Double peaks seen for one component

[eliza2005]

Hi,

I hope you can help me with my assay query. I am trying to set up an assay using LCMS-ESI detection. The active component is Ephedrine and I am using Phenylpropanolamine as my internal std. I am just puzzled why I obtained two peaks for each component when I run it in LCMS. I only expected one peak for each component. Could you please explain why? I am a new LCMS user.

Thanks

[aldij]

maybe your injectionvolume is to high? try injecting smaller volume.

[MG]

...or your injection solvent is too strong for your mobile phase. What is your injection solvent and injection volume when this happens? Also what are your LC conditions (column dimensions, mobile phase composition, flow rate, etc.)

[eliza2005]

My injection solvent is 0.1% Formic acid. I am using a Hypersil phenyl column, 2.1x100mm, mobile phase is 18%Acetonitrile in water containing 20mM ammonium acetate. The flow rate is 0.3ml/min. Ephedrine produced two peaks and so did my internal standard. I also tried using 18% ACN as solvent for standard and internal std, but I still got the same results. Please advice.

Thanks

[JNF]

Why are you dissolving the sample in formic acid whenthe chromatography is done at another pH? I would dissolve the sample in the same sovent used as mobile phase.

[MG]

Eliza, I didn't see your injection volume listed. Another way to see if this is an injection solvent mismatch (due to the pH difference) is to try injecting a small volume. For that column, 1 or 2 microliters should minimize or eliminate injection solvent issues.

[HW Mueller]

eliza, what pH is your ammonium acetate? A bad buffer + high concentration of an acid injection can give severe mismatch even at very low injection volumes.

[eliza2005]

Hi,

My injection volume is 10ul and my mobile phase pH is about 4.8.

Thanks

[Uwe Neue]

The other possibility is that the column is dead. If both of your double peaks look similar, than this could be the problem.

[eliza2005]

I tried to follow the suggestion of choosing the sample extract solvent to be same pH and same buffer concentration as the mobile phase. There was an improvement. However, I also thought that perhaps my drugs are not very stable in solution itself. So, I added a small amount of reducing substance into the sample/standard mixture prior to SPE extraction. It worked!! I only have 1 peak for each component now.

Thank you all for your helpful suggestions