by
freemab » Sun May 03, 2009 7:52 am
I don't know whether it is a specific requirement of GMP or other "rules," but I think you'll agree that it is essential to know whether your IS is stable. Hence, some test should be done sometime.
One of the simplest tests to determine stability takes advantage of the high stability of modern instruments: Put your sample(s) on the autoinjector and inject them periodically over a weekend or so. If no change is seen, this is pretty good evidence of stability. If change is seen, either the material is unstable, solvent is evaporating, or the detector is unstable. This procedure may not suffice for a validation, but is good for screening work.
If you're lucky, you may have an IS solution that is stable at freezer temperature. In this case, you can use (warmed) freezer-stored IS solution as a standard for comparison to the the room-temperature stored IS solution. Put multiple aliquots (perhaps in autosampler vials) in the freezer and bring them out only when needed. However, if any change is seen in the freezer-stored solution (precipitate, cloudiness, etc.), don't go this route.
Measuring against freshly prepared solution is a good method, providing you take into account the variability of preparation. You may have to prepare several solutions and compare them to see what variability to expect from preparation to preparation. You can estimate this on the basis of accuracy of the various steps in your procedure. Typically, the gravimetric steps are virtually error-free, but the volumetric steps introduce errors that you can estimate on the basis of the accuracy of class-A volumetric glassware. Be leery of air-cushion (e.g., Eppendorf) pipettors. These can give high or miserable accuracy, depending how they are used, and if you don't know how to use them you won't be able to distinguish good from bad.
As to mixing different batches of IS, the problem here is that the tech will often take short cuts. If your standards were prepared with IS batch No. 1 and are stable a week, then you make up new samples with IS batch No. 1 + No. 2, you will not have the same IS in samples and standards. These things do happen in practice and can be very difficult to spot if recordkeeping is not flawless (as it seldom is). This is why the typical requirement is to make fresh IS and fresh standards for each assay. It takes longer but it avoids potential problems that could cost the company more than the shortcuts saved.
