Separation of cannabinoids

Huizing and T. M. Malingre. Use of amberlite XAD-2 columns for the separation of cannabinoids from cannabis extracts. J Chromatogr 1981 205 444—450. 

Method. The derivatives are formed by shaking the sample (dissolved in acetone) for 1 h at 45 °C with a 3-5 molar excess of recrystallized DNS-C1. The reaction is buffered at pH 10.8.0.25 ml of 1N sodium hydroxide is then added in order to hydrolyze the unchanged DNS-C1. The derivatives are extracted with 3 ml of ethyl acetate after addition of 1 ml of a saturated aqueous solution of sodium chloride to the reaction mixture. The organic phase is used for TLC on activated layers of silica gel G. The cannabinoids yield mono-DNS derivatives with the exception of cannabidiol which forms a bis-DNS derivative. The following solvent systems are satisfactory for separation of cannabinoids on silica gel A, benzene-acetone (9 1) B, cyclohexane-ethyl acetate (5 1) C, cyclohexane-acetone-diethylamine (20 4 1) and D, cyclohexane-acetone-triethylamine (20 4 1). The R f values of nine cannabinoids in the above solvent systems are given in Table 4.25. 

Figure 4. Normal phase HPLC separation of cannabinoids (two p-Porasil columns in series) using 5% THF in n-hexane at a flow rate of 0.5 mL/min...

Indeed, the nature of the sample seems to be a critical parameter in the reproducibility equation. Short-term and long-term reproducibility of retention times and peak areas for CEC separation of cannabinoids have been measured by Lurie et al. [66]. The RSDs (seven replicates) for retention times are around 0.5% for short-term and vary from 1.49% to 2.03% for the long-term study, while peak areas showed up to 39% variation. Significantly improved reproducibility values were obtained when one compound, cannabinol, was used as a reference. The RSDs for relative peak areas varied from 2.3% to 3.95% for short-term and 1.65% to 8.1% for the long-term study. 

Hajos N, Freund TF (2002a) Pharmacological separation of cannabinoid sensitive receptors on hippocampal excitatory and inhibitory fibers. Neuropharmacology 43 503-510... 

Later et al. analyzed steroids, antibiotics, and cannabinoids on a methylpolysiloxane (SE-33) capillary column using carbon dioxide as the mobile phase [27]. Figure 7.16 shows the separation of tetrahydrocannabinol and its six metabolites in less than 20 min. 

Figure 6. GLC (FID) of an extract of 2 mL of plasma containing A9-THC, Peak I (200 ng/mL), and ll-hydroxy-A9-THC, Peak II (200 ng/mL), before (A) and after (B) reversed-phase HPLC separation of both cannabinoids over a range of predetermined collection volumes.

Figure 7. Separation of natural, neutral cannabinoids on MicroPak CN-10 column with 0.25 mh/mvn 3% B isocratic solvent A = hexane solvent B — 5% methanol in dichloromethane.

In summary, RIA has two applications in the screening of plasma and urine specimens for cannabis use. Direct analysis of a sample gives an indication of the presence of cannabinoids on a semi-quantitative basis. Quantitation may then be expanded further and more specifically following HPLC separation. The identification of THC and its metabolites in established patterns could form the basis for an estimate of both the quantity of THC absorbed and the time of intake. 

Figure 6. Separation of neutral dansyl cannabinoids by one-dimensional TLC. Developing solvent cyclohexane/ethyl acetate (95 5). The striped spots indicate breakdown products of DANS-Cl. The left side of the plate shows the products of derivatization of standards. The right side shows a blank reaction mixture.

An excellent demonstration of the concept of niche application in CEC was offered by Lurie et al. in a study of CEC feasibility for analysis of seized drugs [66], In (he past, standard separation methods such as GC, GC/MS, HPLC, and CE have been employed in the analysis of cannabinoids, weakly acidic... 

