Sudden Unknown Peak During HPLC Validation – Need Advice

[henry_55]

Hello everyone,
I'm validating an HPLC method for a pharmaceutical sample and have encountered a strange situation. During one of the precision runs, a small unknown peak appeared at approximately 5.2 minutes. This unknown was not present in the blank or in a few injections I ran previously. I also make all diluents fresh and mobile phases fresh, and I flushed the column before starting the sequence.

At this point I have ruled out carryover and injection error. I am curious if this is maybe a degradation product or possibly related to the column itself. I am curious if anyone else has experienced this seemingly unknown peak during their method validation? What steps did you take to try and isolate the cause of such peaks?

It was interesting to me this topic came up as I was brushing up on some data analysis fundamentals in a generative ai training and thought to myself if there is any advantage to AI-based chromatogram pattern recognition for identifying such issues. Has anyone gone down this route yet?

I would appreciate any suggestions or literature references relating to how to proceed with consideration of an unknown peak, because I want to feel confident I have a robust method before I submit it.

I appreciate your time and support!
henry

[DR]

Try rinsing your sample vials with a little sample filtrate before filling them.
Some vial vendors do not do as good a job of cleaning them as they would have you believe.