Different peak shape in std curve and in urine

[xy]

*Analyte: mox-derivatised prostaglandin metabolite
*mobile phase: 20% MeOH containing NH4OAc, pH5.2
*Injection solvent: 1. mobile phase; 2. 10% MeOH containing NH4OAc, pH5.2
*Standard curve prepared in 1x PBS
*problem: double and board peak in standards only. Excellent peak shape for urine samples.
*Current thinking: (1) purchase synthetic urine to prepare std curve, (2) low flow rate and temperature to increase organic composition in mobile phase.
*Help: (1) if synthetic urine will fix it? (2) why it happened: different behavior on binding or viscosity?

*Thanks very much!

[JGK]

Can you not prepare the Std in something closer in composition to the sample injection solvent.

What pH is the PBS? could the pH and/or lack of methanol be the cause of the poor peak shape?

[xy]

Thanks for comments. All samples (std curve and urine) are undertaken SPE extraction. After dried down, all samples are re-consituted in the same injection solvent. Sorry for not being clear in previous message.

Some things in urine result in the different peak shape?

[Don_Hilton]

Beware on the synthetic urine. You will find more than one formulation out there - and it may or may not contain compunds that would affect your chromatography - assuming that you have some compound coming through to affect your chromatography.

Can you describe the SPE steps and derivitization? I am wondering if there is somethign in the original solutions of standards coming through the SPE that is not coming through with the urine. What is your source of standard materials?

[xy]

The mox derivalization is conducted by adding 1:1 of methyloxime.HCl into samples. Sample are either 1ml of urine or 1ml of standards in 1x PBS. Methyloxime.HCl is prepared in water in 1g/mL. After incubation for 15 min at RT, the mixture is loaded into pre-conditioned 3mL SPE cartridge. Analytes are eluted into ethyl acetate following water and hexane wash. Finally, dried residues are re-solved in mobile phase (20% MeOH containing NH4OAc pH5.2). I also tried use 10% ACN as injection solvent.

When I increase MeOH composition in mobile phase to 30%, I got improved peak shape from standards but separation is wrose than that from 20% MeOH mobile phase.

20% MeOH should not result in phase collapse. Interesting matrix effect. What compositions cause it?