Quantitation of drugs of abuse in body fluids is challenging due to the varying concentrations and substantially different chemical and physical properties of the drugs, interfering matrices, occasionally small volumes of sample to test, and the presence of many similar compounds. Further, constant evolution of drugs makes it harder to identify and quantitate them. Currently, most of the quantitative analysis is done using a selected reaction monitoring (SRM) approach on a triple-stage quadrupole mass spectrometer. This approach is inherently targeted and misses new compounds. Also, 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 spectrometry based on Orbitrap™ technology offers a practical solution for the challenge at hand. The high resolution of up to 140,000 FWHM 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. 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 quantitation of drugs and metabolites in urine, plasma, whole blood, and oral fluid matrices.
For additional resources, search the Orbitrap Science Library
Overview
Workflow Overview for Opiates
Quantitation of opiates in urine and blood is performed in many forensic toxicology labs. The opiates and their metabolites are present in urine in conjugated form. The molecules 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.
The workflow described here uses a simple dilute-and-shoot, solid-phase extraction (SPE) or liquid-liquid extraction (LLE) for sample preparation. It uses full-scan MS at 70,000 to 100,000 FWHM resolution. The main opiates, among other molecules include Morphine, Hydromorphone, Oxymorphone, Codeine, Dihydrocodeine, Oxycodone and Hydrocodone.
Resources
Quantitative Analysis of Opiates in Urine using Exactive LC-MS System
Clinical Research Applications Group
Slide Presentation
Johnson K.
Slide Presentation
Sample Preparation
Sample Preparation Workflow for Opiates
Resources
Quantitative Analysis of Opiates in Urine using Exactive LC-MS System
Clinical Research Applications Group
Slide Presentation
Johnson K.
Slide Presentation
Mass Spectrometry
Mass Spectrometry Workflow for Opiates
A Thermo Scientific Exactive Plus mass spectrometer with a HESI source was coupled to a Thermo Scientific Accela 1250 uHPLC system. The MS was operated in Full Scan MS mode at a resolution of 70,000 – 100,000 FWHM. The column eluent was diverted to waste for a specified period of time after the sample injection 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.
Resources
Quantitative Analysis of Opiates in Urine using Exactive LC-MS System
Clinical Research Applications Group
Slide Presentation
Johnson K.
Slide Presentation
Data Analysis
Data Analysis Workflow for Opiates
Thermo Scientific TraceFinder software was used for quantitation. The software extracts chromatograms using accurate m/z, identifies detected peaks using retention time and quantitates by using the area under the peaks.
Resources
Quantitative Analysis of Opiates in Urine using Exactive LC-MS System
Clinical Research Applications Group
Slide Presentation
Johnson K.
Slide Presentation
Thermo Fisher Scientific
United States
