Bioequivalence and Bioavailability Forum

Dear Ohlbe!

❝ ❝ Ohlbe, I’m not sure whether you are talking about duplicates?

❝ I was talking about single measurements, where you sometimes have an IS response which is very different from what you have for other samples (for instance half the "usual" response).

Thanks for the clarification; I wasn't sure.

❝ ❝ If measuring singlets, we don't know whether or not the ratio is affected; we only may notice an 'unusual' IS response.

❝ True, and that's really a complex issue.

❝ Actually I've come across different situations, which may have different causes.

❝ One situation is when all samples from one or several given subject(s) have an IS response which is significantly lower, or higher, than the response of the calibration and QC samples. I've seen runs where subject samples all had an IS response 20 to 30 % lower than cal and QCs. There you may suspect matrix effect (this was LC/MS/MS) and investigate whether it affects your analyte and your IS in a similar way, and thus whether the ratio is modified or not.

This is the ‘classical’ example of a matrix effect in LC/MS. Although LC/MS was just fashionable a decade ago, it became the standard in routine analyses for many compounds. Unfortunatelly many analysts still are not aware of the potential ion suppression/enhancement by co-eluting compounds and follow the ‘precipitate-and-inject’ approach propagated by instrument manufacturers/vendors.
With rare exceptions 'cleaner' extracts are a prerequisite for LC/MS compared to sample pretreatment needed for 'old-fashioned' HLPC/GC (OK, electrochemical detectors in HPLC are also quite unforgiving).
Everybody should remember that 'not seeing' a signal does not mean 'nothing' is there - analysts can physically touch it sticking in the ion trap after 500 biosamples…

❝ Another situation is isolated IS "abnormal" response. HS, you have given a number of reasons which may result in a decrease in IS and may, or may not, affect the ratio.

Not me, this was another guy.

❝ This makes it important to note any abnormal event during sample processing (such as a blocked SPE column). The only way to check whether the ratio was affected or not is to repeat the assay for that sample...

Agree.

❝ […] there are different ways to deal with [re-anyalsis], for instance:

❝ - define criteria in a general SOP (for instance, reassay the sample if the IS peak area differs by more than (50 % ?) from (the mean IS peak area in your calibration samples ? in your calibration and QCs ? In the run ?) (many possibilities !)

Oh yes, fully agree! But since the topic is IS variation, I’m having my doubts whether anybody right now looks at the actual peak area of the IS during method development and/or validation. Normally it’s recorded and stored in the depths of the data system and only the ratio analyte/IS is used.
But as Jaime just stated in his post in another context:

❝ Nothing has to be done in terms of additional laboratory work; it's just a little bit sitting by the desk working on your PC.

In every lab there’s freetime; it should be possible to re-evaluate existing methods in that respect and come up at least with some ideas.
Just my 2 cents (only gut feelings, haven't done it myself): 50% seems to be high (30%?); I would go with calibrators only (because QCs come from a frozen stock, and including the unknowns also will be 'masking' a matrix effect). But this is just a quick shot.

❝ Whatever you do, should be done consistently…

You should have formatted it consistently! Your examples are scarry ones; trouble is preassigned.
Being a certified cave diver we like this one (to be followed in case of problems):

Stop! - Think! - Act!

The order has to be kept, and no step has to be omitted.