Dried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may significantly impact DBS-based quantitation. In this review, an overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis. The different strategies that try to cope with this problem are discussed, along with their potential and limitations. Implementation of some of these strategies in practice may help to overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioanalysis.

Synthetic cannabinoids (SCs) are the largest group of compounds currently monitored in Europe by the EU Early Warning System on new psychoactive substances. Emerging recreational use of these products has led to multiple cases of adverse health effects and even death. In contrast to marijuana, where Δ⁹-tetrahydrocannabinol (Δ⁹THC) is metabolized to only one major active metabolite, it has been reported that several major phase I metabolites of SCs remain biologically active, exerting cannabinoid (CB) receptor affinity, potency, and efficacy greater than those of Δ⁹THC. It is therefore reasonable that more SCs can also be biotransformed into molecules with various levels of CB activity. Here, we developed and applied a new G-protein coupled receptor (GPCR) activation assay based on NanoLuc binary technology (Promega). More specifically, by demonstrating CB1 and CB2 receptor activation by JWH-018 and a selection of its metabolites, we are the first to show the suitability of the newly developed bioassay for monitoring GPCR-mediated activity. We also successfully applied this reporter system to evaluate the in vitro activity of JWH-122, JWH-210, and PB-22, their 5-fluoro analogues (MAM-2201, EAM-2201, and 5F-PB-22, respectively), and their main phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at cannabinoid receptors. All of these active metabolites may prolong the parent compound’s psychotropic and physiological effects and may contribute to its toxicity profile. We also demonstrate a proof of concept of the applicability of the newly developed bioassay for screening urine for CB receptor activity exerted by SCs.

About a century after its first described application by Ivar Bang, the potential of sampling via dried blood spots (DBS) as an alternative for classical venous blood sampling is increasingly recognized. Perhaps best known is the use of DBS in newborn screening programs, ignited by the hallmark paper by Guthrie and Susi half a century ago. However, it is only recently that both academia and industry have recognized the many advantages that DBS sampling may offer for bioanalytical purposes, as reflected by the strong increase in published reports during the last few years. Currently, major DBS applications include newborn screening for metabolic disorders, epidemiological surveys (e.g. HIV monitoring), therapeutic drug monitoring (TDM), as well as toxicology. In this review, we provide a comprehensive overview of the distinct subdisciplines of toxicology for which DBS sampling has been applied. DBS sampling for toxicological evaluation has been performed from birth until autopsy, aiming at the assessment of therapeutic drugs, drugs of abuse, environmental contaminants, toxins, as well as (trace) elements, with applications situated in fields as toxicokinetics, epidemiology and environmental and forensic toxicology. We discuss the strengths and limitations of DBS in the different subdisciplines and provide future prospects for the use of this promising sampling technique in toxicology.