*** Mass Specs on CBL HGH ***

[Brolloks]

There are no charts whatsoever

Correct. Doc Jim will now avoid this thread like all the others where his comprehension failed.

 

[janoshik]

Jano, can you explain to us what these graphs mean?

I'll go into depth a little bit, because I got plenty of time in the standstill traffic.

To put it into physics terminology, those graphs are absorbance plots over time.

As you know, if you mix 90% red color and 10% water, it will be MORE red than mix of 50% red color and 50% water. The relationship (Lambert-Beer law) is linear - so 25% red color will have half of absorbance of 50% red color mix. Perfect for determining concentration.

HGH doesn't have any visible color, however it absorbs light in UV region. Those graphs are absorbance plots at 214 nanometer wavelength, which is universal to all peptides and proteins, as it's the absorbance of the peptide bond.

Now you might be thinking - but if it's universal property - then any protein, even protein impurities would show in concentration of HGH! (This is how Analyzer tested mg/vial [total protein assay], because he didn't have standards)

Easy fix would be to separate the mix - that's what HPLC is used for - various components of a mix are separated on basis of any of the countless physical properties and they show on detector in different time. So for each compound that can be detected at the said wavelength you have a peak on the graph at different time.

To determine concentration precisely you need a standard - the principle is simple. You run standard of known concentration through the HPLC and compare it to the unknown. You compare the integrated areas under the peak which appeared at the same time. Height of the peak can be used at times too, but it's use is extremely limited.

So for example you have:
5 mg/ml = area of the peak 10 000
then you have unknown sample
x mg/ml = area of the peak 20 000

So you can safely conclude (under some conditions, like linearity of the detector) conclude that the unknown sample is 10 mg/ml.

 

[janoshik]

Jano, can you explain to us what these graphs mean?

To talk about the graphs, one of them depicts HGH standard and one of them unknown sample.

You can notice that there is no concentration mentioned anywhere in the sheets - that's because I dissolve all samples of HGH in 2 ml of H2O, unless noted otherwise, so I don't write that down.

So if area 11992924 = 3.33 mg vial, with the peak, at corresponding time on the unknown sample sheet having area of 17354557 we can do the math like this: 17354557/11992924*3.33 mg = 4.81 mg

----

Now, this is possible only because:
1. the linearity of the detector in this region had been confirmed and validated (without that if 1 mg = 10 000 and unknown = 20 000 it's entirely possible the unknown is 100 mg, not 2 mg....)

2. The standard is known to be good - the standard I use is derivative and traceable to European Pharmacopoeia Standard. Without good standard one, or more will apply: a) the results are complete trash b) additional tests for identification must be ran in order to get ID (mass spec) c) concentration can be only estimated

3. The method is specific enough - if all impurities are not separated out, then false readings are obtained.

I'm sure I've forgotten some, but that gives you about the right idea.

 

[Doug_S]

I'll go into depth a little bit, because I got plenty of time in the standstill traffic.

To put it into physics terminology, those graphs are absorbance plots over time.

As you know, if you mix 90% red color and 10% water, it will be MORE red than mix of 50% red color and 50% water. The relationship (Lambert-Beer law) is linear - so 25% red color will have half of absorbance of 50% red color mix. Perfect for determining concentration.

HGH doesn't have any visible color, however it absorbs light in UV region. Those graphs are absorbance plots at 214 nanometer wavelength, which is universal to all peptides and proteins, as it's the absorbance of the peptide bond.

Now you might be thinking - but if it's universal property - then any protein, even protein impurities would show in concentration of HGH! (This is how Analyzer tested mg/vial [total protein assay], because he didn't have standards)

Easy fix would be to separate the mix - that's what HPLC is used for - various components of a mix are separated on basis of any of the countless physical properties and they show on detector in different time. So for each compound that can be detected at the said wavelength you have a peak on the graph at different time.

To determine concentration precisely you need a standard - the principle is simple. You run standard of known concentration through the HPLC and compare it to the unknown. You compare the integrated areas under the peak which appeared at the same time. Height of the peak can be used at times too, but it's use is extremely limited.

So for example you have:
5 mg/ml = area of the peak 10 000
then you have unknown sample
x mg/ml = area of the peak 20 000

So you can safely conclude (under some conditions, like linearity of the detector) conclude that the unknown sample is 10 mg/ml.

I'm sure DRJIM would explain this better

 

[Thaistick]

To talk about the graphs, one of them depicts HGH standard and one of them unknown sample.

You can notice that there is no concentration mentioned anywhere in the sheets - that's because I dissolve all samples of HGH in 2 ml of H2O, unless noted otherwise, so I don't write that down.

