María del Mar Aguilera-Luiz

Multi-residue determination of veterinary drugs in milk by ultra-high-pressure liquid chromatography-tandem mass spectrometry.

A simple, selective and fast multi-residue method was developed to determine 18 veterinary drugs in milk by ultra-high-pressure liquid chromatography coupled to tandem quadrupole mass spectrometry. The selected antibiotics include quinolones, sulphonamides, macrolides, anthelmintics and one tetracycline. An extraction procedure based on QuEChERS methodology (quick, easy, cheap, effective, rugged and safe), consisting of a liquid extraction of the milk samples with acetonitrile without sample clean up was performed. The extract was centrifuged and the supernatant was filtered prior to chromatographic analysis. Several chromatographic conditions were optimized in order to obtain a fast separation (less than 10 min). The antibiotics were detected by electrospray ionization in positive ion mode with multiple reaction monitoring (MRM) and mass spectrometric conditions were optimized in order to increase selectivity and sensitivity. The developed method was validated in terms of linearity, trueness, precision, limits of detection (LODs) and quantification. Mean recoveries ranged from 70% to 110% and interday precision was lower than 21%. LODs ranged from 1 to 4 microg/kg. Finally, the method was applied to real samples and only traces of tylosin and fenbendezol were detected in two samples.

Comparison of several extraction techniques for multiclass analysis of veterinary drugs in eggs using ultra-high pressure liquid chromatography-tandem mass spectrometry.

This study compared four extraction methods for the simultaneous determination of tetracyclines, macrolides, quinolones, sulphonamides and anthelmintics (including benzimidazoles and avermectins) in eggs by ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Solvent extraction, solid-phase extraction (SPE), matrix solid-phase dispersion (MSPD) and modified QuEChERS procedure were compared in terms of recovery and number of veterinary drugs extracted. The solvent extraction procedure with a clean-up step provided better results than the other tested procedures. The QuEChERS procedure was simpler and faster, but extracted fewer compounds than solvent extraction. MSPD did not extract tetracyclines and quinolones, whereas macrolides and tetracyclines were not extracted when SPE was applied. The solvent extraction procedure was validated, obtaining recoveries ranging from 60% (sulfaquinoxaline) to 119% (levamisole) with repeatability values (expressed as relative standard deviations, RSDs) lower than 20% at two concentration levels (10 and 100 microg kg(-1)), except for erythromycin, emamectin and ivermectin that showed RSD values close to 25% at 10 microg kg(-1). Limits of quantification (LOQs) were always equal or lower than 5 microg kg(-1). Finally the method was applied to egg samples, and erythromycin, enrofloxacin, difloxacin, thiabendazole, emamectin and fenbendazole were detected in four samples.

Multiclass analysis of antibiotic residues in honey by ultraperformance liquid chromatography-tandem mass spectrometry.

A method has been developed and validated for the simultaneous analysis of different veterinary drug residues (macrolides, tetracyclines, quinolones, and sulfonamides) in honey. Honey samples were dissolved with Na(2)EDTA, and veterinary residues were extracted from the supernatant by solid-phase extraction (SPE), using OASIS HLB cartridges. The separation and determination was carried out by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), using an electrospay ionization source (ESI) in positive mode. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two ion transitions per compound to provide a high degree of sensitivity and specificity. The method was validated, and mean recoveries were evaluated at three concentration levels (10, 50, and 100 microg/kg), ranging from 70 to 120% except for doxycycline, erythromycin, and tylmicosin with recovery higher than 50% at the three levels assayed. Relative standard deviations (RSDs) of the recoveries were less than 20% within the intraday precision and less than 25% within the interday precision. The limits of quantification (LOQs) were always lower than 4 microg/kg. The developed procedure was applied to 16 honey samples, and erythromycin, sarafloxacin, and tylosin were found in a few samples.

Application of conventional solid-phase extraction for multimycotoxin analysis in beers by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

A new analytical method has been developed and validated for the simultaneous analysis of mycotoxins (aflatoxins B1, B2, G1, G2, and M1, fumonisins B1 and B2, deoxynivalenol, ochratoxin A, HT-2 and T-2 toxins, and zearalenone) in beers. Mycotoxins were extracted by solid-phase extraction (SPE) using C18 as the cartridge. Several parameters such as type of sorbent, elution solvent, and dilution of the sample were evaluated. The separation and determination were carried out by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was validated, and mean recoveries ranging from 70 to 106% were obtained. Repeatability and intermediate precision, expressed as relative standard deviations, were lower than 21% for all mycotoxins and levels assayed. The limits of quantification were lower than 0.5 microg/L. The developed method has been applied for the analysis of several types of beers with different alcoholic content (nonalcoholic, normal, and special), and T2, HT-2 toxins, aflatoxin B1, and fumonisin B2 were detected. This methodology combines the simplicity of SPE using conventional cartridges and UHPLC-MS/MS, producing a rapid, sensitive, and reliable procedure.

