Carrying out test injections prior to running sequence.

[pontiuspilot]

Hi guys,

I've recently taken up a role with a biopharm company. Much to my surprise they don't carry out any test injections prior to running a sequence on their LCs.

I've never encountered this and have always (sometimes painfully) carried out multiple injections of my blanks and standards.

My supervisors are saying that test injections introduce the potential for extra regulatory scrunity and I am blue in the face in trying to convince them otherwise. It is bizarre behaviour from what is a major multinational.

Would you guys be able to point me in the direction of any comprehensive documentation to present to my supervisors to stop this irrational practice?

Thank you for any help you can offer.

Regards,
Kevin.

[kubowicz.tomasz]

Hello

If your column is fully equilibrated with mobile phase test injection (or injections) is not really necessary.
I always recommend solvent (mobile phase) injection at the beginning of the sequence to check pressure profile and baseline but I wouldn't treat it as something you need to do.

Regards

Tomasz Kubowicz

[Kreall]

As Tomasz said, there is no need to make a test injections if the whole system is equilibrated.

Futhermore I also think that the mutliple injection before every analysis to check the RSD of peak area is not necessary if the HPLC system is systematicly checked for injection precision and repeatability. If there is something wrong with the system itself the results will make no sense, but this is propably another matter.

[Peter Apps]

What do the test injections test for that is not also tested by, for example, the calibration standards and bracketing standards that are part of the analytical sequence ?

There might be an argument that running the tests saves launching a sequence on an instrument that was not working properly, and subsequently having to re-run the sequence, but whether that is cost effective will depend on how well the system suitability is maintained.

Peter

[HPLCaddict]

The previous (valuable) posts concentrated on test injections of references, SST solutions or calibration solutions, if I understood them correctly.
Just as an addition: Don't even think of doing "test" injections of your actual sample (!) solutions! That's a practice that will give you a BIG slap in the face by FDA or similar folks.

[pontiuspilot]

Thanks everyone for their input.

I think in a high volume qc lab where systems are constantly being changed for different products and the demand to get things right first-time is significant it is essential to carry out preliminary injects.

There are a number of justifiable reasons (certainly none include injecting a sample vial). Removing any doubt of atypical chromatography in your quantifying std , ensuring your diluent is clear of impurities, ensuring that you meet spec on your recovery std and ensure column efficiency, tailing, resolution etc. I think you can remove all uncertainty , in a pressurised production driven environment, that there may be an unforseen issue with your system.

I'm really going for it now!!! but I think it is good practice!!!!

I'm going against the grain on this thread but I just don't see it as anything but helpful as long as you don't inject your sample!!

Thanks,
Kevin.

[Peter Apps]

Hi Kevin

The question is not whether extra pre-sequence tests are helpful, but whether they are helpful enough to justify the extra cost in time and consumables. Beware the infinite regress of checking whether all the vials are clean, all the SPE tubes are working right, all the sample prep measurements are properly calibrated against properly certified measurement references, all the reagents are the purity that it says on the bottle, the water purifier is producing pure water, and so on ad infinitum. By the time you have checked everything you have done so many test injections that the column is worn out and you have to install a new one that needs a set of tests to prove that it is working right.

In the end all that matters is the accuracy and precision of the measurements of target analytes. That should be taken care of by the standards and replicate samples that should be part of the analytical sequence.

Peter

[pontiuspilot]

Apologies for the delay in replying Peter.

Thank youfor the post. It has made me concede my pursuit of this point. My boss has the very same views as your goodself.
Regards,
Kevin.

[DR]

Going with the grain... we equilibrate and let fly. If SST fails, any subsequent injections are ignored until whatever problem is resolved, as demonstrated by a passing SST portion of the same sequence, restarted.

The key is in documenting
1)that there was an issue justifying scrapping of the previous run and
2)corrective actions to prevent more of the same

[Peter Apps]

Apologies for the delay in replying Peter.

Thank youfor the post. It has made me concede my pursuit of this point. My boss has the very same views as your goodself.
Regards,
Kevin.

It's not often that I agree with the bosses, I must be getting old !

Peter

[KM-USA]

We inject twice on the first line of the Sample Table (Agilent) using Calibration:Replace. Then the next line is 4 injections using Calibration:Average, so the last line of Replace and the next four injections are used for system suitability and also averaged and used for quantitation.