Sciencemadness Discussion Board

Compounds (API's) with various hydrate forms - what testing can differentiate these

RogueRose - 9-3-2018 at 11:46

I am curious about how GCMS/HPLC or LCMS (and other testing methods I guess) would identify a compound that has various hydrate forms and anhydrous forms. For example, if a pharmaceutical ingredient in it's anhydrous form had a MW of 246g/mol but it has 2 hydrates, a tri hydrate and a decahydrate, with corresponding MW's of 300g/mol and 426g/mol

So the decahydrate weighs 73% more than anhydrous and the trihydrate ~22% more than anhydrous - or conversely, the anhydrous is about 57% of the decahydrate & anhydrous is 82% of trihydrate weight.

Now I've seen compounds similar to this available in the different hydrates from places like Sigma so it seems that they are used as API's (active pharma ingredient) in various hydrates.

Now if the medication is tested for quant/qual analysis, would it ID the compound as the hydrate or would it see it as the anhydrous in all tests - or would some ID the difference and others miss this?

If a medication were made with the API compound, magnesium stearate, cellulose & color - all ingredients mixed dry then pressed into a pill then, I would assume, the compound would retain it's hydrate form in the medication.

Now it's obvious that if you took equal amounts of the anhydrous vs the decahydrate, you are getting great differences in the active compound, as the water on the molecule doesn't effect it's action within the body.

I'm trying to figure out if 3 medication were made, each in the different hydrate states, but same total weight (so say 200mg of the anhydrous, tri & deca hydrates, each in their own pill), would the testing be able to identify the differences in the total API based on it's anhydrous MW?

The reason I ask is because if you read the FDA guidelines for drugs and generics, it is quite frightening in some cases where there is a +/- 20% of the standard name brand medication and then it gets even worse when it states that "equivalent" compounds can be substituted in some generic medications - and IDK exactly what that means and I'm looking through my old PDF's where I downloaded the FDA docs.

So I hope you see what I am asking with regards to whether a (generic) company could get away with using the decahydrate in place of the anhydrous (name brand) - in equal total weights of the compound (so much less ingredient in the generic with the decahydrate).

I did contact the FDA to get clarification on this and it was impossible to get directed to anyone that may have been able to answer questions regarding this let alone explain what was on their regulations PDF.

This is obviously within the US, but I'm interested in hearing from those in other countries and if they have come across anything such as this or may have insight into this issue in any manner. Thank you for any assistance!

Melgar - 9-3-2018 at 19:32

I took a class on FDA regulations as part of my biomedical engineering degree. Generics would have to take that information into account when developing a functional equivalent, for sure. Hydrate information would be treated the same as salt information; it's an essential part of the drug description. You couldn't fake the test by using, say, the "deoxyribonucleate" salt of a drug, as opposed to its sulfate form or whatever.

[Edited on 3/10/18 by Melgar]

Texium - 9-3-2018 at 19:50

As far as the different analytical techniques go, GC would probably show different peaks for hydrate/non-hydrate since the polarity of each would be different. HPLC would depend on the solvent you're using- if your solvent mixture contains water, it will become the hydrate regardless. MS blows everything apart into ionized chunks, so it should have some pretty clear water peaks in there if the hydrate is present, and for a pure sample, the amplitude of that peak should be proportionate to the number of water molecules in the hydrate.

RogueRose - 9-3-2018 at 22:12

Quote: Originally posted by Melgar  
I took a class on FDA regulations as part of my biomedical engineering degree. Generics would have to take that information into account when developing a functional equivalent, for sure. Hydrate information would be treated the same as salt information; it's an essential part of the drug description. You couldn't fake the test by using, say, the "deoxyribonucleate" salt of a drug, as opposed to its sulfate form or whatever.

[Edited on 3/10/18 by Melgar]


Thanks for the response. I'm not sure I understand what you meant when you said "Hydrate information would be treated the same as salt information; it's an essential part of the drug description. "

Now I know that if I were making the drug and had the three salt forms (from my example), I would just use molar equivalents, which is how all calculations are done on this forum and I'm guessing everywhere else. But the thing is, when taking profit into consideration, corners are often cut and of a test can show 200mg of "salt" anhydrous, could the test also ID if it was actually "salt" decahydrate (when for it to be legitimate it would have to be a higher amount = to the "salt" anhydrous amount)

There are some medications that have numerous different salts available for their composition. Off the top of my head I can think of sulfate, HCl, HBr, bitatrate and I know there was one more. In this example I think there was something like a 73% difference in the weight of the HCl and the bitartrate formulations, and then there were the various hydrates with the sulfate version.

