The Analytical War on Novel Psychoactive Substances

“Have you tried plant food?” Why anybody would eat plant food let alone abuse its use is bemusing. And yet, this is one of the many names given to “legal highs” or, as they’re more formally known, novel psychoactive substances (NPS). These designer drugs are produced in clandestine laboratories and given mercurial names to circumvent drug legislation – and they are not going away. “We have got to the point where young people think they are safe because of the word ‘legal,’” said the mother of a victim of NPS abuse. We tend to agree with her.

Analytical challenges

The nature of their underhanded production, purposely designed to evade international drug legislation means they are intrinsically marketed and sold as legal highs and as such, are considered safe. But there are no assurances to the customer of these NPS products that the contents are the same as advertised. For example, mephedrone was detected in products sold as naphyrone or NRG-1 in the UK even after its ban in 2009. The dangers of NPS abuse become clear when one considers that each new designer drug, designed to circumvent drug legislation, has not been evaluated for its effect on human health – the testing comes in the field, customers are test subjects, guinea pigs, at the mercy of potentially fatal side effects. There are many instances reported in the press where the use of NPS has unfortunately resulted in death (1). Analytical chemistry is key to combating these dangers and thus avoiding preventable deaths, crippling addictions and the ruination of lives.

The United Nations Office on Drugs and Crime (UNODC) and European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) detailed the following sub-categories: synthetic cannabinoids, synthetic cathinones, ketamine, phenethylamines, piperazines, plant-based substances: Khat, Kratom, Salvia divinorum and miscellaneous: aminoindanes, phencyclidine, and tryptamines. Abuse of NPS has been increasing since 2009 and represents a fast-growing market; something reflected in the online marketplace (the number of online vendors in the UK increased by more than 300 percent between 2010 and 2011). New materials made available for abuse appear rapidly and, at times, can gain a ‘foothold’ in the market. Mephedrone is one such example. In 2014, 101 new substances were reported for the first time to the EU early warning system (EWS) run by the EMCDDA up from 81 in 2013 (it was 74 in 2012). The findings of the EWS revealed synthetic cannabinoids are the most frequently discovered, with 102 detected between 2005 and 2013.  

Given their rapid appearance on the market, the principle challenge facing analytical scientists is to be ‘one step ahead’ of the clandestine drug manufacturers. We recently reviewed the current state of the “analytical fight” against legal highs/NPS (2). 

Current approaches, as expected, are generally laboratory based and take advantage of a range of standard analytical approaches, such as LC-MS/MS and so on. These have been applied successfully for the detection of a range of NPS in a number of different matrices and suspected NPS samples (such as powders). Of course, such approaches are useful because they can be applied to diverse samples, being selective with no interferents, and because they provide exact confirmation of the target analyte within the sample. And though this type of analytical approach has its place (and associated pros and cons), there is a growing need for rapid, cheap, small, portable and analytically useful methods where a near-instantaneous response is required, such as in a clinical or law enforcement setting, as noted by Guirguis in "Legal Highs & Lows".

Presumptive testing

One approach to address the growing need is the use of presumptive testing, which has been commonly used for existing drugs of abuse. A recent study explored the presumptive testing of cathinone derivatives as per United Nations recommended guidelines (3). In these approaches, color changes highlight the presence and absence of the target drug/analyte and have been deemed consistently effective for cathinone derivative screening. However, this is not the case for all legal highs/NPS. Selectivity is an issue, so false positives make the analysis of an unknown NPS powder/sample ambiguous. Another approach relies on microcrystalline presumptive tests. A sample of the NPS solution is mixed with a reagent that and then placed onto a glass slide; the resulting crystal structures are then used to identify the unknown NPS. This microcrystalline approach has been successfully used for the identification of MDAI, mephedrone and N-benzylpiperazine (4). The study evaluated purchased “legal high” samples and applied the microcrystalline presumptive test approach, which was then collaborated with GC- FTIR/MS. But there a couple of drawbacks: the reagent solution is costly (sometimes gold chloride is used) and simple changes, such as temperature, can significantly alter the test. Currently, microcrystalline presumptive tests cannot be used for the wide range of NPS. 

Immunoassays have also been explored towards the screening of several NPS. A recent study explored 16 different ELISA reagents to determine the cross-reactivity of 30 designer drugs, including 24 phenylethylamines (including eight cathinone derivatives, three piperazines, and three tryptamines) (5). Cross-reactivity towards most drugs was <4 percent in assays targeting amphetamine or methamphetamine. Compounds such as MDA, MDMA, ethylamphetamine, and α-methyltryptamine demonstrated cross-reactivities in the range of 30–250 percent, but the data was found to be consistent with both manufacturer claims and the published literature. When tested against the commercially available Randox Mephedrone/Methcathinone ELISA kit, cathinone derivatives demonstrated cross-reactivity at concentrations as low as ng/mL levels.

Other approaches recently developed have reported a novel sensing protocol based upon electrochemical methods and are similar to glucose sensors that use disposable carbon-based strips with a small electronic reader.

Researchers have shown the sensing of cathinone substitutes to be comparable to HPLC; detection of mephedrone and 4-MEC was possible in seized street samples (NRG-2), so the method could potentially offer on-the-spot analytical screening (6)(7). However, once again the approach is not without its limitations. If multiple NPS are present, the sensor is unlikely to be able to distinguish between them since selectivity is limited. Even in our recent work, we have now moved to using HPLC with electrochemical detection (8). However, while this provides a new laboratory-based approach, a selective and sensitive NPS sensor is still required and needs developing. 

Work in progress

From the above quick summary, it is clear that a rapid in-the-field sensor covering all subcategories outlined by the UNODC would be ideal; however, this has yet to be realized. And though we’ve seen some promising research, we must not forget that such screening tools need to be accurate – a point that was proven in a recent case in Australia, where a routine roadside drug test reported positive for methamphetamine – a drug the victim said he had never touched (9). The roadside drug test on the victim’s saliva was positive, which allowed further testing in a portable testing station where the second test was negative. However, a further sample was sent for laboratory analysis, which came back as positive two weeks later. Luckily, the accused challenged the result and further retests were negative.

Saliva is a complex matrix and the tests are looking for minute amounts of drugs, which makes the results unreliable. Clearly, development must focus on reducing false positives (and negatives) to ensure robustness. What is surprising in this case is that the laboratory “confirmatory” test also got it wrong...

Whichever analytical protocol is taken forward, the variety within each sub-category of NPS is huge – particularly between different generations. Another issue faced by analytical scientists regarding the detection of NPSs is the presence of adulterants within a sample. Our research showed that “street samples” contained caffeine and benzocaine in addition to the active ingredients. Other groups have also found novocaine, lidocaine sugars and many other compound classes. These ingredients can be randomly changed, which adds to the complexity of testing – in most cases, real sample testing and the effect of interferents is merely an afterthought. In summary, there is much work for analytical scientists to do in combatting the war on NPS. In particular, a portable, rapid, cheap yet sensitive and selective sensor is still required. Sadly, we are aiming for a moving target. As one legal high is made illegal, sensors are developed accordingly, and these are rapidly changed as others emerge. On a high note, it certainly means that this area of research remains both exciting and challenging...