Benjamin De Backer

Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material

GC is commonly used for the analysis of cannabis samples, e.g. in forensic chemistry. However, as this method is based on heating of the sample, acidic forms of cannabinoids are decarboxylated into their neutral counterparts. Conversely, HPLC permits the determination of the original composition of plant cannabinoids by direct analysis. Several HPLC methods have been described in the literature, but most of them failed to separate efficiently all the cannabinoids or were not validated according to general guidelines. By use of an innovative methodology for modelling chromatographic responses, a simple and accurate HPLC/DAD method was developed for the quantification of major neutral and acidic cannabinoids present in cannabis plant material: Delta9-tetrahydrocannabinol (THC), THC acid (THCA), cannabidiol (CBD), CBD acid (CBDA), cannabigerol (CBG), CBG acid (CBGA) and cannabinol (CBN). Delta8-Tetrahydrocannabinol (Delta8-THC) was determined qualitatively. Following the practice of design of experiments, predictive multilinear models were developed and used in order to find optimal chromatographic analytical conditions. The method was validated following an approach using accuracy profiles based on beta-expectation tolerance intervals for the total error measurement, and assessing the measurements uncertainty. This analytical method can be used for diverse applications, e.g. plant phenotype determination, evaluation of psychoactive potency and control of material quality.

Differential effects of cocaine on dopamine neuron firing in awake and anesthetized rats.

Cocaine (benzoylmethylecgonine), a natural alkaloid, is a powerful psychostimulant and a highly addictive drug. Unfortunately, the relationships between its behavioral and electrophysiological effects are not clear. We investigated the effects of cocaine on the firing of midbrain dopaminergic (DA) neurons, both in anesthetized and awake rats, using pre-implanted multielectrode arrays and a recently developed telemetric recording system. In anesthetized animals, cocaine (10 mg/kg, intraperitoneally) produced a general decrease of the firing rate and bursting of DA neurons, sometimes preceded by a transient increase in both parameters, as previously reported by others. In awake rats, however, injection of cocaine led to a very different pattern of changes in firing. A decrease in firing rate and bursting was observed in only 14% of DA neurons. Most of the other DA neurons underwent increases in firing rate and bursting: these changes were correlated with locomotor activity in 52% of the neurons, but were uncorrelated in 29% of them. Drug concentration measurements indicated that the observed differences between the two conditions did not have a pharmacokinetic origin. Taken together, our results demonstrate that cocaine injection differentially affects the electrical activity of DA neurons in awake and anesthetized states. The observed increases in neuronal activity may in part reflect the cocaine-induced synaptic potentiation found ex vivo in these neurons. Our observations also show that electrophysiological recordings in awake animals can uncover drug effects, which are masked by general anesthesia.

Evolution of the content of THC and other major cannabinoids in drug-type cannabis cuttings and seedlings during growth of plants.

Abstract:  In Europe, authorities frequently ask forensic laboratories to analyze seized cannabis plants to prove that cultivation was illegal (drug type and not fiber type). This is generally done with mature and flowering plants. However, authorities are often confronted with very young specimens. The aim of our study was to evaluate when the chemotype of cannabis plantlets can be surely determined through analysis of eight major cannabinoids content during growth. Drug‐type seedlings and cuttings were cultivated, sampled each week, and analyzed by high‐performance liquid chromatography with diode array detection. The chemotype of clones was recognizable at any developmental stage because of high total Δ9‐tetrahydrocannabinol (THC) concentrations even at the start of the cultivation. Conversely, right after germination seedlings contained a low total THC content, but it increased quickly with plant age up, allowing chemotype determination after 3 weeks. In conclusion, it is not necessary to wait for plants’ flowering to identify drug‐type cannabis generally cultivated in Europe.