Rapid and sensitive detection of synthetic cannabinoids JWH-018, JWH-073 and their metabolites using molecularly imprinted polymer-coated QCM nanosensor in artificial saliva
Graphical abstract
Introduction
There has been a proliferation at an unprecedented scale of new drug discoveries and syntheses over the last decade, culminating what can be considered to be an issue necessitating global attention and serious public health hazard. Consequently to this rise, a substantial quantity of novel psychoactive substances (NPSs) have become obtainable in illicit drug markets with negligible or non-existent prior knowledge and/or practical experience about their associated health risks, such as side effects or toxicity profiles [1]. Analytical laboratories in the modern age are thus confronted by the challenge of having to account for and adapt to the appearance of such NPSs, which bear the advantage over conventional drugs that they may remain undetected in standard drug testing procedures. Such compounds are also characterized by a scarcity of reliable pharmacological and/or toxicological data, such as their toxicokinetic profiles or detectability in many body matrices. The risks posed do not end with the user either, as many NPSs can also pose risks to non-users, such as in the case of the operation of vehicles or machinery. The operation of vehicles under the influence of drugs (commonly referred to as DUI or DUID) can result in hazardous circumstances and has been indicated as a possible reason for the increase in road accidents [2], [3], [4]. Screening tests applied to the detection of NPSs are generally sensitive, inexpensive and rapid but lack high selectivity and thus may be subject to false positives associated with cross-reactivity to similarly-structured by stander drugs [5,6].
Urine is currently the matrix of choice for the evaluation and monitoring of recent drug exposure. It has several advantages, such as an extended envelope of detection, where drug exposure can be detected over the length of several days, and advantages resultant from the characteristics. However, the saliva has been proposed as an alternative matrix that has attracted rising interest in clinical and forensic toxicology, such as workplace drug testing, DUID programs, pain mitigation and management, epidemiological studies [7], [8], [9], [10], [11], [12]. Saliva as an analytical matrix bears the advantages of ease of collection, low biohazard risks, and gender neutrality in procurement and monitoring during sample acquisition [13]. Studies conducted on synthetic cannabinoids indicate that only a minor fraction of the parent compound is excreted in unchanged form, where the vast proportion is metabolized into more hydrophilic metabolites [14,15]. Saliva has an additional advantage in that the proportion of parent compounds is more predominant in its matrices, allowing for faster identification of new compounds [16]. Although saliva concentration is not considered to be viable to determine blood concentrations [17], [18], [19], [20], the shortness of the detection time in saliva [20], observed after a single intake renders saliva more applicable to identify recent exposure than urine [16]. Saliva can oftentimes be used in DUID determination, workplace drug testing, and monitoring of drug abstinence [21,22]. The primary advantage of saliva in such a testing set is that it allows the setting of the parent compound as a major analytical target. Immunological testing methods utilizing cross-reactions with JWH-018 are already under development for synthetic cannabinoids detection [23,24]. JWH-018 is among the targets of multi-target screening methods employing liquid chromatography–mass spectrometry [25,26]. However, quantitative data are scarce on saliva specimens [23,27,28]. Despite having been isolated first in 2008 [24], JWH-018 was detectable in specimens obtained as late as 2015 [29], therefore it retains its relevance [30]. Among the compounds classified as NPSs, synthetic cannabinoids are considered to hold importance and new members have been continuously introduced [31].
Synthetic cannabinoids (SCs), which mimic the influence of Δ9-tetrahydrocannabinol (Δ9-THC), are chemical compounds a major active content of cannabis. In the beginning, they were used as therapeutic agents for pain cure and are linked to cannabinoid receptors in the brain [32]. Unlike the common utilize of marijuana, which poses only relatively limited acute toxicity, serious adverse effects, often requiring medical attention, are not uncommon with SC consumption. Indeed, the relative risk of seeking emergency medical treatment following the use of SCs has been reported to be 30 times higher than that associated with the use of natural forms of cannabis [33]. JWH-018 and JWH-073 are the most well-known aminoalkylindol structure SCs that have appeared as new drugs of abuse being promoted as “legal” marijuana and sold under brand names [32]. The detection of alkyindole derivatives and their metabolites in biological fluids is quite complicated for the low concentration of these compounds [34]. There are several reports about the side effects of SCs including agitation, confusion, dizziness, fast heart beating, and sickness. In human matrices, an assay of SCs is of special importance in areas of clinical and forensic toxicology [35]. Although traditional methods are priceless for conclusive recognition of emerging drugs of abuse, the devices used are naturally expensive and bulky and are not fit for field analysis. There is a requirement for alternative general screening methods that would be sensitive enough for SCs detection in body fluids.
