Lincomycin by HPLC

[angupa]

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

[Mark Tracy]

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?

[angupa]

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

[Mark Tracy]

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

[angupa]

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)

[Mark Tracy]

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.

[Peter Apps]

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

[angupa]

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.

[JM]

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

[angupa]

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