Chromatography Forum

I'm trying to quantify lincomycin by HPLC with an isocratic method that has a mobile phase that consists of 2.25% (v/v) acetonitrile, 5% (v/v) phosphate buffer (2.72% m/v KH2PO4 adjusted to pH 5.0 with 3.48% m/v KH2PO4) and 0.067% v/v methanesulfonic acid and water to 100%.
In the chromatograma obteined the peak of lincomycin B is perfect, but the peak of lincomycin is veri strange: the front is perfect but the tail goes down in a way that the peak seems a right triangle.
Can anyone help me? What can I do to keep a well shaped peak

This sounds like a case of mass overload. What happens if you dilute the sample, say 5x? Does the retention time shift later and the symmetry improve?

I'll try to dilute my sample but, because is a method of Pharmaeuropa, I need to apply it in the way that it has been described

This is more of an experiment to determine the source of the poor peak-shape, not a recommendation to alter your SOP.

I've made the experiment diluting my sample and the result is the same: lincomycin B perfect and Lincomycin as a right triangle. The retention time has not been modified.
Is it possible that the methanesulfonic acid is not working properly? (is an old reagent in my lab)

There are not many ways to cause the peak form you describe. The only others I can think of are either a contaminated/damaged column, or the the standard is degraded.

Methanesulfonic acid is quite stable, but it does pick up water, and few suppliers make it in HPLC quality.

Hello Ana

Could you post a copy of the chromatogram or a more detailed description of the peak shape - it is not clear where the triangle makes a right angle. What is the width of the peak compared with the lincomycin B peak ?

Thanks Peter

At the beginning of the experiment I tested three column models: an Agilient Hypersil ODS 5 µm, a thermo hypersil BDS 5 µm and a tekcnokroma ODSB 3 µm. All three column show the same profile. I choosed the last one because the lincomycin B peak was narrow.
The right angle is at the beginning of the peak and it seems like a right angle because of the "tail" of the peak starts in the apex of it.
The lincomycin B peak has a width of 1.453 min and the lincomycin has 5.474 min.

Why not use the same column as recommended by EP? -supelcosil DB LC-18 and also check if you are using column oven at 45 °C.
Have you tried method given in USP?


JM

EP is European Pharmacopeia? Because I think that in this Pharmacopoeia the determination of Lincomycin is by GC.