Food contaminant analysis at high resolution mass spectrometry: Application for the determination of veterinary drugs in milk

Veterinary drugs (VDs) can remain in milk as a consequence of their use in livestock. In order to control the levels of VD residues in milk, screening methodologies can be applied for a rapid discrimination among negative and non-negative samples. In a second stage, non-negative samples are classified as negative or positive samples by using a confirmation method. Pre-target screening methods in low resolution MS (LRMS) are normally applied, but the number of analytes is limited, whereas the information obtained by full scan acquisition in high resolution mass spectrometry (HRMS) is improved. Here, three screening methods (running time<4 min) based on Orbitrap, quadrupole-time of flight (QqTOF) and triple quadrupole (QqQ) have been compared, using in all cases ultra-high performance liquid chromatography (UHPLC). For HRMS, the identification of the VDs was based on retention time (RT) and accurate mass measurements. Confirmation was based on the monitoring of fragments generated without precursor selection. The performance characteristics of the screening method provided reliable information regarding the presence or absence of the compounds below an established value, including uncertainty region and cut-off values. Better results in terms of cut-off values (≤ 5.0 μg kg(-1), except for spiramycin with a cut-off of 13.4 μg kg(-1) for milk samples and 43.1 μg kg(-1) for powdered milk based, emamectin with a cut-off of 42.2 μg kg(-1) for milk samples and doxycycline, with a cut-off value of 15.8 μg kg(-1) in powdered milk-based infant formulae) and uncertainty region were obtained using the Orbitrap-based screening method, which was submitted to further validation and used to analyze different real milk samples. The proposed method can be used in routine analysis, providing reliable results.

Determination of ochratoxin A and T-2 toxin in alcoholic beverages by hollow fiber liquid phase microextraction and ultra high-pressure liquid chromatography coupled to tandem mass spectrometry

A new method for the determination of ochratoxin A and T-2 toxin in alcoholic beverages (wine and beer) by hollow fiber liquid microextraction was optimized. The extraction step was followed by ultra high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The extraction procedure was based on the extraction of mycotoxins from the sample to the organic solvent (1-octanol) immobilized in the fiber, and afterwards, they were desorbed in a mixture of acetonitrile/water (80:20, v/v) at pH 7 prior to chromatographic determination. Different variables affecting the extraction process such as organic solvent, salt content, extraction time and desorption solution were studied. The developed method was validated in wine and beer, using white wine and alcoholic beer as representative matrices for both types of samples. Relative recoveries higher than 70% were obtained for the selected mycotoxins. Good linearity (R(2)>0.993) was obtained and quantification limits (0.02-0.09 microg L(-1)) below European regulatory levels were achieved. Repeatability, expressed as relative standard deviation, was always lower than 12%, whereas interday precision was lower than 21%. The proposed method was applied to the analysis of several types of wines and beers and ochratoxin A was detected in a rosé wine at 1.1 microg L(-1).

Rapid and semiautomated method for the analysis of veterinary drug residues in honey based on turbulent-flow liquid chromatography coupled to ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (TFC-UHPLC-Orbitrap-MS).

A simple and rapid method is described for the determination of veterinary drug (VD) residues in honey samples using turbulent flow chromatography coupled to ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (TFC-UHPLC-Orbitrap-MS). Honey samples were diluted with an aqueous solution of Na(2)EDTA (0.1 M). Then, they were injected into the chromatographic system including a TFC column. Afterward, the analytes were transferred to an UHPLC analytical column, where they were determined by UHPLC-Orbitrap-MS. Mean recoveries were obtained at three concentration levels (5, 10, and 50 μg/kg), ranging from 68 to 121% for most compounds. Repeatability (intraday precision) and interday precision (expressed as relative standard deviation, RSD) were <25% for most compounds. Limits of quantification (LOQs) ranged from 5 to 50 μg/kg and limits of identification (LOIs) from 0.1 to 50 μg/kg. The developed method was applied in honey samples, and it was fast and nonlaborious.

Fast analysis of polyphenols in royal jelly products using automated TurboFlow™-liquid chromatography-Orbitrap high resolution mass spectrometry.

This study describes the development of a novel, simple and fast analytical method for the detection and quantification of polyphenols in royal jelly products, using an in-house database containing more than 50 compounds. The extraction method consisted of sample dilution, followed by a fast on-line system composed of turbulent flow chromatography (TurboFlow™) coupled to liquid chromatography (LC)-Exactive-Orbitrap analyzer. The total run time was 18min, including automated extraction, analytical chromatography and re-equilibration. The method was validated obtaining limits of quantification (LOQ) ranging from 10 to 150μg/kg. The linearity range was up to 2000μg/L and determination coefficients (R2) were higher than 0.994. Adequate recoveries were obtained at three concentration levels (500, 1000 and 2000μg/kg). This method was applied to the analysis of nine samples and the concentration of polyphenols ranged from 14 (apigenin) to 18,936μg/kg (ferulic acid).