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 Benzodiazepines
Quantitation of benzodiazepines in urine and blood is performed in many forensic toxicology labs. The benzodiazepines are present in urine in conjugated form. The analytical workflow needs to handle the 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 simple SPE or LLE for sample preparation. It uses full-scan MS at 70,000 to 100,000 FHWM resolution. Approximately 24 benzodiazepines molecules are routinely quantitated.
Resources
Analysis of Benzodiazepines in urine using the Thermo Scientific Exactive Mass Spectrometer
Johnson K.
Slide Presentation
Johnson K.
Slide Presentation
Sample Preparation
Sample Preparation Workflow for Benzodiazepines
The sample preparation for benzodiazepines analysis involves enzymatic hydrolysis followed by SPE or LLE extraction. For fully automated online sample preparation TurboFlow™ chromatography can be effectively employed.
Resources
Analysis of Benzodiazepines in urine using the Thermo Scientific Exactive Mass Spectrometer
Johnson K.
Slide Presentation
Johnson K.
Slide Presentation
Mass Spectrometry
Mass Spectrometry Workflow for Benzodiazepines
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 Benzodiazepines in urine using the Thermo Scientific Exactive Mass Spectrometer
Johnson K.
Slide Presentation
Johnson K.
Slide Presentation
Data Analysis
Data Analysis Workflow for Benzodiazepines
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 Benzodiazepines in urine using the Thermo Scientific Exactive Mass Spectrometer
Johnson K.
Slide Presentatio
Johnson K.
Slide Presentation
Thermo Fisher Scientific
United States
