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rapid/in vivo salvinorin analysis? Options
 
no_thing
#1 Posted : 11/14/2018 12:28:49 AM
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Greetings, big brains!

When planning a plant breeding program, one would like a rapid method of phenotyping. Does anyone know or have suspicions about a quick method to quantitatively compare salvinorin a content between (many) Salvia sp. leaves?

In vivo methods would be preferable due to speed.

I see from pubchem that the substance has an optical absorption peak around 5800nm; unfortunately mid-IR spectroscopy is not easy to do for cheap. I am not sure what other options are likely.

I am unfortunately not a chemist - please forgive any ignorance on my part.

Many thanks for any thoughts!
 

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endlessness
#2 Posted : 11/14/2018 10:43:44 AM

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TLC can work for semi quantification in a pretty simple way. Most basic way is simply getting a sample from different leaves of same weight, dissolve in same amount of methanol and run each side by side with TLC, the bigger spots will mean more of that substance, so that's the genetics you select, regardless of the exact number.

You can go a bit more complex to get some aproximate numbers, by doing a calibration by using some reasonably pure extracted salvinorin and doing serial dilution by dissolving it in different known amounts, and getting a number by simply comparing the size of your calibrated spots with your sample's spot sizes and deciding which one is more similar, like this I did to get an approximate idea of mimosa hostilis dmt content, or you can go a bit more complex and use a software to measure it for you like ann1ca beautiful did with mescaline in cactus.
 
no_thing
#3 Posted : 11/14/2018 4:26:01 PM
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I appreciate your helpful comments, and that may indeed be the best way to go. "Localization of Salvinorin A..." (Siebert 2004) gives a method for TLC analysis using vanillin to develop the plates.

That same paper suggests that vanillin solution applied directly to un-extracted plant material makes salvinorin visible under microscopy. Accordingly, I am wondering if I could skip TLC, apply the vanillin directly to a quick extraction, and quantify with visible-band spectrophotometry. The plants I will be working with have not been shown to have a high amount of salvinorin b, which would be a confounding factor.

Any thoughts on that idea? The best approach may be to try both with an initial sample for verification.

As mentioned I had hoped to find an in vivo method, but the only promising lead I've found is atr-ftir, which is out of both my budget and diy capabilities.

Thanks again!
 
Loveall
#4 Posted : 11/16/2018 3:46:34 AM

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no_thing wrote:
I appreciate your helpful comments, and that may indeed be the best way to go. "Localization of Salvinorin A..." (Siebert 2004) gives a method for TLC analysis using vanillin to develop the plates.

That same paper suggests that vanillin solution applied directly to un-extracted plant material makes salvinorin visible under microscopy. Accordingly, I am wondering if I could skip TLC, apply the vanillin directly to a quick extraction, and quantify with visible-band spectrophotometry. The plants I will be working with have not been shown to have a high amount of salvinorin b, which would be a confounding factor.

Any thoughts on that idea? The best approach may be to try both with an initial sample for verification.

As mentioned I had hoped to find an in vivo method, but the only promising lead I've found is atr-ftir, which is out of both my budget and diy capabilities.

Thanks again!


I think this is a good idea. I've read that salvinorin is on the outside of the leaf (in little hair like structures called Trichomes). If you have a microsocope you may be able yo see the hairs and compare their color/density/width/lenght.

I also seem to remember that sulfuric acid was part of the vanillin reagent. That last ingredient can be found in car part shops and is sold as battery acid.

I've attached moar info you may have already seen (if not I think it may be useful). Good luck.
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no_thing
#5 Posted : 11/19/2018 10:07:36 PM
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Thanks, those papers are very helpful! I had found some but not all previously.

After reading those, I am thinking the best approach will be a 1-minute acetone extraction, TLC, derivatization by dipping in some reagent (vanillin/h2so4?). If visual comparisons prove insufficient a computerized approach should be easy enough to implement.

A few questions, again with my apologies for ignorance:
- are there easily obtainable reagents other than vanillin that are known to work for visualizing salvinorin a? I can certainly do the vanillin/sulfuric acid approach, but am curious what the options are.

- I am restricted to easily available/non-specialist chemicals. Does acetone sound appropriate for the mobile phase, or would something like methanol likely work better?
 
downwardsfromzero
#6 Posted : 12/18/2018 6:48:51 PM

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Quote:
Does acetone sound appropriate for the mobile phase, or would something like methanol likely work better?

The Siebert paper, you've probably read that he used 1:1 ethyl acetate/hexane:
Quote:
Thin layer chromatography (TLC) was performed using
Whatman silica gel plates (catalogue no. 4410222). The
plates were developed in ethyl acetate/hexanes (1 : 1).
Terpenes were visualized by spraying the plates with
vanillin reagent, and then heating at 110 °C. The vanillin
reagent was prepared by mixing 50 mL ethanol, 0 ́3 mL
sulfuric acid, and 1 g vanillin. The salvinorins react with this
chromogenic reagent to produce pinkish-purple spots on the
plates.

You'd need something less polar than methanol, are there any other solvents you can get? Acetone is too good of a solvent for salvinorin A for TLC to work properly, I think.
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no_thing
#7 Posted : 12/20/2018 1:51:51 AM
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Greetings,

After some further search I've found an interesting master's thesis, "Characterization and colorimetric analysis..." (Carter 2014) which includes UV/Vis spectra for salvinorin a and b. From this thesis and from some of the papers cited earlier in this thread, it sounds like few substances besides salvinorins are extracted from leaves if the extraction time is short enough, and that this may accordingly be a usable method.

UV/Vis is relatively cheap, fast, and easy. It may be a few months before I can find the time to get to it, but I will test this method against S. officinalis leaves as a control. It's not as good analytically as TLC, but it's very easy and won't require any special equipment beyond the spectrophotometer.
 
 
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