HPLC Validation Reproducibility Sample Prep

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

7 posts Page 1 of 1
I am writing an HPLC method validation protocol. I am wondering if anyone knows for a fact if it is acceptable by current guidelines for one person to prepare the samples for an inter-lab reproducibility study, then divide these preps between the two labs rather than each lab preparing their own samples. I am trying to avoid differences (and possible failures) due to sample prep error since they want us to show reproducibility at the specification limit of the related substances in addition to the assay level. Thanks.
The whole sense of an inter-lab trial is to compare the results of the same method run at both labs. And the sample prep is an inherent and important feature of each method. Sometimes probably even the most important feature, running the HPLC method itself often (not always, unfortunately) is rather easy, but with sample preparation there might be a lot of pitfalls.

Running the same samples at both labs will basically only show that both labs are able to run the HPLC method and evaluate the raw data correctly. It does NOT show that both labs are able to handle the whole procedure and produce equivalent results. So, no, this is definitely not acceptable. This would be a procedure to validate only the HPLC method - you want to validate the whole analytical method, and the HPLC is only a part of it.

Preparing one set of samples and running the same samples at both labs can be one method of troubleshooting if you actually see that results from both labs (with individually prepared samples) are different. But it's definitely against the sense of an inter-lab trial.
Concerning related substances, you definitely should be able to produce reproducible results at the specification limit; I mean, this is the concentration range where the serious stuff happens - is the results out-of-spec or not?
Things can be quite different when looking at lower concentrations. A customer once wanted us to check reproducibilty at both the specification limit and the lower limit of quantitation - at was completely stunned that reproducibility was significantly worse at the quantitation limit. What a surprise.
Hi, Mike S.,

I am with HPLCaddict--the validation (transfer) is to show the precision between two labs...having one individual prepare the samples to be tested between two labs does not agree with the spirit or the letter of this notion.
MattM
Hi, Mike S.,

I am with HPLCaddict--the validation (transfer) is to show the precision between two labs...having one individual prepare the samples to be tested between two labs does not agree with the spirit or the letter of this notion.
MattM
I'm a little confused. Our cGMP test methods were developed and R&D, and validated at R&D as per cGMP requirements. Part of that includes additional analyst(s) to prepare and assay samples.

We never "validated" an assay using data from a facility like the manufacturing site QC lab. We did to cGMP test method transfer, sent three blinded samples to QC labs, each operator there had to assay each sample in multiple (doing their own sample preparations, standard preparations, etc.) and then send the data and chromatograms to us. Their equipment was the same, the columns the same, etc., and we rarely had issues with test method transfer.
I did not word my question very well. What I am proposing for the related substances level is one person making a stock solution of the related substances, then using that same stock solution in both labs. So, the analysts are making their own sample preparations, but using the same stock solution for the preps so that the spiked concentration is exactly the same. I have seen this done in the past. What do you think? Thanks.
Hi Mike _S.,

I think I understood your original post as it is. I'm sticking to my guns--I have Not seen this done in my past--always six separate weighments per each RS per lab. It is not a perfect or "waste-free" system, but it resembles most closely the assessment of RSs in real, live samples, I think.
MattM
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