Accurately Analyzing Drugs Like Spice and K2 May Now be Possible

Synthetic cannabinoids and other novel psychoactive substances (NPS) aren’t just a problem for health officials, but for lab analysts, too. 

Due to the ever-shifting, clandestine nature of production, the compounds are often impossible to separate and identify using traditional techniques such as gas chromatography. 

Now, researchers from Florida International University have used 2D liquid chromatography to successfully separate the small molecules and compounds of structurally related synthetic cannabinoids. 

If widely adopted, this new technique could advance the burgeoning field of novel psychoactive substances forensics and become a valuable tool for its investigators. 

The results of the research were published in the Journal of Analytical Toxicology.

A Brief History of Novel Psychoactive Substances 

Informally known as ‘legal highs’, novel psychoactive substances are designed to replicate the effects of traditionally illicit drugs, like cannabis, cocaine, and ecstasy. Partly due to their unregulated production, partly to keep forensic investigators guessing, the substances’ ingredients are constantly changing. One week an NPS could be blue, the next it could be red and constitute several new functional groups. 

This ‘underground’ nature of manufacture lends many products to contain dangerous and life-threatening additives. Only in July of this year, the US Food and Drug Administration warned about the risks of consuming synthetic cannabinoids contaminated with rat poison. Half-jokingly, many products are often labeled "not for human consumption."

Synthetic cannabinoids, such as Spice and K2, are usually either solids or oils. Products typically contain around 3 g of dried vegetable matter that has been sprayed with multiple synthetic cannabinoids (and other mystery compounds). 

Originally developed for research purposes, synthetic cannabinoids were used to study the body’s endocannabinoid system and whether it could be triggered by synthetic compounds. 

As a group of cannabinoid receptor agonists, the drugs mimic the effects of THC and anandamide by interacting with CB1 receptors in the brain. Certain in vitro studies have even shown that synthetic compounds can bind more strongly to this receptor than THC, as measured by the affinity constant Ki.

However, these studies only scratch the surface of the science of NPS. In reality, precious little research has been undertaken into the effects of NPS and synthetic cannabinoids on the human body. It’s even possible that as well as having a high potency, some cannabinoids could have particularly long half-lives, which could lead to the prolonged and dangerous psychoactive effects many users exhibit. Thus, there is far higher potential for overdose than with cannabis.

The Problem with Separating Spice

More than any other NPS, synthetic cannabinoids have quickly become a nuisance for drug analysts. Due to the large number of compounds, metabolites, and isomers the products can contain, they pose a significant challenge to chromatographic separations. 

For example, some of the most popular synthetic cannabinoids at the moment include compounds such as JWH 018, JWH 019, JWH 080 and JWH 250, all of which would be identical if not for a differing indole alkyl side chain. Most synthetic isomers also have similar chromatographic retention times, which also makes their separation more difficult. 

Of course, many of the compounds a lab analyst would co-elute from these substances would also be unknown to them. 

Prior to the Florida University group’s research, 2D liquid chromatography (2D-LC) had been used successfully to separate pharmaceuticals and small molecules such as methamphetamine, antiretroviral drugs, and traditional Chinese medicinal preparations from complex samples. Taking these results as a promising sign, the researchers decided to use the technique to separate and identify NPS. 

The method’s proficiency in analytical separation is down to many of its unique features. These include increased greater resolving powers, peak capacities, separation of isomers and isobars, and better separation of compounds and metabolites, particularly in complex mixtures. The 2D-LC method’s increased separation power is due to its combination of high resolution and peak capacities of the two orthogonal dimensions.

This improved separation power alone doesn’t directly lead to identification, but it can provide additional, orthogonal information to help analysts properly identify compounds. This extra information is particularly vital for identifying compounds from MS data such as fragmentation patterns, which might contain previously unreported analytes. 

What the Researchers’ Results Mean for NPS Identification 

To test the utility of their method, the researchers created three mixes of synthetic cannabinoids, which each contained five compounds that were unresolvable in a traditional, 1D-LC separation. 

Consisting of columns, UV detection, diode arrays, and QTOF-MS, the group’s 2D-LC method had a high enough selectivity to effectively separate and identify isomeric and structurally related synthetic cannabinoids. Contour plots of UV absorbance in 1D and MS ion intensity in 2D proved that all components in each mixture were successfully separated and identified using the 2D-LC separation method.

The results of the study serve as a proof-of-concept for the application of 2D-LC to the separation of isomeric and structurally related SC. The group hopes that with further investigation, optimization, and validation, their 2D-LC technique will become a viable tool for drug and forensic analysts to accurately report the ever-shifting map of novel psychoactive substances.