High Area

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hi guys. can you help me with my QA? I have performed an HPLC analysis with validated method.
I have been doing this analysis for years without any problems.

Yesterday I analyzed many batches (2 injections of the same solution) and one injection of the two came out too high.
How can I justify with my Quality Assurance?

I can't get them to understand me.
etaxene wrote:
Hi guys. can you help me with my QA? I have performed an HPLC analysis with validated method.
I have been doing this analysis for years without any problems.

I can't get them to understand me.


I can't seem to understand you either - we need much more information.

We'll assume that autosampler is being used.

"one injection of the two came out too high" - do you mean for each autosampler vial of sample that one was always high? Was this always the second injection, or did it vary? What about the system suitability injections at the end and at specified intervals during the sequence? What is the solvent used for the samples? What is the time interval from first injection to second injection from the vial? Are you using screw-on caps or crimp-on caps on the autosampler vials?

Might have more questions later....
Thank you. I will be clearer. Solvent is Water. I use screw-on caps vials.

All system suitsab. are in compliance.

I have performed 20 injections.
The rsd% is compliant for all injections.

From one batch I have done two injections.
The first result is 117%.
The second one is 100% (theoretical).

What can I say to justify an instrumental error on the first injection? The original solution was the same
If two injections of the same material result in two different results, then the analysis must stop. Results are invalid as clearly there is a problem with the method, instrument, sample and/or procedure used.

BTW: Two samples are not enough statistically to draw any conclusions from. Depending on the requirement, 3, 5 or 7 would be better.

Many reasons exist for one result to be "high" (area counts). A common reason for this is caused by a worn injector resulting in carryover. Maybe some instrument maintenance is in order. Flushing down an HPLC system is not the same thing as carrying out regular maintenance. The injector rotary valve seals and surfaces wear out over time. They trap sample material in the valve. This results in extra sample eluting out during an analysis (or wash step). You can read more about how to troubleshoot them in this free article, "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html
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