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Different peak shape in std curve and in urine
Posted: Mon Mar 22, 2010 6:36 pm
by 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!
Posted: Mon Mar 22, 2010 9:02 pm
by 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?
Posted: Mon Mar 22, 2010 9:25 pm
by 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?
Posted: Mon Mar 22, 2010 11:15 pm
by 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?
Posted: Wed Mar 24, 2010 8:02 pm
by 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?