Bioequivalence and Bioavailability Forum

Hi Ionsource!

❝ Please let me know if this difference in the method of solution preparation is significant enough to invalidate our previously done studies?

Duno. The inspector is right since depending on the compound in the worst case you may face volume contraction/dilation. Although rare if dissolving a solid (more likely mixing two liquids), but existing. Talk to an oceanographer about sea salinity.
On the other hand you can easily compare your previous method to the one he suggested.

❝ Secondly, which method is more practical to work with?

Use volumetric flasks in the future – actually I never have seen anything else in the past. In my experience the 10 mL ones are terrible to handle. If possible weigh in more and use a 20 or 25 mL flask.

Dear ionsource,
Pl read my response in blue.
Regards
Chirag

❝ In our lab when we prepare stock solution we weigh an approximate quantity of standard and add an equivalent amount of solvent in it. For example, we weigh about 10.3 mg of reference standard (when trying to weigh 10.00mg )and add 10.3 ml of solvent with a calibrated pipette to prepare a 1mg/ml solution. From that solution we prepare our calibrators.

To the best of my understanding this practice is not acceptable. Volumetric flasks have to be used upto their graduation mark to get accurate dilutions.

❝ However, an auditor raised the objection that in our case the final volume of the solution would change since the volume occupied by solute also adds to the final volume and hence the strength we finally get is different from what we intended. They suggested using a volumetric flask (say 10ml), weigh an approximate quantity (say 10.3mg as in our case) but make up volume to 10ml so final concentration is 10.03mg/ml which should be used for preparation of further dilutions.

The volume of stock can be adjusted for preparing subsequent dilutions keeping the final volume according to the graduation mark of the volumetric flask used

❝ Please let me know if this difference in the method of solution preparation is significant enough to invalidate our previously done studies?

If the subsequent dilutions are prepared in a similar way as indicated, the concentrations of the calibrator, QCs and unknown samples may be incorrect

❝ Secondly, which method is more practical to work with?

The one suggested by the auditor

Hi Ionsource,

❝ Please let me know if this difference in the method of solution preparation is significant enough to invalidate our previously done studies?

In my opinion both methods are acceptable and GLP compliant.
Having said that, I would also like to say:
1. I understand the comments from other posters in this thread: The more pipetting that takes place the larger the risk of an error being carried forward. But om the other hand the same holds true for every step involving dilutions. And a normal standard curve or QC prep. involves "a lot" of such steps.
2. I am aware of the source of your doubt. In some cases I have witnessed inspectors or auditors being upset by the fact that analytical results are given with e.g. 4 decimals or 5 significant digits or whatever, while at the same time, if the assay developer weighs "approximately XY.Z mg" into a vial and dilutes from there to a known volume then the LLOQ cannot necessarily be generally (yes, that term was generally) specified in the same fashion and we end of up with an assay having an LLOQ of "about ABC ng/mL" etc. This is due to the fact that the lowest calibrator conc. on standard curves may differ a little. It simply seems unpalatable to some auhtorities (whether formal or self-appointed ) that the LLOQ is given approximately while results are given with higher accuracy.