Chromatography Forum

Dear forum members,

I am developping a LC-MS/MS screening method for analysis of forensic drug and blood samples.
The LC-MS/MS method was published before and it was working in the lab.

Because of sensitivity we decided to directly inject the supernatans of urine (after centrifugation). 30 µl is injected on a PFP column, 5 µm, 2.1*50 mm. Before this, we diluted the urine 1/10. This worked fine for about 500 injections. Then, when we switched to undiluted urine, we saw a decrease in retention time after several injections.

Because we suspected column ageing, we tested a new column: 10 injections of 1/10 samples: constant RT, 4 injections of 1/1 urine: decrease in RT! So already a change in RT after only 15 injections?! The pressure is unchanged! This phenomenon was seen for 10 compounds, both at the beginning and the end of the run.

Has anyone experienced this before? How can we solve this?

Thank you!

I forgot to mention:

A filter and a 1 cm guard column are both used to protect the column

degass your moblie phase......filter your mobile phase.........if there is a column heater make sure it working and not fluxuating....make sure all the wire entering and exiting the column is within the column heater...and not sticking...or a portion of it sticking out causing changes in temperture of samples.is the LCMSMS in a temperature controlled room? if so is the temperature of the room stable or changing?....make sure you filter your sample with 0.22um or 0.45um filter.

The mobile phase is degassed & filterd.
The column temperature is controlled (40°C) and not fluctuating.
The room is supposed to be temperature controlled, but can however vary. But in the 5 years I am using this room there never has been a problem regarding this.

I didn't filter the sample because I experienced that some compounds stick to the filter and dissappears from the sample after filtration. I have to admit the urine samples can be quite dirty... But can this cause a decrease in retetion times, even after only five injections of such a dirty sample??

Thanks!

In order to solve the problem and to restore the retention times, I washed the column:
10% ACN 90%H2O
100%ACN
75%ACN 25% isopropanol
100% isopropanol
100% CH2Cl2
100% isopropanol
75%ACN 25% isopropanol
100%ACN
10% ACN 90%H2O

This didn't help, any other suggetsion to repair the column?

Thanks!

maybe you can try other type column (temporarily forget about the resolution of some target analytes) such as C18 to test the stability of the LC-MS/MS system.
we have similar experience when we used different batch PFP column, sometimes the phenoma you described happened.

if c18 indicated that your LC-MS/MS is satble. then continue to injection some sample by using PFP column, then may injection calibration solutions again to check whether RTs of samples and standrad solution will be matched.

hope it is useful

It has been a while for me but I recall stuff precipitating out of urine when it is mixed with certain solvents (may have been methanol). You may need to mix the urine 1:1 with your mobile phase before filtering?

Perhaps the pure stuff has dropped a load of gunk at the top of the column.

Tim

What's your mobile phase? Are you running a buffer? Are your compounds ionizable? Maybe the 1:1 urine has too high an ionic content, and this is affecting RT, and you need to either dilute into a strong buffer (or dilute out ionic content) or run a buffer to keep compounds at a desirable pH/ionization level?

If I don' inject undiluted urine anymore, the retention times stay stable (at their new decreased value obtained after injection of some undiluted samples). Any idea how I can restore this? (I washed the column as mentioned above and also with 100% hexane).

@s2008: Other columns work fine, but I didn't inject the undiluted urine on them. Problem is specific for the PFP propyl column.

@Yama001: I don't think there is debris at top of the column, because the pressure is unchanged. The pressures is stable, while retention times decrease...

@bisnettrj2: Mobile phase is water and acetonitril, both with 2 mM ammoniumacetate and 0.2% acetic acid. Compounds are good ionizable! A 1:10 dilution of the urine gives repeatable results instead of decreasing retention times with 1:1 urine. However, because of sensitivity of detection I prefer not to dilute...

Thanks for your help!

ok try this
run 1 hour of warm water 50c through your lc for about an hour.....then run 3M nitric acid through your system for about an hour and then again water for a hour to wash out the nitric acid. when your running the water and acid separatley also prime your system and purge injector too...do all those litlle cleaning steps your lc should allow you to do. obviously dont have your column attached....i bet you this will work.it will cleam out your whole LC