THE new "Generic" HGH Assay PAGE! AAA testing

Doug_S

Member
Split was done on PM.com 1st round of HGH testing - immunoassays were carried out by 3rd party lab and came in very similar.

View attachment 71777
I see no bias high or low. Back in the day for some compounds we had to immunoassay everything. I. Now routinely detecting organics in ng/L, literally parts per trillion.

Amazing how the technology has adapted.
 

janoshik

Member
I would love to see your duplicate and split sample QC. Essentially to look at precision and accuracy

I think a peek into 25ul injections of standard might be nice.

Shows duplicates and interday


Is this what you were interested in, or is there some more data I can provide?

Looking forward to your input
 

janoshik

Member
I see no bias high or low. Back in the day for some compounds we had to immunoassay everything. I. Now routinely detecting organics in ng/L, literally parts per trillion.

Amazing how the technology has adapted.
The final results were provided to general public and the request was to make them as easy to read, as possible.

Immunoassays are awesome, currently working on huge project regarding them to help general public.
 

Doug_S

Member
I think a peek into 25ul injections of standard might be nice.

Shows duplicates and interday


Is this what you were interested in, or is there some more data I can provide?

Looking forward to your input

That's nice. What kind of columns do you use for these runs?
 

janoshik

Member
That's nice. What kind of columns do you use for these runs?
For RP-HPLC method I got a set of 300A, 250*4.6mm, 5um C8 YMC columns. (Or maybe it was C4? Not sure, I got the one specified in EU Pharmacopoeia).

They have proven to be very reliable and they don't lose resolution even after prolonged exposition to very high % TFA I've used for some experiments.

I'm eyeing equivalent, but 3um, columns from ACE - their ultracore have impressed me compared to kinetex, so I think I might as well try out their 300A line.

I've also done SEC and for that Yarra line from Phenomenex had been the winner by far.
 

Doug_S

Member
For RP-HPLC method I got a set of 300A, 250*4.6mm, 5um C8 YMC columns. (Or maybe it was C4? Not sure, I got the one specified in EU Pharmacopoeia).

They have proven to be very reliable and they don't lose resolution even after prolonged exposition to very high % TFA I've used for some experiments.

I'm eyeing equivalent, but 3um, columns from ACE - their ultracore have impressed me compared to kinetex, so I think I might as well try out their 300A line.

I've also done SEC and for that Yarra line from Phenomenex had been the winner by far.
I remember the old days when you had to pack your own.:D
 

Naugahyde

Member
For RP-HPLC method I got a set of 300A, 250*4.6mm, 5um C8 YMC columns. (Or maybe it was C4? Not sure, I got the one specified in EU Pharmacopoeia).

They have proven to be very reliable and they don't lose resolution even after prolonged exposition to very high % TFA I've used for some experiments.

I'm eyeing equivalent, but 3um, columns from ACE - their ultracore have impressed me compared to kinetex, so I think I might as well try out their 300A line.

I've also done SEC and for that Yarra line from Phenomenex had been the winner by far.
Can I Google Translate this?
 

janoshik

Member
I remember the old days when you had to pack your own.:D
There's actually company walking distance from me (if you're up for long walks) where they do custom packing on demand without any additional charge.

I don't think there's any city in the world where there are more companies related to liquid chromatography than Prague, so it has its perks.
 

janoshik

Member
Can I Google Translate this?
For analysing (or rather separating) you need a chromatographic column.

This video is definitely worth your 2 minutes if you are interested in this:
So, for separating different stuff you need different columns - so for example if you separate on basis of molecular mass (or rather hydrodynamic diameter) you can easily separate HGH from HGH dimer (which is basically an impurity consisting of two HGH molecules aggregated together).

However, then there is no way for you to determine if for example only a single functional group switched place, because then the mass remains the same. So you have to separate it based on something else - with RP-HPLC it's polarity.

