Scrutinizing Strains with Science : An Objective Discussion

Hi guys! I thought starting a new thread for this discussion, in the right place, would be a good idea. Here it is!

For reasons which should be obvious, serious scientific analysis of MJ, particularly in relation to strain, and the levels of active compounds, has been limited. Given recent political developments, this analysis is now available to everyone in legal areas, not just govt mandated tests. We can finally start seeing what makes the best strains so good, and determine what is there without having to go through the highly subjective method of just smoking it(nonetheless an important part of any analysis )

So how do they do that anyways?
There are two basic methods in use to look at the profile of compounds in mj:
Gas Chromatography with Mass Spectroscopy(GC/MS), and
Thin Layer Chromatography(TLC)

GC/MS provides actual number based data, and is more useful for actual analysis. It is not a cheap test to run. I dont have actual pricing data, anyone that has done testing, feel free to chime in with what you paid!
TLC provides a visual means of seeing what your weed is made up of, and is of limited value from a measuring aspect. It is however very affordable per test, and could be used as a somewhat reliable method of fingerprinting a strain, and could be useful in this aspect for dispensaries to check they are getting what they expect and not a substitution.

OK thats a start. I will add more detailed explanations of each method at a later time.

The results of these analysis can be useful in many ways:
-Developing a rational database to help medical users find the right plant for what ails them.
-Helping breeders narrow selection with great surity than can be had with smoking alone. (for example 2 plants up for breeding are both excellent, to the point of being hard to choose. if analysis shows one is .5% stronger in all categories, mystery solved.)
-helping us growers choose the strains we'd like to grow!
-one thing I always wanted to do with access to Gas Chrom/Mass Spec, is to do several harvests spread out by one week(i.e harvest some at 8 wks, some at 9, some at 10), and have each batch analyzed to help find the best cut day. If you spread the cuts out over the period of trichs changing from mostly cloudy over to amber, the results of analysis could be very interesting. Great way to find the point of peak THC or CBD content for a strain...

Obviously these tests are not within reach of everyone. I didn't start this thread to have any measuring contests or potency battles. I'd just like to discuss the analysis objectively. Please keep that in mind.

Keep it Green
MGT

PS I'm no expert on this stuff, though I am well read on the topic. Feel free to note any necessary corrections, or alternatives I may be unaware of.

bump good thread start. i'll be back

also I would be very interested in seeing lvpk under a lab test. the strain has been floating around for like a decade, but still kicks everything else's ass imho. i just burned a bowl and am baked as a cake, and I have been smoking at least a gram a day plus of the stuff pretty exclusively for like 6 months now and it still does the trick.

Per the OP's request...

CBD predominates over THC in cannabis that grows wild (ditchweed) and plants grown for fiber (hemp). When plants are bred for psychoactivity CBD is replaced by THC because the same gene codes for one or the other cannabinoid. According to research done in Europe and Israel, CBD has anti-inflammatory, anti-convulsant, anti-psychotic, anti-oxidant, and neuroprotective properties. It also has a direct inhibitory effect on certain cancer cells.

Biologists at California Pacific Medical Center, Sean McAllister and Pierre Desprez, have determined that CBD inhibits breast cancer metastasis by suppressing a gene called Id-1. This winter they started working with mouse models of breast cancer, and if all goes well, they will be conducting clinical trial at CPMC in less than two years.

A British company, GW Pharmaceuticals, has developed a high-CBD strain that it mixes with a high-THC strain to make Sativex, a plant extract formulated for spraying under the tongue that has been approved in Canada and elsewhere to treat neuropathic pain. CBD evidently bolsters the pain-killing effects of THC while moderating its psychoactivity. In various studies, patients with severe pain have reported getting significantly more relief from Sativex, the mixture, than from GW's high-THC extract.

