The history of cannabinoids detection & quantification with Chromatography :

Chromatography : which literally means marked color, is the general term used to define laboratory techniques to separate complex mixture.

The  Thin Layer Chromatography (TLC) is first ever analytical techniques used to separate and detect compounds in natural products. The TLC technique has been use for analytical process in quality control in many industry, like agro-food, neutraceutical, pharmaceutical, etc… For almost a century, it was the prefer method to primarily recognize active ingredients present in product manufacturing process and in the end product itself.

Mikhail Tsvet
Mikhail Tsvet (1872–1919)

1900 : Official Birth of Modern Chromatography

Modern chromatography began more than 100 years ago when Russian botanist and plant physiologist Mikhail Tswett developed his differential migration method of liquid-solid chromatography, which he published in 1906. He succeeded in resolving complex mixtures of structurally similar yellow and green chloroplast pigments of various leaf extracts by using pure solvent to develop the chromatogram. The colored pigments Tswett studied formed readily visible bands, which evolved from the application of selective adsorbents and extraction to purify the chlorophyll; hence he coined the term “chromatography.” Chromatography—“color writing” (translated from the Greek roots chroma and graphein)— earned Tswett the cognomen “father of chromatography.”

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1950s : Perfection of the method and terpenes separations

The Thin Layer Chromatography evolved from the experiments of a Dutch biologist M. W. Beyernick in 1889, with later improvements in World War II by American scientists J. E. Meinhard and N. F. Hall used the technique to separate terpenes found in essential oils. Building upon that work, Justus G. Kirchner and colleagues at the U.S. Department of Agriculture’s fruit and vegetable laboratory in California improved TLC in a technique that used adsorbent-coated glass plates that Kirchner called “chromastrips.” Throughout the 1950s and into the 1960s, Kirchner and colleagues also perfected TLC techniques using square coated plates, carried out two-dimensional chromatography, and introduced the use of reactions on plates for identification purposes.

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1960s : The technique became quantitative

From its beginning, the technique was an inexpensive, reliable, fast, and easy to perform means of distinguishing different compounds from each other. The method was qualitative, it showed the presence of a compound but not how much of the compound was present. In the late 1960s, TLC was refined so that it could reliably measure the amounts of compounds. In other words, the technique became quantitative. Further refinement reduced the thickness of the support material and increased the amount of the separating material that could be packed into the support. In High Performance TLC (HPTLC) the resolution of chemically similar compounds is better than with conventional TLC, and less sample is required. HPTLC requires specialized analysis equipment, and so is still not as popular or widespread as conventional TLC. TLC is useful in detecting chemicals of forensic concern, including chemical weapons, explosives, and illicit drugs . Advances in TLC technology, largely driven by the efforts to quell terrorism, have benefited forensic science. As one example, the Forensic Service Center of Lawrence Livermore National Laboratory has designed a computerized and portable TLC machine that can be taken to the field, and which has the ability to analyze 20 samples at a time. Analysis can be completed within 30 minutes. This allows an analysis that previously required a dedicated laboratory to be done at the scene.

Source : Thin Layer Chromatography” World of Forensic Science. 2005.

1980 : The UNODC explain how TLC help to determine illegal cannabis origin based on cannabinoids presence

UNODC recommended TLC for cannabinoids detection in the 80’s

In 1980 the United Nations of Drug and Crimes (UNODC) published a paper from Baker et al based in United Kingdom, called « Illicitly imported Cannabis products: some physical and chemical features indicative of their origin ».

This study was conducted on illicit cannabis and cannabis resin samples seizures from know geographical location,  samples were examined by TLC. The chromatographic characteristics were considered in conjunction with the gross physical appearance of the materials and it was found possible to discriminate between samples of different origin. Thus by carrying out thin-layer chromatography and a visual inspection on a sample of unknown provenance, an opinion as to its geographical origin can be offered, which can be use as reference collection of samples.

Source : Illicitly imported Cannabis products: some physical and chemical features indicative of their origin

Animated Anandamide
Animated Anandamide Molecule”

1991 : Isolation and first described structure of anandamide

In 1991 Lumir Hanus William Anthony Devane isolated and first described the structure of anandamide, an endogenous cannabinoid neurotransmitter by the used the TLC in their lab as first screen testing to target deeper analysis led by Raphael Mechoulam at the Hebrew University of Jerusalem

See the book : Endocannabinoids: The Brain and Body’s Marijuana and Beyond by Emmanuel S Onaivi, Takayuki Sugiura, Vincenzo Di Marzo.

1995 : Birth of the first European Cannabis patient program with the strain “SIMM 18”

Franco Loja SIMM
Franco before Strain Hunter at the SIMM facility

In 1995 in Netherlands a new medical cannabis program started to emerge to provide and assist patient with cannabis treatment. The company Maripharm B.V directed by Marcel de Wit.

It was  the first Dutch company researching and delivering cannabis to patient by pharmacy, coffee shop and patient associations. Maripharm mission was to make medical cannabis safe, accessible and affordable. They worked extensively on analytical method to  detect cannabinoids as other plant ingredients and contaminants. The TLC technique was their method of choice for day to day testing. In 2002 the medical cannabis growing licence was granted. For this occasion, Maripharm and GreenHouse seed company joined force under the company Sitching Institute of Medicinal Marihuana (SIMM) directed by James Burton who obtain the first Dutch growing permit to produce high THC strains for medicinal purposes.

