New psychoactive substances pose a challenge for drug testing laboratories

New psychoactive substances (NPS) reach the recreational drugs market at a fast pace and are of concern because of potential health risks. In addition to not being legally regulated, NPS escape detection in standard drug tests. Drug testing laboratories, therefore, must adapt their analytical methods to also cover these new substances. For screening and confirmation of NPS, mass-spectrometric multicomponent methods are useful.

by Prof. Olof Beck and Prof. Anders Helander

New psychoactive substances
The emergence of new drugs of abuse that are designed to circumvent narcotics legislation by slight chemical structural modifications of already classified drugs represents an ever increasing problem [1, 2]. Nowadays, this phenomenon is commonly termed ‘new psychoactive substances’ or ‘NPS’, but also other names such as designer drugs, legal highs, research chemicals, smart drugs, bath salts, and spice have been and are used. The NPS problem is of global concern but may vary in extent between countries, partly due to national differences in legislation and drug culture. Statistics from the EU Early Warning System operated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol on the number of NPS reported for the first time in Europe on a yearly basis gives a good insight on the progress of this phenomenon (Fig. 1) [2]. Over the past 6 years particularly, it has escalated to the level of more than 100 new substances in 2014 (i.e. about two new substances each week on average). The NPS market was long dominated by stimulants and synthetic cannabinoids but currently comprises all classes of abused substances [2].

Problems related to NPS
NPS are of particular concern because they can be sold openly in web-based shops and elsewhere and thereby reach new drug users that are attracted by their ‘legal’ status. Of public concern are the unforeseen toxic effects of NPS, as using these uncontrolled and unsafe substances and products may lead to severe intoxication and even death [1, 3]. In Sweden, the progress of the NPS phenomenon and associated harmful effects has been followed in a collaborative project between the Department of Laboratory Medicine at the Karolinska University Hospital and the Karolinska Institutet, and the Swedish Poisons Information Center [3, 4]. This project, named STRIDA, enrolls patients with suspected NPS intoxication presenting in emergency departments all over the country. By combining the results from laboratory investigations of serum and urine samples with clinical information, new knowledge about NPS prevalence and toxicity is compiled. Since the start in 2010, the STRIDA project has documented over 2000 non-fatal but often severe acute intoxication cases involving a large number of different NPS. Polydrug use is commonly seen in these cases [3].

NPS in drug screening
One reason for using NPS instead of conventional drugs of abuse may be that NPS often remain undetected in standard drug testing procedures. Accordingly they are especially attractive alternatives for individuals who want to minimize the risk of being detected, such as in workplace drug testing and drug rehabilitation programmes.

The established procedure for drug testing is to use initial screening by immunoassays and then to confirm positive samples using methods based on the more sensitive and selective mass spectrometry (MS) technique. On one hand, the NPS present a challenge for the immunoassay screening, as available methods are typically directed only towards the conventional substances, e.g. amphetamines (amphetamine and methamphetamine), tetrahydrocannabinolcarboxylic acid (THC, cannabis), morphine (heroin), and benzoyl ecgonine (cocaine). On the other hand, as NPS are often designed to mimic and are chemical derivatives of conventional drugs, there is a possibility that certain NPS will also bind to (i.e. cross-react with) the antibodies used in immunoassay screening methods. And this is indeed the case. However, when these ‘false-positive’ screening results are subjected to confirmatory analysis by methods based on MS detection, they will turn out negative (i.e. ‘false-negative’ for drug use), if the MS method is only directed toward the standard set of abused drugs.

Cross-reactivity of NPS in immunoassays
When ecstasy (3,4-methylenedioxymethamphetamine, MDMA) became established as a street drug, interest emerged to detect it in immunoassay screening. MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were found to be detectable in existing assays for amphetamine and methamphetamine, due to a high degree of cross-reactivity for these compounds [5]. Likewise, also other new amphetamine-like substances were detectable [6].

