Decreasing Peak Area, poor reproducibility

[MaddyMadeline]

Hi I'm having a problem with a current LCMS method that has been established and validated by a more senior chemist who has since left. I took over this assay 4 months ago and it was running fine until recently whereby whenever I run the SST or system suitability testing, I noticed that the peak area for the injections decreasing greatly in particular the last 2 injections.

What I typically run for the SST is 8 injections from the same vial same volume 5ul every injection. What I noticed was the first 6 injections are typically fine, it was the last 2 injections that have a significant drop in peak area resulting in very poor RSD% or CV%

I can't afford to let the CV% go above 5% for any of the analytes as I am running clinical samples.

This assay runs on isocratic mode for 8 mins in each run. We are quantifying 4 analytes including internal standard for each run.

I'm using an AB Sciex Qtrap 5500 that is linked to an agilent 2960 LC system.

What I have tried so far is making fresh standard, fresh mobile phase and also changed the guard and analytical column but still the problem persisted. Have checked with the field specialist who insists that it must be my mobile phase or column. An engineer has came down previously and assured me that the MS is fine. The retention time for all the analytes are also pretty constant, they do elute out at the correct retention times.

The strange thing is sometimes 3 of the analytes the peak areas will be fine while one the last 2 peak areas are severely low, other times 3 analytes can have low peak areas in the last 2 injections while the other analyte is fine for all injections. This is the inconsistency that I am really perplexed about.

Have also flushed the system many times with ACN and water. Also the peak areas do bounce back to normal whenever I inject blanks or re equilibrate the system each day. The peak area does not decrease day by day but rather just the last 2 injections

[tkubowicz]

Hello

I'd check LC sampler - if the problem with consistence peak area is for last 2-3 injections it looks like problem with injection volume. I'd check:
-method settings - washing needle/washing solvents etc
-rotor seal - perhaps is worn out
-sample loop/needle/needle seat/metering device seal

Please provide info what sampler you have - is it LC 1260 or 1290?

Of course it is possible that problem is with column so if you want to narrow it down I'd recommend to prepare simple checkout sample (caffeine or acetone in water) and run it on your column and water:ACN (methanol).

Regards

Tomasz Kubowicz

[MaddyMadeline]

Hi thanks so much for the reply. I'm at my wits end really

The autosampler is an Eksigent NanoLC Ultra 2D system. I made a mistake with the LC system, its an agilent 1290 Infinity system with 2 binary pumps coupled to a 1260 agilent isocratic pump. It is then linked to an AB Sciex Qtrap 5500 MS.

The autosampler looks the same as this : http://www.medicalexpo.com/prod/sciex/p ... 23379.html

I also just changed a brand new column and the problem is still the same. This is the second brand new analytical and guard column I have changed so I doubt its the column anymore.

[tkubowicz]

Hello

I think it is sampler problem.
You need to check your method for all intermediate steps like:
-number of washes for syringe between injections
-drawing speed
-injection speed
-numbers of "pumps"

If you have those parameters set up incorrectly the result is random injection volume (random peaks areas). So I could imagine that for first 2-3 injections everything is fine and then because of air bubble in syringe (the reason can be wrong drawing speed or number of "pumps") you have wrong sample/std volume in syringe.

Try to observe sampler ans syringe during all steps. If you'll see air gap (or bubbles) in syringe you need to reset parameters. Of course air gap can be set up as parameter but try to set up method without it so you will be able to see if you have air in syringe.

Regards

Tomasz Kubowicz

[tkubowicz]

Hello

If you have fixed sample loop (attached to valve) perhaps there is no enough sample to load full sample - the best way is to over fill sample loop with factor x1.5 or even x2

Regards

Tomasz Kubowicz

[Peter Apps]

Taking 8 samples from one vial may be creating a vacuum in the vial that prevents the autosampler loading the full volume for the last two injections.

How much liquid do you put in the vial ?

Does the same pattern (last two injections out of spec) happen if you do four injections from each of two vials.

Peter

[MaddyMadeline]

Hi thanks so much for the reply. I checked the autosampler and there weren't any air bubbles in the syringe but I can't tell if it is the valve giving the problem instead.

The paremeters on the autosampler is the default method that is set on the autosampler. The only parameter changed is the injection volume.

When you said overfill the sample loop do you mean physically flushing the loop or valve with a syring while bypassing the autosampler needle? The loop is attached to the valve by the way.

I did continue to update the field specialist regarding the issue and also to the engineer and they said now to pull the rail and clean the MS from within as it could be contamination. This is the last thing I want to do as I'm afraid of handling the MS. Plus I haven't ran any other assays since the engineer assured me it was ok so I don't know what the contamination can be.

Thanks i really appreciate this as Im actually quite new to LCMS. I took on this job only about 6 months ago.

