Derivatized cannabinoids

A typical separation which can be achieved is shown in Figure 4.1, while the mass spectra of the trimethylsilyl (TMS) ethers of the derivatized cannabinoids are shown in Figures 4.2-4.S. 

N,0-bistrimethylsilylic acid (BSA) is commonly used to derivatize cannabinoids, opiates and cocaine analogs (ecognine, etc., cocaine shows good chromatography). 

Mass fragmentography. Mass fragmentography was carried out using an LKB 9000 GC-MS instrument. The column was a 1.4 m x 2 mm i.d. silanized glass column containing 3% OV-17 on Gas Chrom CLP 100/120 mesh. Temperatures were in the column 180-210 , flash heater 250 , and source 290 . Helium was carrier gas (25 ml/min) and typical retention times for non-derivatized cannabinoids are ... 

Sanofi-Synthelabo researchers discovered pyrazole 53 and analogs to have potent Cannabinoid receptor-1 (CB-1) antagonist/inverse agonist activity and have progressed 53 into development for treatment of obesity and alcohol dependence. The synthesis of 53 was accomplished by heating the diketone sodium salt 51 with the aryl hydrazine hydrochloride in acetic acid to provide the intermediate 52, which was further derivatized... 

It was clear that a milder form of derivatization was required if we were to be successful in carrying on with our original philosophy of analyzing the cannabinoids as their 1-0-alkyl derivatives. We found that the technique of phase transfer catalysis suited our requirements. 

Our first two techniques determined these compounds but by different extraction and derivatizations. Therefore, these techniques lacked general applicability to the cannabinoids. Our philosophy held true but our chemistry did not. 

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.

Summary of Derivatization and Detection Mode for the Analysis of Cannabinoids... 

It should be noted at the outset that gas chromatography of the cannabinoids may present problems in that if derivatization is not employed, then the carboxylic acid compounds will decompose, since they are thermally labile. 

Given the structures of the cannabinoids below, why is derivatization necessary ... 

Figure 4.6 Gas chromatogram of cannabinoids from a herbal sample of cannabis after derivatization with N,0-BSA.

HPLC as a comparative technique has a number of advantages, including the fact that it does not require the analyte to be volatile, it does not require any pre-treatment of the sample prior to analysis, such as derivatization, it can be automated and can be used quantitatively. The HPLC technique used is a reversed-phase system and employs ion-suppression. Cannabinoids are weakly acidic drugs due to the presence of phenolic moieties on the aromatic rings. 

This chapter has concentrated on only the major derivative classes employed in GC-MS, and only a few examples of each could be mentioned. However, the literature, including the other chapters of the present handbook, contains an abundance of references and descriptions of other derivative types, many of which will have hidden mass spectrometric potential for particular applications. Trace analyses have been covered in depth for steroids [202] and cannabinoids [203] and in the application of NICI to drugs [204] and neurotransmitters [205]. A recent volume [206] includes a number of specialist chapters devoted to the application of mass spectrometry in different areas of biomedical research. It can be consulted for more in-depth information of work done with particular analyte classes, and contains a short overview of chemical derivatization for mass spectrometry, including some GC-MS examples [207], Comprehensive reviews by Evershed, covering developments and new applications of GC-MS, contain many references to derivati-... 

Many drugs can be chromatographed using GC directly howeveq a number of compounds may give rise to problems such as thermal decomposition (cannabinoids and cocaine alkaloids), reactions within the instrument between compounds in the injected mixture (morphine), adsorption, or coelution. In order to improve chromatographic results some drug compounds require derivatization where -OH,... 

Alemany et al. (1993) resolved a mixture of cannabinoids in human plasma by extraction with a C-18 Sep Pak cartridge, derivatization with dansyl chloride and scanning at 340 nm. Jain (1993) studied interferences of adulterants on TLC... 

There are numerons reports for the gas chromatographic determination of THC and its metabolites, ll-nor-A-9-tetrahydrocannibinol-9-carboxylic acid (THC-COOH) and ll-hydroxy-A-9-tetrahydrocannibinol (11-OH-THC) in urine and blood. THC is not normally found in urine, so it must be determined in blood at levels around 2-4 ng/mL. The TMS derivative is the most widely used derivati-zation procedure with GCMS for the determination of cannabinoids. In addition to the obvious advantages of derivatizing the THC metabolites, the acidic constituents of cannabis mnst be derivatized because they can easily decarboxylate above 80°C. Almost aU gas chromatographic procedures today use fused-silica capillary columns for this analysis. Determination of THC in blood is routinely done in forensic toxicological samples, and the detection and quantification of the two THC metabolites in mine is a routine procedure for proof of cannabis use in workplace testing. Several of the procedures used for this type of analysis are listed in Table 16.9. 

Some compounds can be easily detected by both CGC and high-pressure liquid chromatography (HPLC), for instance, cocaine, which has characteristic electron-impact mass and UV fluorescence spectra (Fernandez et al., 2009). Inert nonpolar or low-polar silicone phases have chromatographic features suitable for separation and analysis by CGC. In addition, dedicated base-treated stationary phases are now used to improve the identification and quantification of compoimds. HPLC analyses are done by reversed-phase elution with buffered water/organic solvent mixtures. In contrast, numerous native drags and almost all metabolites require chemical derivatization before analysis [e.g., with trifluoroacetic anhydride, A -methyl-7V-trimethylsilyltrifluoroacetamide, or iV-(r-butyldimethylsilyl)-Af-methyltrifluoroacetamide, which is usually used for cannabinoids] this improves the analytical performance of the method and drastically reduces the risk of misinterpretation and misidentification of the compoimds. 

Because marijuana is the most widely used illegal drug of abuse in this country there is great interest in detection of cannabinoids and their metabolites [38,39]. Initial extraction from blood, hair, and body tissues is typically by LLE [38,40-44]. Goodall and Basteyns [40] used BSTFA for derivatization and found LODs of 0.2 ng/ml for A tetrahydrocannabinol (THC) and 11-hydroxy-THC (11-OH-THC) and 2 ng/ml for 11 nor-9-carboxy-THC (THCCOOH) in blood, using deu-terated internal standards [40]. For MS detection, El was used with SIM. This method of analysis is proposed as an alternative to more costly detection methods involving negative-ion Cl GC-MS or tandem MS. 

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