With a few notable exceptions the California cannabis samples tested to date have contained only trace amounts of CBD. The first notable exception exception occurred in late February when D.L. saw a spike on a computer-generated graph indicating a high level of CBD in one of the samples provided by Harborside. After some additional testing he confirmed that this strain, produced indoors in San Francisco, contained 4.2% CBD (and 8.9% THC) by weight.

DeAngelo promptly made arrangements with the grower to rev up production. Buds and clones from the strain of interest should be available at Harborside within months. "It would be immoral to try to hoard the genetic material," says Deangelo. As this story goes off to CounterPunch undetermined time 12, a second high-CBD strain has been identified, grown outdoors in Mendocino County. It is a little more than five percent CBD by weight.

Thus the medical marijuana movement/industry is entering a new stage. Growers will develop strains with higher CBD to THC ratios. Pro-cannabis doctors, who have long awaited high-CBD strains, are already planning rudimentary clinical trials to determine whether and in what ways high-CBD cannabis is beneficial.

Because CBD counters the anxiety induced by THC, a high-CBD strain might prove palatable to many people who dislike the way marijuana makes them feel. High-CBD strains might also enable patients who need megadoses to ingest them while remaining functional. According to Jeffrey Hergenrather, MD, "Patients with certain cancers, ulcerative colitis and Crohn's Disease, seizure disorders... they all need to maintain a higher blood level of cannabinoids than is convenient with our high-THC strains. For them, development of a high-CBD strain could be a life or death matter."

Whatever the outcome of clinical trials involving CBD, the effort alone -the attempt to produce and evaluate less psychoactive strains of marijuans- will refute the image of stoners paying lip-service to medical use that has tarnished the industry. And if and when the effectiveness of high-CBD cannabis in treating, say, rheumatoid arthritis, can be established, a wave of older Californians will be asking their doctors if cannabis is right for them

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If anyone has had anything tested, feel free to post up the results here, along with the strain name and where it came from(cut/seed/seed from where, etc).

GC/MS is a highly analytical device capable of measuring down to 1/1000000 of a decimal place. Just to properly calibrate it, pretty much takes a PhD...

^ lol,

so, where does one get these sort of tests done?

Finding the cannabinoid profile of samples could be easily done, as there are many labs that can do it. I am in Canada and here it is illegal for any lab to be in possession of cannabis without authorization. However there are labs that will and are doing it. Pharmaceutical companies are very actively investigating cannabiniods, they are just doing so very quietly and not publishing their results. I think that is the problem, the work is being done just no one is talking about what they are finding. If government allowed any lab to handle cannabis for testing purposes it would be very easy and really not that expensive to do it. Maybe a little out of reach of the average person but definitely feasible for a dispensary. In terms of testing to help breeding I don't think it would really help produce the "optimum strain" for everyone. I think it would definitely help find other cannabiniods that may have different effects in medical applications and how they effect the "high". I feel we are really good at breeding the "ideal" strain. Just need to find the right plants for you.

Breeding CBD back into medical strains should not be that hard. If it is a co-dominate trait then all you would need to do to get equal CBD to THC was cross a homozygous CBD plant to a homozygous THC plant. It really wouldn't be that hard to breed a line of plants homozygous for CBD (thats what hemp breeders are doing to get ride of THC). I just feel that since breeding is still risky adventure for most people, trying to keep CBD lines to do this just hasn't been a priority. As most people who use cannabis still use it for recreational purposes and just want to get "high" they are not as concerned if what they get has CBD in it. The fact that it is co-dominate means that if you try and breed using heterozygous plants some of the offspring will be homozygous for CBD which would not be good for growers. I think if you asked the growers on here if they would want to take that risk they would say no it is not worth it. I feel that many would be mighty pissed to have a beautiful frosty plant ready to harvest only to find out after trying it, that it only had CBD.

elmanito said:

Difference with a TLC test
There is a little mistake in the full cannabinoid fingerprint
CBG=CBD, CBC=CBG, CBD=CBC

Different Cannabinoids

Different terpene profile

Namaste

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I might as well add this too. This is analysis commissioned from AN, it's 20 pages. Im not really a fan of AN, ive tried 5-6 of their supplements without noticeably effect. Feel free to believe as much or as little based on who's $ is behind it.

