On-Column Degradation (continue from 11-29-2004)

[Lucy]

Thanks a lot for all suggestions.

I should make the case more clearly. The active pharmaceutical ingredient API(A) and API (B) are two different compounds. API(A) is sodium salt and unstable at pH <3. API (B) is sulfate salt and unstable at pH>8.

I tried to separation API(A) and API(B) at pH=9 with a YMC Pro-Pack, C18 column, but no separation was achieved. Since I am also concern with the degradation of compound B at high pH, I chose to use acidic mobile phase to satisfy the separation first, and meanwhile to overcome the on-column degradation of compound A.

Initial condition: C18 column 150x 4.6 mm,3u,isocratic, mobile phase of acetonitrile: water: TFA (70:30:0.05), pH =1- 2, flow rate at 1ml/min, API(B) elutes faster than API(A). Under this condition, I can separate A from B and other 7 known impurities which reached my first objective. However, an on-column degradation impurity peak from API(A) was detected.

To confirm the impurity peak is an on-column degradation product, I tested API(A) alone under another chromatographic condition (pH=5-6, compound A is stable, and it is well separated from the known degradation impurity) with a shorter column, then compared the chromatogram obtained from previously described conditions. No degradation impurity peak was observed as using the less acidic mobile phase. The same sample was injected onto the pH=2 mobile phase. I found the baseline is elevated after the elution of compound A, and the degradation impurity peak (<0.4%) was eluted. It appeared that the degradation happened as soon as API(A) was exposed to acid, the API (A) and the degradation product move along the column and are separated.

As Bill suggested, the key point to control the on-column degradation is time and temperature. All tests were performed at room temperature. I do not have column chiller so lower temperature was not further pursued. I will try a short column, such as 10 cm or 7.5 cm column to reduce the run time. Any suggestions? Thanks.

[Bill Tindall]

I went from sparce information to information overload, but I have studied your new information. Let me repeat to see that I got it right........

Lets call this stuff Na+A- , where A- is anion of the compound that is unstable in acid (unstable when protonated, presumably).

So, Na+A- injected by itself at acidic pH and you see a second peak (assigned to be degradation product, which we shall call D) and an elevated baseline after A elutes-right? I expect that who ever made the stuff knows that it is unstable in acid, so the concern for the separation is well founded.

From the information provided it is not a convincing case that peak D is the degredation product of A. Degradation during elution will not produce a peak. But likely you may have more information to make the case. If not, make up A in acid mobile phase, let it sit a bit and inject to see where indeed the degradation product will elute. In fact it would be well to get some degradation kinetics by making A- up in mobile phase and see how fast it degrades (analyze at higher pH). Keep in mind that the compound only spends the void volumn time in the mobile phase and the rest of the time for the separation in the stationary phase where degredation may be more or less.

The rising baseline after A in acid eluant is more consistent with on-column degredation. To be consistent the rise must occur between where peak A and D elute, once you know for a fact where D elutes.

There is a big pH range between your failures. Could a solution to this problem be to just run at pH 3.5, or 4 or some such between 3 and 9? If not then the only recourse will be to derivitize A into something stable and analyze that. If A to D is clean reaction, make it up in acid and analyze for D.

[Uwe Neue]

Here are a few suggestions: pH 4.75 with ammonium acetate buffer, or, if you do not need to use MS detection, pH 7.0 with a phosphate buffer. Either pH will get you into the pH range where the compound is stable. Next, you may or may not have lost your separation, menaing that some of your peak might coelute. If this is the case, I would substitute acetonitrile with emthanol and see what is happening. If you end up with a situation, where some peaks are resolved in acetonitrile, and some are resolved using methanol. you can try a mixture of both. Usually this strategy gets quick results.

[Steve]

One thing to remenber going from an Acetonitrile(70/30) mixture to methanol(70/30) mixture the viscosity is changing from 0.7 cP to over 1.5 cP which will effect the pressure on the column.

[Lucy]

Thanks a lot for all comments!

To confirm the degradation peak, I dissolved the API(A) (0.1mg/ml) in acid mobile phase acetronitrile:water:TFA (70:30;0.05), tested it at different time intervals up to 60 minutes. I found compound A degrades 2% in five minutes. The retention time of the degradation product matches that of the on-column degradation peak. In addition, I have the pure “markerâ€

[Bill Tindall]

Good experiment. Now I am convinced that peak D is degradation product. The degradation that resulted in this peak has occured before compound A- started moving down the column or you would not have a peak. Do you understand that degredation from a moving parent peak can not cause a degredation peak, only a tail or front to the parent peak?

The worrisome problem is that you can estimate how much degradation will occur during the time the compound spends in the mobile phase. This time, T, is simply the time to elute an unretained peak, probably a minute or two. And you have an estimate of this degradation rate, which seems to me insignificant, given that you can up the flow rate to 2 or 3 mil/ min and maybe shorten column.

Time T subtracted from the total elution time is the time A- spends in the stationary phase and there is no easy way to measure the degradation rate in this environment. It may be more or less than the rate in mobile phase.

The degradation you need to worry about is the tail or front that will extend from the A- peak to peak D. This area results from degradation of A- to D that occurs as A- moves down the column. If you don't see any significant (say 5%) area as a front or tail then you don't have a degredation I would worry about. If you have a big tail or front, then the problem needs fixing.

So what if 5% degrades so long as the peak A- can be reliably integrated? Unless something strange happens both standards and sample will degrade the same fraction during elution and the error will calibrate out. Even if there is a 50% error in this assumption, a 50% error on a 5% degradation is not an error I would normally fret over.

Can you simply raise the pH some? If the degredation is acid catalyzed you can slow the rate to insignificance with even a .25 pH increase.

[Lucy]

Thank you every one for your suggestions. Bill, please accept my special appreciation for your comments and suggestions, I find them very helpful.

Luckily I do not have any problem with the main peak (A) although the baseline slightly rise, the peak shape is fine and the injection precision was <0.6%. However, I am still concerned with the on-column degradation peak (D) because this degradation product is a major one, which will be used as a stability indicator. I will continue to try different approaches to minimize the on-column degradation as Bill and others suggested.

1)shorten the run time using shorter column

2)Minimize the void time by fast injection so the degradation will be minimized before bedding on column.

3)Raise pH of mobile phase to pH=3 , 3.5


Any additional suggestions and comments are welcome.

[Uwe Neue]

If you only want to go to pH 3.5, the best buffer there is a formate buffer.. if you need MS compatibility, use either formic acid, or ammonium formate buffer at pH 3.75.

[Bill Tindall]

the easiest thing to do is simply increase flow rate. Increasing flow rate often has a barely noticable effect on resolution. Increase until the pressure is at your upper comfort level or some critical resolution becomes unacceptable. 2 or more mL/min is not unreasonable. The decrease in degradation (decreased time on column) will be proportional to the increase in flow rate.