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Demand more from your Sponsors! Peptide Testing Explained
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Demand more from your Sponsors! Peptide Testing Explained - 02-15-2013, 11:00 AM

Alright i am really tired of all the posts and emails i get from different sponsors that claim "receptor grade", "99.xxx purity" and a whole host of other useless claims. I am here to tell you that most of this is pure garbage. These last few weeks have been very eye opening. Most sponsors (and i mean owners of the companies) are completely clueless on testing methods or even how to read their own tests they post on their websites.

And i am here to tell you that not ONE sponsor is innocent of this. When i did the recent LR3 analysis i had a chance to discuss the testing methods and procedures with many of the sponsors and was shocked that most did not even know what should be the actual procedure for testing their peptide or protein. Even the manufacturer that sells them peptide provides them with whatever BS tests that mean nothing. So i am going to explain some tests and graphs you see and what they mean and what they dont mean.

This is meant to be strictly educational so you guys understand what you are looking at and dispel some myths and mis-information.

HPLC:

This is a method where a mixture of compounds are studied and analyzed and broken down to see how many different components it is made up of. This method does not identify the specific compound in hand at all. It does not give you the mass of the compound in daltons for you to cross match with what you are looking for. If you already have proof that you have something in that mixture it will show you purity but or not if there are several compounds are there. For example you can do an HPLC on a perfume to see how many different actual perfumes it is made up of etc. But for our purpose HPLC is pretty useless because it does not tell you anything useful. So when you see a sponsor post an HPLC of a so called product you need to be ware.

This is what a typical HPLC looks like. Notice the X axis is always in time and not weight so it does not tell you anything. The Y axis is the concentration but then again does not identify the product.






SDS-PAGE:


This is a sodium gel electrophoresis and used heavily in genetics, forensics and chemistry where proteins are separated based on their ELECTROPHORETIC mobility which is a function of their polypeptide chain length and its electric charge. This shows up as a band on the gel. This is pretty decent in identifying if a certain protein exists at a certain weight range we are looking for. For example if we know that IGF LR3 has a 9111 dalton weight we should expect a thick heavy band in the 9000 range. The major issues with this procedure is that it is very limited and not very accurate down to the dalton weight. You may see a band in the ~9000 range but you dont know if it is 8800 or 9200 etc. And this is a huge issue when you are actually identifying a specific product. It also DOES NOT tell you how much total of a compound you have to begin with.

This what a typical SDS-PAGE looks like. You can see bands showing up but there is nothing specific or accurate about it. For example, look at column 2. We can see a thick band showing up between 50-75kda but where does it actually fall? It is 58K or 63K? Who the hell knows. You also see lighter shadows around the thick band but doesnt tell you how bad the other impurities are.






MASS SPEC:


Mass spectrometry is where singular spectrum of the masses of the molecules comprising a sample of material are displayed. It really doesnt get much more accurate than this. It is basically used to determine the elemental composition of a sample based on the molecules mass weight. This will tell you not only if you have a pure sample but what the actual mass weight of the sample is and if there are other molecules that weight more or less than what you are looking for. Once again this will not tell you what is the total weight of your sample (as in total product weight). What i mean is that if i turn over a vial of IGF LR3 to the lab and they do a mass spec and it comes back with a perfect spike at 9111 daltons. All it means is that there was LR3 in there. It wont tell me if there was 0.1mg or 1mg or 10mg of LR3 in that sample. Hope that makes sense.

This is what a typical MALDI TOF mass spec looks like. You can see peaks at the different exact mass where the compounds were detected. There is no guess work.





So we have discussed the potential ways to identify a compound with a certain mass weight and discussed how mass spectrometry is the best method. But none of the above have verified what we actually have short of what is the mass weight of the molecule. Let me explain further. Since we have discussed IGF LR3 we will just stick with that. We know it has a mass weight of 9111 daltons. I can piece meal together enough crap protein in a chain in a lab to give you A SAMPLE that has a mass spec that will show up in the 9100 range. But does that mean just because you have a sample that weights close to 9111 that it is actually IGF LR3? HELL NO.

The only way is to do a full amino acid analysis. There is a two fold reason for this. One it will show if the percentage of amnio acids found in the sample match the % of amino acids that SHOULD be in the LR3. Because we know from the sequence how many glutamaine, leucine, aspartamate etc are in one chain of LR3. So when you do an amino acid analysis those percentages should match up. For example, if your sample shows up with an amino acid that doesnt even exist in the LR3 chain you know something is screwy.

