Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

Ádám Tölgyesi, Virender K. Sharma, Loréna Kovacsics, J. Fekete

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Abstract

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.

Original languageEnglish
Pages (from-to)1471-1479
Number of pages9
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume878
Issue number19
DOIs
Publication statusPublished - Jun 1 2010

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Solid Phase Extraction
Liquid chromatography
Reverse-Phase Chromatography
Tandem Mass Spectrometry
Mass spectrometry
Adrenal Cortex Hormones
Urine
Flumethasone
Triamcinolone
Methylene Chloride
Mass spectrometers
Ion sources
Prednisolone
Organic solvents
Atmospheric pressure
Ionization
Methanol
Cleaning
Signal to noise ratio
Buffers

Keywords

  • Corticosteroid
  • Ion-suppression
  • Liquid chromatography-tandem mass spectrometry
  • Urine
  • Validation

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Cell Biology
  • Clinical Biochemistry

Cite this

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title = "Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry",
abstract = "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.",
keywords = "Corticosteroid, Ion-suppression, Liquid chromatography-tandem mass spectrometry, Urine, Validation",
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T1 - Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

AU - Tölgyesi, Ádám

AU - Sharma, Virender K.

AU - Kovacsics, Loréna

AU - Fekete, J.

PY - 2010/6/1

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N2 - 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.

AB - 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.

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