DOI 10.1515/labmed-2012-0059 J Lab Med 2013; 37(4): 167–180 Drug Monitoring und Toxikologie/Drug Monitoring and Toxicology Redaktion: W. Steimer August Goebel , Marcus Boehm , Hartmut Kirchherr and W. Nikolaus K ü hn-Velten * Simultaneous identification and quantification of synthetic cannabinoids (cannabimimetics) in serum, hair, and urine by rapid and sensitive HPLC tandem mass spectrometry screenings: overview and experience from routine testing Simultane Identifizierung und Quantifizierung synthetischer Cannabinoide (Cannabimimetika) in Serum, Haar und Urin mittels schneller und sensitiver HPLC-Tandem- Massenspektrometrie-Screenings: Ü bersicht und Erfahrungen aus der Routineanalytik Abstract: Detection and quantification of synthetic can- Keywords: cannabimimetic; cannabinoid receptor nabinoids (synonym: cannabimimetics) used as a substi- agonist; designer drug; drug screening; LC-MS/MS; spice; tute for natural cannabis has been a real toxicological and synthetic cannabinoid; tandem-mass spectrometry. forensic issue since 2008. On the basis of a short overview of the pharmacological principle, chemical classification, Zusammenfassung: Der Nachweis und die Quantifizierung and legal situation in Germany, the development of sev- synthetischer Cannabinoide (Synonym: Cannabimime- eral analytical and screening approaches is presented. tika), die in Form von R ä uchermischungen als Cannabis- The paper further describes and validates a novel method ersatz in Gebrauch sind, ist seit 2008 ein aktuelles Thema for the simultaneous identification and quantification of der toxikologischen und forensischen Analytik. Auf der JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH- Grundlage einer kurzen Ü bersicht ü ber das pharma- 200, JWH-210, JWH-250, WIN48,098, WIN55,212-2, AM-694 kologische Prinzip, die chemische Klassifikation und die and CP47,497 by means of liquid chromatography-tandem gesetzliche Situation in Deutschland wird die Entwick- mass spectrometry (LC-MS/MS) in human serum and lung verschiedener Screeningmethoden dargestellt. Es hair using JWH-018-d11 and THC-d3 as internal stand- wird eine neue Methode zur simultanen Quantifizierung ards. Detection limits ranging from 0.018 to 0.192 ng/mL von JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH- for serum and from 0.140 to 0.820 pg/mg for hair, as well 200, JWH-210, JWH-250, WIN48,098, WIN55,212-2, AM-694 as very short retention times (up to 2.4 min), qualify this und CP47,497 in Serum und Haaren mittels Fl ü ssigchroma- method, which presently includes 11 further compounds tographie-Tandem-Massenspektrometrie (LC-MS/MS) mit on a semi-quantitative basis, for rapid screening. The same JWH-018-d11 und THC-d3 als internen Standards beschrie- method, using the deuterated 3-hydroxybutyl derivative of ben und validiert, die aufgrund der Nachweisgrenzen JWH-073, was applied for detection of 14 urinary metabo- von 0.018 bis 0.192 ng/mL im Serum und 0.140 bis 0.820 lites after enzymatic hydrolysis of conjugates. Observation pg/mg Haar sowie der besonders kurzen Retentionszeiten from routine analysis of 359 serum and 84 hair specimens (maximal 2,4 Minuten) als Screeningmethode geeignet reveals that concentrations of cannabimimetics in posi- ist. Die Methode inkludiert derzeit 11 weitere Substanzen tive samples may differ by factors of at least 400 and 3000 auf semiquantitativer Basis und ist nach enzymatischer with median values of 0.5 ng/mL and 9 pg/mg for serum Hydrolyse der Glucuronide unter Verwendung des deu- and hair, respectively. Conclusions regarding consump- terierten 3-Hydroxybutyl-Metaboliten von JWH-073 als tion behaviors and strategies to decipher metabolic routes internem Standard f ü r die Suche nach 14 Urinmetaboliten of these substances and possible implications are also verwendbar. Eine konsekutive Analytik der Routineana- discussed. lytik von 359 Serum- und 84 Haarproben ergibt, dass die 168 Goebel et al.: Simultaneous identification and quantification of synthetic cannabinoids gemessenen Konzentrationen in positiven Proben um or alterations in motor control [8 – 10] . Neuroprotective einen Faktor von 400 (Serum; Median 0.5 ng/mL) bzw. effects are currently discussed. Furthermore, receptor 3000 (Haare; Median 9 pg/mg) differieren k ö nnen. M ö gli- activation improves appetite and weight gain, so that it che R ü ckschl ü sse auf das Konsumverhalten und Ans ä tze can be expected that blockade of the CB1 receptor system zur Aufkl ä rung von Stoffwechselrouten und ihre Bedeu- leads to appetite loss. This has been successfully tried for tung werden abschlieβ end diskutiert. overweight patients with the drug rimonabant until 2008, although obese patients did not benefit from rimonabant Schl ü sselw ö rter: Cannabimimetika; Cannabinoid- treatment in