Tracking a Moving Target: Advancing Surveillance of Designer Drugs with Accurate Mass Scanning in Forensic Samples
For Research Use Only. Not for use in diagnostic procedures.
Using high resolution mass spectrometry to screen for novel psychoactive substances in forensic samples
Keeping pace with the ever-shifting spectrum of designer drugs has always been a challenge for forensic toxicologists, who regularly test for low-levels of recreational substances–whether legal or illegal—against the complex backdrop of biological components in blood or urine samples. Designer drugs are novel, synthetic chemicals that are closely related to known psychoactive compounds and provide the same high, but have a slightly altered composition that makes these analogues difficult to recognize in routine screens (Table 1). Because designer drugs are continually evolving –frequently incorporating structural changes that allow them to skirt regulations and complicate law enforcement efforts—drug screening laboratories are often left in the dark on what compounds to target, with few details to guide their efforts.
Traditionally, drug tests employ targeted LC-MS/MS-based methods, but these are only able to analyze substances with established structures, limiting drug detection to compounds found on lists of pre-characterized analytes. By integrating minor adjustments to side-chains or functional groups, designer drugs essentially can outwit these types of targeted drug screens, while retaining similar pharmacodynamic properties to the controlled parent substance. A designer drug may even have multiple sub-species or highly varied concentrations of psychoactive components between lots, creating endless possibilities of substances to pursue. Thus, the ideal screening tool would be one that can scan suspicious samples for all unusual components even with little or no prior knowledge of their structural identities.
To tackle this challenge of discovering never-before-seen drugs, researchers at SCIEX have developed a novel, all-inclusive testing approach that makes it possible to detect synthetically-produced drugs, their metabolites, and any other unusual sample components—with the high sensitivity needed to locate even trace amounts in a biological sample. This new LC-MS/MS-based method relies on accurate mass detection of every sample component using a hybrid triple quadrupole/time-of-flight (TOF) mass spectrometer, the TripleTOF® System, which simultaneously collects MS/MS fragmentation data, providing enough detail to pin down the structure of unknown compounds.
To sort through the very complete, but immense compilation of information, data mining tools built into PeakView® and MasterView™ Software can collectively evaluate a vast number of spectra, searching for peaks of interest and establishing a structural identity for newly-emerging compounds. Finally, drug testing samples can be further scrutinized against a spectral library of known compounds generated at high resolution—the High Resolution Spectral Library for Forensics Version 1.0—a solution that combs through urine or blood samples to confirm the presence or absence of well-characterized drugs and their metabolites.
"Typical workflows in the forensic toxicology lab are mostly targeted analyses, routine assays, where we search for knowns from a list of compounds. With the emergence of designer drugs, that is changing. Nowadays, you have to do more surveillance screening, [and] we don’t have any pre-existing knowledge of [the designer drugs] that we are looking for, so it makes it very tricky to [analyze for them]," said Adrian Taylor, the technical marketing manager for forensic toxicology at SCIEX who helped develop the accurate mass-based method for detecting unknown compounds.
Isolating designer drugs with high resolution mass spectrometry
The gold standard for quantitating and detecting substances in a complex matrix is the triple quadrupole mass spectrometer, which uses multiple reaction monitoring (MRM) transitions pre-calculated from a known structure to track well-characterized drugs and their metabolites. But in the absence of this specific information, researchers need a system with the capacity to explore the full range of compounds in a sample, while at the same time be able to distinguish between normal and atypical components. Instead of matching a suspicious peak to a known list of drugs, the accurate-mass approach measures the signals of all eluting peaks and provides high-resolution MS spectra along with information dependent acquisition (IDA) enabled MS/MS data collection for every component. In turn, these information-rich scans provide the details needed to build the identity of shadowy substances that have never before been characterized.
"Finding all these potential compounds in complex samples is very difficult," remarked Taylor, "but accurate mass/high resolution LC-MS/MS hardware is powerful enough to acquire all the data on unknown samples." Because of this very fast instrument speed, over 100 MS scans per second can be generated, leaving enough time for 20 to 30 TOF MS/MS survey scans to be collected that can support compound identification through MS/MS library searches.
Comprised of a triple quad instrument equipped with the Accelerator TOF™ analyzer, the TripleTOF system measures analytes over a wide dynamic mass range, capturing analyte information with high mass accuracy. This high-resolution detection allows for needle-sharp signals that can be extracted using a very narrow mass range and easily permits the exclusion of non-specific peaks corresponding to background or co-eluting contaminants that may obstruct accurate peak integration or decrease the signal-to-noise ratio (Figure 2). "We have a lot of interferences and a lot of background at low resolution. If we go down to higher resolution (such as a 0.1 Da XIC window), all of those [interferences and background signals] are removed, so our signal-to-noise is increased," said Taylor. "That really shows the power of TOF technology." This high-resolution data mining can then permit an increased level of specificity when searching for a designer drug with very slight variations in structure.
