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 Synthetic Cannabinoids
Quantitation of synthetic cannabinoids, such as the JWH class of drugs Spice and K2, in urine and other body fluids is being performed at increasingly higher rates in forensic toxicology labs. The drugs in this class metabolize to form many different products depending on the specific drug and the body fluid sampled. The metabolites are mostly hydroxylated and carboxylated forms of the drug and present in their glucuronide and sulphated forms. The molecules include isomers that need to be identified by chromatographic separation.
As there are large numbers of these drugs that are constantly evolving and their usage varies depending on the geography, it is extremely difficult for analytical labs to identify and quantitate them. 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. Ideally, the method should be able to quantify the known drug metabolites and also qualitatively capture the new ones.
The workflow described here uses a simple dilute-and-shoot method, solid-phase extraction (SPE) or liquid-liquid extraction (LLE) for sample preparation and a Thermo Scientific Exactive Plus MS operated in Full Scan MS mode at 70,000 to 100,000 FWHM resolution. The ultra-high resolution and accurate mass with the calibration curves of the known metabolites are able to quantitate and simultaneously identify new drugs and metabolites. In many cases, the identity of the new drugs or metabolites can be confirmed using a slightly different data acquisition routine in which alternating full-scan MS and HCD MS are performed. The fragmentation data obtained with high resolution and accurate mass in this fashion enables unambiguous confirmation of the metabolites in the same run.
Resources
Analysis of Synthetic Cannabinoids (Spice) in Urine Utilizing HRAM
Van Natta K.
Slide Presentation
Sample Preparation
Sample Preparation Workflow for Synthetic Cannabinoids
Resources
Analysis of Synthetic Cannabinoids (Spice) in Urine Utilizing HRAM
Van Natta K.
Slide Presentation
Mass Spectrometry
Mass Spectrometry Workflow for Synthetic Cannabinoids
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
Analysis of Synthetic Cannabinoids (Spice) in Urine Utilizing HRAM
Van Natta K.
Slide Presentation
Data Analysis
Data Analysis Workflow for Synthetic Cannabinoids
Thermo Scientific TraceFinder software was used for quantitation. The software extracts chromatograms using accurate m/z, identifies detected peaks using retention time, and quantitates using the area under the peaks.
Resources
Analysis of Synthetic Cannabinoids (Spice) in Urine Utilizing HRAM
Van Natta K.
Slide Presentation
Thermo Fisher Scientific
United States
