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Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS

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Abstract

A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single 13C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d9-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g−1) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized 13C1-clenbuterol and a commercially available d9-clenbuterol. The detection limit of the method in human urine was 0.050 ng g−1 with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the 13C1-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD.

A procedure for the determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry has been developed. In this method IDMS is employed in combination with minimal labelling and isotope pattern deconvolution.

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Acknowledgements

The authors are grateful for financial support from the Spanish Ministry of Science and Innovation through project refs. CTQ2009-12814 and CTQ2007-61126/PPQ. A.G.-A. acknowledges her doctoral grant to FICYT (Asturias). P.R.-G. and I.L. acknowledge their research contracts from the Spanish Ministry of Science and Innovation through the Ramón y Cajal Program.

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Authors and Affiliations

  1. Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería 8, 33006, Oviedo, Spain

    Ana González-Antuña, Pablo Rodríguez-González & J. Ignacio García Alonso

  2. Department of Organic and Inorganic Chemistry, Faculty of Chemistry, Instituto Universitario de Biotecnología de Asturias, University of Oviedo, Julián Clavería 8, 33006, Oviedo, Spain

    Iván Lavandera & Vicente Gotor

  3. ISC-Science, Innovative Solutions in Chemistry, S.L. Edificio Científico-Tecnológico Campus de “El Cristo”, 33006, Oviedo, Spain

    Giuseppe Centineo

Authors
  1. Ana González-Antuña
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  2. Pablo Rodríguez-González
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  3. Iván Lavandera
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  4. Giuseppe Centineo
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  5. Vicente Gotor
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  6. J. Ignacio García Alonso
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Correspondence to J. Ignacio García Alonso.

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González-Antuña, A., Rodríguez-González, P., Lavandera, I. et al. Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS. Anal Bioanal Chem 402, 1879–1888 (2012). https://doi.org/10.1007/s00216-011-5611-1

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  • DOI: https://doi.org/10.1007/s00216-011-5611-1

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