Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Bioanalysis of new designer drugs

Since the late 1990s the illicit drug market has undergone considerable change: along with the traditional drugs of abuse that still dominate, more than 100 psychotropic substances designed to bypass controlled substances legislation have appeared and led to intoxications and fatalities. Starting from the huge class of phenylalkylamines, containing many subgroups, the spectrum of structures has grown from tryptamines, piperazines, phenylcyclohexyl derivates and pyrrolidinophenones to synthetic cannabinoids and the first synthetic cocaine. Due to the small prevalence and high number of unknown substances, the detection of new designer drugs is a challenge for clinical and forensic toxicologists. Standard screening procedures might fail because a recently discovered or yet unknown substance has not been incorporated in the library used. Nevertheless, many metabolism studies, case reports, screening methods and substance-profiling papers concentrating on single compounds have been published. This review provides an overview of the developed bioanalytical and analytical methods, the matrices used, sample-preparation procedures, concentration of analytes in case of intoxication and also gives a résumé of immunoassay experiences. Additionally, six screening methods for biological matrices with a larger spectrum of analytes are described in more detail.

Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents

The Cannabis plant and its products consist of an enormous variety of chemicals. Some of the 483 compounds identified are unique to Cannabis, for example, the more than 60 cannabinoids, whereas the terpenes, with about 140 members forming the most abundant class, are widespread in the plant kingdom. The term “cannabinoids” [note: “ ” represents a group of C21 terpenophenolic compounds found until now uniquely in Cannabis sativa L. (1). As a consequence of the development of synthetic cannabinoids (e.g., nabilone [2], HU-211 [dexanabinol; ref. (3), or ajulemic acid [CT-3; ref. 4]) and the discovery of the chemically different endogenous cannabinoid receptor ligands (“endocannabinoids,” e.g., anandamide, 2-arachidonoylglycerol) (5,6), the term ’“phytocannabinoids’” was proposed for these particular Cannabis constituents (7).

Synthetic-echo time postprocessing technique for generating images with variable T2-weighted contrast: diagnosis of meniscal and cartilage abnormalities of the knee

To assess the feasibility and accuracy of a synthetic-echo time (TE) magnetic resonance (MR) postprocessing technique for the diagnostic evaluation of abnormalities of menisci and articular cartilage in the knee.

Hair analysis for Delta9-tetrahydrocannabinolic acid A--new insights into the mechanism of drug incorporation of cannabinoids into hair

Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Delta9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10 pg/mg) than the corresponding THC concentrations. Delta9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a 'precursor' of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the 'non-artefact' THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Design and construction of a new Drosophila species, D.synthetica, by synthetic regulatory evolution

Here, I merge the principles of synthetic biology1,2 and regulatory evolution3-11 to create a new species12-15 with a minimal set of known elements. Using preexisting transgenes and recessive mutations of Drosophila melanogaster, a transgenic population arises with small eyes and a different venation pattern that fulfills the criteria of a new species according to Mayr's "Biological Species Concept"7,10. The genetic circuit entails the loss of a non-essential transcription factor and the introduction of cryptic enhancers. Subsequent activation of those enhancers causes hybrid lethality. The transition from "transgenic organisms" towards "synthetic species", such as Drosophila synthetica, constitutes a safety mechanism to avoid hybridization with wild type populations and preserve natural biodiversity16-18. Drosophila synthetica is the first transgenic organism that cannot hybridize with the original wild type population but remains fertile when crossed with other transgenic animals.

Effects of dietary supplementation with synthetic vitamin D-3 and 25-hydroxycholecalciferol on blood calcium and phosphate levels and performance in laying hens

We investigated the effects of different dietary vitamin D regimen on selected blood parameters in laying hens. Supplementation with vitamin D-3 only was compared with a combination of vitamin D-3 and its metabolite 25-hydroxy-cholecalciferol (25(OH)D-3). Blood concentrations of total calcium, phosphate and 25 (OH)D-3 were determined. Four thousand one-day-old LSL chicks were split in two treatment groups and distributed to eight pens. The control group was given a commercial animal diet containing 2800 IU synthetic vitamin D-3 in the starter feed and 2000 IU synthetic vitamin D-3 in the pullet feed. The experimental group was fed the same commercial diet in which half the synthetic vitamin D-3 content had been substituted with 25(OH)D-3 (Hy center dot D (R)). At 18 weeks of age, pullets were transferred to the layer house. At the ages of 11, 18 and 34 weeks, between 120 and 160 blood samples were collected from both the control and the experimental groups, respectively. The experimental group had higher levels of 25 (OH)D-3 than the control group at all three ages. Serum calcium levels did not differ between the treatment groups at any age. With the onset of laying, calcium levels rose significantly. Whereas blood serum concentration at 18 weeks was 3 mmol/L in both treatment groups, it increased to 8.32 mmol/L in the control group and to 8.66 mmol/L in the experimental group at week 34. At weeks 11 and 34, phosphate was significantly lower in the experimental group. In conclusion, HyD (R) significantly affected serum phosphate and 25(OH)D-3 levels. No effects of (25(OH)D-3 supplementation on performance, shell quality and fractures of keelbones were found.

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.