Is it me or is this subreddit dying out?

Hyperforin is on our list!

It might seem like it is not as crucial, but it is even more so. You are talking natural things growing in various environments. Species, cultivar, plant part, extraction method, growing conditions, soil/growth medium content, etc. all play a massive role in what you are getting as an end product. The chemistry is also much more complex because of that. A pure synthetic compound? That's usually easy. FTIR or NMR for ID, then HPLC for purity analysis. Maybe you need a mass spec for some extra verification, and ICP-MS for heavy metals. However, they are likely not there. A natural extract? You need to get creative for ID. This usually means HP-TLC. Then you can use the marker bands to compare to known botanical reference standards to determine species, plant part used, and extraction methods. You have to build all that ahead of time, though. Sometimes you get lucky. Sometimes you have to get creative with your chromatography to separate things out. Content analysis? Probably some advanced UPLC methods. However, when you say "withanolides" or "bacopasides" that is a group of things. What are you considering as part of that group? Do you have reference standards for all of them? Do you know the response factors for all of them in your methods? Did you know that there is no terminology for that grouping? That's fucking crazy to me! There is no universal terminology for what you are considering as part of the grouping for a specific standardization! So when one place says "5% withanolides" you have no idea what they mean by that. You don't even have a name for the grouping they chose for their standardization. Me and my team created a new term for it called STAG, or Standardized Target Analyze Grouping. So when you set your standardization for withanolides, you need to set your STAG first. USP's monograph looks at 8 withanolides, but there are over 30 of them. So if you use USP methods, you are only looking at 8, and really only measuring 2 of them. That's right, USP's methods only specifically measure 2 of the withanolides. You calculate the others using math based off their assumed response factors. That's crazy to me, too! Nobody know this! People just see "5% withanolides" and think nothing else of it, but it is so much more complex than that!

Okay, so you set your STAG. Now you need reference standards for everything in your STAG. Ohh shit, they don't exist! Nobody makes those reference standards! Hell, they don't even make all of the 8 in the USP monograph. So what do you do? Well you have to make your own! That requires a flash chromatograph or prep HPLC system, a rotavap, a lyophillizer, then a mass spec and NMR for verification. That's at minimum. To get the standards validated, you need to send off to multiple other labs for them to confirm your work. Let's say you do all that. You set your STAG and you either buy or make all the reference standards in it. Well then you have to create your analytical methods to measure them. So you go back to your UPLC and start setting up chromatography. Well that's harder than it sounds. It's probably months of work to do, which you then need to validate. Well you can't do that on your QC machines. You have every day samples coming in that need to be tested of your normal stuff. So you need a second UPLC to do the R&D work. Well sometimes UV peaks are hidden in the chromatography. So you should probably put a mass spec detector on the end of your PDA detector, so you can isolate the fragmentation patterns and see any signals hidden in the UV peaks. Let's say you do all that, and you get assay data for your STAG. Cool, now you know it is ashwagandha root extract, and has 5% withanolides that you have defined in your STAG. Then you have to move on to heavy metals.

So heavy metals are much more critical to measure in natural extracts. This is because plants like to absorb things from their environment, and heavy metals are in the soil and waters around the world. We almost NEVER see elevated heavy metals in any synthetics we deal with, but we reject natural extracts all the time for them. So you need to do ICP-MS on every batch to determine what the heavy metal content is, and if it meets your specs. Well what are your specs? You will never have 0 heavy metals in a natural extract. That's impossible. There will always be some detectable amount. So what are your acceptable levels? Well that's your responsibility to set. So you have to go through the research, and apply that to what the expected dosages and daily limits will be. If you suggest something be taken 3 times a day, you have to make sure your heavy metals limits you set are 3X less than if you only say once per day. So we set VERY tight heavy metals specs on our stuff, and look at every single product on an individual basis to set them. So cool, now you have your heavy metals limits set, you ICP-MS the sample and they come in below the limits. Now you have to move on to other contaminants.

So natural extracts are extracted with something, right? Usually that is water or ethanol, but you can usually get better yields if you use less safe solvents to do it. That's not really allowed, but some shitty suppliers do it. So how do you test to ensure no harmful solvents were used in the extraction process? You have to do residual solvent testing via headspace GC. This essentially creates a vacuum in your sample to pull off the residual gasses, and passes them through a flame ionization detector, or FID. This allows you to see what residual solvents are left, and at what concentration. At least USP has specified limits here, and standardized methods. So this one is not too difficult once you have your machine and methods setup. So that's good.|

Then you have other methods you might need to use in the process. Sometimes you have to us LC-MS on the QC sample, because the analytes don't have a chromophore that can be seen on a UV detector. Sometimes you have to use chromatography to separate things, then use NMR to identify them. HPLC/UPLC can see things with chromophores, which are usually ring structures. FTIR can see things with carbon. P-NMR can see things with hydrogens. Mass specs can see ion fragmentation patterns. FIDs can measure the current made by passing through a hydrogen flame. Polarimeters see how light bends through a sample. Not one single methodology can tell you everything. So you have to use orthogonal techniques to get the full picture. Getting the full picture is MUCH easier with a known synthetic compound compared to a natural extract. If you get into more complex biological samples, it gets even more difficult.