different recovery of repeating injection

[ym3142]

the same sample was injected twice consecutively and the recovery were 104% (1st inj) and 98%(2nd). simple thought would be injector malfunction. but my system suitability was OK(RSD=0.9 for 5 inj). So any other possible reasons?
A(3M KH2PO4):B(MeOH)=55:45, Gemini C18, 150x4.6, 1.75ml/min, 40C

[pve]

hello,
in our lab, we consider the system as OK when the RSD for 5 inj is under 0.5.
after those 5 injections, we can start the sequence to determine the title of the product.
concerning the non-reproductibility of the injections, who is the vendor of your HPLC system?

Pierre

[DR]

Sample prep exactly the same as Standard prep?

[hokie]

Were your two injections injected from the same vial or different vials?

If they were from the same vial, it could be several things depending on what type of LC you are using. Certainly you could have an injector problem, you might have air in your syringe, or it could even be related to vial septa. I've actually seen poor reproducibility caused by septa that seal too tightly around the needle combined with vials that were too full. A small vacuum was being created in the vial causing problem with pulling sample.

On the simple side, if they were from separate vials, it might be contaminated vials. Inject the two vials on a different LC to see if you get area counts that are in the same proportion to each other compared to the original area counts.

There are certainly other possibilities, but these should get you started.

[ym3142]

thanks for all who replied my question.
I am sorry I should have said that two injections from the same vial.

[Rob Burgess]

My 1st thought would be to run your assay again: do you get poor reproducibility again from injection from the same vial?
It is a strange one... the good precision of your stds. suggest there isn't a problem per se' with your LC instrument...

[Alex Buske]

Sometimes strange things happens on the first and second injection from one vial. we have had needles that "cut" pieces from septa and got blocked after few injections; we have had times when we used to punch each septum befor placing the vials in the autosampler. During my very first expieriences with autosamplers I learnd to like "pre cut" snap caps.

alex