I only have pictures of the first 4 pages, the last 8-10 i think are gas chromatography.
http://www.growersunderground.com/PhosphorusMyth.pdf

I heard Sam Skunkman has bought a Gas Chromatograph.In Sam we trust!
You know it's not just the presence of certain cannabinoid, it's the coactivity between them that determines the effects of marijuana.

You can use a GC alone, only you will need the right pure (99.9%) cannabinoid reference standards, so i was wondering what sort of cannabinoid reference standards certain labs in the US are using.Is it made by themselves or did they bought it.Cannabinoid reference standards are controlled by the DEA, even in Europe.

Namaste

My Doctor recommended "Cannatonic" from Spain apparently. It's CBD, if I recall right is at 6% or higher. As a grower I would be interested in these higher CBD strains because I would rather go the medical route then the recreational route if prop 19 passes here in California.

Dr_Tre said:

You know it's not just the presence of certain cannabinoid, it's the coactivity between them that determines the effects of marijuana.

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that is one of the most fun things about growing. you can grow the same strain and grow/harvest it differently and get a whole new high.

TLC can be used for much more than suggested in the OP.

The biggest difference between TLC and GC is the former is qualitative and semi-quantitative and the latter is quantitative.

To me, the main attraction to TLC is comparative results using spot density scanning and analysis (measurement) software. For example, that way one can use semi-quantitative (comparative) testing between bud A and B to see what one has higher amount of specific cannabinoids. This shows what bud (or extract, or whatever) has more of a looked-for substance, not how much of the substance is present.

This type of testing using good-and-repeatable methodologies, good silica plates, fast blue BB reagent, dipping (not spraying) the plates and using JustTLC software and a good flatbed scanner (for spot density scanning, measurement and comparison) provides analytical result accuracy on par with those provided with GC and HPLC.

I believe comparative TLC is the best testing method to use when breeding, or when testing to see when to harvest, or when choosing what bud to buy (ex. from a dispensary); the last reason would be in lue of GC.

Advanced usage of TLC would be the usage of standards to make 'benchmark' plate references. For example, a known quantity of pure cannabinoid (standard) is used to make a spot on a plate. That spot destiny (and Rf value for redundancy) can be used to cull plants for breeding that do not meet the benchmark.

I think using GC for testing buds for market is a good idea, and when breeding too. The issues with GC are sourcing quality standards, cost of standards, effort to run a test and cost/maintenance of equipment. Sourcing pure standards can be hard from the U.S., but generally not from other countries. In the U.S. a few sources sell cannabinoid standards, but AFAIK the DEA isn't cool with cannabis labs trying to use normal routes to acquire standards. For those living in the U.S. making a pure standard for various cannabinois is possible using TLC (and extracting away the silica from the pure cannabinoid spot) or using HPLC. Another option is using DEA exempt cannabinoid standards that any company can legally buy; they are supposed to provide the same results as non-DEA exempt cannabinoid standards.

The biggest reason more people do not use GC or HPLC from home is cost and a learning curve and sourcing standards (for GC). But I think mainly it's cost.

What I really like about TLC with spot destiny scanning and analysis is the low cost (< $200-300), easy DIY at home nature, low learning curve, and accuracy. I think DIY TLC with spot density measurement will start to become the norm in the future.

Anyone can use TLC with spot density analysis from home for a couple of hundred dollars (max) to get all setup for many tests.

Here are two good reads about the high and effects associated with whole cannabis (buds, extracts, etc.) vs. mono-or-poly cannabinoid extracts like Sativex (50/50 THC/CBD):

"Cannabis and Cannabis extracts: Greater than the sum of their parts"
John M. McPartland and Ethan B. Russo
http://www.omma1998.org/McPartland-Russo-JCANT 1(3-4)-2001.pdf

And...