terms of progression of carotid atherosclerosis Rezeptor-Agonisten; Designerdrogen; Drogenscree- [11] . By contrast, CB2 receptors are mainly associated with ning; LC-MS/MS; Spice; Synthetische Cannabinoide; different cells of the immune system and are expressed in Tandem-Massenspektrometrie. the cell membrane of B lymphocytes but mostly intracellu- larly in T cells and monocytes [12] . Their agonistic ligands exert anti-inflammatory and immunosuppressive activ- *Correspondence: Prof. Dr. W. Nikolaus K ü hn-Velten, Medizinisches ity via modulation of cytokine release. A recent approach Labor Bremen, Haferwende 12, 28357 Bremen, Germany, Tel.: + 49 421 2072107, Fax: + 49 421 20727107, integrating both receptor systems focuses on cannabi- E-Mail: [email protected] noid modulation of neuroinflammatory diseases [13] . As August Goebel, Marcus Boehm and Hartmut Kirchherr: could have been expected, a huge amount of non-natural Medizinisches Labor Bremen, Bremen, Germany cannabinoid receptor agonists (also known as cannabi- mimetics) were synthesized over the years in order to dif- ferentiate receptor systems by selectivities and potencies, Introduction to develop pain modulators, and, more recently, to find compounds with a potential to interact with the appetite Δ 9 -Tetrahydrocannabinol (THC), the pharmacological regulation network [3, 8] . principle of cannabis (marijuana), is the best-known The heterogeneous expression of CB receptors exogenous psychoactive cannabinoid with its effects enforces the presence of one or more endogenous physi- being known for many centuries. As a recreational drug, ological ligands. The search for candidate molecules THC is used in the form of chopped parts or pressed finally resulted in the identification of anandamide resin (hashish) of female plants [1] . For pharmaceutical (synonym: arachidonoyl ethanolamide) and related non- purposes, THC can be extracted by solvents from respec- classical derivatives of (poly)unsaturated fatty acids such tive plant material with the consequence that additional as 2-arachidonoylglycerol or oleamide [8, 14] . In addition psychoactive compounds are likewise present as in, for to receptor-mediated neuroprotective and immunosup- example, nabiximols which is used as supportive treat- pressive effects, anandamide may similarly act through ment of spasticity in patients suffering from multiple scle- modulation of ion channels, of vanilloid TRPV receptors, rosis [2] . By contrast, semi-synthetically produced THC is or of peroxisome proliferator-activated receptors, which used in certain countries as a pharmaceutical agent, such belong to the family of nuclear receptors [3, 8, 15] . From a as dronabinol for treatment of hyperemesis and appetite pharmacological point of view, the local impact on anan- loss for patients suffering from the AIDS wasting syn- damide synthesis and of anandamide degradation, which drome or side effects of chemotherapy [2, 3] . is predominantly catalyzed by fatty acid amide hydrolase The search for molecular transducers has led to the [16] , are promising approaches with regard to pain, appe- detection and cloning of two subtypes of cannabinoid tite, and immune cell modulation [8] . receptors, namely CB1 (with two subpopulations) and Cannabimimetics have been synthesized for several CB2 [3, 4] . Both are predominantly coupled to heterotri- years and have emerged on the market, which are offi- meric inhibitory G proteins with the consequence that cially marked “ not for human consumption ” , although, receptor activation results in diminished cAMP accu- not officially, designed as recreational hallucinogenic mulation, although the receptors express significant drugs and distributed in headshops or, predominantly, on intrinsic (or constitutive) activity due to rab-dependent internet platforms. These preparations are usually herbal endocytic cycling [5, 6] . The CB1 receptor predominates in mixtures to which one or more synthetic cannabimimetics the central nervous system where it mediates retrograde are added; one of the first brands was called “ Spice ” [17 – signaling at GABAergic and glutaminergic synapses [7] . 20] . Initially, the search of active components focused on It mediates psychoactive effects such as modulation of secondary plant ingredients such as salvinorin A before emotional control, impairment of cognition and memory, the principle became public [21] . The success of such Goebel et al.: Simultaneous identification and quantification of synthetic cannabinoids 169 preparations resulted, at least in part, from the fact that Makriyannis); (iv) aminoalkylindoles (subgroups consist it was initially possible to circumvent positive results of of naphthoylindoles, phenylacetylindoles, naphthylmeth- routine immunoassay-based