In recent years, the number of novel psychoactive substances has grown significantly, especially synthetic cannabinoids, as pictured here. Photo: DEA

With the emergence of novel psychoactive substances as an alternative high, forensic laboratories are forced to employ alternative strategies to detect these new derivatives. Advances in high-resolution full-scan mass spectrometry (MS) are helping laboratories do this more quickly and accurately, while improvements in data sharing allow authorities to track their spread. Here, we take a look at the untargeted MS technology helping enforcement agencies in the fight against drug trafficking.

Both forensic toxicology and controlled substance analysis laboratories continue to see a rise in first-time identified novel psychoactive substances (NPSs). A recent study by the U.S. Drug Enforcement Administration’s National Forensic Laboratory Information System found that, of 400 U.S. forensic laboratories surveyed, around 30 percent reported their drug chemistry caseloads increasing year on year. Approximately 60 percent of those questioned said the influx of newly identified drugs was a major contributor to caseload backlogs.

Take the recent U.S. influx of the synthetic opioid fentanyl, for instance—what some are calling the “new heroin” epidemic. Fifty times more potent than heroin, fentanyl was originally developed as a prescription painkiller for terminally ill cancer patients and those with severe debilitating pain. Yet, some drug traffickers are now using fentanyl to lace diluted heroin in order to cut costs. As smaller amounts can be manufactured and shipped, its potency also makes it easier to evade detection. In the U.S., fentanyl drug seizures increased from 618 in 2012 to 4,585 in 2014, according to figures published by The New York Times, an increase of more than 640 percent in just two years.

What’s more, the illegal trade in fentanyl has evolved to stay ahead of drug detection and import/export laws. Illegal drug manufacturers are routinely modifying the chemical structure of synthetic designer drugs, meaning they are not readily detected by traditional targeted analysis methods such as immunoassays or conventional single or triple quadrupole MS.

The emergence of new drug variants such as these isn’t just a problem in the U.S. According to figures reported by the United Nations Office on Drugs and Crime (UNODC), the number of NPSs reported worldwide has grown significantly, with the largest classes of NPSs being synthetic cannabinoids, stimulants and hallucinogens.

One reason for this influx of designer drugs may be the success of forensic screening and the UNODC’s Early Warning Advisory (EWA) system, which enables greater sharing of information on NPSs between countries. As more regions regulate and ban substances, and advances in analysis technology make drugs easier to identify, suppliers are forced to reformulate to evade detection.

However, with so many NPSs emerging on the scene, forensic laboratories are under increasing pressure to cast their net wider. It takes time and resources to identify new compounds, develop new screening procedures and revalidate methods. Fortunately, advances in MS and cloud-based libraries are helping labs expand their capabilities and screen for unknown analytes more efficiently.

Targeted analysis approaches

S methods for detecting or simply confirming controlled substances in border patrol or DEA labs are largely based on targeted approaches, using instruments manually configured to quantitatively detect specific, known drug targets. Such labs have traditionally deployed gas chromatography single quadrupole (GCMS)-based systems for these analyses.

For forensic toxicology investigations, liquid chromatography coupled with highly targeted tandem MS (LCMS/MS) has become a method of interest, due to its excellent analytic specificity and ability to more easily manage biological sample matrices. Triple quadrupole MS is one such tandem approach that employs three sets of quadrupole rods to filter and identify analytes based on the way their ions fragment. The first set of quadrupole rods filters analytes, allowing only ions with a specific mass into the system. The second set acts as a collision cell to fragment this precursor ion, while the third filters the resulting fragment ions, allowing only specific daughter ions to reach the detector. Using this knowledge of a molecule’s fragmentation pattern, coupled with its retention time obtained by chromatography, LCMS/MS can identify specific analytes with very high precision.

or this reason, LCMS/MS is beginning to replace traditional two-step analyses methods that involve immunoassay screening and gas chromatography. Such methods display poor substrate specificity, low throughput and can be costly in cases where a laboratory needs to outsource testing with a particular assay containing hundreds of compounds.

However, while LCMS/MS is capable of identifying hundreds of analytes in a single run to exceptional levels of precision, it is inherently targeted—that is, you have to know what you’re looking for. With such an influx of new drugs of abuse and ever-changing “drugs of choice,” labs are increasingly unaware of what they might find in samples. Targeted methods are therefore unable to identify recently discovered NPSs, causing delays as samples need to be re-run. Such a challenge requires a different analytical approach.

The opioid fentanyl was originally developed as a prescription painkiller, but drug traffickers are now using it to lace diluted heroin. Photo: DEA

Growing need for untargeted analysis

In contrast to targeted analysis, untargeted MS approaches are essentially able to detect all species in a sample. Using full-scan MS techniques, coupled with up-to-date databases of analytes of interest, untargeted methods are capable of detecting new drug variants that may not have been identified in a routine scan limited to a relatively small number of known analytes.

While recent innovations have made untargeted analysis more useful and cost-effective, full-scan MS is not a new concept. Early untargeted techniques based on GCMS have been used for screening drugs and pesticide residues since the 1980s. However, the poor sensitivity, selectivity and cost of early instruments, based largely on time of flight (TOF) MS analysis have meant limited adoption.

Over the last five years, advances in high resolution mass spectrometry (HRMS) have made significant progress in overcoming these challenges. Affordable benchtop, hybrid MS instruments combine full-scan capability with high resolution—facilitating both targeted and untargeted screening. Using an ion trap that can store and analyze multiple ions simultaneously, these instruments capture all the information about a sample’s contents in one go, allowing tomorrow’s drugs to be found in today’s samples. Additionally, existing samples can be retrospectively analyzed for new drugs without the need to re-run.

Such hybrid systems can be coupled with both GC and LC separation methods and alternative ionization techniques, allowing a whole range of drug compounds to be investigated. New direct ionization methods, such as direct analysis in real time (DART) and paper spray, allow labs to obtain more information from a much wider range of samples. Such techniques can instantaneously ionize gaseous, liquid and solid sample matrices under ambient conditions, facilitating rapid access to results. DART can, for instance, be used for full-scan MS analysis of cocaine residue on paper money, without the need for any sample preparation steps.

Spectral sharing is key

Key to the continued success of untargeted screening is the sharing of information on new analytes. Such systems help forensics labs in one state share information with another state, or even globally, to catch new drug variants. These collaborative approaches enable drug enforcement authorities to trace points of origin and map distribution networks, which help in the fight against drug trafficking.

MS data acquisition and processing tools utilize local and cloud-based databases to perform targeted analyte quantitation as well as screen for untargeted unknowns. Online spectral databases are regularly updated with spectral information on authentic samples, and contain highly curated information on analyte fragmentation patterns. Algorithms allow structural prediction of unknown analytes based on similarities with the fragmentation patterns of structurally related analytes. This ability to quickly recognize structural derivatives allows authorities to keep up with new threats.

New hybrid MS instruments capable of full-scan HRMS permit forensic laboratories to screen for controlled substances and their variants more efficiently and effectively. Such technology, in combination with greater sharing of information through cloud-based databases, helps laboratories detect new drug threats as they emerge. The ability to locate new drugs today using full-scan approaches and perform retrospective sample analysis as needed helps enforcement authorities map the spread of NPSs more quickly, while simultaneously reducing laboratory workloads and operating costs.