"Cannabis is more than simply ^9-tetrahydrocannabinol"
Ethan B. Russo and John M. McPartland
http://www.springerlink.com/content/gefrp0xg76d72926/

excerpt from paper:

Terpenoid cannabis components probably also contribute significantly to clinical effects of cannabis and boil at comparable temperatures to THC (McPartland and Russo 2001). Cannabis essential oil demonstrates serotonin receptor binding (Russo et al. 2000). Its terpenoids include myrcene, a potent analgesic (Rao et al. 1990) and anti-inflammatory (Lorenzetti et al. 1991), betacaryophyllene, another anti-inflammatory (Basile et al.1988) and gastric cytoprotective (Tambe et al. 1996), limonene, a potent inhalation antidepressant and immune stimulator (Komori et al. 1995) and anti-carcinogenic (Crowell 1999), and alpha-pinene, an anti-inflammatory (Gil et al. 1989) and bronchodilator (Falk et al. 1990).

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along with testing for normal secondary metabolites like cannabinoids, testing for some terpenoids and flavonoids should be the norm IMO. Terpenoids such as myrcene, etc., can be tested with GC, HPLC, TLC; as well as some flavonoids. Finding standards for those substances (for GC), or finding Rf values or spot color (for TLC) is the tricky part. I think I have info about extracting and testing myrcene with TLC but I am not sure.

The biggest obstacle I see in terms of testing for terpenoids and flavonoids is knowing what ones to test and the effect they have when smoked (or ingested). There are a few terpenoids known to have worthwhile medical efficacy, and a few that affect our high, etc., but I do not know of a large list.

I also think expanding the cannabinoids tested is a good idea, sometimes a freak of nature can happen worth trying to work with. Testing for the normal THC, CBD, CBN is fine, but I think labs and individuals should also test for CBC, CBG, etc., and THC-V, CBD-V, CBC-V, along with other varin (-V) cannabinoids.

spurr that's an excellent post! I will be editing my first post to better reflect my new understanding of TLC(thanks to you). To be perfectly honest I threw this together with zero research in order to get the discussion away from the drama of another thread.

If you have anything else to add, please do, as you clearly know alot more about TLC than I do!

spurr said:

I also think expanding the cannabinoids tested is a good idea, sometimes a freak of nature can happen worth trying to work with. Testing for the normal THC, CBD, CBN is fine, but I think labs and individuals should also test for CBC, CBG, etc., and THC-V, CBD-V, CBC-V, along with other varin (-V) cannabinoids.

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I absolutely agree on this 150%! It's the reason I have yet to write a section on "what we test for, and why". What we test for is not the whole picture at all. If it were, we wouldnt see anywhere near the level of nuance and difference from strain to strain that we do. There's more to this equation than 4 simple compounds. Lumping all THC into one figure isn't a great idea either, as far as I'm concerned.

The main problem I see with trying to label a race (ex. old-school never hybridized indigenous kush), or variety (ex. NL#5), or strain (ex. elite mom of NL#5 variety) with X or Y amount of THC is that secondary metabolite amount in final bud is a phenotype that is heterogeneous in terms of impacts from growing environment and methods and even harvest time...

Here is something I wrote at another place and time, but I think it's worthwhile to post here:

I don't know about all of you, but I have read the debate over when to harvest according to the color of the trich heads for ages. Everyone has their own opinion, but it's all subjective, I had yet to see data showing cannabinoid levels at various stages of trich development.

I tend to harvest when most trichs are cloudy/translucent and it looks like the head has stopped swelling. I know others like to harvest with clear trichs or amber trichers.

The data in the table below is from a study using drug bio-type cannabis we grow and non-drug bio-type (hemp). The researches found highest levels of cannabinoids (THC and CBD) when trichs are cloudy/translucent (called "mature" in the study). It seems that mature trichs has the highest THC.

