I have a new molecule for which I have initially developed an isocratic LC-MS/MS method (Synergi Fusion column, H2O/ACN 20/80 (+0.1% HCOOH)). Analysis of this method gives limited sensitivity because of broad peak shape (but it was sufficient for the time being), and with no traces of carry-over.
Later on I needed a more sensitive method, so I switched to a gradient method to sharpen up the peak. Now I have great sensitivity but a carry-over of 50%!
Gradient:
A: H2O+0.1% HCOOH
B: ACN + 0.1% HCOOH
t %A %B
0 90 10
3 90 10
3.1 20 80
5 20 80
5.1 10 90
8 10 90
Rinse solvent: ACN
Injection of a standard solution followed by a series of blanks showed that the peaks decrease somewhat evenly by about half, after each injection. It will be impractical to therefore injection 10 blanks after each standard solution and sample.
Injection of a completely fresh blank vial (injected as a 'sample) after injection of a standard gave a peak, suggesting that the analyte does not stick to the outside of the needle. Use of an external wash vial showed no improvement in the carry-over. So the problem must be somewhere in the system.
I tried a number of things already, none of which had any effect:
- Tried a different column (Synergi Polar instead of Synergi Fusion)
- raise the gradient to 100% ACN and keep it for an extra minute
- rinse solvent: ACN/H2O 50/50, and MeOH/H2O 50/50 + 1% acetic acid
- used an external wash vial with ACN/THF 90/10
I then went back to using the isocratic method, and no carry-over was observed. But it is not sensitive enough, so I really need this gradient method to work.
What other rinse solution combinations could work? Perhaps something more similar to the mobile phase?
- H2O/ACN 20/80 + 0.1% HCOOH ??
- H2O: ACN: MeOH: Isopropanol (25: 25: 25: 25 V/V) ??
The LC-MS/MS system is an Agilent Series 1290 Pump and Autosampler, with an API 5500 MS
Any help would be appreciated!
It is likely this carryover is from the injected samples that have been selectively retained and need a stronger organic solvent to 'boot' them off the column. So instead I would try as your Mobile Phase B H2O: ACN: Isopropanol (25: 25: 50 V/V) +0.1% HCOOH (we don't want a pH gradient).
I am not sure at all, but do You wash Your needle/Injector? with our systems we wash those routinely 3 times before and after the injections with pure solvent.
However if You have that much left in the loop/needle it smells like a major part of your sample is not getting into the main column....
If You have alread tried to go up to 100% ACCN without effect I do not believe the compound is on the main column...
Hi Phil.
I think you could switch auto injector valve( for by pass/ inject) for remove possible analyte residue.This is very easy configurable in Analyst software. I had same problem with similar equipament in my work. Some times loop remained analyte residue in your loop injection. You could try wash needle with a solvent in wich your analyte is more soluble.
Regards
The sample injection protocol in Agilent 1290 bypasses the needle for a few seconds only for drawing the sample and then switches back to the mainpass position, in which the mobile phase always continuously flow through the needle to the column, for minimizing carryover. So, there is a little chance of carryover from needle internal.
A couple of things. One: have you reduced the injection volume or better yet diluted the sample? Two: I have an Agilent QTOF in which I experienced the sample attaching itself to the plastic lines.... Lastly have you varied the gradient to have more organic in the initial steps?
Here's my few cents, if it is the autosampler issue, then why he did not have issue with isocratic mode? I assume that the autosampler program (no of rinse and type of solvent is the same). I had this issue before (see my research gate on the question that I asked). The solution that I had is from an engineer, not chemist, from Shimadzu. The column has some imperfection and retains your analyte. You can test by programming as usual but after you equilibrate the column at 8 min, bumping the mobile phase to 80% B immediately for 5 min and dropping to 10% B immediately and staying for 5 min. Repeat the 80% B again and 10% B again one more time and still collecting the data. If you column is good, you should not see the analyte peak after the two cycle because it should be eluted at the first gradient. However, if you see two more peaks with lower response because of the carry-over, it means you need another column that will not give you that. Try this test and tell me the result.
I don't see a cycle time to re-equilibrate to 90:10 water:ACN. Did I miss it? It will take some time to go from 10:90 to 90:10; I'd guess 5-7 minutes at least, depending on your flow rate (you didn't give us flows or column dimensions). if you don't properly re-equilibrate the column you can get all kinds of wacky things occurring.
Thanks for all your input!
In the end it turned out to be very strong carry-over due to the physical/chemical properties of the analyte. The analyte showed very strong affinity to virtually all column phases, which means residual amounts remained stuck on the solid phase of the column which kept on eluting in small fractions with each subsequent blank run. I have found a highly deactivated C18 column that showed minimal carry-over with which the analysis could be adequately performed.
Hi Phil.
Carryover is two types one is system carryover while another is column carryover. If carryover comes from column than change the column. If your compound has high affinity to your stationary phase it will take more time for complete elution so better the change the column chemistry to resolve the problem. If carryover comes from system than increase the rinsing volume, rinse dip time, change the rinsing programme, change the composition of rinsing solvent( rinsing solvent must be dissolve your compound).
Next time you have a carryover issue, please read this article to troubleshoot and understand the reason for it. Better yet, read it and avoid having the problem at all.
"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"
https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html
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