Hey folks!

Via a friend, I got access to do some analysis in the near future with HPLC and GC, if I want. But I not yet totally prepared.

I have done some analysis using modern machinery some ten years ago, bit I have to refresh a bit about preparing and evaluating the results.

I wanted to start with some homemade Ayahuasca-brews - my friend said that there is no problem using aquaeous extracts to pass into the HPLC but could I just use pure, well filtered brew to pass into the columns?

I don't want to be responsable to crash a several thousand euro machine by putting brown ayahuasca-brew inside. After all, with fine filtration, there shouldn't be any issue?

Another problem of fine filtration is, that there is a lot of material in the bottom of the bottles, so a only liquid phase wouldn't get 100 % results.

How do scientists process, who bring samples from the amazon for analysis? Is there any need to reextract from the aquaeous phase with a solvent, who fits better for this tye of analysis?

And there is also the question about detection and quantification of alcaloids inside - there could be Harmine, Harmaline, THH, NMT and DMT besides other stuff. For quanification wouldn't it be better to extract each compound separately?

Seems kinda hard, but I just came about the ocassion and need to think more on it.

In the future, I would like to analyse Acacia confusa and Caapi extracts, too - this finally seems much easier, but for the moment, a full brew analysis is more interesting to improve brewing skills, quantities and effects of homemade-aya.

Any opinions on this topic?

I will come up with a paper, including the molecular mass of each alcaloid, the nm-lenght desired to look at and so on...

Just to be clear: my friend is doing routinely analysis in a huge lab, so she is quite familiar with what she is doing. But she knows nothing about alcaloids, so I just wanted to present her quick and easy to-go samples to pass in the HPLC and better understand myself also.

She usually analyizes pesticide-residues in vegetables.

There is always a safety column ahead in a HPLC to catch micron-scale solids, but still should not be a reason to deploy anything in here

If you want to have a better stand you should use some real lab filter systems for syringes:

https://www.fishersci.co...6PM/syringe-filters.html

Can be quite a pain to squeeze stuff through them, if there are a lot of substrate particles, but then you will be on a much safer side.

Of course solids on the bottom cant be deployed into the machine, but are you sure that these are really a part of the brew?

I dont have any clue about aya, but from what I thought is that the shaman is cooking his plant material in a big pot and when he deliveres his medicine, then any plant material would sink to the bottom anyways, making him only deliver the liquid portions of his soup, wouldnt he? Then it may not even be off from reality if you deploy a filterate of the liquid only.

No idea how Scientists would handle retrieving samples from the Amazon though ...

But redarding quantification:

A problem is that you will just get an outcome signal of the detector, measuring stuff like the refracting index or heat convection or absorption of the liquid, that comes out of the column. You will get the integral of each peak, knowing how much % a peak has based on all other peaks.

But that output signal (being it whatever kind of physico-chemical detection) is not generated in the same magnitude for different compounds. This means the peak-% is not the purity already. To get from that % to the real purity one has to use a standard. Sadly the outcome signal is different from any device and therefore you need to make a manual calibration of all compounds you want to measure, IF you want to do an exact quantification measurement.

I guess your friend will know how to do it, but then you would need some (preferably) super pure sample of all of the components you would need to quantify precisely. Should not be a problem with DMT, but I dont know if the pH-precipitation is a 100% separation regarding Harmalas, so you may need a preparative HPLC for this (this ones are not actually only for analytical purposes, but also separate a sample for you, which can be collected afterwards).

Depending on potency you will likely want to dilute the sample prior to HPLC or GC analysis. For HPLC would likely want to dilute the sample in a solvent such as methanol or acetonitrile or a mixture of 50% of either solvent mixed with water. This will depend a bit on the mobile phase. I'd suggest starting out with a 10x dilution (100 uL ayahuasca liquid into 900 uL diluent). You should then filter this solution through a 0.22 micron or 0.45 micron filter depending on HPLC system set-up although really it doesn't matter much (syringe filter set-up).



Well it depends on your detector. But in general the best is to have reference material to compare. Basically if you have pure dmt you can analyze that and then you'll know where the dmt signal is in the sample. There are ways to figure out what is what without reference material but it gets more complicated. Are you dealing with a UV or PDA type detector or a mass spectrometer?



There is a lot of good literature on this. If we know more about details of techniques she is using HPLC with which detector or GC with which detector it will be easy to point you in the right direction.


Also be aware depending on country sometimes you can legally buy small amounts of reference material as an analytical laboratory. Depending on local laws you may or may not need special permit. I know there are exemptions for small quantities pre-diluted in solvents too so I'd suggest checking local laws and there are chemical supply companies that specialize in reference materials for all sorts of compounds.

some analysts centrifuge, but I typically use the 0.45 um pall syringe filters.
If I'm really lazy, I'll just stuff a piece of kimwipe in a 1000 uL tip, and pipette raw extract through that.