So if area 11992924 = 3.33 mg vial, with the peak, at corresponding time on the unknown sample sheet having area of 17354557 we can do the math like this: 17354557/11992924*3.33 mg = 4.81 mg

----

Now, this is possible only because:
1. the linearity of the detector in this region had been confirmed and validated (without that if 1 mg = 10 000 and unknown = 20 000 it's entirely possible the unknown is 100 mg, not 2 mg....)

2. The standard is known to be good - the standard I use is derivative and traceable to European Pharmacopoeia Standard. Without good standard one, or more will apply: a) the results are complete trash b) additional tests for identification must be ran in order to get ID (mass spec) c) concentration can be only estimated

3. The method is specific enough - if all impurities are not separated out, then false readings are obtained.

I'm sure I've forgotten some, but that gives you about the right idea.

That actually makes sense.

 

[master.on]

C18 and H2O + MeCN. C18 I've used either Ace SuperC18 2.5um 4.6*100mm superficially porous or Kinetex 2.6um CoreShell 4.6*150mm. Not 100% sure, but the difference is very little.

Jano, can you explain to us what these graphs mean?

To talk about the graphs, one of them depicts HGH standard and one of them unknown sample.

You can notice that there is no concentration mentioned anywhere in the sheets - that's because I dissolve all samples of HGH in 2 ml of H2O, unless noted otherwise, so I don't write that down.

So if area 11992924 = 3.33 mg vial, with the peak, at corresponding time on the unknown sample sheet having area of 17354557 we can do the math like this: 17354557/11992924*3.33 mg = 4.81 mg

----

Now, this is possible only because:
1. the linearity of the detector in this region had been confirmed and validated (without that if 1 mg = 10 000 and unknown = 20 000 it's entirely possible the unknown is 100 mg, not 2 mg....)

2. The standard is known to be good - the standard I use is derivative and traceable to European Pharmacopoeia Standard. Without good standard one, or more will apply: a) the results are complete trash b) additional tests for identification must be ran in order to get ID (mass spec) c) concentration can be only estimated

3. The method is specific enough - if all impurities are not separated out, then false readings are obtained.

I'm sure I've forgotten some, but that gives you about the right idea.

Can you please show the calibration curve?

Chart peak heights don't automatically correlate with concentration.
You can have a large percentage of an impurity that absorbs little UV light, and it will show as a small peak, but the impurity is found at a large percentage.
So you can't skip doing a calibration curve.


Also, @mercury do you think the peaks aren't as sharp as they should be?
IMO wide peaks aren't acceptable, even if they are tall.
Tall as tall tales.

 

[janoshik]

Can you please show the calibration curve?

Chart peak heights don't automatically correlate with concentration.
You can have a large percentage of an impurity that absorbs little UV light, and it will show as a small peak, but the impurity is found at a large percentage.
So you can't skip doing a calibration curve.


Also, @mercury do you think the peaks aren't as sharp as they should be?
IMO wide peaks aren't acceptable, even if they are tall.
Tall as tall tales.

The calibration curve was literally written in the post you had quoted. You just don't really understand what exactly it is.

17354557/11992924*3.33 mg = 4.81 mg is a great example of a single point calibration curve (3.33 mg being ie. X axis and 11992924 being an Y axis, intersecting [god I hope that's the right word] zero).

Now you might try objecting that you'd like to see 3, 5 or however-many point calibration curve. That is not necessary as long as linearity of the method is confirmed and method is validated for use of a single point calibration curve. Which is quite common indeed.

However, if you need to see the 5 or 6 point calibration curve, look around, I'm fairly certain I've uploaded an excel file containing it over here on Meso somewhere.

Regarding the peak heights just a post before the one you had quoted I had written: "You compare the integrated areas under the peak which appeared at the same time. Height of the peak can be used at times too, but it's use is extremely limited."

However I will assume you mean peak area. First thing first.
1. Disparity in absorbance of compounds has literally absolutely nothing to do with calibration curve of the standards and generally it's nonsense what you have written.
2. With GH only impurities that are taken into the account are impurities of protein nature. You might notice that they indeed have something in common. To quote myself again:
"Those graphs are absorbance plots at 214 nanometer wavelength, which is universal to all peptides and proteins, as it's the absorbance of the peptide bond."


Regarding the peak width, your opinion does not really matter, because again, you don't really understand it.

This is high resolution targeted analysis specifically for GH. The whole method is developed so that the impurities, which differ from GH just in couple of atoms ( in a molecule of 22 thousand molecular weight! ). It doesn't matter how wide the peak is, as long as impurities of interest get separated.

And they do. Check the unknown sample.

Thin peaks are great. Thin peaks are necessary in many cases. Not this analysis.