The reason I'm asking is because there have been a number of people complaining about what seems to be a major decline in potency/length of effect/etc in some medications on another forum I go to for a health condition. The reports seemed to start happening at once with members of 10-15+ years who have been on the same brand/dose for the entire time. It seems odd that you would have 30%+ of the people experiencing the same thing at all within a month or two. There were supposedly no reformulations, no changes, nothing, so we are a little concerned about what might be the reason.

RogueRose - 9-3-2018 at 22:14

Quote: Originally posted by Texium (zts16)  
As far as the different analytical techniques go, GC would probably show different peaks for hydrate/non-hydrate since the polarity of each would be different. HPLC would depend on the solvent you're using- if your solvent mixture contains water, it will become the hydrate regardless. MS blows everything apart into ionized chunks, so it should have some pretty clear water peaks in there if the hydrate is present, and for a pure sample, the amplitude of that peak should be proportionate to the number of water molecules in the hydrate.


THANK YOU! That clears up how that process would work. Having never used any of these machines, it all kind of nebulous while trying to picture it in my mind. Thanks for the explanation.

Tsjerk - 10-3-2018 at 04:59

As far as I know (although out-dated knowledge; summer 2013) quantitative MS is very hard, and not really workable to do. Only if there is no other choice.

Molecules influence each others ionization in a unpredictable way.

I remember getting it done to determine the percentage of a synthetic peptide in a sample consisting completely of any peptides. Here we used the total protein as an internal standard, assuming the other peptides would get ionized to the same extend as the target peptide. It is however known peptides don't ionize in the same way, and they influence the way other peptides get ionized. The combination of these two factors makes it very hard to develop a algorithm to calculate quantitative numbers out of peaks.
You need known purity samples diluted with known purity impurity controls, which we didn't have as the peptide was firstly synthesized and the impurities where unknown and probably impossible to reproduce in a know purity fashion.

With water it is a little more straight forward as at least the impurity (water) part is relatively easy to reproduce, but you still need known standards, and with the peak area/amount of water being far from linear it is hard to confidently extrapolate found peak areas to known peak areas; this means you have to reproduce the hydrate you measured while not knowing if it is a mixture (you will likely find these) of e.g. mono- di- and/or three hydrates.
The three different molecules most likely will ionize in a different way, thereby effecting the water ionization as well, and as it is unknown what the 1/2/3 hydrate ratio is, you need to reproduce the result with known samples, which you will need to confirm in another fashion.... which makes the MS obsolete as you can just as well use the other fashion to determine the mixture** that is going to give you headaches if done with a MS.

with LC or GC you can easily determine the amount of water in a sample, which is linearly proportional to the peak area. Given that they do not differentiate between e.g. mono, di and/or three mixtures, they just give an absolute value for the amount of water. This would I believe be the only pro for using a MS; the fact it can differentiate between different mixtures of hydrates (but being a pain to calibrate).

Edit: MS can even discriminate between different kinds of hydrates of the same molecule, e.g. large molecules with multiple water binding pockets. Here the extrapolation game is big, translating into big errors, but cool nonetheless.

Quote:
For those who are more accustomed to perfectly linear calibration plots obtained by ultraviolet absorption spectroscopy, the fluctuating data points in MS-derived plots (figure 1b) may look somewhat awkward. After more than three decades of intensive research, we still cannot cope with that signal variability, at least in many of the ‘real-word’ applications involving complex samples. The difficulties associated with quantitative MS analysis do not deter the ever growing family of users who find ingenious ways to solve the notorious technical problems. The benefits of MS as a universal chemistry tool—including its superior chemical selectivity—are just too huge to give up using this technique.


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031646/

Edit: ** : the details of the mixture of hydrates, although scientifically interesting, is unimportant when determining the amount of water when making a medicine, as it will dissolve in the body anyway, removing the hydrate water.

[Edited on 10-3-2018 by Tsjerk]

LearnedAmateur - 10-3-2018 at 09:34

Quote: Originally posted by Tsjerk  

Edit: ** : the details of the mixture of hydrates, although scientifically interesting, is unimportant when determining the amount of water when making a medicine, as it will dissolve in the body anyway, removing the hydrate water.


