Clomipramine HCl Method Development Issues

[jmmunson]

I am trying to develop a stability-indicating method for clomipramine.

The HPLC parameters are as follows:
Mobile Phase Preparation: (A) Dissolve 5.5 grams of Na 1-Heptanesulfonate in 50 mL of H2O, then bring to volume with GAA in a 100 mL VF. In a 1 L bottle add 40 mL of the Na 1-Heptanesulfonate solution + 4 mL TEA + 960 mL H2O, pH 3.2. (B) Use 100% ACN

Gradient: 0 - 5 min 50% B; 5.5 - 6.5 min decrease MP B to 20%; 6.5 - 8 min ramp MP B back to 50%, hold for 8 min to re-equilibrate.

Column Temp = 45 C; Injection Volume = 10 uL; Wavelength = 254 nm; Diluent = MeOH

Column: Agilent Poroshell EC-C8, 2.7 um, 150 x 4.6 mm.

The issue I have ran into is during initial method development and running my stressed samples my Retention time was 7.7 mins, when I ran my next sequence my retention time shifted to 6.8 mins (keep in mind this is the same column and mobile phases from the same bottles). I thought the column was going bad so I purchases a new one. When I used the new column with the same mobile phase as used above the retention time was 6.3 minutes, when I went back to the previous column it also shifted forward to 6.3 minutes.

I have determined to separate the placebo peaks from the API I need to use a C8 rather than a C18, and that I obtain the best peak shape with a smaller particle size column.

Any thoughts as to how I should proceed or what may have caused the huge retention time shift would be greatly appreciated.

[Gerhard Kratz]

Obviously it is an equilibration Problem. When you start with the Methode development did you use a brand new column? Keep in mind that pH of your MP A will Change at 45°C. Can you try to dilute your API in the Initial MP composition rather than in MeOH?

[HPLCaddict]

- Gradient with ion-pairing reagents is generally not the best thing to do.
- You're running a gradient from high ACN to lower ACN content in RP?? Why?
- Maybe your reequillibration time is not long enough. It's ion-pairing!

[jmmunson]

Gerhard Kratz: I did not start my method development on a brand new column. It was a column we already had in- house. When I bought a new column after I thought it was a column issue I allowed it to equilibrate for over an hour. Which I do with all brand new columns. Even after that equilibration it gave the same 6.3 RT as the older column which didn't give that until after 3 sequences (50-100 runs each). I could try to dissolve in the initial MP composition and see what happens.

HPLCCaddict: I re-equilibrate for 8 minutes before each run which I had to increase during the initial development. However if that is the case that would not explain why during my first 100 injection sequence that my retention time did not shift at all. The reason why I am going from high organic to low organic is because that is the only way I was able to initially separate the API from the placebo and degradant peaks.