The column is characterized by these:
300A - pore size, if it's too small large molecules like HGH won't get into the pores and won't interact with the column well, if it's too large, you fit 'less pores' into the column, so resolution suffers

250*4.6mm - length and diameter - the longer the better resolution, but the more backpressure rises, after some point, resolution gains are diminishing, 4.6mm is common analytical diameter


5um - particle size

C8 - polarity of the column C18 retains stuff more strongly under RP-HPLC conditions (so you have to wait a looooong time for something to come out compared to C8) C4 retains stuff less strongly, so stuff comes out way sooner under same conditions



Edit: And it's not like wife lets me enjoy the perks of the nightlife here, so that goes around me, haha
 

Naugahyde

Member
For analysing (or rather separating) you need a chromatographic column.

This video is definitely worth your 2 minutes if you are interested in this:
So, for separating different stuff you need different columns - so for example if you separate on basis of molecular mass (or rather hydrodynamic diameter) you can easily separate HGH from HGH dimer (which is basically an impurity consisting of two HGH molecules aggregated together).

However, then there is no way for you to determine if for example only a single functional group switched place, because then the mass remains the same. So you have to separate it based on something else - with RP-HPLC it's polarity.

The column is characterized by these:
300A - pore size, if it's too small large molecules like HGH won't get into the pores and won't interact with the column well, if it's too large, you fit 'less pores' into the column, so resolution suffers

250*4.6mm - length and diameter - the longer the better resolution, but the more backpressure rises, after some point, resolution gains are diminishing, 4.6mm is common analytical diameter


5um - particle size

C8 - polarity of the column C18 retains stuff more strongly under RP-HPLC conditions (so you have to wait a looooong time for something to come out compared to C8) C4 retains stuff less strongly, so stuff comes out way sooner under same conditions



Edit: And it's not like wife lets me enjoy the perks of the nightlife here, so that goes around me, haha
I absolutely appreciate you taking the time to attempt to teach me about this. Thanks mate.
As for Prague, well it is a beautiful city in many other ways too...not just for the women!
 
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Doug_S

Member
There's actually company walking distance from me (if you're up for long walks) where they do custom packing on demand without any additional charge.

I don't think there's any city in the world where there are more companies related to liquid chromatography than Prague, so it has its perks.


Blow a column, pack a column....

Kids today have no idea how easy they have. We used to use glass knives on our transmission electron microscope too. Now they have diamond ones...
 

Marcus

Member
Blow a column, pack a column....

Kids today have no idea how easy they have. We used to use glass knives on our transmission electron microscope too. Now they have diamond ones...
99% of people, kids and adults, have literally no fucking idea what you are talking about
 

janoshik

Member
Blow a column, pack a column....

Kids today have no idea how easy they have. We used to use glass knives on our transmission electron microscope too. Now they have diamond ones...
We've always had this sweet lady do the cuts. She knows her magic, because anytime somebody else tries to make a nice cut it gets fubar. Don't think she changed equip in the last 30 years.

Once she retires there will be a huge problem, as nobody else in the dept. has any idea how to cut those ones properly.
 

Doug_S

Member
Lol I sure as hell dont
In the old days, there were no pre-packed columns. You essentially built your own. I did a lot of environmental sampling, especially fuels and volatile organics and you never knew how "hot" the sample was when it came in. If it was too concetrated, it would ruin the column, just overwhelm it or "blow" it, as the industry slang.

You would then have to start over and pack new columns, cussing like a sailor. Dilute your sample to a safe range, then re-run.

It set you back hours and was tedious and frustrating.
 

Doug_S

Member
Yeah, I wonder what kind of objections can be raised against that data :)
I am impressed with the fact that you used real pharmacological standards, employed real data quality objectives (including split samples and dupes), and performed validation.
 
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janoshik

Member
I am impressed with the fact that you used real pharmacological standards, employed real data quality objectives (including split samples and dupes), and performed validation.
Thank you.

Do you have any suggestions what could be improved in the future?

I can't really think of anything else, my plans consist mostly of increasing resolution for RP-HPLC method, be it by further method improvement or by investing into additional columns. However, even as it is it's exceeding EU pharmacopoeia requirements for resolution by far as far as I'm aware.
 
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