LC column saturation ????

[Maristela]

Dear Friends

I am working with a drug analysis in plasma samples by LC-MS-MS.
I extract 50ul of sample with 1 ml of diethylether-hexane (8:2 v/v). My samples concentration are between 100 ng/ml to 40000 ng/ml.
The mobile phase is acetonitrile-ammonium acetate 2 mM (7:3 v/v), the injection volumn is 10ul and the run time is 3.5 min.

I have a decreased of 20% of the analyte signal on a list of 50 samples injected.

The internal standard did not decreased at the same way. (it is not a deuterated internal standard and I think it does not respond the same way)

After each 50 samples I wash the column with acetonitrile during 40 min and it return to the same response of the beggining.

Is it a column saturation problem ? Is methanol better than acetonitrile to avoid this " accumulation " in the column ???

I hope to hear from you very soon......

[james little]

Interesting. Methanol is definitely better for eluting the phospholipid endogenous material in the plasma samples.

see http://littledomain.com/james/files/matrix_effects.htm

The lipids could enhance or decrease the analyte response. Normally when using some methanol in the acetonitrile (see above link), the response for the analyte will level out after 5-7 injections of the anlayte.

I have also noted on my instrument at times that the response does drift with time during the firt 6-8 hours and then tends to flatten out. The unlabelled internal standard in my experiences didn't always follow that of the analyte. The labelled standard helped a lot as long as the amount added was chosen not to suppress the unlabeled analyte.

[Maristela]

Interesting. Methanol is definitely better for eluting the phospholipid endogenous material in the plasma samples.

see http://littledomain.com/james/files/matrix_effects.htm

The lipids could enhance or decrease the analyte response. Normally when using some methanol in the acetonitrile (see above link), the response for the analyte will level out after 5-7 injections of the anlayte.

I have also noted on my instrument at times that the response does drift with time during the firt 6-8 hours and then tends to flatten out. The unlabelled internal standard in my experiences didn't always follow that of the analyte. The labelled standard helped a lot as long as the amount added was chosen not to suppress the unlabeled analyte.

I tested a the same mobile phase (ammonium acetate 2mM:acetonitrile 3:7 with 5% of isopropanol, but the results are the same, in other words, the response of my analyte decreased after 50 injections.

Should I test methanol instaed of isopropanol ???
Do you have any other idea to help me on this point ?

[Uwe Neue]

I am not sure that phospholipids are the problem. The Sailor has more experience with phospholipids than me, but I have my doubts that they extract that easily into your extraction solvent.

I would consider the possibility that hexane or diethylether or both accumulate on the column. Either way, a brief wash with acetonitrile should remove the organic solvent. 1 mL of acetonitrile over the column every 20 runs or so should make the problem disappear, if this suspicion is correct.

[Maristela]

I am not sure that phospholipids are the problem. The Sailor has more experience with phospholipids than me, but I have my doubts that they extract that easily into your extraction solvent.

I would consider the possibility that hexane or diethylether or both accumulate on the column. Either way, a brief wash with acetonitrile should remove the organic solvent. 1 mL of acetonitrile over the column every 20 runs or so should make the problem disappear, if this suspicion is correct.

Dear Friend

I will test one injection of 50ul of isopropanol every 10 injections and I will let you know.
Thanks

[Uwe Neue]

I would have appreciated a larger volume, but if you want to do this with injections, I suggest to use acetonitrile instead of isopropanol. Reasons: it's in your mobile phase anyway; it is more hydrophobic and probably is better at removing hexane; it has a lower viscosity.

[Maristela]

I would have appreciated a larger volume, but if you want to do this with injections, I suggest to use acetonitrile instead of isopropanol. Reasons: it's in your mobile phase anyway; it is more hydrophobic and probably is better at removing hexane; it has a lower viscosity.

Dear Uwe

I read this suggestion after test IPA. It works well, but I will test extraction with pure ether instead of hexane-ether.
If the results are better, I will make faster runs (as I dont have to put IPA or acetonitrile after 10 runs).
I will let you know.
Thank you.

[Peter Apps]

Am I right to undertsnad that you are injecting your samples in hexane-ether ?

