Chromatography Forum

Hi, I am new on the forum and looking for same help
I am working on EP gradient method for related substances with high pH MP (8.9) and high temp (60°C).
I have CRS for peak identification to identify 13 peaks. At the beginning of work I am able to receive excellent results so I believe in what EDQM says that XTerra MS is suitable for that analysis.
My problem is number of injections I can do on the column. I tried two XTerras and one XBridge, and I was able to work only for a few days making 60-80 injections on each. After that I can see fronting of the main peak and the column collapses.
I went through a lot of talks with local Waters people (they suggested precolumn, which I was using) and other manufacturers (they suggested changes in the method I am not allowed to do...) and general conclusion was "such combination high pH and high temperature must kill every column".
Do you have any experience with such conditions? 80 inj-is that all I can receive?
Can anybody help? Thaks in advance.

XBridge C18 is likely to be more stable than XTerra MS C18. If the cause of the rapid column death is a problem with the stationary phase, XBridge C18 should last at least 10 times longer than XTerra MS C18. You also should use a precolumn of the same material, and not a random precolumn, since unstable precolumns could cause the problem observed.

Nice to hear from you Uwe! I've read somewhere that XTerra is "your baby" I even tried to reach you via local Waters representative but it failed. Anyway I am still looking for some information about working with high pH and high temperature together.
about my work:
I"ve been using C18 XTerraMS and XBridge, both with suitable precolumns. My chromatogram differs a little on XBridge, so I came back to XTerraMS on which it was perfect (according to chromatogram supplied with CRS). I've "killed" four with about 80injections so far and I do not have any idea if it is so or I am doing something wrong
Full conditions:column:
-C18 size 250x4.6mm,5 µm [EDQM suggests XTerraMS ]
-temperature: 60°C
mobile phase:
A: 1.80g/l disodium hydrogen phosphate pH=8.9
B: methanol, acetonitryle (250:750 V/V)
linear gradient program from 50%A to 25%A for 80min, than 10min eqlibration 50%A
flow rate: 1.0 ml/min, injection: 50ul
solvent mixture: 1.73g/l ammonium dihydrogen phosphate pH=10.0 Mixed 350ml of buffer to 300ml of acetonitryle and 350ml of methanol.
test solution: 200mg of the substance in the solvent mixture, diluted to 25ml.

I've read that phosphate is not right choice for HPLC at such pH, but it's EP monograph... I feel puzzled. What happens inside the column if I see fronting peaks? Any opinion?
One more: I wash columns with 10%acetonitryle.

Multiple elements in this procedure go against our standard recommendations. We have found that phosphate buffers are more aggressive than a bicarbonate buffer or ammonia alone. We commonly use ammoniumbicarbonate around pH 10.

If we know that something is problematic at room temperature, we do not recommend it for use at elevated temperature. The dissolution of a stationary phase increases roughly 3-fold for every 10 degrees Celsius.

In our experience, the XBridge packing is (under alkaline conditions) over 10x more stable than the XTerra packing, everything else being equal. You could move from some 80 injections to some 800 injections switching the packing.

Fronting peaks are a clear sign that the packing is dissolving, and a void is forming by a dissolution mechanism.

My suggestion is to depart from your execution of the EP procedure. I assume that the person who developed it was successful. This may be explainable, if the real temperature was lower than 60 degrees, due to insufficient preheating of the solvent. Consider not to preheat the solvent, but to keep the column in an oven at 60 degrees. If the procedure prescribes bad chromatography, and you are successful with the bad chromatography, you should follow the procedure...

Thanks a lot Uwe! I've just talked to my boss about that issue and with your opinion it was much easier .

But the problem with EP monographs is worth to be discussed anyway. Previous monograph (EP 5.3) for the same substance wanted keeping column at 70°C temp, and XTerra was called "suitable". Manufacturer's recommendation is 20-60°C!
10°C above upper limit is not save area, I think...

Open :
why it is possible that the analytical procedure becomes a part of EP official monograph while it is against columns' manufacturer recommendation or some scientific information (temp, phosphates for pH~9 etc.) without any explanation or a hint?
It's part of my job to give my opinion about HPLC methods from different DMFs, I check everything, ask for each indefinite issue.
What EP experts do?
Am I the only person who feels puzzled with that issue?
Does anybody have an experience with making complaint to EP authorities?

I have a lot of contacts in the pharmaceutical industry here in Bulgaria and I can say that here the situation is the same. Many of the monographs in the USPh or EP ot BP are quite strange from chromatographic point of view. For example I saw 2 months ago a monograph in which a pH 11 was ponted to analyze a compound that doesn't have any basic functionalities. I think that some regulations in the industry have to be changed and the only thing that have to be uniformed is the validation of the method, the validation parameters and the way for expressing the results. All other method development or adaptation have to be performed by R&Ds in the companies - that is part of their job...