Testing Your Supplements

That depends on what you are looking to get. Proper analytical testing is HARD. It's infinitely harder than anyone here thinks. We've even stopped using multiple well-known labs that many others use because their data was either faulty, fake, or poorly acquired. I've caught multiple instances from multiple labs giving completely fabricated data. So even getting a lab to test something doesn't always mean you are getting real information. There are very few labs in the United States doing things properly, and even fewer that are willing to test the more uncommon things. Anyone here that thinks they can just get an uncommon chemical synthesized in China, then send it to a common lab spoke about here, and be confident in the identity, purity, and safety of said compound is in waaaaay over their heads.

I'll pick a compound to illustrate my point. Let's say you are looking to get GL-II-73. That's a novel investigative benzodiazepine derivative with potential nootropic and anxiolytic actions. Well first off, there are no reference standards for that. So right off the bat you are going to have trouble with ID and purity analysis. So you are going to have to start from scratch. It has a molar mass of 386.430 g/mol and a couple benzene rings. So it is a good candidate for liquid chromatography like HPLC/UPLC, as it has a chromophore and can be seen by the UV detector in those machines. You have no reference standard, though. So you are likely going to need to get on a triple quad LC-MS/MS. Your analytical chemist would need to design the chromatography methods in such a way to separate that compound out from whatever else is in your sample, then see what mass it is. If the mass matches 386.430 g/mol, you know you are on the right track. So then you would use those methods and a sample collector on your LC to get a pure sample of just that compound. That's much much harder than it sounds. Then you would take that sample to an NMR. Now a useful NMR is in the 400 MHz range. That's a $500,000-$1,000,000 machine, and uses a lot of gas each time it runs. So you are going to have to probably do that at a larger university, as most labs do not have an NMR. Then you run your NMR and get results for how many hydrogen signals are in the sample. Then you need to interpret those results, and most analytical chemists are not very good at that. If everything went perfectly, you should see the appropriate number of signals for the hydrogens you know are in the structure. However, it's not going to go perfectly. You are going to have extra signals, missing signals, and split signals that you are going to have to scientifically explain. Sometimes you are just not going to be able to do it with just the NMR data.

I was just interpreting two NMR results yesterday, and there are two signals we cannot account for, even though the IR, UPLC, and melting point data are all conclusive that this compound is what it is supposed to be, and pure. However, the NMR is giving us extra peaks. Could it perhaps be a residual solvent, or a salt residue left from the synth at sub-mass-percent concentrations? Who knows. The NMR is just giving you hydrogen signals. The IR is looking at carbons. The UPLC is seeing chromophores. The melting point is probably the most indicative, as everything melts at different temperatures, and you can't really hide things in the methodology there. Still, we want to figure out the extra hydrogen signals. So we decide to send off for a headspace GC-MS, to test for residual solvents. Now we wait for those results before making a determination on that batch.

Back to the hypothetical story. So let's say you get a couple extra signals in your GL-II-73 NMR data. Well my next step would be FTIR-ATR. That will shine a laser into the sample and read the mid/far range of the infrared spectrum. Then you can look at the spectrum and make a determination of the functional groups in it. You don't have a reference standard, though. So you are going to have to manually read the results. Let's say that all goes perfectly. You get a good spectrum, and all the functional groups you think should be there are. Well IR can only see carbons. What if there is a salt or metal in the sample? Onto melting point! Let's head to the data to figure out what the melting point should be! Ohh shit, there is no data. This is too new of a compound, so there is no melting point data for that chemical yet. Then sometimes you get conflicting melting points on different sites. Who do you trust? You can sure a shit know that USP has no data on GL-II-73. So you run the melting point to at least see how it melts. Does it melt quickly all in a tight range, or does it melt poorly over a large range? You can't make definitive conclusions from that, but it is good to have data. So you write it down and move on to headspace GC for residual solvents. Let's say that goes perfectly, and there are no known class 1 or class 2 residual solvents. Great! That's good. Now what about heavy metals? Well then you move on to the ICP-MS. That's at least an easy method. Same thing every time. It just ionizes the sample and looks for the mass of the known metals. Let's say that goes great. There is a little bit more chromium than you would like, but it is still sub-toxic levels. So you move on.

So now you know that what you have is probably GL-II-73. You know there are no real dangerous levels of heavy metals or class 1/2 residual solvents. There might be other solvents, but you are only using the USP list to test for bad ones. There is an extra signal on the NMR, but you are not super concerned with it. There's something else in there, but it is going to be really hard to figure out exactly what it is. So as long as the purity is high, you can ignore it. So you move onto a purity assay. Well shit, there's no reference standard. So properly doing an HPLC or UPLC is not possible. You can build some methods on UPLC, and hopefully you have a PDA detector so that you can see the whole spectrum (many places don't, and just pick a single wavelength to look at, which can hide many things). So you run your UPLC methods with your PDA detector and get some data. Now let's say everything goes perfectly there. You get some peaks. Maybe you even get a clean big peak with some smaller ones. Well you have no reference standard available. So all you can do is guess on the concentration. Let's say it is an ideal scenario, and you have one big peak and one small peak. Let's say the big peak is a 0.80 AU and the small one is 0.05 AU. Well you can sort of guess the purity is fairly good, as long as the impurities don't lack a chromophore. Then you have no idea, because the UPLC is blind to them. That's not an issue with the reference standard, since you can do mass percent calculations off the same methods with your sample vs. the reference standard. Without a standard, you are shooting in the dark. You do some area percent calculations and just hope that is somewhat accurate.

So now you are left with what you think is GL-II-73. You are fairly sure it has over 80% purity by the results, but you are making huge leaps on assumptions. However, you know it doesn't have heavy metals that are going to harm you, and you know that the most dangerous residual solvents are not present. So you decide that is good enough. It has taken you 3 months of work to get to this point, and going further would require tens of thousands of more dollars to create a reference standard and run more testing. So you say that's good enough and take your GL-II-73. Hopefully you don't die because you totally missed something, but you are likely going to be fine with only a single dose. Then you realize there is no real human data on dosage. So either you wing it, or you give up. Well you put too much into this to give up, so you pour through any animal studies you can find, and come up with some justification for the dose you are going to try. You try it, and you don't die! YAY! You don't really notice anything, either. Shit, all that work and it doesn't really do much! Sucks... Welcome to nootropics!

So you can see it is difficult. I didn't even get into isomers/enantiomers, different salts like HCL, anhydrous vs. hydrate/dihydrate, or other harder things to test. I also greatly dumbed things down a bit, and made assumptions that things went right. They won't. They never go right. The likelihood of hitting a dead end is at least 50%. Even if the methods all go right, what happens if you definitively find an impurity? You going to get a refund from mr. China middle man supplier you bought from? Hah! Not likely. They will argue with you on methods, or sample prep, or wavelengths, or some other dumb reason that makes no sense, but what are you going to do? You are a single person with no pull. So you are out whatever you spent on the compound plus whatever you spent on the testing, which would probably be around $10,000 if you contracted it all out, if not more.