If you are there is probably some material precipitating out as the sample mixes with the mobile phase and things that are soluble in non-polar organics drop out of solution.

It would be a better procedure to evaporate off the organic solvent and then take the sample up again into the same volume of mobile phase. You might find that you rpeak shapes improve as well.

Peter

[Maristela]

Am I right to undertsnad that you are injecting your samples in hexane-ether ?

If you are there is probably some material precipitating out as the sample mixes with the mobile phase and things that are soluble in non-polar organics drop out of solution.

It would be a better procedure to evaporate off the organic solvent and then take the sample up again into the same volume of mobile phase. You might find that you rpeak shapes improve as well.

Peter

No, you are wrong.
I am extracting 50ul of plasma sample pH3 with 1 ml of hexane-eter (2/8). Afetr evaporation of organic residue, I ressuspended the sample with 200 ul of acetonitrile-ammonium acetate 2mM and inject 20 ul in the LC-MSMS (API 2000)

Maristela

[meillid]

[[size=9]color=orange][color=orange] "No, you are wrong.
I am extracting 50ul of plasma sample pH3 with 1 ml of hexane-eter (2/. Afetr evaporation of organic residue, I ressuspended the sample with 200 ul of acetonitrile-ammonium acetate 2mM and inject 20 ul in the LC-MSMS (API 2000)

Maristela "[/color][/color][/size]


Then where the hexane come from if it is the hexane accumulate on the coulumn that account for the response decrease? Am I lost here?

[Maristela]

[color=orange] "No, you are wrong.
I am extracting 50ul of plasma sample pH3 with 1 ml of hexane-eter (2/. Afetr evaporation of organic residue, I ressuspended the sample with 200 ul of acetonitrile-ammonium acetate 2mM and inject 20 ul in the LC-MSMS (API 2000)

Maristela "[/color]


Then where the hexane come from if it is the hexane accumulate on the coulumn that account for the response decrease? Am I lost here?

The residue(???) of hexane could come from the extraction procedure, but as it was evaporated until dry, the hexane could extract something undesirable from plasma that can accumulate in the column, maybe the lipoproteins.....

[Peter Apps]

Hexane - ether will extract things that are not soluble in ACN - ammonium acetate. When you resuspend your samples in mobile phase for injection, do you get a clear solution ? My guess would be that you do not, and that there are insolubles suspended in your samples that accumulate at the head of the column.

Try filtering the resuspended samples.

Peter

[Maristela]

Hexane - ether will extract things that are not soluble in ACN - ammonium acetate. When you resuspend your samples in mobile phase for injection, do you get a clear solution ? My guess would be that you do not, and that there are insolubles suspended in your samples that accumulate at the head of the column.

Try filtering the resuspended samples.

Peter

Peter

I get a very clear solution.
I did the ion supress test again and I notice that after few injections the supression in the region of my IS increase and that is why I had my initial problem
I tested an acidic mobile phase and had a better separation between the analyte and the internal standard . The supression region stayed before the peaks. I think that the problem is solved. To confirm my supposition,
I exctract the same spiked sample 8 times and ressuspended all together in the same vial. I will make 60 injections of the same vial to see if there is any significant variation between them.
I will let you know.

[Uwe Neue]

I was the one that assumed that you inject out of the ethylacetate hexane solution. Since this is not the case, I do not think any more that hexane is the probelm. However, if a wash every X injections with isopropanol works and solves the problem, I would not go further. Or do you still have a problem?

[Maristela]

I was the one that assumed that you inject out of the ethylacetate hexane solution. Since this is not the case, I do not think any more that hexane is the probelm. However, if a wash every X injections with isopropanol works and solves the problem, I would not go further. Or do you still have a problem?

Dear Uwe

Yes, it works washing every 7 samples, but as I have 2000 samples to inject, I will try 2 or 3 days more to find out anyother way to avoid this increasing of my runs.
As I had a problem only with the IS (I also tested other 2 transitions and the variation between 50 injections is always between 11 and 15%) I will make this last test, changing the IS.
Thanking you very much for your help and I will let you know as soon as I finish it.