Screening for unknown drugs with high throughput software
In a typical drug test with the accurate mass workflow, injecting and collecting data is rapid and fairly routine; most biological components are easily ionizable, and no additional sample prep steps are required for LC-MS/MS analysis. However, a large number of peaks are generated when analyzing blood or urine samples, and researchers are quickly faced with the complex task of locating minor amounts of a toxic compound from amidst a sea of normal, endogenous components. Extraction of every peak individually would be too time-consuming, so researchers used high-powered data analysis tools—PeakView and MasterView Software—to decipher and quickly screen results for unusual components in large batches of MS/MS data.
The first challenge is to identify which components are normal background and which are atypical. To zero in on drug-related components, a background subtraction method is used to remove sample peaks that are also found in a clean sample, thereby reducing the peaks to a more manageable number and highlighting those most likely to correspond to drug-related material or metabolites (Figure 2). Once the field is narrowed, researchers can begin to focus on these "standout" peaks and extract them using the non-targeted peak finding algorithm within PeakView Software.
Finally, the detailed mass and fragment data collected for each peak can then be used to characterize the peak. The software evaluates the TOF-MS data and the high resolution fragment data to assign an empirical molecular formula based on the extracted accurate mass and the isotope pattern. These derived formulas are then automatically compared to similar structures in the ChemSpider database, whose MS/MS fragmentation patterns are matched up with the accurate mass MS/MS scan generated for the unknown. Multiple structures may exist for one particular accurate mass, so the fragmentation data contributes heavily to the structural assignment process for a particular peak. "It takes a lot of time to go through all those structures, and so we do have a link to the ChemSpider website, which helps you narrow down what you want to look at and search for," explained Taylor.
Once an unknown designer drug compound is fully characterized, the new structure can be added to a list of pre-identified compounds that complement the screening efforts for unexpected sample components (Figure). SCIEX has created a new solution especially for accurate mass workflows, and has recently released an inclusive list of high resolution MS/MS spectra for 370 commonly tracked drug entities –the High Resolution Spectral Library for Forensics Version 1.0. As a final step in the workflow, the suspicious sample is re-evaluated and compared to the high resolution list to ensure that no known drug is present in the sample; and if a known compound is detected, then it can also be quantitated and quantitatively compared to a selected standard. To keep up with the changing landscape of designer drugs, this list is continually expanded with new compounds detected by law enforcement agencies, ensuring that individual laboratories have the most up-to-date information for the most effective screens.
Benefits of general unknown discovery with accurate mass technology
Accurate mass workflows on the TripleTOF System extend significant benefits for the detection of unknown recreational drugs and offer a leave-no-stone-unturned approach for gathering all-inclusive data that is also sensitive enough to detect very low-levels of unexpected compounds. This collection of high-resolution detail can help structurally define compounds with a murky past and provide a more accurate characterization of unknowns. Additionally, the accurate mass data files are preserved after acquisition, so it is possible to go back and re-interrogate a high resolution scan for unanticipated compounds, a very powerful option that allows for a sample to be re-evaluated for novel drugs that emerge after an initial screen. "[To discover a designer drug], we need to do unknown and retrospective analysis, and we really need accurate-mass, high-resolution technology in order to do that for structural elucidation purposes," said Taylor.
Forensic toxicologists can now access a complete scope of compounds with this efficient accurate mass method for screening known and unknown samples simultaneously. No longer do researchers have to go all over the map to track designer drugs, and discovery of unknowns is easily facilitated, leaving little room for these synthetic drugs to hide. With all the right information in hand, researchers can ask all the right questions to get all the right answers about what is lurking in a suspicious sample.
Table 1 A partial list of the types of common designer drugs that have been identified through screening methods.
Figure 2 A series of extracted ion chromatograms shows a peak extracted over a range of decreasing extraction widths (from 0.7 to 0.01 Da, left panel). An extracted ion chromatogram shows the removal of interference from a peak extracted at 0.01 Da (middle panel). A series of peaks extracted at very high resolution (0.005 Da) are shown for a series of drug-related compounds (right panel).
Figure 3. A pictorial representation of comparative unknown screening highlights how background subtraction can reveal small differences between multi-component samples.
Figure 4 An example of the MasterView Software results window illustrating the multiple steps of the unknown screening workflow is shown here. The total ion chromatogram (TIC) for both the sample and control indicates which peaks overlap in both samples (upper panel). After background subtraction using the control, peaks unique to the sample reveal potential drug-related material (middle panels). Accurate masses from isolated peaks are used to determine the molecular formula, which is evaluated for potential structures on ChemSpider (lower left panel). Fragmentation data for the isolated peak is compared to TOF-MS/MS data in ChemSpider to verify the structure of unknown peaks (lower right panel).