pharmaceuticals by HPLC-MS/MS, presence of impurities

[carol]

Hi all,

I am using for the analysis in paracetamol a HPLC-MS/MS (ZQ).
I am working on a method for the analysis of paracetamol and I am also using a compounds called Acetophenetidine, I use this compounds as an internal standard (but I am not sure that I am allowed to use no isotope for an internal standard) but my problems is; I have a compounds X which I don’t know which one is . It appear at the same Rt than the internal standard but it has the same two transitions that I use for the identification of paracetamol . Because the internal and paracetamol have very similar structure. Do you think that internal standard suffer a transformation on the probe of my MS and change to paracetamol? Is an isomer of paracetamol? I know it came from the internal standard because it doesn’t appear on the blank but if I spike internal standard on the blank I have the peak. The concentration of X doesn’t change if you increase the concentration of paracetamol or concentration of the internal standard….Any idea???

Thanks in advance.
Carolina

[MG]

If you look at the product ion spectrum of your ISTD (acetophenetidine), is one of the product ions the same m/z as paracetamol? Looking at the structures, it looks like this could be possible. If so, then paracetamol could also be formed as an in-source fragment of acetophenetidine, and then you'd see signal for the paracetamol transition at the retention time of the acetophenetidine. I've seen this sort of behavior before when working with structurally similar compounds. Since your analyte and ISTD are chromatographically separated, it is nothing to worry about, as long as your chromatography is stable. The only thing that worries me is your last statement:

The concentration of X doesn’t change if you increase the concentration of paracetamol or concentration of the internal standard

I assume you are referring to the response of X. I would expect the response of X to increase with increasing acetophenetidine, if indeed X is an in-source fragment of acetophenetidine.

[Kostas Petritis]

Carol,

Two contradictory things... The compound X seem to come from your internal standard but when you change the internal standard concentration the response of the compound X does not change...

I do not have in mind the exact chemical structure of acetophenetidine but it is possible that due to in-source fragmentation of your internal standard you might have the same transitions as paracetamol. However, if this is true, for the same MS parameters you should observe the same areas ratios between your iternal standard and compound X for different concentrations of your internal standard...

[Kostas Petritis]

Well...

It seems that MG beated me by replying 3 minutes earlier... fortunately we just say the same thing in different words

[carol]

Hi

Thanks for yours replies, yes, if I increase the concentration of the internal standard X the concentration of X does increase, maybe I should put a lot more.

Them, do you think that it is possible that the internal standard suffer a transformation on between the HPLC and the MS. I just want to be sure about what I say and try to give good data.

Thanks again for your ideas,

Carol

[Kostas Petritis]

Carol,

No it is not possible as if you had a tranformation/degredation during storage or during the run, you would have either different retention time or just an increase in the baseline for that specific transition respectively. What you get is propably in source fragmentation that you can maybe minimize by changing the MS settings.

I do not see why you are saying that you should put a lot more...?

[carol]

Hi,

What I am trying to say is that if I put more internal standard the concentration of X doesn't increase clearly them if my compounds X come from the internal standard why the response doesn’t increase proportional to the concentration of the internal standard? I running an experiment to see how stable is the Rt of X. I don't know what to think or do.

[carol]

I am here aging,

I don't remember if I had mentioned that before. I run MRM them I look for two transition of paracetamol don't remember the value but if you what them I will give you them but as far as I know MRM look transition from a parent to daughter and the MW of the internal standard is different than the one of paracetamol...I can give you the structure also if you want.

Thanks again,

Carol

[MG]

I am confused. In one post above, it seems like you are saying that increasing the concentration of internal standard increases the signal of X. Then in a following post, it seems like you are saying that there is no increase in the signal of X.

I suggest the following experiment to help us understand what is going on: Do a product ion scan (not MRM) of your internal standard (acetophenetidine), but use the same collision energy that you normally use in your MRM experiment. Use a fairly high concentration of internal standard for this experiment, so that you can be sure to see all the product ions. Does one of the product ions have the same mass as your paracetamol precursor (152)? If so, it is possible that this same fragment ion is being formed in your ion source. Specifically, it can be formed in the region between your electrospray source and Q1. Some people call these "in-source CID fragments". If it bothers you, there are instrument parameters that can be adjusted (as Kostas said) to minimize in-source fragments, but you may also affect the sensitivity for your internal standard by doing so. The above experiment does not completely rule out the possibility of another cause, such as an impurity in your ISTD having the same precursor and product ions as paracetamol and same retention time as acetophenetidine. However the latter possibility seems less likely than the former.

If you developed this method in your lab, you probably already have a product ion scan for your ISTD on-file somewhere, and don't need to repeat the experiment.

[Kostas Petritis]

MG's suggestion is a good one.

Actually you can also vary the collision energy and make breakthrough curves for your molecular ion and the product ions. Normally you should observe decreasing M+H and increasing the 152, in higher collision energies the 152 should go down and your paracetamol transition higher.

Actually, as you normally have in source fragmentation you could infuse your internal standard and look in full scan for the ion 152. If it is there you can fragment it, and it should give (also) the transition you see when you fragment your paracetamol...

[carol]

Hi,

You are very good help!!
The engineer was here two days ago and I asked him that if the
in-source CID fragments (but without using this terms) can be possible and the said no. He said to me two fragmentations define a compounds and transformation doesn't happen. I am doing my PhD and I am the only one how know this instrument, at the moment in my lab, them I thought that he know a lot more than me.

Thanks gays,
I would do all those experiments to find out more and yes, I said the concentration of this compound X doesn't increase when you increase the concentration so the ISTD.

Thanks a lot you can imaging how useful it is all of that for me.
I will keep you inform.

Carol

[carol]

Sorry, I am spanish and my speling it is not very good.

I mean guys!!I am very sorry...

Carol