The second reason for the amino acid analysis is that it will tell you within 10micrograms what the total weight of the protein you are testing for in that vial. What is the point of having a pure IGF LR3 but it really is 0.1mg vs 1mg or 10mg.



AMINO ACID ANALYSIS NOW WILL CONFIRM AND BACK UP WHAT YOU FOUND IN THE MASS SPEC BUT ALSO TELL YOU THE TOTAL WEIGHT OF PRODUCT IN GIVEN SAMPLE.

This is what a typical amino acid analysis looks like. I have taken out the name of the sponsor because this is not meant to be an advertisement and strictly educational. But you can see that we have the different amino acids listed on the left and what the known percentages should be vs what is the calculated from the sample. There is always a deviation allowed up to 1.0-1.5% considered very acceptable. You can see in this report that at the bottom there was a total of 952micrograms of LR3 found which is 0.952mg that is as close as to the 1.0mg advertised on the vial as you can get. You can also see that there was a slight amount of Histidine found in this sample. If you look at the sequence of IGF LR3 there is no Histidine found anywhere so to find it means that a very small sample had Histidine attached that were not cleaved off during the purification process. This is why amino acid analysis is so important.




CONCLUSION:

So what do we learn from all the above? Not to be a sheep and just swallow everything that is fed to you. The only way any sponsor can prove to you what they are selling as far as a peptide/protein of any sort is to show you a proper Mass Spec report WITH a full amino acid analysis. One without the other is worthless. Just showing you a mass spec is worthless. Showing you HPLC reports and SDS PAGE reports shows that they really have no idea what they are doing. Please pass this information on as this should be a must read for everyone researching. Good luck.
   
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02-15-2013, 11:11 AM

For some reason the last amino analysis chart didn't show? Is that my pad or do others see it? Interesting and informative read that I myself am into.thanks.
   
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02-15-2013, 11:14 AM

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For some reason the last amino analysis chart didn't show? Is that my pad or do others see it? Interesting and informative read that I myself am into.thanks.
If you are within a network that is blocking sites such as photobucket etc the pics may not show up. I just tried two PCs that are not blocked and it shows up fine.
   
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02-15-2013, 11:16 AM

This is great information that everyone should be aware of! Thank you for sharing!!




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02-15-2013, 11:32 AM

Great read! Thanks!


Disclaimer: The articles and conversions posted are for entertainment and informational purposes only - experiment at you own risk.
   
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02-15-2013, 03:55 PM

I will definitely share this on the Swedish boards!
Good work Alpha!
   
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02-15-2013, 04:18 PM

thank you for this post
   
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02-18-2013, 02:40 PM

Great post!


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Thank you! Very informative.
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Thumbs up Thank you! Very informative. - 02-16-2014, 03:57 AM

Finally, someone who tells it like it is in a way ANYONE (even a RC/Peptide seller) can understand.
The science behind the testing and the testing itself means absolutely nothing if you don't have a clue.

VERY INFORMATIVE.

I could not have explained the testing methods and processes any better myself. My background is in theoretical physics and cosmology, not chemistry or chemical engineering but I do understand the testing methods and do run mass spec quite often. I have never run an amino acid analysis but it would be a must to determine weight and concentration with peptides.

I agree the 98.xxx purity and "100% US Made" and other claims made by peptide sellers (who don't manufacture it themselves and are at the mercy of what their suppliers tell them) are just hot air.

Unfortunately, trial and error seems to be the only way without performing these tests yourself, the sellers aren't going to go to the expense when people keep buying. I have had questions completely ignored when asking for genuine test results in the past, even worse, I've seen so many made up, nonsensical BS answers to questions that just confirm what you're saying...they're clueless.

If you don't mind, I would love to share your post on a couple other sites...with full credit to you, of course.

I have had positive lab results myself (bloods and mass spec (no AAA) for the most part but I have had less than stellar results from some compounds (sponsors on other boards) and choose not to do business with those particular sellers anymore.


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08-09-2016, 02:01 PM

Sir, I mean no disrespect, but that post is incorrect in many points both unimportant and important.
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Originally Posted by janoshik View Post
Sir, I mean no disrespect, but that post is incorrect in many points both unimportant and important.


Good to see you here Janoshik.




