Chromatography Forum

Hi everyone,

I'm currently setting up an HPLC assay for paracetamol (254nm, MP -KH2PO4, 1.5% propanol, 0.1% THF, run time ~20 min., 5um C18 15CM X 4.6mm) column) and in the fullness of time I'm hoping to have it set up for metabolites as well however I'm struggling to find an appropriate internal standard. I have tried phenacetin however it is not showing a peak anywhere up to 90min. I have also tried salicylic acid which is good except that the peak is broad (3 min) and weak. (My paracetamol peak is quite broad too at ~1min). Any suggestions for an appropriate IS.

Cheers,

Merrin

A bunch of questions:

1. None of your peaks should be broad. What is the solvent, in which you make up your sample, and what is the mobile phase? If you make the sample up in a solvent that is stronger than the mobile phase, your peaks may get broad, and you may not see the more retained phenacetin.

2. What are the details of your method, and where did you get it from? Reason for the question: my bet is that 1.5% propanol and 0.1% THF are not going to elute your analytes from a decent C18 column, unless there is some voodoo happening. Such voodoo may depend on the specific C18 that was used in the original method. I find the use of these very small amounts of propanol and THF rather unusual.

Hi again,

At this point I have made up solutions only in water and in a water/ethanol mix. Once established the test will be used for plasma assays (plasma injected directly on to the column as opposed to being extracted). I wasn't seeing phenacetin peaks in either.

The method was taken from the literature (Jensen et al, Journal of Pharmaceutical and biomedical analysis 2002 34:585-593) and so far is running as expected for the paracetamol however no internal standard is used in this paper hence the reason for my query.

As mentioned the mobilt phase is 0.1M potassium dihydrogen orthophosphate, isopropanol, THF (100:1.5:0.1 pH 3.7).

Thanks for your help.

Merrin

Hi,
I agree with Uwe. This eluent seems not to be real common conditions for this kind of analysis.
More "common " HPLC conditions can be found in this reference ( Karthik A, Subramanian G, Kumar AR, Udupa N. Simultaneous estimation of paracetamol and domperidone in tablets by reverse phase HPLC method. Indian J Pharm Sci 2007;69:142-14):

Drugs are chromatographed on a reverse phase Kromasil C18 column using a mobile phase, 20 mM phosphate buffer (pH 7.0±0.1) and acetonitrile in the ratio of 60:40%v/v. Diclofenac potassium was used as an internal standard. The retention time of paracetamol, domperidone, and diclofenac potassium was 2.94, 8.30, 5.67 min, respectively.



A lot of information can be found on the world wide web !

Succes
philippe

Multiple comments...

1. The method prescribes the use of a Platinum EPS column. This is a column with a rather low retentivity, and compatibility with 100% water. Are you using this column, or at least something similar that is compatible with 100% water?

2. The retention of paracetamol on this column is already fairly high (~14 minutes). It is the last peak in the chromatogram, and the author was looking for polar metabolites. The use of a more non-polar compound such as phenacetin will not work terribly well, since phenacetin will have a much larger retention than paracetamol. You need to find a much more polar internal standard.

3. The reason for the high retention of paracetamol is that the author was looking for a method to separate more polar metabolites such as the glucuronide and sulphate.

4. All things considered (including the low retention and the broad peaks that you are reproting), I think that you are trying to do this with the wrong column. You need to do this with a column that works in a high water content. The Platinum EPS column has been used by Jensen, Atlantis T3 is another column that will work well or even better for this assay. Most C18s won't work for this method.

Hey,

Thanks for your suggestions/advice etc. I'm fairly new to small molecule HPLC so all suggestions are definitely welcome!!

I will have a look for a more polar internal standard and hopefully we can overcome that hurdle. At this point I think getting a new column is fairly unlikely but hopefully we can work with the one we have - even if it's not ideal. I don't have the metabolite samples yet but once I do I'll be able to see how they look and then maybe look at altering the mobile phase etc. I may have more questions then!

Anyway, thanks again - and if you have any more suggestions etc I'd love to hear them.

Merrin

How do you know that your broad peak is your analyte? Maybe your C18 column is dewetted and all your important things are at to. Also, is that a restricted access column? If not : It would be fun to find out what happens when you inject plasma on that column.

From your description of the retention data that you gave, you have the wrong column and will not be successfull with the column that you have.

The retention data I have so far is very similar to that reported in the paper I referenced above. I have spoken with a friend in another lab however and they have offered an EPS column should we need it for the development work. If this column gives us what we want then I might be able to persuade people to buy one!!

Thanks again for your help.

You said that your paracetamol elutes at ~ 1 min. The paracetamol peak in the paper elutes around 14 min. I think that this is different. What am I missing?

Oops that was a typo. It elutes at 10 min and the paper says it elutes at 10.5min so they're actually pretty close. Sorry about that!!

What I meant about the 1 min was that it takes around 1 min for the paracetamol to elute. i.e. it is a relatively broad peak.

OK that changes the picture... Sorry about this misunderstanding...

I still think that you will be better off with a packing that has been designed for this environment, you will get better and more reliable retention, and good peak shapes and narrow peaks. I recommend the Atlantis T3.

Adenine, epinephrine, dopamine could all be good internal standards.

Thanks Uwe Neue.

Your suggestions and exlanations have been really helpful. I am agitating for a new column as we speak!

I'll give epinephrine a shot as well!

Thanks again.

Merrin