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 THCA in Oral Fluid
Quantitation of Tetrahydrocannabinol (THC) and Tetrahydrocannabolic acid (THCA) in oral fluid is of great interest to the forensic science community. The non-invasive nature of sample collection makes it an attractive practical approach. The presence of THCA in oral fluid is reported to be a good differentiator between cannabis casual use and abuse. The analysis of THCA in oral fluid is challenging due to very low concentrations and smaller volumes of sample collected (~10 pg/mL). The sample collection devices and preservative buffers further increase the complexity of the sample. The analytical workflow needs to be capable of handling buffers, preservatives, detergents, matrix interferences and ion suppression. Up to now, this analysis has been done by GC-MS/MS or 2D GC-MS, both involving labor-intensive and time-consuming sample preparation. High-resolution MS offers an easier and fully automated alternative.
The workflow described a new methodology involving simple one-step sample preparation followed by automated 2D LC-high resolution MS/MS using the Thermo Scientific Q Exactive MS system. It uses Full Scan MS2 mode at 70,000 FWHM resolution. The LC-MS analysis quantifies THCA at 10 pg/mL level in the presence of very high amounts of THC.
Resources
He K, Kozak M.
Slide Presentation
Sample Preparation
Sample Preparation Workflow for THCA in Oral Fluid
The sample collected in the buffer using various commercial devices is dried, reconstituted, ultra-centrifuged and filtered. Other sample preparation procedures of solid-phase extraction (SPE) and liquid-liquid extraction (LLE) failed to reduce interferences and, in some instances, added contaminants.
Resources
He K, Kozak M.
Slide Presentation
Mass Spectrometry
Mass Spectrometry Workflow for THCA in Oral Fluid
The Thermo Scientific Q Exactive mass spectrometer with a HESI-II source was coupled to a Thermo Scientific Dionex Ultimate 3000 RSLCnano LC system operated in 2 D mode. The first dimension and second dimension column used the stationary phase. The first dimension captured the analytes and washed out the interferents. The second dimension analytical column separated THC and THCA from the other remaining interferences. The Q Exactive™ system was operated in Full Scan MS2 mode at a resolution of 70,000 FWHM. 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 needed for operation over a period of a few days.
Resources
He K, Kozak M.
Slide Presentation
Data Analysis
Data Analysis Workflow for THCA in Oral Fluid
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
He K, Kozak M.
Slide Presentation
Thermo Fisher Scientific
United States
