Rate of THC Decomposition to CBN

G.O. Joe · Dec 17, 2017

MS is no more necessary for LC than it is for GC. If you are not separating at the LC, MS isn't going to help. Analytical standards and following published separation procedures will go a long way just like with GC even before considering FID or UV characteristics. Used 1100 series LCMS have been found for $10,000 - the ready to go part and profit is the extra cost. HPLC can be hacked together under $4000 with a bin pump, manual injector, DAD, and analog output. Or a palmpilot for controlling 1100 components. The old GC's really only need a working 3395-ish integrator and cables, they don't grow on trees just like older LC parts.

WaterFarmFan · Dec 17, 2017

Thanks Joe. I have seen some other threads where the 1100 is rated very highly. This seems to come with a large module stack that can vary (G1311, G1313, G1315, G1316, G1322, G1329, G1330, G1365, G1379, etc.). What is the most basic configuration required just from cannabinoid and terpenoid testing?

G.O. Joe · Dec 17, 2017

Ah well LC is not really recommended for terps though not impossible it's easier to get good separation from GC. The temperature control of the samples and column is not really necessary if you're not sloppy. A bin pump is fine and the degasser module is cheap. The DAD is the most expensive part though I suppose other detector types can be used. Chemstation is not required if you have the palmpilot controller but the old obsolete versions should not be expensive. Module licenses may not have been needed with the very first version but for sure now each module additionally requires a license to work with Chemstation, and libraries needed to make CSI-style matches used to be free maybe but are now expensive and also need license numbers.

Gray Wolf · Dec 17, 2017

G.O. Joe said:
MS is no more necessary for LC than it is for GC. If you are not separating at the LC, MS isn't going to help. Analytical standards and following published separation procedures will go a long way just like with GC even before considering FID or UV characteristics. Used 1100 series LCMS have been found for $10,000 - the ready to go part and profit is the extra cost. HPLC can be hacked together under $4000 with a bin pump, manual injector, DAD, and analog output. Or a palmpilot for controlling 1100 components. The old GC's really only need a working 3395-ish integrator and cables, they don't grow on trees just like older LC parts.

There isn't general agreement on that issue.

There is more than one constituents with the same or close enough to the same peaks, that further definition of those peaks are needed for positive identification. A MS breaks those peaks down into their constituents.

Case in point, in Constituents of Cannabis Sativa, XVII, A review of the Natural Constituents, page 172, says:

Both the Waller and Small systems are useful; however, recent advances in *THC is used when authors referred to THC or the exact structure of A9-THC had not been determined.

2 the quantitation of cannabinoids have shown the gas chromatographic peak normally labelled cannabidiol (CBS) may be a mixture of CBD, cannabichromene (CBC), and cannabivarin (CBT') or any combination of the three (32, 33).

Gray Wolf · Dec 17, 2017

WaterFarmFan said:
Damn, you guys love to crush hope and dreams... Haha So, it seems the software is the most expensive part. Interesting in a fucked up patent kinda way...

Not meant to crush hopes, but to aid you in not being led down the garden path. My first used Perkins Elmer GC ended up a boat anchor, and while our $12,000 SRI GC works, we use it to generally see what is going on, but send our samples to a fully equipped lab for important analysis.

A major issue locally is that even with the right equipment, accurate results proved to be spotty because the labs weren't always using properly trained personnel, and customer pressure for good results. Bad enough for the Oregonian to write up an expose article................

Gray Wolf · Dec 17, 2017

As requested, my favorite Molecular Biologist buried me in papers and books to read on CBN, and as committed, I'll share that information once I've read it all.

G.O. Joe · Dec 17, 2017

Gray Wolf said:
There isn't general agreement on that issue.

There is more than one constituents with the same or close enough to the same peaks, that further definition of those peaks are needed for positive identification. A MS breaks those peaks down into their constituents.

Case in point, in Constituents of Cannabis Sativa, XVII, A review of the Natural Constituents, page 172, says:

Both the Waller and Small systems are useful; however, recent advances in *THC is used when authors referred to THC or the exact structure of A9-THC had not been determined.