However, although many NPS showed low cross-reactivity in commercial immunoassays [7, 8], the stimulant methylenedioxypyrovalerone (MDPV) was reported to cross-react in the CEDIA phencyclidine test [9]. A study from the authors’ laboratory comprising 45 NPS confirmed that several possessed chemical similarities leading to high cross-reactivity in the immunochemical screening tests commonly employed in routine urine drug testing [10]. The detectability of NPS observed to possess cross-reactivity was further confirmed by analysis of urine specimens from authentic intoxication cases included in the STRIDA project (Table 1). Given a more widespread use of new drugs among individuals subjected to drug testing, an increased number of unconfirmed positive screening results may occur.
The cross-reactivity for NPS in current screening assays may be seen as a problem or as a possibility to detect more substances. One possibility for improved drug testing is to include the most common new substances in the confirmation methods. As ecstasy became established as an illicit drug, new immunochemical screening tests for amphetamine/methamphetamine were developed that also included MDMA and MDA. Authentic case samples were used to demonstrate the capability of several commercial amphetamine class screening tests to detect MDMA/MDA. At that time, cross-reactivity towards the new ‘amphetamine’ analytes was wanted [5]. With the advent of the large number of NPS, both legal and illegal, the strategy to also cover new substances in the screening assays for classical narcotic drug substances may not be feasible. For example, the multitude of new synthetic cannabinoids (‘spice’) have not been incorporated in screening tests for THC, but resulted in the development of new independent tests [11].

One approach put forward to understand the potential of immunoassays to detect NPS is to use molecular similarity models [12]. Interestingly, the work of Petrie and co-workers [13] included such a molecular modelling method to predict the cross-reactivity of 261 amphetamine-like compounds. However, when comparing the theoretical data with our experimental data for one compound, the predicted reactivity for butylone was 10 times lower than that observed. In a more recent publication, it was proposed that molecular similarity models could be used to design new immunoassays with sensitivity for a larger number of target compounds [14].

NPS analysis by mass spectrometry
Another analytical strategy to cover NPS in drug testing is to employ MS-based ‘screening’ methods. As part of the STRIDA project, a multicomponent analytical MS method for NPS analysis in urine and serum specimens has been developed [15]. The method uses MS in combination with liquid chromatography (LC-MS/MS in selected-reaction monitoring mode) and is continuously updated as new NPS appear. There are also other methods for multicomponent screening of drugs in urine and plasma/serum, which proves that this technology can be employed in routine drug testing [16].

The LC-MS/MS technique has great potential for drug testing and for clinical laboratories in general. There are examples of laboratories that have already successfully replaced immunoassay screening by MS methods, also for the conventional drugs of abuse [17]. One way to make this possible and cost-effective is to use simple sample preparation procedures, e.g. a simple dilution of urine with internal standards [16]. When studying the cross-reactivity of 30 NPS in commercial ELISA tests for serum and blood, only a few were found to display cross-reactivity, and it was therefore proposed that MS methods should be used in future drug screening [18]. One attraction of MS-based screening is that accurate results are already obtained from the initial analytical step, which may be especially important in cases of acute intoxication (Fig. 2).

Potential of high-resolution MS
One promising technique for drug screening is high-resolution MS (HRMS) [19]. In the HRMS technique, the acquisition of data can be made with an untargeted design. Thousands of substances can be monitored at the same time without the need for optimizing MS parameters for each compound. In addition, new compounds can be searched for retrospectively.

Conclusion
The NPS present a challenge for drug testing laboratories and calls for novel drug screening strategies. It is likely that the current broader spectrum of abused psychoactive drugs will persist in at least in the foreseeable future. This new drug situation has put the performance of drug testing into focus and indicates that drug testing laboratories will play a more important role, as on-site drug screening using dipsticks is likely to lose significance.

References
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The authors
Olof Beck*^1,3 PhD and Anders Helander^2,3 PhD
¹Department of Clinical Pharmacology, Karolinska University Laboratory Huddinge, Sweden
²Department of Clinical Chemistry, Karolinska University Laboratory Huddinge, Sweden
³Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

*Corresponding author
E-mail: olof.beck@karolinska.se