Cannabinoid HPLC analysis

This paper is a progress report on the use of simultaneous dual wavelength (215nm, 280nm)absorbance detection for HPLC analysis of cannabinoids in biological fluids. Work in progress on fluorescence detection of cannabinoids will be the subject of a later report. 

Direct HPLC analysis of urine extracts appears feasible for A -THC. 215nm is the optimum wavelength for detection of THC-class compounds. Dual wavelength at 215 and 280nm serves as a valuable check on cannabinoid retention assignment and as a screen for unknown THC or CBN-class metabolites. The latter feature was demonstrated in the observance of CBN-class peaks in both hexane and E-I extracts. This observation suggests a CBN-metabolic route of A -THC. Evidence of a CBN-metabolic route for A -THC has been reported by McCallum (8) and Green (6) for humans and by Ben Zvi et al (9) for rhesus monkeys. 

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]. 

Whilst for the analysis of plant material for cannabinoids both GC and HPLC are commonly used, in analytical procedures the employment of GC-based methods prevails for human forensic samples. Nonetheless, the usage of HPLC becomes more and more of interest in this field especially in combination with MS [115-120]. Besides the usage of deuterated samples as internal standards Fisher et al. [121] describe the use of a dibrominated THC-COOH (see 7.5). The usage of Thermospray-MS and electrochemical detection provide good performance and can replace the still-used conventional UV detector. Another advantage in the employment of HPLC rather than GC could be the integration of SPE cartridges, which are needed for sample preparation in the HPLC-system. 

High performance liquid chromatography (HPLC) can rapidly separate drugs and metabolites from endogenous compounds in biological fluids. Fractions are readily collected and thus HPLC has been used in cannabinoid work to purify biological fluid extracts prior to analysis by techniques offering either more sensitive or specific detection than has been available for LC. 

HPLC cleanup of biological fluids has been reported prior to cannabinoid analysis by direct mass spectrometry, (1) GC-mass spectrometry, (Wall, this vol., Clarks, this vol.), GC (2) and radioimmunoassay (Teale, this vol.). 

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. 

An earlier study on the ultraviolet absorption properties of the cannabinoids performed in our laboratory gave reason to predict that HPLC-UV analysis of these compounds might be feasible. As shown in Figure 3, this reassessment of ultraviolet spectral properties of I-IV clearly demonstrated a preponderance of absorption characteristics of I at 212 nm as contrasted to 273.7 nm used in our earlier work. Literature values had been reported for absorption patterns of I (15),... 

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... 

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. 

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. 

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