This is only one study and I am not sure how old it is; I am not sure if it's peer-reviewed, so take it FWIW. I just thought it was interesting and it's the first data on this I remember reading. Additional testing and data would be good to have but here.

The table below was from the study below that I think was conducted around the 1980s to 1990s, I'm not sure. The report the article was printed in was from 2001.

AFAIK the reason there is no mention of CBD in the first sample (the drug type) is because the amount of CBD in each trich head was so low the researchers could not (or choose not to) quantify it as nanograms per trich head like they did with THC. AFAIK there is not any data on THC for the hemp plants for the same reason; the amount of THC was so low that the researchers could not (or choose not to) report it as nanogram per trich head.

Tests of cannabinoid profile is usually by percent, ex. THC % of a bud, using whole flower or whole leaf samples for extracting cannabinoids. The table posted below used individually harvested tich heads to find the amount of cannabindinoids by nanogram (ng) per trich head; not % of THC by leaf or flower cannabioid extraction.

b. Effect of gland age on cannabinoid content

We also examined cannabinoid content of stalked gland by age to measure the major cannabinoid components in both a fiber and drug strain (Table 2). Glands, viewed under a microscope, can be classified according to their secretory phases from the color of their contents. Glands most active in secretion (mature) are translucent in appearance, aged glands are yellow in appearance and senescent glands are brown in color. Mature glands possessed the highest content of their major cannabinoid in both the fiber and drug strains. Senescent glands possessed low levels of cannabinoids. The concentration of some components, as CBD in the drug strains, may be so low that is was not detectable in our analysis; similarly, for THC and CBN in the fiber strain. It is unknown where the cannabinoids go during the aging process, but we suggest that it is possible they volatilize into the atmosphere along with the terpenes in glands, as noted later in this report. Nevertheless, this phenomenon of altered content in glands during aging is one that should be studied to gain a more complete understanding of the secretory process of cannabinoids in the cell.

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"THC (TETRAHYDROCANNABINOL) ACCUMULATION IN GLANDS OF CANNABIS (CANNABACEAE)"
by Paul G. Mahlberg and Eun Soo Kim
The Hemp Report, Volume 3, Issue 17, Summer 2001 ISSN 1498-8135
http://www.hempreport.com/issues/17/malbody17.html (full HTML)


I found how the removed only trich heads to be neat:

Whole gland heads were removed individually from leaf or bract surfaces under a stereomicroscope with a tungsten microneedle and placed in vials containing chloroform to extract cannabinoids. The needle was rinsed in chloroform after collecting each gland to avoid cross contamination between collected glands. Samples of twenty and 100 glands were collected for different sampling periods and analyzed by GC as described (Turner, Hemphill and Mahlberg, 1978).

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FWIW, CBG is a pre-cursor for at least CBD and THC. It is not uncommon for mutant plants express a chemotype some have termed "prolonged juvenile" because CBG does not convert into other cannabinoids normally; thus the plant stays in a prolonged juvenile chemotype state (i.e. higher levels of CBG at harvest time). For CBG rich plants the CBG gets converted at much lower levels into other cannabinoids.

CBG > CBD
CBG > THC > CBN

Chemotype (ratio of cannabinoids to each other) is shown to be genotypic and thus minimally affected by environment/development. Quantity of each cannabinoid is a phenotype and thus greatly affected by environment/development.

MyGreenToe said:

I absolutely agree on this 150%! It's the reason I have yet to write a section on "what we test for, and why". What we test for is not the whole picture at all. If it were, we wouldnt see anywhere near the level of nuance and difference from strain to strain that we do. There's more to this equation than 4 simple compounds. Lumping all THC into one figure isn't a great idea either, as far as I'm concerned.

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RE: "Lumping all THC into one figure..."

Do you mean listing both THC-A and THC as (total) THC? Or do you mean THC-V? If you mean the former when the THC dataum is listed it is (generally) 'total' THC, i.e., THC-A + THC. There is very little (to no) THC in buds, only THC-A that gets converted into THC when smoking, baking, etc.