2 the quantitation of cannabinoids have shown the gas chromatographic peak normally labelled cannabidiol (CBS) may be a mixture of CBD, cannabichromene (CBC), and cannabivarin (CBT') or any combination of the three (32, 33).

My hunch is if you emailed Restek for their typical recommendation, it would be GC-FID for terps and HPLC-UV for cannabinoids. Do mono and sesqui terps, as groups, look mostly the same within their group to MS?

Those recent advances were published in 1973. Columns have come so far since then. Everyone was packing columns themselves at the time. Capillary columns did not exist until around 1979, and columns have come so far since then, too. Do not use GC or LC procedures from the 70's except as a reference point. Even if you pack your own columns and few do, there are probably better packings available now too. From ref 32:

Although one published report (6) gives slightly different retention times for cannabidiol and cannabichromene, this laboratory and others (7) have been unsuccessful in obtaining a neat separation of cannabidiol and cannabichromene using only GC.

Turner and Hadley's drawn TLC shows a spot they say is CBL formed from CBC by decarboxylation at 100c for an hour. Interesting. If I wanted to spend all day finding GC and LC separations of CBD and CBC, I'm confident at least 20 different papers could be downloaded no problem.

Have one of many more relevant choices, randomly selected. There must be GC equivalents if I wanted to waste more time on countering the endless tide. Here, they go with formic/MeCN and a standard ho-hum modern column typical for cannabinoids. Acids are standard for cannabinoid separations - usually formic. Control of the pH greatly influences separation. MeOH is often used instead of MeCN. No MS in sight. If this looks more sciencey than Dussy's work, that's because it is.

Determination of 11 Cannabinoids in Biomass and Extracts of Different Varieties of Cannabis Using High-Performance Liquid Chromatography.
Gul W, Gul SW, Radwan MM, Wanas AS, Mehmedic Z, Khan II, Sharaf MH, ElSohly MA.
Journal of the AOAC International
2015 98(6) 1523-1528

Abstract
An HPLC single-laboratory validation was performed for the detection and quantification of the 11 major cannabinoids in most cannabis varieties, namely, cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ9-trans-tetrahydrocannabinol (Δ9-THC), Δ8-trans-tetrahydrocannabinol (Δ8-THC), cannabicyclol (CBL), cannabichromene (CBC), and Δ9-tetrahydrocannabinolic acid-A (THCAA). The analysis was carried out on the biomass and extracts of these varieties. Methanol-chloroform (9:1, v/v) was used for extraction, 4-androstene-3,17-dione was used as the internal standard, and separation was achieved in 22.2 min on a C18 column using a two- step gradient elution. The method was validated for the 11 cannabinoids. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with r2 values of >0.99 for all 11 cannabinoids. Method accuracy was determined through a spike study, and recovery ranged from 89.7 to 105.5% with an RSD of 0.19 to 6.32% for CBDA, CBD, THCV, CBN, Δ9-THC, CBL, CBC, and THCAA; recovery was 84.7, 84.2, and 67.7% for the minor constituents, CBGA, CBG, and Δ8-THC, respectively, with an RSD of 2.58 to 4.96%. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in different types of cannabis plant materials.

Results and Discussion
Several methods have been published for the analysis of cannabinoids in cannabis, including TLC (11), HPTLC (12), GC with and without derivatization (5), and HPLC (13). GC is the most commonly used technique (5); however, GC will not detect cannabinoid acids, which will undergo decarboxylation at the temperature of the injection port. HPLC is a suitable alternative that allows the analysis of both free cannabinoids and their acids in the same run without the need of derivatization. The developed method is used for the analysis of cannabinoids of the high Δ9-THC/low CBD variety, variety rich in both Δ9-THC and CBD, and the fiber-type variety (high CBD/low Δ9-THC; Figures 4–6).

