Selectivity problem in validation

[Karvezide]

I am validating a method to determine the concentration of iothalamate acid (a contrast agent) in serum. To assess the selectivity of the method, I added the iothalamate reference substance in patient serum, delipidated the sample using ACN (2 volume of the sample), and then dried the sample. Following is my HPLC-analysis parameters:
RP-Column: Vydac C18 218 TP (4.6 * 250 mm)
Oven temperature: 37°C
Eluents: 80 mM KH2PO4 : Methanol (90:10)
Flow rate: 1ml/min
UV: 240 nm
Sample buffer: 50 mM KH2PO4
Compared to the blank patient sera sample, there is a minor peak in the retention time of iothalamate (about 4.4 min). I have tried different buffer concentration (20, 50, 80 and 100 mM KH2PO4). Although higher concentration of buffer could shift the iothalamate peak a little bit from the minor peak of the blank sample, it still bothered my determination. Furthermore, there is almost no difference of retention time between 80 mM and 100 mM.
I have also tried NaH2PO4 (50 mM), but it resulted in almost the same chromatograms as those of KH2PO4.
Do you have any idea or suggestion, how can I solve this problem? I have thought about replacement of some portion of methanol by ACN. Could it be helpful?



Thanks a lot

[Uwe Neue]

Usually the best approach to change the selectivity of the separation is to change the organic solvent or the pH. The ionic concentration does very little for improving the selectivity.

[grzesiek]

First things first

Where does this method come from? Who was responsible for this method development? Do you know how the method was developed?

If you want to change the selectivity you should try other parameters such as organic concentration/type, pH of mobile phase, column type but first tell us about method development

[Karvezide]

Firstly, thanks for the suggestion.
I am quite new in HPLC field.
The method comes from a publication. I should establish the method for my lab. The method in this publication did not mention that there was any interference peak in blank serum in the time window where iothalamate was eluted (Acturally I have this problem only in certain patient group). The authors also used other RP-column. And perhaps the patient samples that I tested contained medicine trace that could be eluted in this time window.

[danko]

Karvezide,

The authors also used other RP-column

Yes, but which brand and type? If it isn’t stated, then you should try at least a couple of different brands and see what difference that makes.
Even more important: Since your analyte is ionizable (an acid) you should pay extra attention to the buffer and especially its pH. 4.4 min retention time on a 250 mm column isn’t much. It may indicate an ionized analyte. So, if you don’t know and can’t find the analyte’s pKa, maybe you should just adjust the buffer pH to 2.2 – 2.3 and see whether or not it prolongs the retention time and hopefully improves the selectivity.
Then try some different column temperatures (e.g. 32, 35, 37 and 40° C).
Finally, a well designed gradient can improve the separation considerably, so one shouldn’t forget this option either.

Best Regards

[Uwe Neue]

Dancho is right: the retention is way too low. Add some more water to the mobile phase! You want a retention of at least around 10 minutes with the flow rate and the column that you have.

[Vlad Orlovsky]

You can increase retention if you reduce pH to about 2. I think that pKa of your acid is way below 3. Also reducing MeOH from 10% to 5% will help.

[Karvezide]

That's great.
Now I approximately know in which direction I should go.
Another question was raised:
Can I use orthophosphoric acid to adjust the pH? I have just bought it ...
Thanks over again

[TimB]

That's great.
Now I approximately know in which direction I should go.
Another question was raised:
Can I use orthophosphoric acid to adjust the pH? I have just bought it ...
Thanks over again

o-phosphoric acid should be fine to adjust the pH. Thats what I used to do for phosphate buffers.

[HW Mueller]

There are, in my opinion, better ways to get the desired pH, check the many discussions in this forum.

[grzesiek]

"Can I use orthophosphoric acid to adjust the pH? I have just bought it ... " - sure you can

I have searched some articles and found that chromatographic parameters are quite similar in all methods, you should search some more aricles and do some preliminary studies of your method before attempting validation

In your field I think that analysis time is very important because you get to do a lot of samples, 10 minutes retention that was mentioned could be too much for you, check the literature first

[Uwe Neue]

grzesiek: I suggested 10 minute retention to get him to a decent retention factor. The reason that it will be 10 minutes is the long (25 cm) column that he is using. If he wants to get to a faster analysis, he must throw away the column that he has and get a shorter one wiht a smaller particle size. To just go to a lower retention factor is not going to solve the basic problem.

[Karvezide]

In your field I think that analysis time is very important because you get to do a lot of samples, 10 minutes retention that was mentioned could be too much for you, check the literature first

Most of the publications about iothalamate determination used perchloric acid for deproteination of serum and didn't use any internal standards. Becouse I have observed that strong acid destructed the analyte, I used this present method (ACN for deproteination and addition of internal standard).
Unfortunately, the internal standard would be eluted at about 13 min under the conditions that I have mensioned

[grzesiek]

"grzesiek: I suggested 10 minute retention to get him to a decent retention factor. " - well I know, but bear in mind his situation, this time is probably unacceptable

"If he wants to get to a faster analysis, he must throw away the column that he has and get a shorter one wiht a smaller particle size" - that's why I suggested literature search, seen using shorter columns there

"To just go to a lower retention factor is not going to solve the basic problem." - I never mentioned retention factor

"Unfortunately, the internal standard would be eluted at about 13 min under the conditions that I have mensioned Mad" - gradient elution, shorter column, pH optimisation, please get to know your method first, find the variables that are important and how they influence the separation

[HW Mueller]

For those who have not seen it:

viewtopic.php?t=10339&highlight=buffer+preparation


Also, shouldn´t the time factor be completely subordinate to good chromatography, especially if you have to work with body fluids which show intermittant interference?