[MaddyMadeline]

Hi Peter,

I have been doing SST with 8 injections from the same vial since I took over the assay 4 months ago and have been fine till now.

Typically i use a 1.5ml vial with about 800ul to 1000ul of sample in it.

I don't think it is the vaccum though.


The same pattern does not occur with 4 injections just after 6 injections. I have done 20 injections and noticed that the peak area does go up and down.

Madeline

[Peter Apps]

Hi Peter,

I have been doing SST with 8 injections from the same vial since I took over the assay 4 months ago and have been fine till now.

Typically i use a 1.5ml vial with about 800ul to 1000ul of sample in it.

I don't think it is the vaccum though.


The same pattern does not occur with 4 injections just after 6 injections. I have done 20 injections and noticed that the peak area does go up and down.

Madeline

Hi Madeline

Why do you not think that it is the vacuum ?, and how difficult would it be to load two vials in the autosampler and run an actual troubleshooting exercise to prove it one way or the other ?. "The same pattern does not occur with 4 injections just after 6 injections." makes me think that you did not understand what I was suggesting; put standard into two vials and run an 8-injection sequence with four injections from the first vial immediately followed by four from the second. You admit that you don't want to do maintenance on the MS (which is understandable), and it is good troubleshooting practice to eliminate the simple, cheap things first before tackling the difficult expensive ones - 8 injections from two vials is about as simple and cheap as you are going to get.

Peter

[Steve Reimer]

Madeline
For what Peter is suggesting all it would take to create your problem would be a different specification for the vial septa. You wouldn't even know it, all you would see is that one package of vials works and the next doesn't.
I've been there with VOA vials.

[MaddyMadeline]

Hi Peter,

I did misinterpret your suggestion I will update again when I tried it.

The vial septas do come with a slit though which I thought was suppose to reduce vaccum problem. Can just hope that the problem is as simple as that and I don't have to go to troubleshoot the MS

Thank for the suggestion. Appreciate your help.

Madeline

[James_Ball]

A rail cleaning is not something you want to do unless you have watched and engineer do it before. For that it would be a service call for sure.

On the AB Sciex though, there are the Q0 which is the four U shaped rods just behind the interface cone that can become dirty, you just need to use a swab and some acetonitrile or IPA to clean those.

If the quads are charging, which is what cleaning will fix, they you can test that by infusing a standard with the syringe pump and watch the response over time. If it starts out with high response then slowly drops off over time then it could be dirty quads. But I am with Peter on this one, sounds like it could be too many pulls from a single vial. Even if it doesn't pull a vacuum on the vial, if septa particles fall into the solution, they could absorb some of the target analytes causing the same problem you are seeing.

[MaddyMadeline]

They have told me to do a water injector test which is to measure a vial of water, inject 10 constant injections of 5ul and measure the vial afterwards. I know this might sound stupid but that should indicate whether there is a vaccum in the vial right?

Also i forgot to mention that while i need to keep all the CV% of the analytes below 5%, the CV% of them now are not too high either. They are actually <10% although my most recent run, 2 of the analytes are 12% and 16% respectively.

Which is why the engineer suggested need ti pull the rail as if its a autosampler problem, the deviation will be way higher.

[Peter Apps]

I'm not sure who "they" are, but in this case "they" are not being particularly helpful.

Weighing the vial before and after a set of 10 injections will tell you whether the autosampler is accurate with its volumes, but not whether it is repeatable.

5 ul of water weighs 5 mg, to check repeatability you need to weigh, inject, re-weigh, inject again, weigh again etc etc 8 times (and look for lower than expected weight increments on the past two injections). Your balance and weighing technique need to be good to 3 SDs better than the variation you are seeing in your results, so about 50 ug, so you need a 6-figure balance, although you might get away with 5 figures.

I can't help thinking that a series of two sets of four injections is way less trouble than all that weighing.

Why are you so reluctant to do the 2 x 4 test ?

Peter

[MaddyMadeline]

Hi Peter,

"They" refer to the field specialist and service engineer from AB Sciex. Sadly I have no one else to turn to for advice except them and here

So far what i have done is to change the probe on the Mass spec and flushing the wash line as well as clean the valve at the sample loop of the autosampler.

I did the water injector test but I'm not sure how accurate it is but they want me to do it again since i have purge the wash lines and clean the valve.

As for the 2x4 test, I did it as well and the results are far from ideal. 3 analytes are out with 2 of them going as far with CV% of 14% and 25%. The peak areas are now going high and low between the injections instead of just the final 2 as before.

Here's one of the analytes: http://s44.photobucket.com/user/Madelin ... %20Uploads

Im not sure whether the problem comes from the water source which I used to make the mobile phase as I had that problem previously before which resulted in 2 baselines being very high in intensity. But so far I have checked and the baseline for all analytes are ok.