Molecular imprinting technology has been introduced to usher in the in-built designed selectivity towards target analytes by molecularly imprinted polymers (MIPs). MIPs have become very attractive artificial receptor stages for the selective capture of small molecules of structurally similar and related species [36], [37], [38]. This mimic recognition based technique, being a type of polymerization, employs the use of the fabrication of specific cavities (binding sites) in a highly polymeric matrix, in such a manner as to be complementary to an analyte designated a target [35,39]. This then permits selective binding between the matrix and the target analyte, even in a chemically complex sample. Due to their high selectivity, MIPs have been used to modify already-existent sensor systems to enhance their performance, such as electrochemical sensors [40,41], optical sensors [42,43], and mass-sensitive sensors [44,45]. Quartz crystal microbalance (QCM) nanosensors are mass-sensitive and have important properties high sensitivity, ease of use, affordability, stability, simplicity and portability. A number of strategies can be developed to design a QCM electrode surface, the most viable approach among these methods is a molecular imprinting strategy [35]. The widespread utilization of molecularly-imprinted polymer coated QCM nanosensors have been published the sensing of various analytes such as proteins [46], enzymes [47], amino acids [48], drugs [49], pesticides [50], metals [51], antibiotics [52], biomarkers [53], bacteria [54,55], pathogens [56], vitamins [57] and low molecular weight molecules [58], [59], [60], [61] etc.
The common applications of MIP in the QCM sensor have been reported for different analytes detection but there is no combination of MIP and QCM study to sensing SCs in saliva. Hence, the development of simple monitoring methods for SCs detection in biological materials is required. Herein, we synthesized molecularly-imprinted nanoparticles using mini-emulsion polymerization. Then, the QCM chip surface was coated with SCs-MIP nanoparticles. Detection studies of JWH-018, JWH-073 and their major metabolites were performed using aqueous and artificial saliva solutions. Kinetic results were determined by calculating association kinetics analysis, Scatchard, Langmuir, Freundlich and Langmuir-Freundlich isotherms.
Section snippets
Chemicals
Ethylene glycol dimethacrylate, 2-hydroxyethyl methacrylate, polyvinyl alcohol, ammonium persulfate, sodium dodecyl sulfate, sodium bisulfite and sodium bicarbonate were purchased from Sigma (St. Louis, USA). Naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018); naphthalen-1-yl-(1-(5-pentanoic acid)-indol-3-yl)-methanone (JWH-018 pentanoic acid); naphthalen-1-yl-(1-butylindol-3-yl)methanone (JWH-073); and naphthalen-1-yl-(1-(4-butanoic acid)-indol-3-yl)-methanone (JWH-073 butanoic acid) were
Characterization of nanoparticles and QCM chips
A two-phase mini-emulsion polymerization procedure was used to prepare SCs-MIP and NIP nanoparticles. Characterization studies of MIP and NIP nanoparticles were performed using zeta sizer, SEM, and FTIR-ATR spectroscopy. Results obtained from zeta sizer indicated that average particle size MIP and NIP nanoparticles have in the ranges of 38.5 and 45.6 nm with a polydispersity around 0.10–0.16, respectively (Fig. S2, Table S1). Zeta sizer results were confirmed by SEM images of the nanoparticles.
Conclusions
The toxicity of the SCs appears to be worse than that of natural cannabis likely due to the higher potency. In certain circumstances, acute toxic effects can contribute to death. For this reason, in clinical and forensic settings it is fundamental to supply methods targeting these compounds and their major metabolites in saliva samples as well. In this research, QCM nanosensors coated with synthetic biomimetic recognition nanoparticles have been designed for real-time and highly sensitive
Acknowledgements
This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK); (Project grant no: 215S945).
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