimized on a Poroshell 120 EC-C18 column (100 × 3.0 mm, 2.7 µm) with gradient using methanol and water containing 0.1% of formic acid as mobile phases. The method was validated in bovine muscle in the range 3.3-150 µg kg(-1) for all antibiotics; for some compounds with MRL higher than 100 µg kg(-1), the validation interval has been extended until 1500 µg kg(-1). The studied performance characteristics were selectivity, linearity, precision, trueness (recovery), decision limits, detection capabilities, detection and quantification limits. Satisfactory quantitative performances were obtained for all the analytes. Ruggedness tests demonstrated the applicability to swine and poultry muscle, too. Finally the wide participation in proficiency tests allowed to investigate in deep the method performances.

Determination of α- and ß-boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis.

The debate about the origins of boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-boldenone conjugate, is considered fully compliant up to 2 ng mL(-1). The conjugated form of ß-boldenone must be absent. In recent years, the literature about boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. ß-boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of ß-boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-boldenone sulfate and glucuronide, α- and ß-boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08 ng mL(-1), the detection capabilities (CCß) between 0.08 and 0.1 ng mL(-1). Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of boldenone in a trial on a significant number of animals.

Investigation on the origin of prednisolone in urine and adrenal glands of cows.

Prednisolone is a steroid belonging to the corticosteroid group. The results obtained in the application of the 2008 and 2009 Italian Residue Control Plans show the frequent detection of prednisolone traces in cow's urine. Since most of the positive samples were detected at the slaughterhouse, the researchers hypothesised that, together with an increase of cortisol concentration, traces of prednisolone could be produced endogenously during stressful situations due to transport and handling before slaughter. In the present trial, 52 lactating cows housed in seven different farms in Lombardy, Italy, were studied. Urine samples were collected at the farm (after urethral catheterisation) and immediately after slaughter (from urinary bladder) together with 40 adrenal gland samples belonging to the same animals. All the samples were analysed for the determination of prednisolone and cortisol by LC/MS(n). The results demonstrated that prednisolone can be endogenously produced in dairy cows and, furthermore, its endogenous presence in bovine urine seems to be strongly related to a state of stress in the animals (at the farm and at the slaughterhouse). The data from adrenal glands do not, however, clarify if the endogenous production occurs, partially or totally, in this organ.

Determination of macrocyclic lactones in food and feed.

A confirmatory high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the simultaneous determination of the following anti-parasitic veterinary drugs in foodstuffs (liver, muscle and milk) and feed: abamectin, doramectin, emamectin, eprinomectin, ivermectin and moxidectin. Samples were extracted, purified and analysed by HPLC-FLD after prior derivatisation of analytes with N-methylimidazole, trifluoracetic anhydride and acetic acid to produce stable fluorescent derivatives. Recoveries were in the range of 73-98% (liver), 82-93% (muscle), 77-84% (milk), and 93-110% (feed). In all matrices the coefficients of variation for repeatability and intra-laboratory reproducibility were satisfactory. For emamectin in liver the worst method performance was observed. As this multi-analyte method fully met the required criteria and applicability both to food and feed, it has been successfully applied in National Control Plans in food and feed.

Analytical investigations about the presence of prednisolone in cow urine.

Since 2008, the analyses carried out in the Lombardia region as part of National Residue Control Plans have evidenced unexpected frequent detection of the corticosteroid prednisolone (PRED) in cow urine samples taken to the slaughterhouse. Considering the scarce plausibility of these high frequent findings, analytical investigations were started to ascertain the real presence of this corticosteroid. The applied confirmatory method involved liquid-chromatography low-resolution tandem mass spectrometry (triple quadrupole) as instrumental technique, and it was validated in compliance with the requirements of the Commission Decision 2002/657/EC. However, recently some criticism regarding Commission Decision 2002/657/EC identification criteria has been pointed out, experimentally demonstrating false positive results (wrong identification) although these criteria have been strictly observed. Therefore, considering the serious implications (i.e. the possibility that PRED could be considered endogenous in particular animal conditions), studies were carried out to investigate the reliability of PRED identification through the change of the chromatographic conditions (mobile phases, gradient and analytical column) of the confirmatory procedure routinely applied. Further confirmation came from the application of high-resolution mass spectrometry technique (MS(2) and MS(3) experiments) to analyze incurred cow urines samples. All the obtained results confirmed definitively the real presence of this corticosteroid excluding false-positive findings in routine analysis. In addition, other experiments demonstrated that high-resolution mass spectrometers (Time of Flight and Orbitrap technologies) could be successfully applied to routine determination of steroid residues in biological fluids at very low concentrations (< 1 µg L(-1)).