Optimization Studies
Extraction and chromatography parameters were optimized. For extraction, different solvents, extraction methods, and temperature were tried. For chromatographic conditions, various parameters such as analytical column type and dimensions and mobile phase composition were examined. Good separation of the 11 cannabinoids was observed, and the following approximate retention times (min) were obtained: CBDA (7.77), CBGA (8.68), CBG (9.07), CBD (9.41), THCV (10.16), CBN (13.38), Δ9-THC (15.84), Δ8-THC (16.34), CBL (18.63), CBC (19.18), and THCAA (19.69; Figure 2). Representative chromatograms of extracts of the high Δ9-THC/low CBD, approximately equal Δ9-THC/CBD, and fiber type (High CBD/Low THC) varieties are shown in Figures 4–6, respectively.

Method Validation Results
Specificity.—Sufficient resolution between the different cannabinoids in the chromatogram and peak purity check were used to establish specificity of the method. Resolution (Rs) ≥0.9 of each cannabinoid from the preceding peak was observed.

Linearity.—Five-point standard calibration curves were used to evaluate linearity. The concentration-response relationship of the present method was linear between the concentration and peak area with r2 values of >0.99 for all 11 cannabinoids as follows: CBDA (0.9988), CBGA (0.9997), CBG (0.9955), CBD (0.9990), THCV (0.9999), CBN (0.9948), and Δ9-THC (0.9977) (Figure 4), and Δ8-THC (0.999), CBL (0.9975), CBC (0.9996), and THCAA (0.9998) (Figure 3).

Accuracy.—Accuracy was determined through a spike study, and the average recovery for individual cannabinoids was 90.0% for CBDA (RSD 0.97%), 90.96% for CBD (1.05%), 91.75% for THCV (0.96%), 93.16% for CBN (0.19%), 89.67% for Δ9-THC (1.91%), 105.54% for CBL (6.32%), 94.11% for CBC (1.97%), and 91.49% for THCAA (1.2%). It was 84.7% (2.8%), 84.2% (2.58%), and 67.7% (4.96%) for the minor constituents CBGA, CBG, and Δ8-THC, respectively.

Precision.—The intraday precision was assessed by applying the procedure repeatedly to multiple samplings (n = 6) of a homogeneous sample. RSD was calculated for CBD (0.6%), CBGA (0.9%), CBDA (0.7%), Δ9-THC (0.7%), and THCAA (1.2%). The interday precision was determined by applying the procedure repeatedly to the same sample on 6 different days. RSD was calculated for CBD (2.0%), CBGA (2.0%), CBDA (6.6%), Δ9-THC (3.2%), and THCAA (1.7%). The procedure was stable after minor changes were made followed by reinjection of the same sample preparation (intermediate precision).

LOD and LOQ.—LOD and the LOQ of the 11 cannabinoids are listed in Table 1. (0.3-6.1, 1.0-18.4 microgram/milliliter)

If someone without the background wants to analyze cannabinoids, a system like Shimadzu's cannabis analyzer is the way to go.

Gray Wolf said:
As requested, my favorite Molecular Biologist buried me in papers and books to read on CBN, and as committed, I'll share that information once I've read it all.

You could have had CBN papers 9 years ago. Wood, Todd, and Adams to today were available before sci-hub. Notice how Wood et al. finally obtained their CBN by distilling it at 285c/80 mm. Faucet aspirator vacuum is usually better than that.

Ringodoggie · Dec 17, 2017

OK, it's clear that most of the options available to us for establishing canna profiles is plagued with hurdles.

The world still needs a DIY testing setup that can give, at least, a partial profile.

There are home DIY TLC products but I really have my doubts about thin layer's accuracy. Not to mention, you might only get 1, 2 or maybe 3 cannabinoids separated out. Might be OK for determining relative THC and CBD content but I don't see it being worth much more than that.

How about FT-IR Spec? Again, this appears to be very software dependent for data interpretation. Hardware is less to purchase than GC or HPLC but the software might be limiting. Love to hear your input on FT-IR.