A single change (faster gradient) in the method would make the peak 1/4 as 'thick,' but wouldn't necessarily increase the quality of the method.

 

[master.on]

The calibration curve was literally written in the post you had quoted. You just don't really understand what exactly it is.

17354557/11992924*3.33 mg = 4.81 mg is a great example of a single point calibration curve (3.33 mg being ie. X axis and 11992924 being an Y axis, intersecting [god I hope that's the right word] zero).

Now you might try objecting that you'd like to see 3, 5 or however-many point calibration curve. That is not necessary as long as linearity of the method is confirmed and method is validated for use of a single point calibration curve. Which is quite common indeed.

However, if you need to see the 5 or 6 point calibration curve, look around, I'm fairly certain I've uploaded an excel file containing it over here on Meso somewhere.

Regarding the peak heights just a post before the one you had quoted I had written: "You compare the integrated areas under the peak which appeared at the same time. Height of the peak can be used at times too, but it's use is extremely limited."

However I will assume you mean peak area. First thing first.
1. Disparity in absorbance of compounds has literally absolutely nothing to do with calibration curve of the standards and generally it's nonsense what you have written.
2. With GH only impurities that are taken into the account are impurities of protein nature. You might notice that they indeed have something in common. To quote myself again:
"Those graphs are absorbance plots at 214 nanometer wavelength, which is universal to all peptides and proteins, as it's the absorbance of the peptide bond."


Regarding the peak width, your opinion does not really matter, because again, you don't really understand it.

This is high resolution targeted analysis specifically for GH. The whole method is developed so that the impurities, which differ from GH just in couple of atoms ( in a molecule of 22 thousand molecular weight! ). It doesn't matter how wide the peak is, as long as impurities of interest get separated.

And they do. Check the unknown sample.

Thin peaks are great. Thin peaks are necessary in many cases. Not this analysis.

A single change (faster gradient) in the method would make the peak 1/4 as 'thick,' but wouldn't necessarily increase the quality of the method.

Single point calibration curve = oxymoron.

Have you ever done a calibration curve yourself?
Or it's because you believe that it will remain linear down to zero or the limit of detection?

 

[janoshik]

Single point calibration curve = oxymoron.

Have you ever done a calibration curve yourself?
Or it's because you believe that it will remain linear down to zero or the limit of detection?

1. It's literally explained in the HPLC manual for dummies. I suggest you read that.

https://www.researchgate.net/profil...Of_HPLC_Quantitative_and_Qualitative_HPLC.pdf

Page 16.

Says literally the very same thing I had explained in my previous posts.

2. It sounds funny and a bit rude when you ask me whether I have ever done a calibration myself, when it's you who doesn't even really understand it.

3. Yes, I believe it, because I have experimentally verified it and validated the method. Exactly how it's done.

 

[mtt]

Than you Jano for taking the time explaining all this. It's pretty involved and over my head but I will reread it for a better understanding. Again thanks, you really go out of your way for all of us.

 

[janoshik]

Than you Jano for taking the time explaining all this. It's pretty involved and over my head but I will reread it for a better understanding. Again thanks, you really go out of your way for all of us.

Happy to do so!

If there's anything I haven't been clear enough, let me know. I'm not really sure of my ability to explain rather complicated concepts in English.

 

[mands]

Than you Jano for taking the time explaining all this. It's pretty involved and over my head but I will reread it for a better understanding. Again thanks, you really go out of your way for all of us.

Yeah he goes out of the way for all of us. lol So far out of the way he lies about testing samples and then reports he does(gives false test results). Gets caught and then goes so far out of the way he doesn't admit his lies until a year later to try and gain business over here at meso. Stand up dude right there.

Hey but it worked because people don't research and go into most things blindly.

But, it's okay now because he's better at controlling his anger.

mands

 

[janoshik]

Yeah he goes out of the way for all of us. lol So far out of the way he lies about testing samples and then reports he does(gives false test results). Gets caught and then goes so far out of the way he doesn't admit his lies until a year later to try and gain business over here at meso. Stand up dude right there.

Hey but it worked because people don't research and go into most things blindly.

But, it's okay now because he's better at controlling his anger.

mands

I see you are still set on beating the dead horse, while lacking the integrity to call out your friend Jim ever or admitting the AAA failure of you two.

Whatever suits you Mands, if this is what you want to do with your time, instead of bringing anything of value here, feel free to do so.

Regarding what I bring to the board and the community, I'm sure the people can make up their own mind. Whole lot of them already did, however much you try to antagonize me.