Greenhouse seed company was in charge of the cultivation procedure, Franco Loja now famous for being a Strain Hunter, was the Grower Technician of SIMM facility at that time. This is when Franco working closely with GHSC. On the other hand Maripharm was in charge of lab quality control process, Ronald Glas was the lab technician assisting Marcel de Wit with cannabis analysis at Maripharm.

Simm 18
SIMM 18 Cannabis Flower

2004 : SIMM replaced by Bedrocan

In 2004, SIMM was officially withdrawn and discard from the Dutch program following a series of events, as a restructuration of the Medical Cannabis Bureau. Then there was only company called Bedrocan B.V remained with a licence to grow medical cannabis under Dutch law which gave Bedrocan a total monopoly in the Netherlands and in EU for the past 13 years.

2005 : a First TLC Patent and a first test kit for cannabinoids analysis available in the EU

PatentedAfter Maripharm shut down, its former lab technician Ronald Glas, filed a pending patent in 2005 on a Thin Layer Chromatography procedure which was develop by Maripharm back in the days.

The Patent US20070077660 :
Method for the detection of a cannabinoid, detection kit, and developing solvent

At the time he approached Canna B.V who produceg the famous CANNA fertilizers. Then they started to work together to introduce the first TLC cannabinoid test kit which was called « Cannalyse ».

CANNA made a partnership with Kulu Trading to handle the production and distribution, see article in Spannabis Magazine, 2006 p.84.

2008 : Canalytics Vs Cannalyse (or CANNA Vs Kulu Trading)

Beware of other type of test sold on the internet : They often contain outdated or unfunctionals components
Beware of other type of test sold on the internet : They often contain outdated or unfunctionals components

After some important internal opposition between CANNA and Kulu Trading, both companies decided to quit their collaboration. However Kulu Trading kept on producing « Cannalyse » test kit but they distributed under a new brand name called  « Cannalytics ».

In this context in 2008, CANNA filed a complaint to the justice of Benelux toward the abusive use and confusion brought to their brand CANNA and trademark name « Cannalyse » which were in direct conflict of interest with name « Cannalytics » distributed by Kulu Trading. CANNA won the exclusive ownership of both name meaning that every company who is selling test kit under these name can be directly sue by CANNA. See court case conclusion.

Nowadays you can still find a website hosted in the US selling the Cannalyse test kit version. As well as Cannalytics, with a version first designed almost 10 years ago now !!! Another copy of  the Cannalyse TLC kit is also being sold under the name of GrowBuddy. Because of the blockage situation regarding direct the conflict with CANNA, it will be impossible to sell a TLC test kit like this in EU.

2009 : TLC use as a quantificative method by densitometry

In 2009, Mr Glas was approached by Klaas Hesselink, CEO of Cannatest Lab, based in Seattle. They bought his know how and paid for a final reaserach study at Leiden university to show that TLC can be a quantificative method by densitometry.  The study was conducted by Justin Fischedick and Arno Hazekamp (former Director of Bedrocan R&D) who published the results in 2010.

2010 : Sébastien Béguerie, now Alpha-cat CEO use the TLC method for his thesis in NL.

By that time Sébastien Béguerie was finishing his master thesis sponsored by Bedrocan B.V at Wageningen University. His thesis title was, ‘Yield improvement of Cannabis sativa inflorescences by natural plant growth regulators’. Obviously he needed for his research a method to quick and inexpensive way to run extensive cannabinoid test. After reading Fischdick‘s publication, it revealed that the TLC techniques was best suited for his thesis purposes. However he still needed to find a straight way to semi-quantify his results to have solid date for this stastiscal analysis.

2011 : Birth of the Alpha-cat test kit with updated calibration charts for the 6 main cannabinoids

TLC Calibration Chart
Alpha-cat Calibration Charts

From his previous research study working with image analysis on roots system architecture, Sébastien found a way to see the spot size of each cannabinoid in correlation with its percentage. It turned out that using a scanned plate picture and mesuring the surface area over the number of pixel, Sébastien found a « regression line » meaning that the spots were indeed proportional to content of cannabinoids.

From this regression line, Sebastien updated the cannabinoid calibration chart of the 6 main cannabinoids (CBD,CBN,THC, THCV,CBG,CBC). In 2011 Sebastien re-designed the all process of the TLC test kit for offeriing a new user friendly version with a step by step booklet proven to give reliable data.

Sébastien called the kit « Alpha-cat » (Cannabinoid Analysis Test) given now the possibility to see through the Trichomes with “the eyes of a Lion” to understand the chemistry beyond the bud.

Cannatest was pleased to use the alphaCAT calibration method. After a year of beta testing with Cannatest competing with the most advanced Lab in Washington state.

2013 : A Comparative test between GC-FID and the TLC method.

Sébastien decided in 2013 to run a final comparative test with GC-FID at CannaFundacion, the results were presented at the 7th International Conference on Cannabinoids in Medicine.

IACM, Cologne, Germany, September 27-28, 2013. Since Sebastien  have training and set up an Alpha-cat mobile lab in Cannabis College in Amsterdam. He has accredited GreenHouse Medical and a Seed company in Tenerife Islands called Sick Meds.

2015 : Alpha-cat Lab moved in Praha, to work with patients coalitions and scientific organizations.

Internationnal Cannabis and Cannabinoids Institute

Since 2015 Sebastien have moved his company « alphaCAT Lab » in Prague to work in close collaboration with the International Cannabis and Cannabinoid Institute (ICCI) organization.

He’s also trying to help as his best the “Union Francophone pour l’utilisation des cannabinoïdes en Médecine” (UFCMed), a french cannabis patients organization that is fighting the actual laws and government for their right to choose cannabis as medicine.