This is probably an irrelevant point on my part, but instead of removing hydrates, wouldn’t dissolving something put it in its maximum hydrated state instead? Going back to basics, taking copper sulphate for instance, it is blue when fully hydrated because it forms the ion [Cu(H2O)5]2+. When you dissolve it in water, that species is still present, and if you dissolve white anhydrous CuSO4 then it will obviously take up water and turn the solution blue. If the water of crystallisation is removed in solution, then wouldn’t a species such as this form a clear solution? I’d assume that any other hydrate would behave the same, regardless of whether it changes colour or not, since it is surrounded by water molecules which are attracted via coordinate bonding through the lone pairs.

Tsjerk - 10-3-2018 at 12:04

Yes, you could see it as maximally hydrated on solution. I always think of hydrates as being water which is part of the crystalline structure, which is lost on dissolving.

AvBaeyer - 10-3-2018 at 19:50

In the "old days" hydrate numbers were obtained through regular elemental analysis IF you knew your compound was otherwise pure. Pretty straightforward. You could run a second analysis after subjecting the compound to a drying protocol looking for a change in the numbers.

But that was then...

AvB

LearnedAmateur - 11-3-2018 at 01:04

I guess it just depends on how you look at it and the context really, whether you’re just studying the relevant species present or what it is actually doing when you subject it to these conditions, it’s basically a matter of being pedantic (like me) or straightforward.

Yeah if you have a pure sample and don’t have access to fancy equipment then you can always weigh it, perform a titration if possible, figure out the mass of the dry product through that and then the remainder of the mass will be water which can then easily be converted to the hydrate ratio.

unionised - 11-3-2018 at 01:46

Quote: Originally posted by RogueRose  
Quote: Originally posted by Texium (zts16)  
As far as the different analytical techniques go, GC would probably show different peaks for hydrate/non-hydrate since the polarity of each would be different. HPLC would depend on the solvent you're using- if your solvent mixture contains water, it will become the hydrate regardless. MS blows everything apart into ionized chunks, so it should have some pretty clear water peaks in there if the hydrate is present, and for a pure sample, the amplitude of that peak should be proportionate to the number of water molecules in the hydrate.


THANK YOU! That clears up how that process would work. Having never used any of these machines, it all kind of nebulous while trying to picture it in my mind. Thanks for the explanation.

Unfortunately, it is substantially wrong.

The water of hydration would be lost (along with any solvent) in the injector of a GC, so the only peak would be from the anhydrous form, no matter what hydrate you started with.
Of curse, the size of the peak, for a given weight of material would depend on the degree of hydration.

Similarly, it's kind of hard to define the degree of hydration in solution- it's almost bound to be different from that in a crystalline hydrate so you would get a single peak by HPLC if there was much water in the mobile phase, but it's not clear that it would be present as the hydrate.

Again the size of the peak would tell you what hydrate you had.


unionised - 11-3-2018 at 01:49

Quote: Originally posted by Tsjerk  
As far as I know (although out-dated knowledge; summer 2013) quantitative MS is very hard, and not really workable to do. Only if there is no other choice.



[Edited on 10-3-2018 by Tsjerk]

The lab I worked in has been doing quantitative GC MS routinely since the 1980s.
It's nothing special.

Sigmatropic - 11-3-2018 at 04:42

Water content is checked with Karl Fischer titration. https://en.wikipedia.org/wiki/Karl_Fischer_titration

Tsjerk - 11-3-2018 at 05:43

Quote: Originally posted by unionised  
Quote: Originally posted by Tsjerk  
As far as I know (although out-dated knowledge; summer 2013) quantitative MS is very hard, and not really workable to do. Only if there is no other choice.



[Edited on 10-3-2018 by Tsjerk]

The lab I worked in has been doing quantitative GC MS routinely since the 1980s.
It's nothing special.


Did you guys have known standards? In that case, yes; nothing special. If not, I would love to known how you guys did it.

Edit: Ah, I think I get it now, It is GC MS. I would qualify that as GC, with the MS part only used to confirm what the peaks coming of the GC are. The MS part is still not quantitative.

[Edited on 11-3-2018 by Tsjerk]

[Edited on 11-3-2018 by Tsjerk]