Chromatography Forum

I run L-Cys and L-Met standards, each gave two peaks in HPLC profiles. Who know why? How to calculate or recalibrate them? Using both peaks area? But my real samples showed only one peak.

Please Help Me!

The most likely explanation is air oxidation. Cysteine dimerizes to cystine. Methionine oxidizes to its sulfoxide. Both reactions are slower at low pH, and both can be catalyzed by transition metal ions.

Questions:
How do you prepare and store your standards?
Do you see this problem with freshly prepared standards made from crystalline amino acids?
How are you doing your analysis?

Thank you for your reply!

I forgot to mention that I used performic acid oxidation method according to AOAC method, and then derivatized with PITC. The L-Cys and L-Met should be oxidated to their acidic forms. Was possible partial L-Cys to transfer to D-Cys acid?

Oh, that is different!
I've never done the PITC procedure, and don't know all the tricks and traps. It doesn't distinguish D from L, so I'm guessing it has to do with not matching the reaction conditions between standards and samples.