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Dear All,
We have been experiencing a problem with our API2000 LC MS/MS instrument (Agilent 1100
series HPLC - quaternary pump) for the last two months. We inject samples and observe the
retention time to vary back and forth. The retention time will shift between two specific
times but not consistently (i.e. every second sample). There seems to be no pattern to the
frequency of the shift and it happens with every method used on the instrument.
This prevents automated peak integration, but does not affect the accuracy of the method.
We have tried the following, but to no avail:
1. Flushed the system with isopropanol and performed leak and pressure tests (all good)
2. Swopped out the autosampler
3. Bypassed the degasser and the solvent mixing chamber
4. Replaced the seals in the pump head
5. Changed the active inlet valve and outlet valve
6. Replaced the gold cap and frit at the purge valve
7. Note that there is no column switching valve attached.
The peak continues to shift back and forth.
If anyone has experienced this before, and can suggest how to rectify the situation, it
would be greatly appreciated!
Many thanks,
Jennifer
Jennifer Norman MRQA
BSc (MED) Hons Pharmacology MSc
QA and Logistics Manager
K50.30 Division of Clinical Pharmacology
Old Main Building, Groote Schuur Hospital
Observatory, 7925
CAPE TOWN
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Hi Jennifer,
First, I'm not the greatest troubleshooter in LC/MS/MS. Secondly, you have done
quite a lot. On the other hand, you left some very important things out of your
description.
Having a low pressure pump in your system is not optimal and may cause problems,
but you probably will not change that right now (but consider it in the future).
Gradients are slow (you did not mention what kind of gradient you are using) and
not precise enough. Maybe your proportioning valve is not working well (I did
not understand how you bypassed it)? Try using your gradient by assigning
alternative channels for your solvents. Did you try using other columns
(pre-columns)?
Are you sure your degassing is working OK? Sometimes air bubbles cause these
problems. What is your mobile phase. Is your gradient steep enough? What is your
injection volume relative to your column volume. Sometimes a too large injection
volume causes a gradient by itself. If you are not using a gradient, these
things may easily happen. Then you would need a organic phase flush between the
injections.
Just some thoughts. Good luck anyway!
Stefan Soback
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Jennifer,
My thought is that you may need a little more time after your gradient to let
the column equilibrium to the initial condition.
Thans
changguang
Department of Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Boulevard
Dallas, Texas 75390-9038
Email:chg-wang.-at-.hotmail.com
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Jennifer,
Have you considered increasing the equilibration time at the end of your LC
method? Just a thought.
Sashi
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The following message was posted to: PharmPK
Hi Jennifer,
Which version of the 1100 pump are you using? Some variants of the quaternary
pump have large mixer volumes so gradients don't respond very quickly (and this
may manifest as changes in RT). Agilent may be able to make the change for you,
but for LC/MS applications I've found the 1100 binary pump to be better. If you
only have access to the quaternary pump then don't expect to be able to run fast
gradients, and make sure you give it time to equilibriate at the start of a run.
The quaternary pumps are great for LC/UV or LC/radio applications or for slow
LC/MS (e.g. for MetID).
I hope that helps.
All the very best,
Bernard
Bernard Murray, Ph.D.
Senior Research Scientist, Drug Metabolism
Gilead Sciences, Foster City CA
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Hi,
Please check the splitter part,
Either you clean it or replace the splitter with new tubing, it may sort out your
problem, if you are using splitter, because we have also observed same problem and we
overcome by this means,
Thanks.
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Dear Jennifer,
I think you should call agilent instrument engineer and get maintenance done.
Dr. Mandar Mote
Dy. General Manager Bio-analytical
Bio-equivalence Division
Macleods Pharmaceuticals Ltd.
Mumbai.
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The following message was posted to: PharmPK
First it cannot be passing any suitability lets look at mechanical
Are you getting the delivery from each pump and from the total at each part of
the gradient. Check valves and piston seals can contribute as can chipped or
broken pistons
Are the pistons and piston heads matched for the process
Is there a selection for tuning for viscosity
What is the size and type of mixing device and is it heated. Do you have a pre
heater on your solvent system
What are the swings on the gradient
See if you can reduce the swings such as changing the solvents. Instead of
using portioning valve to give you starting mix of 10/90 make up starting
solvent to be at this level. Be sure to mod your sample as well. Is your
sample in the starting solvent or something different. Matching it to the
starting condition would give you best control
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The following message was posted to: PharmPK
First it cannot be passing any suitability lets look at mechanical
Are you getting the delivery from each pump and from the total at each part of
the gradient. Check valves and piston seals can contribute as can chipped or
broken pistons
Are the pistons and piston heads matched for the process
Is there a selection for tuning for viscosity
What is the size and type of mixing device and is it heated. Do you have a pre
heater on your solvent system
What are the swings on the gradient
See if you can reduce the swings such as changing the solvents. Instead of
using portioning valve to give you starting mix of 10/90 make up starting
solvent to be at this level. Be sure to mod your sample as well. Is your
sample in the starting solvent or something different. Matching it to the
starting condition would give you best control
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The following message was posted to: PharmPK
Hi Jennifer,
You say problem is persisiting since two months. Can you recall any major
changes in instrumentation, which could be creating this issue?
OR, as others pointed out, is gradient too fast for quaternary pump?
To simplify troubleshooting, try out isocratic runs. If you are getting
reproducible RT, then optimize right gradient profile.
Regards,
Vinayak Nadiger
Vinayak Nadiger
Senior Scientist
Forma Therapeutics(Singapore)
11,Biopolis Way ,Helios # 08-05
Singapore 1386607
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The following message was posted to: PharmPK
Jennifer: did you resolve this? If so how?
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