Bacopa Monnieri - Standardised extracts (e.g Synapsa) and HPLC measurements

This is something we have been wrestling with for a long time. Verdure was one of the first ones to move over to HPLC for their standardization on Bacognize bacopa a few years ago. We had a lot of questions from people back then about why it used to say 45% and now it says around 13%. Nothing actually changed with the extract. It's just the testing methodology got better. I'll explain the details a little bit.

So UV-VIS was the standard for analyzing the content of plant extracts for decades. It was easy and everyone used it. There were USP methods for all sorts of plants that made it easy to analyze the ID and content of the extracts in one go. The big issue is that UV-VIS is not a chromatographic methodology. There is no separation of compounds. Essentially you just shine a light with UV and visible spectrums at a sample of plant material dissolved in a specific solvent, then analyze the graph of either the absorbance or reflectance. Everything absorbs or reflects light differently. The big issue is that you have interferences. Many things absorb or reflect similarly to each other. UV-VIS does NOT separate those things out. There is no chromatography happening. So the numbers you get are inflated 99% of the time, due to other things that reflect or absorb light similarly. In the case of bacopa, we are looking at bacopasides, or triterpene saponins that are the main active ingredients in bacopa. The issue is that there is a lot more stuff in bacopa that reflects/absorbs light in the same way as those triterpene saponins. So the 45%, 50%, 55% numbers you see on most bacopa products are not reality. They are grouping ANYTHING together that reflects or absorbs light in the same way as bacopasides, not just the ones we are interested in. This was industry standard forever. Standardizations were set using an (in my opinion) improper methodology. However, everyone did it, so there was no incentive for someone to correct the bad numbers. Enter HPLC/UPLC.

So HPLC/UPLC is a chromatographic methodology. This means we use chromatography to separate compounds from each other so that we can measure them individually. In HPLC/UPLC's case, that is using specific columns and solvents to separate when certain compounds pass the UV detector on the HPLC/UPLC. We now also do the same thing on our HP-TLC as well. However, HP-TLC uses thin-layer chromatography to separate the compounds. Essentially think of the plate on an HP-TLC as an HPLC column that has been unrolled and flattened out. The same concepts are used. So with an HPLC/UPLC we are developing sample prep with certain solvents at certain concentrations, then developing methods that inject various amounts, ratios, and different solvents with the sample through the column. If you are good at designing the methods, those techniques actually separate the compounds from each other so they leave the column at different times. Then they are measured via their absorbance or reflectance by ultraviolet light. So you can see that the HPLC/UPLC is measuring similar things to the UV-VIS. However, it is measuring them SEPARATELY from each other. This is key, as you remove interferences. Also, everything has a specific response-factor in the UV spectrum. Each bacopaside will have a different response factor to a certain wavelength of light, and that will affect how short/tall the peak is. So if you see two peaks the same size as each other, that does NOT mean they are the same concentration. Ohh no, that would be too easy. That's called area percent calculation, and that is bad chemistry. What you need to do is then run a qualified reference standard through the HPLC/UPLC with the same methods. Then you can calculate the actual concentrations in your sample from the known response factors on your machine, with your methods, at that temperature/pressure. THAT'S how you properly analyze the concentrations of active ingredients in a sample.

So in a very simple sense, UV-VIS measures the absorbance/reflectance of light through a whole sample, and tries to give a general idea of concentrations. It's great for that if your sample is simple. So if there are only a few chemicals in the sample, there will be little interference, and you can run a reference standard on the same machine to account for differing response-factors. It is NOT great for complex samples, which botanicals are. There are hundreds of different things in some plant extracts. You need to separate them if you ever want accurate numbers. We use UV-VIS all the time for mushroom analysis, as it comes AFTER we break the sample down using Megazyme kits. Essentially we are breaking down the specific glucans in the mushrooms so that we can accurately measure the beta-glucans in the sample. If we just ran the mushrooms through the UV-VIS without doing the enzymatic part first, we would get no real useful numbers.

HPLC/UPLC measures the absorbance/reflectance of UV light after things have been separated from each other. Chromatographic methods are always going to be more accurate than either dry techniques or simple non-chromatographic techniques. So our FTIR is an example of another non-chromatographic technique that is great for testing single chemicals, but breaks down when you have a bunch of things combined together. If you don't separate the compounds, you can't accurately measure them. HPLC, and now UPLC, are the gold standard for getting accurate measurements on complex samples. I could talk all day on the complexities of the methodologies, but I will leave that for another time.

So how does this all affect bacopa? Well we have tested a LOT of bacopa from all over the world. We have NEVER gotten HPLC/UPLC results anywhere near 50%. It is likely that everyone out there claiming they are selling that high of a standardization is either referencing bad methodologies, are lying, misinformed, or getting bad data from their labs. The highest we have ever gotten was 24% on our own extract we did. Bacognize is like 12.6%. Synapsa is only coming out at like 8-9%, which is surprising to us. There are people out there claiming to have 50% by HPLC. However, their testing methodology is faulty. Actually, we have found that it's not the methods, it's the reference standard everyone in India is using right now. We just went through this whole thing. A lot of the labs in India are using a reference standard that is degraded, so the results are showing double what they actually are. When you run the same methods on the same machine, but you use Sigma Aldrich's USP reference standard, the numbers come out much lower. That 24% bacopa I mentioned is actually sold to everyone as a 50% extract. However, we have proven now it is not. It took us a while to convince them, and a lot of back and forth with the labs in India. However, they have FINALLY admitted that their reference standard is degraded. So we are about to sell that extract, but we are properly labeling it as a 24% extract. This makes it tough for us, because people are going to see the 24% and compare it to 50% or 55% on other people's product, and think ours is less potent. However, it is literally the most potent bacopa we have ever tested.

So that was a long answer, but it is not an easy one. Should you adjust your dose based on the actual concentration of bacopasides? ABSOLUTELY! However, you can't trust any of the numbers out there. I realize that make things seem pretty bad, and frankly they are. This whole industry is built on lies and faulty data (almost every industry is, but that is a whole other philosophical discussion). Then some people ask me why I spent so much money building my own lab... I would not know any of this if we were not in our lab trying to verify all these numbers! 99% of the people in this industry just want to get a number back from a lab that says they can sell the product. They don't want to reject batches. That costs a lot of money. It's much easier to get a number from a lab that gives you plausible deniability to point to if something later comes back wrong. Trust me, I get so upset about this EVERY DAY. We are spending so much time, effort, and money trying to find the truth behind things, but literally nobody else is. Then we lose sales because of it. Saying it is frustrating is putting it mildly. Also, do you think this is only a thing with bacopa?!? Hell no! I am dealing with this with a TON of other extracts right now: ashwagandha, ginger, gotu kola, magnolia bark, etc. I could go on and on about shit like this for days.

So I wish I could just give you an easy answer for how you should adjust, but the reality is much more complex than anyone realizes. If I ran your specific sample through our UPLC to get the real bacopaside numbers, I could tell you how much to take. Without that I am shooting in the dark.