Can HPLC C18 GENERATE IMPURITIES IN API

[jupallisreenivas]

Dear all,

We are in the job to develop a HPLC method for one of API by using following conditions.

Column : Kromasil Eternity C18 250 X 4.6mm,5µm.
Flow rate : 1.0 mL/min
Wavelength : 215 nm
Column temperature : 50ºC
Run time : 37 min
Injection volume : 10 L
Diluent : Acetonitrile: Water (50:50) v/v
Elution : Gradient
Sample concentration : 2.0 mg/mL
Buffer preparation : Take accurately about 3.48gram of Di potassium hydrogen phosphate in1000 mL of Milli-Q-water and filter this solution, through 0.22µm Nylon membrane filter paper.
Mobile phase–A : Buffer: Methanol (95:05)v/v
Mobile phase–B : Acetonitrile:Methanol(80:20)v/v

GRADIENT PROGRAM
TIME(In min) Mobile Phase-A (%) Mobile Phase-B (%)
0.01 55 45
20 30 70
30 30 70
30.1 55 45
37 55 45


Problem-1: When we using the new HPLC column for analysis no impurities observed at non polar side around 26 minutes, after around 60 injections non polar impurities observed around 0.12% (These impurities are matches with base degradation impurities and conformed as dimmers of our API), after 100 injections these impurities raised up to 0.5%, by injecting same sample. Why this behavior was observed and how can we prevent this behavior. No problem with solution stability. This is observed only different sample preparations by different time intervals with same homogeneous sample.

Mobile phase pH is critical for this analysis always we are using basic pH buffer only, other wise good peak shapes can not be observed.

Any one please help me.

Regards
sreenivas

[HPLCaddict]

- Are you sure these degradation peaks are coming from the sample? What does the chromatogram look like if you're doing blank (no injection) runs or just inject the sample solvent? Could these be system peaks?
- Are you definitely sure about sample stability? Do these impurity peaks still occur if you inject a freshly prepared sample on the "used" column?
- Concerning your chromatographic method, are you adjusting the pH of your buffer? You wrote that the method is pH-sensitive, but no adjustment of the buffer is mentioned during preparation. A simple solution of phosphate in water is no buffer...

[jupallisreenivas]

- Are you sure these degradation peaks are coming from the sample? What does the chromatogram look like if you're doing blank (no injection) runs or just inject the sample solvent? Could these be system peaks?
- Are you definitely sure about sample stability? Do these impurity peaks still occur if you inject a freshly prepared sample on the "used" column?
- Concerning your chromatographic method, are you adjusting the pH of your buffer? You wrote that the method is pH-sensitive, but no adjustment of the buffer is mentioned during preparation. A simple solution of phosphate in water is no buffer...

Thanks for your response.

We did degradation study by using 0.05 N NaoH solution, degradation impurities are matches with non polar impurities peak. To conform these peaks injected the blank, no peak at RT of problematic peaks.

We performed solution stability around 12 hrs no peaks are increased during the period of time.

No, we did not adjusted pH of the buffer and pH sensitive means, we did not perform the analysis at neutral and acidic pHs.

regards
sreenivas

[mattmullaney]

Hi jupallisreenivas,

It may be that HPLCAddict is just getting at whether or not the buffer pH is controlled by adjusting it's value toward the second pKa value of phosphate...or if you make note of the buffer's pH value.

Yes, I have seen cases where drug substance and drug products have degraded on C18 and other stationary phases when placed in basic eluents. Silica is an excellent catalyst...this kind of thing isn't unprecedented. The temperature of your separation may aid degradation on-column as well.

A way to check this is to perform an injection, allow the eluent program to run toward mid-completion, turn off the eluent flow, wait for a time and then complete the gradient program. You may find that the degradation product(s) increase in concentration over time.

See what you think, and Best Wishes.

[HPLCaddict]

As Matt already wrote, on-olumn degradation of analytes actually is possible. But this would be a constant phenomenon, it's rather unlikely that you don't see any degradation with the first samples and only later samples decompose on the column. So on-column degradation is not likely to be the cause of your problem.

Let me summarize if I understood everything correctly:
- with a "fresh" column and a fresh sample, you don't see these impurity peaks
- after ~60 sample injections on this column, impurity peaks start to rise
- with this "used" column, during a blank run you don't see any impurity peaks
- with this "used" column, with a freshly prepared sample you don't see any impurity peaks

If these points are correct, could it be that this is simply a matter of sample stability? You said you verified sample stability over 12 hours. With the impurities starting to rise after ~60 injections, at a run time of 37 minutes this means after 37 hours (at least, depending on the speed of your autosampler it's even a bit more)!
So what you're seeing may be simply that your samples should be used for only, let's say, 24 hours or ~40 injections.

[mattmullaney]

OOPS--HPLCAddict properly read the original post where I failed to do so...it would be unlikely that on-column degradation is a true root cause in this case for exactly the reason he notes.