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Originally Posted by janoshik View Post
Sir, I mean no disrespect, but that post is incorrect in many points both unimportant and important.
Well by all means if you see it wrong set it right.....I'm sure there are plenty looking to learn


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08-09-2016, 09:47 PM

Jano knows his stuff. Glad to see you here friend. Talk to you soon
   
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08-10-2016, 02:47 PM

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Well by all means if you see it wrong set it right.....I'm sure there are plenty looking to learn
I will by the end of the week, if it's not an issue - don't have much time these days.

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Good to see you here Janoshik.
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Jano knows his stuff. Glad to see you here friend. Talk to you soon
Hello my friends, thank you for inviting me over! This board seems very interesting
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[QUOTE=janoshik;294896]I will by the end of the week, if it's not an issue - don't have much time these days.[QUOTE]

No rush of any kind I'm just always looking to learn when someone has insight to something and I have no doubt alot of others are here to learn also.

Welcome aboard


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08-13-2016, 01:05 PM

So, HPLC.

Identification in HPLC is absolutely possible, contrary to the statement of Mr. alpha6164.

With HPLC you identify the compound of interest by its retention time - the X axis on the graphs, which is stated to be useless above. You can also use spectra from PDA detector, but what is generally the way of identification is the retention time.

I'm going to simplify a lot here, so please, don't quote me on this.

With HPLC, when you inject the mixture of compounds into the system, the mixture is carried by the system of tubing to the detector - it is carried by liquid pushed by the pump, the liquid is called mobile phase.

Now, to separate this mixture, in the system of tubing there is a column inserted.

To explain what a column is, it is the best (imo) to imagine it as a metal tube that is magnetic. If you throw in another magnet, it will be slowed down by the magnetic forces and will fall out LATER than, for example, a piece of wood would.

Now, with HPLC we are, of course, not talking magnets, but the principle is the same. There are physical forces at play (from hydrodynamic size of the molecule, through the polarity, charge etc...) that will cause different chemicals to come out at different time.

The properties of a single chemical are the same. 1mg of LR3 will ALWAYS act like another 1mg of LR3 if they are identical in their physical properties.

So if you inject LR3 into HPLC that is stable (pump is okay, temperature is stable, column is not old etc... basic stuff), it will come out at the same time... Always.

Some may argue that there might be another chemical that has the same retention time as LR3. That is true.

But if you change the conditions (adjust mobile phase, use different chemistry column, do a PDA scan...) and the properties are still the same, the consensus in the scientific community is that *no further confirmation is necessary.*

The big disadvantage of this method might be the necessity to own a standard (so you have to have a sample of LR3 that's guaranteed to be good for example) beforehands.

There are also many other ways to identify and quantify stuff using the HPLC, but that would be way too much to write down for me. Let's say PDA scans can be helpful, internal standards, reagent methods...




I am going to talk about PAGE methods too much. SDS PAGE can do what Mr. alpha6164 had stated, but as he said it's not very accurate for getting length exactly and neither it is useful for precise quantification (getting content in mgs).

Also SDS is completely useless in this case - it only works with primary structure of proteins/peptides. So if somebody cooked (and therefore made it useless) your LR3 beforehands you'd not notice at all with that test. But without SDS you can't get the length of the protein!

Regular PAGE with a standard is the way I'd do if I had to use electrophoretic methods - but it has literally no benefits over HPLC, as long as you have standard.

Electrophoretic methods can be used also to get precise amino acid composition, but that's so much work it's only used in research... not regular testing. That's way overkill.




The rest of the original post about mass spec is... just...

That amino acid analysis is sweet, but if there was for example alanine and arginine switched somewhere it would render the peptide useless, while still showing correct weight on MALDI-TOF and correct amino acid profile...

The proper way (if you don't have a standard!) is to do a trypsine digestion, so you can actually detect which position is which amino acid. It also leaves much less space for error than method mentioned.

Also, MS (or, well, MALDI-TOF) without HPLC (or other separation method, but GC is useless for proteins/peptides) is not really useful for most purposes.



I'm open to any questions or discussion, but please, try to be as specific with your questions as possible, so I can answer them directly. Hope I helped, guys.
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08-18-2016, 04:49 PM

Quote:
Originally Posted by janoshik View Post
So, HPLC.

Identification in HPLC is absolutely possible, contrary to the statement of Mr. alpha6164.

With HPLC you identify the compound of interest by its retention time - the X axis on the graphs, which is stated to be useless above. You can also use spectra from PDA detector, but what is generally the way of identification is the retention time.

I'm going to simplify a lot here, so please, don't quote me on this.

