The World Anti-Doping Agency (WADA) distinguishes between three forbidden doping methods and eleven classes of prohibited substances. The concentration of a substance that laboratories are expected to detect on a day-to-day basis is the minimum required performance limit (MRPL), which is established by WADA. Since the different classes of doping substances cover over 200 chemically and pharmacologically different compounds, developing high-throughput chromatographic–mass spectrometric methods to screen such a large set of compounds in a single method is a great analytical challenge.
High-resolution, accurate-mass mass spectrometry based on Orbitrap technology offers for the first time an efficient and easy-to-use solution for the challenge at hand. The high resolution of up to 140,000 FWHM enables separation of drugs from interferences. Fast positive/negative switching catches acidic, basic and neutral drugs. Better than 3 ppm mass accuracy assures confidence in identification, and fragment ions from the HCD cell further confirm the identity beyond a reasonable doubt. All of this is made simple by using Thermo Scientific TraceFinder and ExactFinder processing software designed specifically for this purpose.
Here we describe application of the Orbitrap technology-based workflow solutions for screening and quantitation in urine.
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Sample Preparation
Sample Preparation Workflow for Human Urine Screening
The sample preparation for forensic screening for more than 300 drugs in urine involved a simple dilute and shoot approach.
Literature Highlights
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Meyer MR, Du P, et al.
J Mass Spectrom. 2010 Dec;45(12):1426-42.
Schwaninger AE, Meyer MR, et al.
J Mass Spectrom. 2011 Jul;46(7):603-14.
Mass spectrometric detection of siRNA in plasma samples for doping control purposes.
Kohler M, Thomas A, et al.
Anal Bioanal Chem. 2010 Oct;398(3):1305-12.
Mass Spectrometry
Mass Spectrometry Workflow for Human Urine Screening
The Thermo Scientific Exactive Plus mass spectrometer with a HESI source is coupled to a Thermo Scientific Accela 1250 UHPLC system. The MS was operated in Full Scan MS mode at a resolution of 70,000 FWHM at m/z 200 with alternate positive/negative switching and all ion fragmentation during the entire LC run. The column eluent was diverted to waste after the sample injection for a specified period of time before being sent to the MS. The Exactive Plus™ MS was calibrated at the beginning of the day and used for the entire analysis. No lock mass or internal calibration was used.
Literature Highlights
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Meyer MR, Du P, et al.
J Mass Spectrom. 2010 Dec;45(12):1426-42.
Schwaninger AE, Meyer MR, et al.
J Mass Spectrom. 2011 Jul;46(7):603-14.
Mass spectrometric detection of siRNA in plasma samples for doping control purposes.
Kohler M, Thomas A, et al.
Anal Bioanal Chem. 2010 Oct;398(3):1305-12.
Data Analysis
Data Analysis Workflow for Human Urine Screening
Thermo Scientific ExactFinder software, which supports screening using high-resolution MS systems, was used for automated processing and reporting. The identification method for targeted screening makes use of exact m/z, retention time and fragment ion matching.
Literature Highlights
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Meyer MR, Du P, et al.
J Mass Spectrom. 2010 Dec;45(12):1426-42.
Schwaninger AE, Meyer MR, et al.
J Mass Spectrom. 2011 Jul;46(7):603-14.
Mass spectrometric detection of siRNA in plasma samples for doping control purposes.
Kohler M, Thomas A, et al.
Anal Bioanal Chem. 2010 Oct;398(3):1305-12.
overview
Workflow Overview for Human Urine Screening
Screening of a wide range of drugs in urine is challenging due to the presence of many similar molecules in the sample, the matrix interference, and the significant sample-to-sample differences due to the varying nature of the matrix. For faster analysis time, very high resolution, accurate-mass measurements and fast polarity switching are necessary to identify the drugs and metabolites.
The workflow described here supports simple dilute-and-shoot, solid-phase extraction (SPE), liquid-liquid extraction (LLE), and online sample extraction methods using Thermo Scientific TurboFlow technology for a large number of drugs and metabolites. It uses simple full-scan MS at 100,000 FWHM resolution in alternating positive/negative polarity and HCD fragmentation. The MS and fragmentation data acquired in Full Scan mode is processed by Thermo Scientific TraceFinder or ExactFinder software. The software supports both targeted and unknown screening. It does a very thorough interrogation of the data by making use of the built-in database and mass spectral library of over 1400 compounds, retention times, isotope pattern matching, elemental composition determinations and ChemSpider searches to identify and confirm drugs and metabolites in the sample.
Literature Highlights
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Meyer MR, Du P, et al.
J Mass Spectrom. 2010 Dec;45(12):1426-42.
Schwaninger AE, Meyer MR, et al.
J Mass Spectrom. 2011 Jul;46(7):603-14.
Mass spectrometric detection of siRNA in plasma samples for doping control purposes.
Kohler M, Thomas A, et al.
Anal Bioanal Chem. 2010 Oct;398(3):1305-12.
Thermo Fisher Scientific
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