 

[master.on]

Three simple questions:

1 What testing method did you use?
Please post the method.

2 Did you devise the method yourself, or did you find it elsewhere?
Please specify where did you get the method.

3 What reference standards did you use?

 

[janoshik]

Three simple questions:

1 What testing method did you use?
Please post the method.

2 Did you devise the method yourself, or did you find it elsewhere?
Please specify where did you get the method.

3 What reference standards did you use?

1. Method as in HPLC or method as in gradient composition et cetera?
2. -
3. Certified European HGH standard. Look around, there are enough pictures of it posted.

 

[master.on]

1. Method as in HPLC or method as in gradient composition et cetera?
2. -
3. Certified European HGH standard. Look around, there are enough pictures of it posted.

1 The whole method
mobile phase composition including buffers, pH,
gradient changes if any,
flow rate,
column choice,
pump pressure limiting, if any

2 Who's the manufacturer for the standard?

Sorry if that was stated before, would you mind repeating that, please?

 

[janoshik]

1 The whole method
mobile phase composition including buffers, pH,
gradient changes if any,
flow rate,
column choice,
pump pressure limiting, if any

2 Who's the manufacturer for the standard?

Sorry if that was stated before, would you mind repeating that, please?

1. I will not be posting that. With some luck you might find I have posted that at some point, but I won't be sharing valuable know how.

2. My apologies, I missed a word there. Certified European Pharmacopoeia HGH standard.

 

[mands]

I see you are still set on beating the dead horse, while lacking the integrity to call out your friend Jim ever or admitting the AAA failure of you two.

Whatever suits you Mands, if this is what you want to do with your time, instead of bringing anything of value here, feel free to do so.

Regarding what I bring to the board and the community, I'm sure the people can make up their own mind. Whole lot of them already did, however much you try to antagonize me.

I have brought more to this community and board than you ever have or will and I've done it on my own dime and don't do it to make money.

You are here to sell your services that's it? Meanwhile tooting your own horn pompously to stroke your own ego. If that's what you call bringing to the board so be it.

Your spectrum is limited to a few things. That's not a bad thing so don't take that wrong. I've never denied your knowledge or capabilities. You can ask anyone that's ever inquired about you.

It's your integrity and deceitfulness that gets you in trouble. What you did will happen again when you get into a desperate situation. Someone like you can't change who you are. A liar is and always be in a liar. It's in your blood. Most likely occurs on a daily basis.

Don't you have testing to do. Maybe more testing and less playing on the forums contributing. lol.

And about my failures. I have far less failures in my life than you had in the past two years. Keep making money off the community. I know you see that as a contribution. If you were contributing and educating this board you would do it for cost, not a profit.

contribute
[kuhn-trib-yoot]
See more synonyms for contribute on Thesaurus.com
verb (used with object), con·trib·ut·ed, con·trib·ut·ing.

1. to give (money, time, knowledge, assistance, etc.) to a common supply, fund, etc., as for charitable purposes.

Now go play in traffic.

mands

 

[master.on]

1. I will not be posting that. With some luck you might find I have posted that at some point, but I won't be sharing valuable know how.

2. My apologies, I missed a word there. Certified European Pharmacopoeia HGH standard.

You don't want to share the method?
Or is because you don't have any method whatsoever?

An HPLC machine is much more valuable than a method
cromatographers often share their methods with colleagues for free.

 

[Carlito]

I have brought more to this community and board than you ever have or will and I've done it on my own dime and don't do it to make money.

You are here to sell your services that's it? Meanwhile tooting your own horn pompously to stroke your own ego. If that's what you call bringing to the board so be it.

Your spectrum is limited to a few things. That's not a bad thing so don't take that wrong. I've never denied your knowledge or capabilities. You can ask anyone that's ever inquired about you.

It's your integrity and deceitfulness that gets you in trouble. What you did will happen again when you get into a desperate situation. Someone like you can't change who you are. A liar is and always be in a liar. It's in your blood. Most likely occurs on a daily basis.

Don't you have testing to do. Maybe more testing and less playing on the forums contributing. lol.

And about my failures. I have far less failures in my life than you had in the past two years. Keep making money off the community. I know you see that as a contribution. If you were contributing and educating this board you would do it for cost, not a profit.

contribute
[kuhn-trib-yoot]
See more synonyms for contribute on Thesaurus.com
verb (used with object), con·trib·ut·ed, con·trib·ut·ing.

1. to give (money, time, knowledge, assistance, etc.) to a common supply, fund, etc., as for charitable purposes.

Now go play in traffic.

mands

I always knew that. He is just getting paid to do a bogus test paid by a bunch of sore VIPs who not only sell GH on their VIP rooms but also recs and meds, I already know it and these guys wouldn't even believe one of their own told me.