SickMeds Seeds

AlphaCAT, Cannabinoids Analysis Test for pharma-breeder.

Cannabis contains more than 425 chemical compounds which are produce in the trichomes (figure.1). More than 70 of them belong to the family, so called terpenophenolic compounds or cannabinoids. The most important and prevalent cannabinoids with pharmacological activities are ∆9-THC, THCV, CBD, CBN, CBC and CBG. These cannabinoids are present in the plant in forms of acids (-COOH) group when unheated.

Glandular trichome
  • Figure 1: Glandular trichome present in different forms in aerial parts of Cannabis. It produces the cannabinoids and the terpenoids.

  • The cannabinoids are viscous hydrophobic, colorless and odorless oils, only solvable or extractable in organic solvents. The characteristic odor of Cannabis is due its total mixture volatile compound called terpenoids, like beta-pinene, beta-myrcene, terpinolene, and many others (Pate, 1994).

    The alphaCAT® method is based on the Thin-Layer Chromatography (TLC) analysis.

    TLC is a chromatographic technique used by many phyto-pharmaceutical and analytical research laboratories intended for separation and identification of organic compounds. AlphaCAT® has a simple and rapid protocol that uses adsorption and capillary action to separate complex mixtures into discrete zones. AlphaCAT® can tell about how many cannabinoids are in a cannabis mixture. AlphaCAT® can identify the cannabinoids by their specific color (chroma = Greek for color) and specific location (Rf value) comparing it with the Rf value from the literature, Fishedick et al, 2010. In our case, the cannabinoids can be identified (Rf value) in order of appearance on the TLC plate to give rise to Cannabinoids Finger Print (CFP) which characterize a cannabis mixture with its medicinal value.

    After several years of research and development, alphaCAT® was able to comply the HPTLC analytical procedure into a mobil laboratory test kit. Thanks to its unique protocol, cannabinoids analysis is now making a step forward as a major breakthrough giving the opportunity to cannabis specialist to unserstand the synergy there is between cannabinoid combination and medicianal value. The different cannabinoid combination is called chemotype.

    Therefore alphaCAT® scientists gave for the first time a complete chart reference to specify chemotype with strain characterization by its trivial name. In this matter with alphaCAT ® expertise, ones can indentify cannabis sub-type and with its corresponding biotype e.g. Mexican haze type, Hashplant African type, Thailandese type …

    In total alphaCAT® was able to classify major cannabis subtypes into 15 chemotypes and cannabinoids profiles. Combines with the latest scientific publication each chemotype can be coupled with specific medicinal properties.

    ChemoTypes - alphaCAT

    ''... Breeding for cannabinoid content and the eventual characterization of varying highs produced by Cannabis is total subjective guesswork without the aid of modern analysis technique. A Chromatographic analysis system would allow the selection of specific cannabinoid types, Especially staminate pollen parents ...''

    Robert Connell Clarke, 1981, Marijuana Botany

    Additionally alphaCAT was able to develop a protocol to quantify the different cannabinoids spot appearing on the CFP. The protocol is based on pixilation which enables to get percentages measure comparable with common laboratory analysis tool like Gas-Chromatography (GC) or High Performance Liquid Chromatography (HPLC).

    THC Calibration Chart

    AlphaCAT® is affordable to pharma-breeders in high through put screening program for strains selections. AlphaCAT ® is used for selection of the starting material as the foundation of any breeding program to determine the cannabinoids profile of the pistillate and staminate male female plant. With alphaCAT®, pharma- breeders can now monitor the variation that occur in the cannabinoids profile after change of one or more environmental factors such as photo-period, temperature, moisture, nutrition regime, topographic location, stress, etc…

    Additionally with alphaCAT® one can determine the best harvesting time by comparing the CFP. It helps to control the presence of desire cannabinoid profile by selecting the cross products and to access ethno botanical aspects of Cannabis.

    '' ... In general, it is considered most important that the genotype of the plant, or a resulting seed selection, is the primary factor which determines the THC levels. Environmental factors however depicts true expression of cannabinoid phenotypes and expression ...''

    Robert Connell Clarke, 1981, Marijuana Botany

    Thin Layer Chromatography (TLC)

    HPLC and GC are methods that require complex and expensive equipment for the analysis. TLC, on the other hand, can be performed with simple and inexpensive instruments. Nevertheless an enhanced version exists, called HPTLC, which utilizes machines to obtain higher quality results and to make the measurements more reproducible. HPTLC is performed for the analysis of cannabinoids. The instruments used in this case are an automated sample applicator, to avoid variance in spot size and location, and a densitometer and UV scanner for plate analysis (Fischedick et al., 2009). Densitometry is used to quantify results (Fischedick et al., 2009). The densitometer measures the optical density on the plate, which is higher if more light is absorbed. When it is desired to obtain results on both neutral and acid cannabinoids, the sample should be measured two times, once with and once without decarboxylation. The decarboxylation is done before the cannabinoids are extracted (Fischedick et al., 2009). While these instruments enable higher quality measurements, they are not required for TLC analysis.

    AlphaCAT kit is for determination of cannabinoid composition of cannabis samples. This kit is a form of TLC. It is developed because no scientific background and no expensive instruments are needed to perform the analysis. Therefore it can be used by anyone who would like to analyze the composition of their cannabis, for instance breeders, pharmacists, coffee shops and patients. A fingerprint, which is a qualitative result, can be obtained without decarboxylation. But when quantitative results are preferred, each sample should be measured twice, once with and once without decarboxylation. An exemplary result is visualized in Figure 10a. Instead of the automated sample applicator, the kit contains capillary tubes which take up 2µl when they are placed on a piece of cotton that was dipped in the sample. With the capillary tube the sample is spotted on the plate, one centimeter from the bottom and at least half a centimeter from the edge. The plate is placed in a developing chamber with three milliliters of developing solution, this is the mobile phase. The plate develops in approximately twenty minutes (AlphaCAT, 2011). The detection is done with a dye, selective for the visualization of cannabinoids. The cannabinoids that can be detected with this kit are THC, THV, CBD, CBN, CBG, CBC and their corresponding acids. Every cannabinoid reacts differently with the dye resulting in distinctive colours. The exact list of detectable cannabinoids and respective colours are presented in Figure 10b (AlphaCAT, 2011).

    Cannabinoid Profile