Table I.

Summary of the key recommendations for stability assessment in regulated bioanalysis

Item	Remark*
General
• Stability assessment should cover all relevant conditions encountered in practice	A
• Storage duration should be at least equal to the maximum storage period for any individual study sample	A
• Deviation of the result for a stored sample from the reference value should not exceed 15% (chromatography) or 20% (binding assays)	S/L
• Two concentration levels (low and high) suffice for stability assessment; stability at an over-curve level is not necessary unless scientifically called for	A
• A single time point suffices for each stability assessment with an appropriate number of replicates	A
• Stability results should generally not be extrapolated to other storage conditions	A
Reference values
• Results for stored samples can be compared to nominal or t = 0 values	A
• Fresh calibrators are essential for long-term stability assessment, but calibrators stored frozen suffice for other stability determinations as long as stability under frozen conditions is confirmed	A
Transferability
• Stability results obtained in other laboratories can be used if storage conditions are similar and if the assessment has been performed in an acceptable way	A
Failing results
• Stability results should be rejected in the case of an analytical error or failing calibration or QC results and be accepted otherwise	A
• Stability results not meeting the criteria indicate that investigated storage conditions are unsuitable	A
• Possible analytical outliers can be investigated by re-analysis in duplicate	A
Bench-top, freeze/thaw and long-term frozen stability
• Storage and analysis conditions should mimic the situation for study samples	A
• Frozen stability at a lower temperature is not needed if already demonstrated at a higher temperature, unless scientifically required	S
Stock stability
• Stability assessment is needed for lowest and highest concentrations which will be stored in practice	A
• Stability assessment is needed for the conditions used for long-term storage and for bench-top use	A
• Deviation of the result for a stored sample from the reference value should not exceed 10%	S
• Stability assessment for internal standards is only required when scientifically called for	S
Extract stability
• Assessment of relative stability (against stored extracts of calibrators) suffices as long as extracts of calibrators and QCs will be stored together with study samples	S
Whole blood stability
• Stability assessment is generally not necessary if stability in plasma/serum has been demonstrated under the same conditions, unless the analyte is known to behave differently in the presence of blood cells	A
• Stability assessment can be performed by direct analysis of whole blood or by harvesting and subsequent analysis of plasma/serum	A
Tissue stability
• Stability cannot be demonstrated for intact tissue, but only for tissue homogenate	A
• Storage of study samples in the form of homogenate is recommended	A
Endogenous analytes
• Stability assessment should be similar to that for xenobiotics, whenever possible	A
• Stability should be assessed using the authentic matrix and the authentic analyte	A
Deviating matrix types
• Additional stability assessment should be considered if the biological matrix in a particular study deviates considerably from a normal control matrix and if this deviation in composition is likely to impact analyte stability	A
Incurred sample stability
• Stability in incurred samples should be considered in case of possible differences in stability in spiked and incurred samples	S

QC quality control

*Relevant for small molecules (S), large molecules (L), or all types of molecules (A)