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.

GC vs. HPLC in quantitation of CBD, CBG, ∆9-THC and CBN in plasma using different sample preparation methods.

The sensitivity of complex analytical procedures depends not only on the sensitivity of the analytical instrument used, but also on the recovery degree of the examined analyte by the employed sample preparation method. The recovery degrees of individual cannabinoids reported in literature, estimated using the same sample preparation method, are unexpectedly divergent. Therefore, the aim of this study was a thorough assessment of the most commonly used sample preparation methods, such as protein precipitation, LLE, QuEChERS and SPE, in the context of the reliability of the obtained results. The presented report shows that the highest sensitivity, precision and reliability of the chromatographic analysis of CBG, CBD, ∆9-THC and CBN in human plasma can be obtained using SPE. The recovery degrees of these cannabinoids by SPE are highly repeatable and exceed 95 %, while they are significantly lower for such sample preparation methods as protein precipitation, LLE and QuEChERS (ca. 80, 65 and 87, respectively). Moreover, the supernatants obtained by the latter methods contain interferents evoking matrix-effect, which makes reliable quantification of the listed cannabinoids by GC difficult. To our knowledge, the paper is the first such extensive comparison of sample preparation procedures used for the determination of cannabinoids in plasma by GC-MS and HPLC-MS. The presented results and the discussion allow to understand why different recovery degrees for the same xenobiotic can be find in literature despite they have been estimated using the same or different sample preparation method or different chromatography types.

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.

Oleamide as analyte protectant in GC analysis of THC and its metabolites in blood.

Methods for the analysis of cannabinoids in bio-matrices are continually being developed, to achieve a proper sensitivity required for their detection and accuracy in their quantification. The presented paper shows that the analytical sensitivity of GC-MS to THC and its metabolites in blood samples can be significantly increase by oleamide (OLA) addition to the examined sample, which evokes the matrix effect of transient character. The magnitude of signal increment resulting from oleamide presence in the examined sample is the greatest for THC metabolites and depends on oleamide concentration in the examined sample. The use of transient matrix effect to increase the sensitivity of the analysis can be applied not only in QuEChERS procedure, which is applied in the described experiments, but also in other blood sample preparation methods. Evoking the transient matrix effect by means of OLA in the experimental analytical quantitation of THC and its metabolites in blood allowed to lower limit of detection (LOD) approximately by 20.5%, 87.6% and 90.1% in the case of THC, THC-OH and THC-COOH, respectively.

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.

Improving the sensitivity of estimating CBD and other xenobiotics in plasma samples: Oleamide-induced transient matrix effect.

The paper discusses the matrix effect evoked by oleamide (OLA), a compound frequently found in samples processed and/or stored in lab polypropylene vials or disposable syringes. In the case of many substances a higher response for their samples containing OLA than for net solutions is observed. The analyte signal gain resulting from OLA presence in the examined sample depends on the ratio of OLA concentration to analyte concentration. A characteristic feature of the matrix effect evoked by oleamide is its short duration, which makes the chromatographic data (retention value and signal magnitude of examined compound) repeatable and reproducible. The identified "transient matrix effect" may significantly increase the sensitivity of many analytical procedures employing GC. Evoking the transient matrix effect by means of OLA in the experimental analytical quantitation of cannabidiol in plasma allowed to lower its limit of detection (LOD) by more than 50 %.

Conversion of cannabidiol (CBD) to Δ9-tetrahydrocannabinol (Δ9-THC) during protein precipitations prior to plasma samples analysis by chromatography - Troubles with reliable CBD quantitation when acidic precipitation agents are applied.

The growing popularity of supplements containing cannabidiol (CBD), mainly CBD oils, in self-medication of humans and the increased interest in this compound in different preclinical and clinical trials stimulates the development of procedures of CBD analysis in plasma for the study of CBD pharmacology in people and animals or in establishing dose-therapeutic effect relationships of this compound. Preliminary removal of protein by its precipitation from plasma is still one of the willingly applied plasma sample preparation methods in many analytical procedures estimating plasma drug concentration, including CBD. The present paper shows that a significant amount of CBD transforms to Δ9-tetrahydrocannabinol (Δ9-THC) in a hot GC injection system when acidic precipitation agents, such as TFA, TCA, HClO4, H2SO4, ZnSO4 or CHCl3, are used for plasma protein precipitation. The transformation degree depends on the temperature of the GC injector, the concentration of the precipitation agent and the incubation time of plasma with the precipitating agent. At the CBD plasma concentration equal to 50 ng/ml, which is approximately the mean level for patients treated for epileptic syndromes, the CBD transformation degree can exceed 20%. For a reliable estimate of CBD in blood plasma, neutral precipitation agents (e.g. ACN, MeOH, acetone) should be used when plasma deproteinization precedes GC analysis. The presented results are important not only for analysts cooperating with pharmacologists and for medicine doctors examining the activity of CBD-containing drugs in the therapeutic process, but also for forensic scientists who may erroneously find innocent people guilty of using marijuana or its preparations.