Co-eluting peak when performing cleaning validation study.

[poli275]

Hi, I'm performing a cleaning validation study and is experiencing peak-splitting issue.

We have an analyte eluting at 3.3 minutes. The standard and spiked standard solution gave one, sharp peak at 3.3 minutes. But, when we spiked materials with a spiked standard solution, we saw a peak eluted earlier than our main peak and lies close to it, please see the attached images.

I've checked the UV spectrum of both peaks and they are highly similar. Any idea what can be the root cause of it?

I've tried:
1. Swab blank
2. Placebos blank
3. Matrix Blank
4. Standard only solution
5. Swab and standard only solution
6. Swab and spiked standard only solution without swabbing.
No co-elution nor presence of a peak at that time.

These are performed on Agilent 1290 binary pump HPLC
Mobile Phase: A- 0.05% TFA in purified water
B- 0.05% TFA in Acetonitrile
Column: Zorbax Eclipse Plus C18 RRHD 1.8um, 2.1mmX50mm
Gradient mode of 0.6ml/min, 6.4 mins run time, 1.5 min post time

[HPLC chemist]

The answer is 'It depends'! Most APIs are esters (like an Acetate,
RCO-OAc) for stability purposes. If the cleaning solution is acidic,
de-esterification may occur, so you may be detecting the organic acid (RCOOH).

[HPLC chemist]

The question is whether this peak is relevant and is larger than your limit threshold (typically 0.05% of the API peak as per ICH Q3).

[HPLC chemist]

If I am right, try increasing and decreasing the amount of TFA to 0.10% and 0.01%. These amounts should adjust the pH of your mobile phases and thus change the de-esterification process.

[poli275]

If I am right, try increasing and decreasing the amount of TFA to 0.10% and 0.01%. These amounts should adjust the pH of your mobile phases and thus change the de-esterification process.

Do you mean to increase TFA concentration of Mobile Phase A to 0.1% while decreasing that of Mobile Phase B to 0.01%?

[HPLC chemist]

Yes and no! Try %TFA;

Mobile A; 0.10% AND 0.010%

Mobile B; 0.10% AND 0.010%

Thus, you will have 4 combinations of Mobile Phases and a gradient of TFA and pH during your runs.

Keep track of the Resolution between these peaks. Baseline resolution is 1.5 (if I recall).