Cannabigerol (CBG) signal enhancement in its analysis by gas chromatography coupled with tandem mass spectrometry.

PURPOSE: According to recent reports, cannabigerol (CBG) concentration level in blood and body fluids may have forensic utility as a highly specific albeit insensitive biomarker of recent cannabis smoking. While the analytical sensitivity of cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC) or cannabinol (CBN) estimation by gas chromatography-mass spectrometry (GC-MS) is similar and sufficiently high, it is exceptionally low in the case of CBG (ca. 25 times lower than for the other mentioned cannabinoids). The purpose of this study is to explain the reasons for the extremely low analytical sensitivity of GC-MS in estimating CBG and to present possible ways of its improvement. METHODS: Nuclear magnetic resonance (NMR) data and GC-MS responses to CBG and its various derivatization and transformation products were studied. RESULTS: The validation data of individual derivatives of CBG and its transformation products were established. CBG silylation/acylation or hydration allows to decrease LOD about 3 times, whereas the formation of pyranic CBG derivative leads to 10-times decrease of LOD. The paper enriches the literature of the subject by providing MS and NMR spectra, not published so far, for derivatives of CBG and its transformation products. The most likely cause of low GC-MS response to CBG is also presented. CONCLUSIONS: The presented results shows that although the signal increase of CBG can be obtained through its derivatization by silylation and/or acylation, the greatest increase is observed in the case of its cyclization to the pyranic CBG form during the sample preparation process. The CBG cyclization procedure is very simple and workable in estimating this cannabinoid in blood/plasma samples.

Determination of 3- and 4-chloromethcathinone interactions with plasma proteins: study involving analytical and theoretical methods.

PURPOSE: The purpose of this paper was to determine 3- and 4-chloromethcathinone (3- and 4-CMC) binding degree and possible binding interaction modes with human serum albumin (HSA) using analytical and theoretical methods. METHODS: Experimental determination of 3- and 4-CMC binding degree with HSA was performed using gas chromatography-tandem mass spectrometry preceded by the equilibrium dialysis (ED) and ultrafiltration (UF). Nuclear magnetic resonance (NMR) spectroscopy was used to determine 3- and 4-CMC epitope-binding maps and possible binding sites in HSA. The molecular docking and molecular dynamics were employed to obtain detailed information about binding modes of 3- and 4-CMC enantiomers in HSA. RESULTS: As follows from the presented data, the degree of binding of 3- and 4-CMC is at a similar level of approx. 80%. This indicates a relatively strong binding of CMC to plasma proteins. The model studies employing the NMR spectroscopy and molecular simulations indicate that both CMCs bind to HSA. The whole 3- and 4-CMC molecules are embedded in the binding sites, with aromatic moieties being in the closest contact with the HSA residues. Moreover, conducted experiments show that  Sudlow site II is the main binding center for 3- and 4-CMC and  Sudlow site I acts as the secondary binding site. CONCLUSIONS: Although many studies describe pharmacological and toxicological properties of synthetic cathinones (SC), the data taking SCs binding in plasma into consideration are scarce. To our knowledge, this is the first report presenting comprehensive experimental and theoretical characterization of 3- and 4-CMC binding with plasma proteins.

Unexpected formation of dichloroacetic and trichloroacetic artefacts in gas chromatograph injector during Cannabidiol analysis.

The knowledge about the stability of compounds and possible ways of their transformation in the process of sample preparation for analysis and during analysis itself is very helpful in the assessment of possible errors which can appear when an accurate and precise estimation of compound concentration in tested samples is attempted. The present paper shows that a significant amount of CBD present in the blood/plasma sample analyzed by means of GC transforms in the hot GC injector not only to 9α-hydroxyhexahydrocannabinol, 8-hydroxy-iso-hexahydrocannabinol, Δ9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, and cannabinol but also to the trichloroacetic esters of Δ9-THC and Δ8-THC and, unexpectedly, to their dichloroacetic esters when trichloroacetic acid is used as protein precipitation agent. The increase of GC injector temperature favors the formation of dichloroacetic esters of Δ9-THC and Δ8-THC in relation to their trichloroacetic ones. The appearance of dichloroacetic esters of Δ9-THC and Δ8-THC among CBD transformation products is probably the result of the thermal decomposition of their trichloroacetic esters. The transformation of trichloroacetic derivatives of organic compounds into their dichloroacetic derivatives in GC injector has not been reported yet. The instability of trichloroacetic derivatives of Δ8-/Δ9-THC during their GC analysis is probably accounts for the lack of their GC-MS spectra in the databases. NMR, GC-MS and LC-MS spectra of the newly discovered derivatives constitute an important element of the work. The obtained results demonstrate why the use of trichloroacetic acid for plasma samples deproteinization should be avoided when CBD and/or THC are determined by GC.

Chromatographic analysis of CBD and THC after their acylation with blockade of compound transformation.

METHODS: for the analysis of cannabinoids in bio-matrices are continually improved to achieve best possible sensitivity in their detection and accurate quantification. It has been well documented that CBD cyclizes to Δ9-THC and Δ9-THC isomerizes to Δ8-THC under acidic conditions by means of a Lewis-acid-catalyzed process, causing difficulty in accurate quantification of Δ9-THC in the presence of CBD, of CBD itself and of Δ9-THC itself when these compounds have to be derivatized by acylation. The present paper shows that CBD cyclization and Δ9-THC isomerization can be blocked by tertiary amines or azines, which capture protons appearing in the derivatizing mixture during acylation. The efficiency of the described acylation of CBD depends on the time and temperature of the derivatizing process, whereas the degree of CBD acylation, i.e. the synthesis of mono- or di-acylate CBD derivative, depends on the mutual ratio of the cannabinoid, the acylating agent and the proton binding compound. The way of mono- and di-acyl CBD derivatives formation described in the paper has not been reported yet. The paper contains a comprehensive analytical characterization of two types of CBD acyl derivatives, CBD-TFA and CBD-Ac, obtained by NMR, GC-MS and LC-MS.

Formation of trifluoroacetic artefacts in gas chromatograph injector during Cannabidiol analysis.

The knowledge of compounds stability in the process of sample preparation for analysis and during analysis itself helps assess the accuracy and precision of estimating their concentration in tested samples. The present paper shows that a significant amount of CBD present in the blood/plasma sample analyzed by means of GC transforms in the hot GC injector not only to 9α-hydroxyhexahydrocannabinol, 8-hydroxy-iso-hexahydrocannabinol, delta-9-tetrahydrocannabinol, Δ8-tetrahydrocannabinol, and cannabinol but also to the trifluoroacetic esters of Δ9-THC and Δ8-THC, when trifuoroacetic acid is used as protein precipitation agent. The amount of those newly revealed CBD transformation products depends on the GC injector temperature and on the extrahent type when extracts of the supernatants centrifuged from human plasma samples are analyzed after their preliminary protein precipitation by trifuoroacetic acid. Although trifuoroacetic acid as a protein precipitating agent has many disadvantages, it is quite often used for this purpose due to its very high protein precipitation efficiency. The results presented in the study demonstrate why the use of trifuoroacetic acid for plasma samples deproteinization should be avoided when CBD is determined by GC.