Chromatography Forum

I am in the process of trying to develop a stability-indicating method for Cisapride. I am having a major issue of either peak fronting or splitting after approximately 50 injections depending on which column I use. Mobile phase precipitation has been ruled out. All method parameters are within the specified column ranges. Does any one have any suggestions as to why the API peak begins to split or front over a certain amount of injections. Degradation has also been ruled out.

Mobile Phase:
A: 20 mM NaH2PO4 + 1.4 mL TEA, pH 7.0 (1 liter)
B: Acetonitrile

Gradient:
0-25 minutes (20 - 75% B)
25-25.5 minutes (75 - 20% B)
25.5-35 minutes (20% B)

Column: Agilent Poroshell 120, EC-C8, 2.7 um, 150x4.6
Column Temperature: 45 C

Flowrate: 0.8 mL/min

Simultaneous stability-indicating HPLC method for the determination of
cisapride, methylparaben and propylparaben in oral suspension
Jutima Boonleang* and Chanpa Tanthana
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Hat Yai, Songkhla, 90112 Thailand.
Received 15 February 2010; Accepted 14 July 2010
Songklanakarin J. Sci. Technol.
32 (4), 379-385, Jul. - Aug. 2010
In this publication a C18 column is used with pH8, and it Looks very good. Fronting is always an indication that some dead volume at the column head is responsible for that. Please Google for this application. Good luck.

Thank you for the help, however I had originally tried to use this method but when preparing the mobile phase it would not stabilize at the pH of 8. It took my lab tech approximately 45 minutes before I told her to give up on that mobile phase.

Good news is, I was able to develop a method.

Thank you!

The main cause of fronting , splitting and also tailing of peaks is the presence of sodium phosphate salt in the mobile phase because it may precepitate inside the column so you must wash the column with distilled water after certain number of injections inorder to get rid of any precepitated salt in the column .

ph/Amr Tarek
Instrumental Unit Head at Otsuka pharmaceuticals

The main cause of fronting , splitting and also tailing of peaks is the presence of sodium phosphate salt in the mobile phase because it may precepitate inside the column so you must wash the column with distilled water after certain number of injections inorder to get rid of any precepitated salt in the column .

Such a generalization is nonsense. It's true that phosphate will precipitate if you go to high % acetonitrile, but in this case it will rather precipitate before the column, in the mixer or the capillaries. If there would be a general problem with phosphate, why would it be a standard buffer for HPLC? As long as you're not going too high with the ACN content, there won't be any problems.
And btw washing hydrophobic C18 columns with pure water may lead to phase dewetting...

Concerning the original question, I'd supect that with phosphate buffer pH 7.0 at 45°C you might be well over the pH tolerance of that column. These conditions are quite harsh for a standard silica based C18. Dissolution of the base silica can occur, leading to a void at the column head which causes fronting. I'd work at a lower pH or use some sort of high-pH-stable column.
BTW with a modern column you usually don't need any TEA in the mobile phase. That was used decades ago to supress peak tailing...