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Tolgyesi T.,Food Toxicology National Reference Laboratory | Tolgyesi T.,Budapest University of Technology and Economics | Sharma V.K.,Florida Institute of Technology | Fekete J.,Budapest University of Technology and Economics
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2011

A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5. min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub μg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9. μg/kg for MRL compounds and varied from 0.1 to 0.2. μg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3. μg/kg. © 2010 Elsevier B.V. Source


Tolgyesi A.,Food Toxicology National Reference Laboratory | Tolgyesi L.,Eotvos Lorand University | Tolgyesi L.,Kromat Ltd. Co. | Sharma V.K.,Florida Institute of Technology | And 2 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2010

A new method was developed to identify and quantify corticosteroids (prednisolone, methylprednisone, flumetasone, dexamethasone, and methylprednisolone) in raw bovine milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing mixed-mode polymeric strong cation exchange and reversed-phase (MCX) solid-phase extraction (SPE) to reduce ion effects in a multimode ion (MMI) source. The main advantage of this method over other commonly used methods includes the use of a single SPE cartridge with a low volume for sample preparation and fast separation on the HPLC system with reduced ion suppression. This study is the first to report the determination of methylprednisone, a metabolite of methylprednisolone, in bovine milk. This method was validated in accordance with the European Union (EU) Commission Decision 2002/657/EC. The recoveries vary between 90% and 105%. The within-laboratory reproducibility (precision) is less than 30%. The decision limits and detection capabilities were calculated along with LODs, which ranged from 0.02 to 0.07 μg/kg. The method was further enhanced by its successful adaptation to other LC-MS/MS systems equipped with the newly developed ion source, Agilent Jet Stream (AJS). After optimization of the AJS ion source and MS parameters, even lower LOD values were achieved (0.001-0.006 μg/kg) for the corticosteroids. Analytical results obtained with the AJS were characterized by an enhanced area response and similar noise level comparable to those obtained with conventional orthogonal atmospheric ionization (API). © 2010 Elsevier B.V. Source


Tolgyesi T.,Food Toxicology National Reference Laboratory | Sharma V.K.,Florida Institute of Technology | Fekete J.,Budapest University of Technology and Economics
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

This paper describes a new liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the analysis of three stanozolol metabolites (16β-hydroxystanozolol, 3'-hydroxystanozolol, and 4β-hydroxystanozolol) in urines of animal origins. The solid-phase extraction (SPE) clean-up procedure was optimized to reduce the matrix effects in the LC-MS/MS analysis and to enhance recovery. Four different approaches were tested to prepare the sample, which include anion, and cation mixed-mode ion exchange, reversed-phase and normal-phase SPE cartridges. Mixed-mode anion exchange Strata-XL-A SPE column with diethyl ether elution yielded the best values. The separation of metabolites was optimized on Kinetex XB column using isocratic elution. The best mobile phase composition was achieved at the acidic pH with 0.1% (v/v) formic acid in water and methanol composition. The main advantages of the approach applied in the present study over other known methods include the single step SPE clean-up, relatively fast separation on HPLC column packed with core-shell particles, and lowering the limit of detection of target metabolites to the range between 0.05 and 0.15. μg/l. Additionally, the developed method was successfully validated for the first time for three species in accordance with the European Union (EU) 2002/657/EC decision. Finally, the efficiency of method was demonstrated by analyzing incurred samples. © 2013 Elsevier B.V. Source


Tolgyesi A.,Food Toxicology National Reference Laboratory | Sharma V.K.,Florida Institute of Technology | Kovacsics L.,Food Toxicology National Reference Laboratory | Fekete J.,Budapest University of Technology and Economics
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2010

This paper presents the development of a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0-9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC-MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1-1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L. © 2010 Elsevier B.V. Source


Tolgyesi A.,Food Toxicology National Reference Laboratory | Tolgyesi A.,Budapest University of Technology and Economics | Fekete J.,Budapest University of Technology and Economics | Fekete S.,Budapest University of Technology and Economics | And 3 more authors.
Journal of Chromatographic Science | Year: 2012

A fast liquid chromatographytandem mass spectrometry (LCMSMS) method is developed to determine lincomycin (LM) in honey, muscle, milk, and egg. Samples are cleaned-up at pH 4.7 using Strata-X-C mixed-mode polymeric strong cation exchange solid-phase extraction (SPE) cartridges, which could selectively adsorb the lincomycin from matrices under the acidic condition. LM is separated on the recently introduced Kinetex XB core-shell type HPLC column using isocratic elution mode with a mobile phase containing 0.1 formic acid in water/acetonitrile (93/7, v/v, pH 2.6) at a flow rate of 0.7 mL/min. The subsequent MS/MS detection has decreased ion effect, which allows the limit of detection (LOD) of LM for honey to be 0.05 g/kg for honey and 0.5 g/kg for muscle, milk, and egg. These LODs are much lower than those reported previously. The other main advantage of the developed method is the analysis time of only 3.5 min, which is about three times shorter than other reported LCMSMS methods. Recoveries varies between 94.2 and 125.2 and in-house reproducibility ranges from 3.7 to 28.7. The developed method is validated according to European Union (EU) Commission Decision 2002/657/EC using a matrix-comprehensive validation strategy. All studied analytical parameters fulfills the EU guidelines. © 2012 The Author. Source

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