G.O. Joe · Dec 17, 2017

https://www.icmag.com/ic/showpost.php?p=7686199&postcount=251

G.O. Joe · Dec 17, 2017

Forgot about this, which has come up as something for the herd to follow. It does and says Although HPLC is the method of choice for cannabinoids, it is fairly well established that GC is the method of choice for small volatile organics such as the terpenoids

Ringodoggie · Dec 18, 2017

Thanks, Joe. That flyer from PerkinElmer is the info that more or less put me on the path to FT-IR a while back.

However, from what I am reading, this method presents the one major hurdle that seems to exist in most methods..... operator efficiency. Any method that is going to be suitable for DIY home use has to take into account the abilities (or, rather lack thereof) of the end user.

I am trying to get a copy, or at least some solid info on the Spectrum 10 software used to determine exactly how user friendly it is.

This method will, at least provide a partial profile. Certainly adequate for most of the experiments that I intend to be involved in. GC would be awesome but is perhaps outside the scope of the typical cannabis user.

Has anyone here owned or operated a FT-IR setup? Comments?

EDIT: http://www.perkinelmer.com/lab-solutions/resources/docs/PRD_Spectrum10Software.pdf

superglue · Dec 18, 2017

cbc and cbd can be seperated with a gc-fid if you ramp the temp at much slower rate and while a heated injector would certainly help the separation, it isn't required as cbc will elute just after the cbd with the mxt-500 column
thr mxt-500 is in the right range for cannabinoids and sesquiterpens, some of the monoterpenes will get caught up in the solvent peak so using a mxt-wax column is the better choice for doing terpene analysis as it will slow down the elution speed
most of this is mute though as most cannabis in the usa is cbg,cbd,thc9 and 8 and cbn plus assorted terpenes and anything else is usually just a trace

from my experience
fwiw

WaterFarmFan · Dec 18, 2017

Really great thread! Most informative! I found this article that seems to confirm what Joe was saying about gc-fid for terpenes:

https://www.cannabisindustryjournal.com/column/instrumentation-used-for-terpene-analysis/

"Terpenes are a group of volatile, unsaturated hydrocarbons found in the essential oils of plants. They are responsible for the characteristic smells and flavors of most plants, such as conifers, citrus, as well as cannabis. Over 140 terpenes have been identified to date and these unique compounds may have medicinal properties. Caryophyllene, for example, emits a sweet, woody, clove taste and is believed to relieve inflammation and produce a neuroprotective effect through CB2 receptor activation. Limonene has a citrus scent and may possess anti-cancer, anti-bacterial, anti-fungal and anti-depression effects. Pinene is responsible for the pine aroma and acts as a bronchodilator. One theory involving terpenes is the Entourage Effect, a synergistic benefit from the combination of cannabinoids and terpenes.

Many customers ask technical service which instrumentation is best, GC or HPLC, for analysis of terpenes. Terpenes are most amenable to GC, due to their inherent volatility. HPLC is generally not recommended; since terpenes have very low UV or MS sensitivity; the cannabinoids (which are present in percent levels) will often interfere or coelute with many of the terpenes.
Figure 1: Terpene profile via headspace, courtesy of ProVerde Laboratories.

Headspace (HS), Solid Phase Microextraction of Headspace (HS-SPME) or Split/Splitless Injection (SSI) are viable techniques and have advantages and disadvantages. While SPME can be performed by either direct immersion with the sample or headspace sampling, HS-SPME is considered the most effective technique since this approach eliminates the complex oil matrix. Likewise, conventional HS also targets volatiles that include the terpenes, leaving the high molecular weight oils and cannabinoids behind (Figure 1). SSI eliminates the complexity of a HS or SPME concentrator/autosampler, however, sensitivity and column lifetime become limiting factors to high throughput, since the entire sample is introduced to the inlet and ultimately the column.

The GC capillary columns range from thicker film, mid-polarity (Rxi-624sil MS for instance) to thinner film, non-polar 100% polysiloxane-based phases, such as an Rxi-1ms. A thicker film provides the best resolution among the highly volatile, early eluting compounds, such as pinene. Heavier molecular weight compounds, such as the cannabinoids, are difficult to bake off of the mid-polarity phases. A thinner, non-polar film enables the heavier terpenes and cannabinoids to elute efficiently and produces sharp peaks. Conversely the early eluting terpenes will often coelute using a thin film column. Columns that do not contain cyano-functional groups (Rxi-624Sil MS), are more robust and have higher temperature limits and lower bleed.