With HPLC, when you inject the mixture of compounds into the system, the mixture is carried by the system of tubing to the detector - it is carried by liquid pushed by the pump, the liquid is called mobile phase.

Now, to separate this mixture, in the system of tubing there is a column inserted.

To explain what a column is, it is the best (imo) to imagine it as a metal tube that is magnetic. If you throw in another magnet, it will be slowed down by the magnetic forces and will fall out LATER than, for example, a piece of wood would.

Now, with HPLC we are, of course, not talking magnets, but the principle is the same. There are physical forces at play (from hydrodynamic size of the molecule, through the polarity, charge etc...) that will cause different chemicals to come out at different time.

The properties of a single chemical are the same. 1mg of LR3 will ALWAYS act like another 1mg of LR3 if they are identical in their physical properties.

So if you inject LR3 into HPLC that is stable (pump is okay, temperature is stable, column is not old etc... basic stuff), it will come out at the same time... Always.

Some may argue that there might be another chemical that has the same retention time as LR3. That is true.

But if you change the conditions (adjust mobile phase, use different chemistry column, do a PDA scan...) and the properties are still the same, the consensus in the scientific community is that *no further confirmation is necessary.*

The big disadvantage of this method might be the necessity to own a standard (so you have to have a sample of LR3 that's guaranteed to be good for example) beforehands.

There are also many other ways to identify and quantify stuff using the HPLC, but that would be way too much to write down for me. Let's say PDA scans can be helpful, internal standards, reagent methods...




I am going to talk about PAGE methods too much. SDS PAGE can do what Mr. alpha6164 had stated, but as he said it's not very accurate for getting length exactly and neither it is useful for precise quantification (getting content in mgs).

Also SDS is completely useless in this case - it only works with primary structure of proteins/peptides. So if somebody cooked (and therefore made it useless) your LR3 beforehands you'd not notice at all with that test. But without SDS you can't get the length of the protein!

Regular PAGE with a standard is the way I'd do if I had to use electrophoretic methods - but it has literally no benefits over HPLC, as long as you have standard.

Electrophoretic methods can be used also to get precise amino acid composition, but that's so much work it's only used in research... not regular testing. That's way overkill.




The rest of the original post about mass spec is... just...

That amino acid analysis is sweet, but if there was for example alanine and arginine switched somewhere it would render the peptide useless, while still showing correct weight on MALDI-TOF and correct amino acid profile...

The proper way (if you don't have a standard!) is to do a trypsine digestion, so you can actually detect which position is which amino acid. It also leaves much less space for error than method mentioned.

Also, MS (or, well, MALDI-TOF) without HPLC (or other separation method, but GC is useless for proteins/peptides) is not really useful for most purposes.



I'm open to any questions or discussion, but please, try to be as specific with your questions as possible, so I can answer them directly. Hope I helped, guys.
First of all, thanks for coming over to the board and providing a technical and scientific analysis of peptide information. It's much appreciated, even if it's not completely understood. Which brings me to the important part.

Since no one else wants to do it, I guess I'll have to be the stupid kid in the back of the class that raises his hand and says what everyone else is already thinking. I have no idea what anything in your last post means, or how it applies to me as a potential consumer of peptides. My concern, as a consumer, is how do I look at whatever testing data has been supplied by the peptide dealer I'm potentially purchasing from and determine whether or not I am buying the actual product that I think I'm supposed to be buying?

If I were more technically educated, your last post might have helped me, but honestly it just confused the topic more. I'm a layperson, and my understanding of testing procedures and the ability to interpret the given data is limited, at best. So, if I'm looking at a potential supplier's analysis of a peptide, and the only information they provide me with is an HPLC analysis, can I reliably use that one analysis to know if the peptide I'm purchasing is in fact the peptide that has been represented to me?


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08-19-2016, 05:02 AM

Thank you for appreciation and it is my pleasure!

I'm sorry that I've forgotten to do a short overview what that really means for average customer. Feel free to remind me any time, it is one big fault of mine.

Basically, this means, that demanding more than a HPLC tests from sponsors might not be necessary at all, as HPLC test should be capable of providing all the informations that are necessary.

Of course, there's no guarantee the test is not forged / the test applies also to the batch that is being sold right now - but so it wouldn't be with any other test.



Also, the best feature of HPLC tests is that they are much faster and cheaper than the method suggested by the original poster... Basically allowing everybody to have their products tested cheaply enough.

I'm hesistant to shamelessly advertise myself here, but for example I am one provider of such service.
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