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of nine corticosteroid residues in bovine liver samples.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory method for the simultaneous determination of nine corticosteroids in liver, including the four MRL compounds listed in Council Regulation 37/2010, was developed. After an enzymatic deconjugation and a solvent extraction of the liver tissue, the resulting solution was cleaned up through an SPE Oasis HLB cartridge. The analytes were then detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry, using deuterium-labelled internal standards. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results showed that the method was suitable for statutory residue testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCß) and ruggedness. All the corticosteroids can be detected at a concentration around 1 µg kg(-1); the recoveries were above 62% for all the analytes. Repeatability and reproducibility (within-laboratory reproducibility) for all the analytes were below 7.65% and 15.5%, respectively.

Development and validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs.

A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5-1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2-174 µg kg(-1), depending on the coccidiostat). The repeatability (CV(r) in the range of 1.1-19%) and within-laboratory reproducibility (CV(Rw) in the range of 1.8-30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.

Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography-tandem mass spectrometry.

A confirmatory method for the simultaneous determination of nandrolone (alpha and beta) and trenbolone (alpha and beta) in urine samples by liquid chromatography electrospray mass spectrometry (LC-MS-MS) was developed. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography-positive ion electrospray tandem mass spectrometry using deuterium labelled internal standards. The analytical procedure was applicable to bovine and swine urine samples. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results obtained showed that the method was suitable for statutory residues testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCalpha), detection capability (CCbeta) and ruggedness. The decision limits (CCalpha) obtained, were between 0.54 and 0.60 microg L(-1); the recovery was above 64% for all the analytes. Repeatability was between 1.6% and 5.7% and within-laboratory reproducibility between 1.6% and 6.0% for all the steroids.

Development and validation of a confirmatory method for the determination of sulphonamides in milk by liquid chromatography with diode array detection.

A simple and rapid multiresidue method for the determination of seven sulphonamides residues (sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfadimethoxine and sulfaquinoxaline) in milk samples was developed and validated. The drugs were extracted with a mixture chloroform/acetone and simply cleaned up on a cation exchange solid phase extraction column. The analytes determination was carried out using liquid chromatography with diode array detection (DAD). The procedure has validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The developed method shows good linearity, specificity, precision (repeatability and intra-laboratory reproducibility), ruggedness and is able to confirm each sulphonamide residue above 30mugkg(-1). Decision limits (CCalpha) around 110mugkg(-1) and recovery above 56% were obtained for all the analytes. The results of the validation process demonstrate that the method is suitable for application, as confirmatory method, in European Union statutory veterinary drug residue surveillance programmes. In addition, a hypothetical situation of sample judgement (compliance or not) in the case in which, at the same time, two different sulphonamides are found, is discussed.

Confirmatory method for the determination of resorcylic acid lactones in urine sample using immunoaffinity cleanup and liquid chromatography-tandem mass spectrometry.

The presence of zeranol (alpha-zearalanol) in urine samples due to natural contamination or illegal treatment is under debate within the European Union. The simultaneous determination of zeranol, its epimer taleranol (beta-zearalanol), zearalanone and the structurally related mycotoxin zearalenone with the corresponding alpha- and beta-zearalenol metabolites appears to be critical in deciding whether an illegal use has occurred. The aim of this study is to develop and validate a simple analytical procedure applicable to bovine and swine urine samples for the determination of all six resorcylic acid lactones. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography-negative-ion electrospray tandem mass spectrometry using deuterium-labelled internal standards. The method was validated as a quantitative confirmatory method according to European Commission Decision 2002/657/EC. The evaluated parameters were: linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit, detection capability and ruggedness. The decision limits (CCalpha) obtained, were between 0.56 and 0.68 microgL(-1); recovery above 66% for all the analytes. Repeatability was between 1.4 and 5.3% and within-laboratory reproducibility between 1.9 and 16.1% for the six resorcylic acid lactones.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the separation of conjugated and unconjugated 17alpha- and 17beta-boldenone in urine sample.

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17alpha-boldenone (alpha-BOLD) and 17beta-boldenone (beta-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid-liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC-MS-MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17beta-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCalpha) smaller than 0.5 ng mL(-1) were obtained for each analyte.

Rapid determination of the antiparasitic drugs flubendazole and febantel in feeds by HPLC with ultraviolet detection.

A rapid and very effective analytical procedure for the simultaneous HPLC determination of two anthelmintics, Flubendazole (FLUB) and Febantel (FEBA), in swine and poultry feeds was developed and tested. The ground feed samples were extracted using dimethylformamide. The extracts were directly analyzed, without any purification, on a reversed-phase ODS column (150 mm x 4.6 mm, 5 microm) with acetonitrile-phosphate buffer (pH 3; 0.017 M) as mobile phase. Ultraviolet detection of FLUB and FEBA was carried out at 300 nm. The method was validated for specificity, linearity, accuracy, repeatability, limit of detection and limit of quantification. The limits of detection (LOD) of FLUB and FEBA in feeds, based on a detector signal-to-noise ratio of 3, were 3 mg kg(-1) and the lowest levels tested in feeds by this procedure (limit of quantification, LOQ) were 10 mg kg(-1). The mean recovery of FLUB and FEBA from spiked samples, in a concentration range of 10-200 mg kg(-1), was 98% with a CV% of 4% and 99% with a CV% of 2%, respectively.