Screening and quantitation of a wide range of drugs in horse 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 solid-phase extraction (SPE), liquid-liquid extraction (LLE), and online sample extraction using Thermo Scientific TurboFlow technology for many classes of drugs and metabolites. It uses simple full-scan MS at 100,000 FWHM resolution in alternating positive/negative polarity and HCD fragmentation for screening and targeted MS2 for quantitation. The MS and fragmentation data acquired in Full Scan mode is processed by Thermo Scientific ExactFinder software for screening and Thermo Scientific TraceFinder software for quantitation. ExactFinder™ 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.
For additional resources, search the Orbitrap Science Library
Overview
Workflow Overview for Horse Urine Quantitation
Currently, urine is a matrix of choice for quantitative analysis in equine drug testing. Many drugs and their metabolites are present in conjugated form in urine 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 Opiates workflow). 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 software, which helps attain high productivity and confidence in routine quantitation.
Here we describe the application of Orbitrap technology-based workflow solutions for the quantitation of drugs and metabolites in urine (see the Opiates workflow). 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 at m/z 200 resolution.
Literature Highlights
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Ho EN, Kwok WH, et al.
Drug Test Anal. 2012 Sep 3.
Sample Preparation
Sample Preparation Workflow for Horse Urine Quantitation
Literature Highlights
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Ho EN, Kwok WH, et al.
Drug Test Anal. 2012 Sep 3.
Mass Spectrometry
Mass Spectrometry Workflow for Horse 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
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Ho EN, Kwok WH, et al.
Drug Test Anal. 2012 Sep 3.
Data Analysis
Data Analysis Workflow for Horse Urine Quantitation
Literature Highlights
Moulard Y, Bailly-Chouriberry L, et al.
Anal Chim Acta. 2011 Aug 26;700(1-2):126-36.
Ho EN, Kwok WH, et al.
Drug Test Anal. 2012 Sep 3.
Thermo Fisher Scientific
United States
