Replicates in Bioanalysis

From the following:  Stability: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

Nico van de Merbel, Natasha Savoie, Manish Yadav, Yoshiaki Ohtsu, Joleen White, Maria Francesca Riccio,Kelly Dong, Ronald de Vries, and Julie Diancin

 A stability assessment at a single time point per storage condition per concentration level is considered sufficient, although it should be realized that analysis after multiple time points will provide more detailed information, which can be helpful to interpret the stability profile of an analyte. A sufficient number of replicates should be performed to obtain a reliable average result for any stability assessment. Current practice appears to be that each stability assessment is typically performed in triplicate as a minimum. For methods with a large intrinsic variability, increasing the number of replicates could be considered to increase the level of confidence in the result and avoid drawing incorrect conclusions.

As Clinton stated “it all depends on what the definition of is is.” Here the concern is the definition of replicates.  Muddy waters seemingly even in an era of where all water is opaque.  What is a replicate? At a 2016 meeting, one CRO was awarded a sanction by the FDA on their interpretation that a replicate could be an injection or multiple injections from the same container-tube. This was for long term stability and the majority of other CRO representative present agreed that replicate did not mean injection but rather container.  There still exists confusion over what the definition of replicate is. To avoid confusion perhaps we use the term tube as:

Current practice appears to be that each stability assessment is typically performed using preparations from triplicate tubes (samples) as a minimum. For methods with a large intrinsic variability, increasing the number of samples could be considered to increase the level of confidence in the result and avoid drawing incorrect conclusions.

Increasing the number of injections would seem to help but it does not, since only one sample is tested multiple times, and does not give us a real assessment of stability.

Ligand binding assays take this a bit further than MS assay do, since here a replicate indicates that a sample (contents of a tube) were analyzed in duplicate- one sample, two intermediate results, one final reported result (if all goes well).   

In most freeze thaw experiments there are three tubes for each concentration time point. For MS there are 3 results, for ligand there are six intermediate and three final.  There are two replicates (repeats or analyses for each tube in ligand binding but only one in MS. 

Let’s better define “is” or “replicates.”