Changing retention times

[Merrin]

Hey everyone,

I have been working on an assay for paracetamol and it's metabolites and (thanks to some help here!) I have managed to get it working pretty well but I have noticed that when I run my standard curve the retention time gets progressively longer (by <1-3 minutes depending on the position of the peak - the last peaks to elute change the most).

The HPLC conditions are as follows:
Flow rate 0.8mL min. using 4.6 x 250mm 5um c19 column, mobile phase KH2PO4:Isopropanol:THF 97.4:1.5:0.1 (I know it's unusual but it works well for the polar metabolites).

If anyone can suggest a way to get around this problem that would be great.

Thanks.

Merrin

[Dan]

This may be related to the solubility of paracetamol (or acetaminophen as we call it in the USA).

About 10 years back, I assisted a colleague in the development of an assay method for a cold/flu product that contained acetaminophen (ACE), pseudoephedrine HCl (PSE) and chlorpheniramine maleate (CPM). The amount of the APIs were: 250:15:1 for ACE:PSE:CPM. There was a single sample prep and all APIs were quantitated from the same solution/injection.

What we noted that, over time, there were changes in the chromatogram. If I remember correctly, a retention time drift was a problem. The problem may have occured for each injection but it wasn't apparent until after quite a few injections had been made.

We had a phosphate buffer:acetonitrile mobile phase (also used as the sample solvent). The ACE was soluble at the concentration in the sample prep, but we thought that solubility of the ACE may be an issue that caused the problem (the ACE concentration in the sample prep was about 1/3 of the maximum solubility limit). Over time, there could be small amounts of ACE precipitating (or somehow being retained) on column. This ACE build up would cause changes to the retention characteristics of the method.

Our solution was to add a wash solution at the end of the run (a run could be anywhere from 20 - 60 injections). The wash had a higher acetonitrile content than that of the mobile phase. This solved the problem. We had good reproducibility and the column lasted for many injections when the wash was used.

Time and resource limits did not allow for us to further investigate the issue. However, the problem was solved and management had us move on as we had a solution and, thus, a working method.

Sorry, for the long discussion but I wanted to cover the important details.

Perhaps you are facing the same issue and you will need a wash solution. Your mobile phase is high in water content and low in organic, so maybe it is the same issue. (I am assuming that your method has a high concentration of ACE relaitve to the impurities so you are facing the same solubility issue.)

I don't know if you need a wash after each injection or just after a given number of injections. The wash can be easily programmed on a modern HPLC system.

Regards,
Dan

[Bryan Evans]

Some suggestions:

- Equilibration - the column will equilibrate with more injections

- Guard column - similar to what Dan was saying. If a contaminant is leaching on to the packing material,
guard column will help. Especially for highly aqueous isocratic runs

- Double check to make sure you're in the right buffering range of
the phoshpate buffer (c.a. 2.5)

- Increase ionic strength (e.g. 25-50mM buffer)