Chromatography Forum

Hi again everyone!

The reason why I'm looking for alternative IS here in the forum is because of the probrematic peaks of Gabapentin and Phenyl Glycine IS which I was able to separate at low pH. of around 3.1 I'm getting a tail like peak after the main peak for both analytes.

M.P. 0.02 M KH2PO4/ACN, 50:50, pH 3.1
Sample Prep: GBP is spiked in Plasma and precipitated with 20% Perchloric acid. 100 uL of Borate buffer, pH 10 is added to 10 uL of the supernatant followed by addition of derivatizing agent OPA-MPA solution.
50 uL is injected to LC with Fluorescence detector.
Column: C18, 150 x 4.6 (stable at pH range 2-7.5)

The reference journals i got even set the pH at around 2 but they did not get this tail like peak after the main peak of the analytes. If I increase the pH at 6.2, the tail-like peak disappeared but I'm not getting a good separation from the artifacts of the plasma though separations of the GBP and Phe-Gly is achieved.

I'm not sure if the tail-like peak is a degradant of both analytes at low pH because some journals used to work at even lower pH range. Pls. help! How will I eliminate the tail like peaks I am encountering in my chromatogram? Can I put anything that will make the peaks fused as one peak? The final product of derivatization is an isoindole ring. thanks!

-ghie-

I suspect that your mobile phase buffer may not have enough capacity to counter the pH 10 buffer in the sample. One way to check: Inject only half as much and see if the peak shape changes. Phosphate buffer is not a good choice at pH 3; formate is much better, and is compatible with your separation and detection system.

You are also likely to get a better peak shape as you get closer to pH 2 with your phosphate buffer, since the phosphate will have a better buffering capacity.

Hi!

Thanks for all your help. I was able to solve the problematic peaks of Gabapentin and Phenyl Glycine Internal Standard. I lowered the pH to 2.1 and it seems that Phospate buffered well at this pH level. But I have another problem though. There is a small endogenous peak in the blank plasma that i cannot resolve with a change of ratio. Since I cannot play anymore with pH because if I raise the pH phenyl glycine will not be retained anymore. It is at pH 2 that the analytes are well resolved. The artifact in the blank moves with the IS Phenyl Glycine when the ratio of the mobile phase is changed. The analytes are OPA-MPA derivatized compounds. Would an addition of TFA or ion pair would resolve the problem? I'm not sure if this would work at low pH level. Any feedback? Thanks!

-ghie-