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.
For additional resources, search the Orbitrap Science Library
Overview
Workflow Overview for Human Urine Quantitation
Currently, urine is a matrix of choice for quantitative analysis in human sports drug testing. Other sampling methods like dried blood spots (DBS) hold promise and have been reported in literature. The success of DBS sampling largely depends on the sensitivity of the LC-MS analysis. Many drugs and their metabolites are present in the sample in conjugated form and include isomers that need to be identified by separation using chromatography. The analytical method workflow must handle a wide range of polarities of many similar molecules, matrix interferences, wide dynamic ranges and significant sample-to-sample differences caused by the varying nature of the urine matrix.
Most quantitative analysis is done using a selected reaction monitoring (SRM) approach on a triple-stage quadrupole mass spectrometer. The SRM duty cycle limits monitoring, and quantitating large numbers of analytes is very difficult, if not impossible.
For the first time, high-resolution, accurate-mass mass spectrometry based on Orbitrap™ technology offers a practical solution for the challenge at hand. The high resolution of up to 140,000 FWHM at m/z 200 enables separation of drugs and metabolites 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 (see the Workflow Overview for Opiates).
The Thermo Scientific Exactive Plus mass spectrometer provides confident quantitative analysis in complex samples, such as urine and other body fluids. The Thermo Scientific Q Exactive mass spectrometer, with MS2 capabilities, offers increased sensitivity and the ability to perform ion ratio confirmation. Ion ratio confirmation may be required depending on the confirmation guidelines. All of this is made simple by using Thermo Scientific TraceFinder and ExactFinder software, which help attain high productivity and confidence in routine targeted and general unknown screening applications.
Here we describe the application of Orbitrap technology-based workflow solutions for the quantitation of drugs and metabolites in urine (see the Workflow Overview for Opiates).
The workflow described below uses a solid-phase extraction (SPE) or liquid-liquid extraction (LLE) method for sample preparation. It uses full-scan MS or targeted MS2 at 70,000 to 100,000 FWHM resolution.
Literature Highlights
Thomas A, Geyer H, et al.
Anal Bioanal Chem. 2012 May;403(5):1279-89.
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
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.
Sample Preparation
Sample Preparation Workflow for Human Urine Quantitation
Literature Highlights
Thomas A, Geyer H, et al.
Anal Bioanal Chem. 2012 May;403(5):1279-89.
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
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 Quantitation
The Thermo Scientific Q Exactive mass spectrometer with a HESI source is coupled to a Thermo Scientific Accela 1250 UHPLC system. The MS was operated in targeted MS2 mode at a resolution of 70,000 FWHM at m/z 200. The column eluent was diverted to waste after the sample injection for a specified period of time before being sent to the MS. The Q Exactive™ 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
Thomas A, Geyer H, et al.
Anal Bioanal Chem. 2012 May;403(5):1279-89.
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
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 Quantitation
Literature Highlights
Thomas A, Geyer H, et al.
Anal Bioanal Chem. 2012 May;403(5):1279-89.
Girón AJ, Deventer K, et al.
Anal Chim Acta. 2012 Apr 6;721:137-46.
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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