My apologies to all...while it may not be the stationary phase that is causing the problem, it May Be that there is something up with the autosampler syringe (and/or needle wash)...this is a more likely root cause in this case since the degradation occurred over time.

Is it possible that an injection of a newly-prepared sample could be carried out, the sample then stored overnight with the autosampler syringe in it, and a second injection of the same sample carried out the next day, to learn if this is a better assessment of the source of the trouble? Once I had a problem with sample vial septa (sample reacted with the rubber of the septum once the PTFE was pierced by the needle...only then) of a vial, too, though this was a redox kind of issue rather than strictly acid-base.

[jupallisreenivas]

Hi jupallisreenivas,

It may be that HPLCAddict is just getting at whether or not the buffer pH is controlled by adjusting it's value toward the second pKa value of phosphate...or if you make note of the buffer's pH value.

Yes, I have seen cases where drug substance and drug products have degraded on C18 and other stationary phases when placed in basic eluents. Silica is an excellent catalyst...this kind of thing isn't unprecedented. The temperature of your separation may aid degradation on-column as well.

A way to check this is to perform an injection, allow the eluent program to run toward mid-completion, turn off the eluent flow, wait for a time and then complete the gradient program. You may find that the degradation product(s) increase in concentration over time.

See what you think, and Best Wishes.

We are studied the analysis with pH adjusted buffers like 8.0 and 9.0 and also same impurities observed.

[jupallisreenivas]

As Matt already wrote, on-olumn degradation of analytes actually is possible. But this would be a constant phenomenon, it's rather unlikely that you don't see any degradation with the first samples and only later samples decompose on the column. So on-column degradation is not likely to be the cause of your problem.

Let me summarize if I understood everything correctly:
- with a "fresh" column and a fresh sample, you don't see these impurity peaks
- after ~60 sample injections on this column, impurity peaks start to rise
- with this "used" column, during a blank run you don't see any impurity peaks
- with this "used" column, with a freshly prepared sample you don't see any impurity peaks

If these points are correct, could it be that this is simply a matter of sample stability? You said you verified sample stability over 12 hours. With the impurities starting to rise after ~60 injections, at a run time of 37 minutes this means after 37 hours (at least, depending on the speed of your autosampler it's even a bit more)!
So what you're seeing may be simply that your samples should be used for only, let's say, 24 hours or ~40 injections.

First three points what you understood is right, but fourth point with this "used" column, with a freshly prepared sample impurities observed.

This is not solution stability issue.

[mattmullaney]

Wow, okay then. Something seems to happen as the column ages. Perhaps it's worthwhile to consider the matrix that the API is within as a potential source of the API degradation (you may have done this already). Is there something in the matrix that could get "caught up" in the column that could be inimical to the API, or could be so in combination with the eluent over time?

Still may be worthwhile to see if the autosampler needle could be a source of degradation as well.

Still agree with HPLCAddict...really not likely that the column itself...at least, a column with relatively new stationary phase within...can be the root cause.

[HPLCaddict]

Hm, strange thing. Two possible reasons I can think of:
Agree with Matt that some placebo compound may get stuck on the column and act as a "degradation catalyst". Did you try to clean such an "old" column with some high% organic? It might be also interesting to see the effect of reversing the column once the impurity peaks appear and then continue the analysis.
As a "quick cleaner", you can try to insert high volume injections of pure methanol (100µL or whatever your autosampler is capable of) in your sequence on a regular basis.

The other possibility, of course, is that your column gets altered by the chromatographic conditions themselves. Do you see any signs of column degradation, e.g. peak shapes getting worse or changed retention times? Your eluent's pH is quite high and upon mixing with organic, the "apparent pH" might be even considerably higher. I know that Kromasil Eternity is supposed to be used at higher pH-values, but maybe these conditions are just too aggressive?

[jupallisreenivas]

Hm, strange thing. Two possible reasons I can think of:
Agree with Matt that some placebo compound may get stuck on the column and act as a "degradation catalyst". Did you try to clean such an "old" column with some high% organic? It might be also interesting to see the effect of reversing the column once the impurity peaks appear and then continue the analysis.
As a "quick cleaner", you can try to insert high volume injections of pure methanol (100µL or whatever your autosampler is capable of) in your sequence on a regular basis.

The other possibility, of course, is that your column gets altered by the chromatographic conditions themselves. Do you see any signs of column degradation, e.g. peak shapes getting worse or changed retention times? Your eluent's pH is quite high and upon mixing with organic, the "apparent pH" might be even considerably higher. I know that Kromasil Eternity is supposed to be used at higher pH-values, but maybe these conditions are just too aggressive?

Thank you,

We find one more new thing, my final gradient ratio '' M.P:A and M.P:B in the ratio of 30:70'' in this ratio some whiteness observed, when i am mixing externally.

I am suspecting this may be reason.