Credit for featured photo: TX Attorney General from Flicker available via Creative Commons (CC).

Colorful packets filled with green vegetation pack evidence receiving rooms, but they are not filled with marijuana? Analysts take a closer look at these substances and find that this vegetation, while not marijuana, contains chemical that are similar to ones found in the better-known drug. These substances, termed novel psychoactive substance(s) (NPSs) are a class of drugs that vary slightly in structure from existing illicit substances and are intended to produce similar psychoactive effects on the central nervous system. By having slight structural differences, these substances try to evade existing drug laws, allowing for legal highs. A common type of NPSs, which is pictured in Figure 1, are synthetic cannabinoid(s) (SCs). SCs are human-made chemicals that bare a similarity to the chemicals producing the high in marijuana, and thus, they produce similar effects. They are often sprayed onto vegetation and ingested similarly to marijuana.

One issue that SCs pose for forensic chemist is that they can be difficult to separate and analyze. Traditionally, methods like gas chromatography or liquid chromatography are employed to separate mixtures of drugs based on chemical and physical properties. These methods are often paired with mass spectroscopy in order to identify the components of the drug mixture. SCs often have remarkably similar properties to one another, due to very slight structural differences, which makes them more difficult to separate from one another. If these substances are not separated and reach the mass spectrometer at the same time, then they may not be identified as individual substances. Thus, forensic chemist may be unaware of what chemicals are present in a sample. This can have public health consequences as the community as a whole may not be aware of the substances that are on the streets. In order to prevent the misinterpretation of data, better separation is needed during chromatography.

One method that may be able to combat the issue of incomplete separation is 2D liquid chromatography (2D LC). Much like traditional liquid chromatography (1D LC), this method involves a sample being carried through a column coted in a solid stationary phase by a liquid mobile phase. As the sample travels through the column it separates based on how attracted it is to the stationary and mobile phase before reaching the detector (which is often times a mass spectrometer).

This method differs from 1D LC in that the sample/mobile phase travels through two columns that are in tandem with two different stationary phases. The additional column allows for further separation of the sample from the first column, which results in better overall separation. This had already been shown to allow for better separation of mixtures of antiretroviral drugs and illicit drugs in complex biological matrices.

Recognizing the potential this method has, researchers from the DeCaprio lab at Florida International University decided to apply 2D LC to the analysis of SC mixtures. In a proof of concept study, they assessed if 2D LC paired with mass spectroscopy could be used to separate mixtures of SCs that were isomeric (which have the same chemical formula but different arrangement of atoms) or non-isomeric but structurally similar. They chose accurate-mass quadrupole time-of-flight mass spectroscopy (QTOF MS) in particular for MS because this form of high resolution spectroscopy allows for better analysis of the SC mixture components.

The researchers first prepared three different SC mixtures, including SCs like the ones described in Figure 3. The first mixture contained SC isomers, while the second and third mixtures contained non-isomeric but structurally similar SCs. The scientist then analyzed the first SC mixtures using three different 1D LC columns in order to assess which of the columns would be best for the 2D LC system. None of the three 1D LC columns could completely separate either mixture (further highlighting the need for 2D LC). However, the Bonus-RP column and biphenyl column proved to be the best for separation. These two columns were then used in the 2D LC system.

After optimizing the 2D LC/QTOF MS method, the researchers analyzed all three SC mixtures using the full system. The first two mixtures exhibited excellent separation, with slight coelution of two components of the mixture. Meanwhile, the third mixture exhibited complete separation of all five components of the mixture. These results highlight that this method is advantageous when compared to traditional 1D LC/MS and can be used to successfully separate and identify mixtures of SCs.

While this method was mostly successful, there was still slight coelution in two of the mixtures. This shows that there is still room for improvement which can be done with further optimization of this method. The scientist behind this study suggested that further investigation and validation is needed in order to ensure that 2D LC/QTOF MS is a more reliable method for the separation and identification of SC mixtures.

Title	Separation and Identification of Isomeric and Structurally Related Synthetic Cannabinoids Using 2D Liquid Chromatography and High Resolution Mass Spectrometry
Authors	Melanie N Eckberg , Luis E Arroyo-Mora , Dwight R Stoll, Anthony P DeCaprio
Journal	Journal of Analytical Toxicology
Year	2019
Link	https://pubmed.ncbi.nlm.nih.gov/30295842/
Citation	Eckber, M. N; Arroyo-Mora, L.E; Stoll, D. R; DeCaprio, A. P; Separation 3and Identification of Isomeric and Structurally Related Synthetic Cannabinoids Using 2D Liquid Chromatography and High Resolution Mass Spectrometry. J Anal Toxicol. 2019, 4 (3), 170-178. doi:10.1093/jat/bky081.