Weeding out cannabinoid metabolites

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  • Published: Aug 18, 2016
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Weeding out cannabinoid metabolites

Panacea or pariah?

In the history of botanical chemicals, perhaps no other has courted controversy as much as marijuana. Should it be decriminalised? Should it be outlawed? Nations worldwide have exerted their stance somewhere on this spectrum, from the renowned liberality of the Netherlands to the outright blanket ban strictly enforced in Singapore.

In the history of botanical chemicals, perhaps no other has courted controversy as much as marijuana. Should it be decriminalised? Should it be outlawed? Nations worldwide have exerted their stance somewhere on this spectrum, from the renowned liberality of the Netherlands to the outright blanket ban strictly enforced in Singapore. But what if marijuana may be the panacea? Should it be allowed for providing relief to many with debilitating ailments and maladies?

Over in sun-kissed California, membership of the Medical Marijuana Identification Program enables patients to consume marijuana for medicinal purposes, sidestepping FDA approval and federal law with a quick flash of the card. Medicinal marijuana, proponents claim, can provide therapeutic relief from chronic pain, asthma, glaucoma and multiple sclerosis. On the other hand, habitual use, even at very low dosages, is linked to cognitive deficits, particularly within developing adolescent users. Panacea or pariah, gathering high-quality data is essential for informing the debate around its legality.

‘The prevalence of illicit use of the cannabis plant, and the need of more data about dose–effect relationships for growing medical applications,’ writes Dr Aizpurua-Olaizola and colleagues in Drug Testing and Analysis, ‘have bought with them a special interest in analysing major cannabinoids and their metabolites in human samples.’ Accordingly, researchers from the University of Basque Country in Spain set out to develop the tools required.

Herbaceous cocktail

The herbaceous Cannabis sativa provides a cocktail of 554 compounds. These compounds, however, are invariably modified. Take the psychoactive tetrahydrocannabinol (THC), for example. Hydroxylated and tagged with a glucuronic acid by our metabolic enzymes, two metabolites are produced—11-hydroxy-Δ 9-tetrahydrocannabinol (11-OH-THC) or 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH)—depending on the type of linkage.

Those studying the cornucopia of cannabis medicines should not ignore the cannabidiols (CBD), cannabigerols (CBG), tetrahydrocannabivarins (THCV), and cannabinols (CBN), the authors advise, which ‘are major cannabinoids with considerable health effects.’ These, too, are extensively metabolised, necessitating costly and time-consuming hydrolysis of glucuronide conjugates for a complete picture of total cannabinoid levels. ‘This way,’ the authors explain, ‘proper correlation between specimen concentration and observed physiological effects could be achieved…’

Given the scarcity of standards for the vast array of these metabolites, Dr Aizpurua-Olaizola and colleagues investigated various permutations of enzymatic and alkaline reactions with the aim of liberating the active THC, 11-OH-THC, THC-COOH, CBD, CBG, THCV, and CBN compounds within human plasma and urine.

These liberated components were then cleansed via a quick and simple solid-phase extraction, loaded and resolved from a Kinetex C18 column over a 20-minute, slightly acidic aqueous-to-MeOH gradient, and analysed by tandem mass spectrometry in a triple quadrupole (QqQ) fitted with an Atmospheric Pressure Chemical Ionization (APCI) source run in positive mode.

Tandem hydrolysis

First and foremost, how best to liberate cannabinoids from the metabolic shackles that confound their measurement? It turns out that, whilst an alkaline hydrolysis was sufficient for unshackling glucuronides fixed to THC-COOH with ester bonds, it was unsuitable for breaking the ether bonds that link glucuronides to THC. Vice versa, enzymatic hydrolysis was sufficient for extracting total THC, but below par for liberating THC-COOH. The solution? A tandem alkaline–enzymatic hydrolysis, which extracted total cannabinoids in both urine and plasma with close to 100% efficiency.

Next, the HPLC-MS/MS identification and quantitation method was put through various tests to validate its performance. Per 1 mL drop of urine or plasma, their strategy could detect less than 1 ng of all cannabinoids, whilst affording researchers the ability to quantitate down to 0.5–2 ng/mL. What’s more, determination of 5, 50, and 200 ng cannabinoids spiked into a 1 mL drop of metabolite-rich urine and plasma matrixes was achieved with 84–115% accuracy, whilst the precisions in measuring these same standards three times over the same and different days were lower than 12%.

Finally, the test was applied for profiling the cannabinoids found in the urine of four regular cannabis users, and those that circulate in the plasma of four medical marijuana patients. Measured in triplicate, these quantitations were consistent within the same user, whilst the cannabinoid profiles highlighted the massive variances arising through the use of different types of marijuana for different pursuits.

Could their strategy inform the hotly contested cannabis debate? The authors are hopeful: ‘The developed method provides the opportunity to assess a broader spectrum of cannabinoids and urine and plasma samples…and this may improve the interpretation of results in clinical and forensic research.’

Related Links

Drug Testing and Analysis. , 2016, Early View paper. Aizpurua-Olaizola et al.. Simultaneous quantification of major cannabinoids and metabolites in human urine and plasma by HPLC-MS/MS and enzyme-alkaline hydrolysis.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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