For the GC detector, a Mass Spectrometer (MS) can be used, however, many of the terpenes are isobars, sharing the same ions used for identification and quantification. Selectivity is the best solution, regardless of the detector. The Flame Ionization Detector (FID) is less expensive to purchase and operate and has a greater dynamic range, though it is not as sensitive, nor selective for coeluting impurities.

By accurately and reproducibly quantifying terpenes, cannabis medicines can be better characterized and controlled. Strains, which may exhibit specific medical and psychological traits, can be identified and utilized to their potential. The lab objectives, customer expectations, state regulations, available instrumentation, and qualified lab personnel will ultimately determine how the terpenes will be analyzed."

WaterFarmFan · Dec 18, 2017

For those in the know, and in a remote effort to justify the costs of these systems as a non-commercial research tool, how much does a licensed lab (of high standing) charge for cannabinoid and terpenoid analysis on flower and/or concentrate? Bulk discounts? Incidentals/Operator Costs/Learning Curve aside, how many samples could you get analyzed for $10k?

WaterFarmFan · Dec 18, 2017

superglue said:
most of this is mute though as most cannabis in the usa is cbg,cbd,thc9 and 8 and cbn plus assorted terpenes and anything else is usually just a trace

Solid insight on topic. Just curious about this comment above. Are you referring to a lack of sativa characteristics in most USA hybrids? Different results from different places?

WaterFarmFan · Dec 18, 2017

Gray Wolf said:
Not meant to crush hopes, but to aid you in not being led down the garden path. My first used Perkins Elmer GC ended up a boat anchor, and while our $12,000 SRI GC works, we use it to generally see what is going on, but send our samples to a fully equipped lab for important analysis.

A major issue locally is that even with the right equipment, accurate results proved to be spotty because the labs weren't always using properly trained personnel, and customer pressure for good results. Bad enough for the Oregonian to write up an expose article................

You are the man, GW! I have a ton of research, reading and learning to do, but I will own a GC & HPLC sooner rather than later. Some people buy motorcycles, boats & RVs, while others...

Zimbogro · Dec 18, 2017

What do you think of the THC/CBN conversion over time (not in lab )? I believe that a more effective conversion can only happen over a long period of time (1 to 2 years). Can anyone verify this?
Thanks

Gray Wolf · Dec 18, 2017

WaterFarmFan said:
You are the man, GW! I have a ton of research, reading and learning to do, but I will own a GC & HPLC sooner rather than later. Some people buy motorcycles, boats & RVs, while others...

Thanks for the good thoughts brother! I can relate to having to have one of "those", as that is how I felt about our GC. I justified the cost based on the $80/test I was quoted at the time. At that point in our experimenting, it was either get one or go home.

We still send out our critical stuff, and as Go Joe noted, there have been advancements, even since buying our GS in 2010, so we did end up replacing our FID column with one SRI more recently developed and recommended for terpene testing.

Having spent yesterday going blind pouring over a pile-o-papers from Dr.F, I'm noting issues where there was confusion regarding overlapping peaks, and enough question on exactly what some of the constituents were even after finding them, to require further testing by methods besides GS/HPLC/MS to verify.

What an in house GS has done for us, is give us affordable answers in about 8 minutes and except for carboxylic acids, serves most of our needs.

Pangea · Dec 18, 2017

You can get some useful inhouse info on carboxylic acids using a GC-FID:

Accurate Quantification of Cannabinoid Acids and Neutrals by GC-FID – Derivatives without Calculus:
https://blog.restek.com/?p=15135

A source for refurbished GCFID's and HPLC with good least to own terms, not sure if they work cross border:

https://www.se-source.com/

greenfox · Dec 20, 2017

Rate of THC Decomposition to CBN

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