In the 1980 s, we showed that THC-type compounds exhibit very high stereospecificity of cannabinoid action [20-24], Some of the previous observations regarding lack of stereospecificity were apparently due to separation problems. The (-)-enantiomer of d8-l 1-hydroxy-THC-DMH (3) (HU-210) (DMH=l,l-dimethylheptyl) was shown to be several thousand times more potent than the (-i-)-enantiomer (HU-211) (4) in a series of animal tests. The synthesis of HU-211 is presented in Figure 5.2 [25]. The intermediate (+)-4-oxo-myrtenyl pivalate (5), and its (-)-enantiomer are highly crystalline and can be obtained essentially stereochemically pure by recrystallization. This lucky observation made possible the ultimate synthesis of both enantiomers, HU-211 and HU-210, ine.e. higher than 99.8%, as determined by HPLC analysis of the respective diastereoisomeric bis-(S) (+)-a-methoxy-a-(trifluoro-methyl)phenyl-acetyl (MTPA) esters [25], Nonclassical cannabinoids were also shown to exhibit high stereospecificity of cannabinoid activity [21, 26]. 

Numerous cannabinoids, anandamides and heterocyclics have been compared for CBj and CB2 binding [87], Most compounds did not differ significantly in their binding values for the two receptors except the specific CBj antagonist SR141716A (see below) which binds weakly to CB2 and the indole compound JWH-015 (20) which showed a CBj/CB2 binding ratio of 27.75. Busch-Petersen etal. synthesized a series of cannabinoids in which rotation around the Cj -C2 bond was blocked [102]. The compound with the cis-heptene side-chain (21) had the highest affinity for CBj (0.89 [iM) with the widest separation between CBj and CB2 affinities. Pertwee et al. showed... 

Cannabis and some cannabinoids relieve pain. However, the therapeutic doses are essentially equivalent to the doses that cause CNS effects and, except in very specific conditions (possibly migraine) they are of limited use. Although several companies have produced a large number of derivatives, as previously reviewed in this series [1] and elsewhere [105,107] the situation has not changed. No practical separation of activity has been achieved, except with the synthetic cannabinoid HU-211 (see below) which does not bind to either CBi or CB2. Hence most of the work reported in the last decade deals mainly with the pharmacology of pain reduction by cannabinoids, rather than with drug discovery and development. 

Figure 4.8 HPLC chromatogram of cannabinoids separated by using the HPLC system described in the text f, cannabidiol 2, cannabinol 3, A -THC 4, A -THC.

The CBi and CB2 receptors (Fig. 2) share only 44% overall amino acid identity, which rises to 68% in the TM domains (Munro et al. 1993). However, most cannabinoid receptor agonists do not discriminate between the receptor subtypes (Felder et al. 1995 Pertwee 1997). There are several ligands which are CBi- or CB2-selective (5- to 60-fold), and a few ligands with a greater separation of activity at each receptor (100- to 1,000-fold) (Griffin et al. 1999, 2000 Hanus et al. 1999 Huffman et al. 1996, 1999 Ibrahim et al. 2003 Showalter et al. 1996 Tao et al. 1999). For example, l-deoxy-A -THC showed no affinity for the CBi receptor but has good affinity (K,=32 nM) for the CB2 receptor (Huffman et al. 1999). However, there is a need for more selective agonists to produce specific receptor-mediated effects for in vivo studies. 

As early as in the 1970s, the potential use of cannabinoid ligands as antihypertensive agents had been considered (Archer 1974 Crawford and Merritt 1979 Varma et al. 1975 Zaugg and Kyncl 1983), in the hope that their neurobehavioral and cardiovascular effects could be separated. Although an early study in normotensive rats indicated rapidly developing tolerance to the hypotensive and bradycardic effects of THC (Adams et al. 1976), a subsequent study in spontaneously hypertensive rats (SHR) found no evidence for tolerance for the same effects during a similar, 10-day treatment period (Kosersky 1978). 

Compared to other drugs of abuse, analysis of cannabinoids presents some difficult challenges. THC and 11-OH-THC are highly lipophilic and present in low concentrations in body fluids. Complex specimen matrices, i.e., blood, sweat, and hair, may require multi-step extractions to separate cannabinoids from endogenous lipids and proteins. Care must be taken to avoid low recoveries of cannabinoids due to their high affinity to glass and plastic containers, and to collection devices for alternate matrices (Blanc et al. 1993 Bloom 1982 Christophersen 1986 Joern 1992). THC and THCCOOH are predominantly found in the plasma fraction of blood, where 95% to 99% are bound to lipoproteins. Only about 10% of either... 

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