Table 2.

Assay Validation Parameters with their Testing Strategy, Acceptance Criteria and Results

Parameter	Testing strategy	Acceptance criteria	TAP	CPA1	CPA2
LLOQ	Standard dilution series in surrogate matrix	Accuracy ±25%, precision ≤25%, total error ≤40%	0.537 ng/mL	25.9 ng/mL	25.7 ng/mL
ULOQ			1170 ng/mL	18900 ng/mL	18800 ng/mL
Inter assay accuracy	standard dilution series in surrogate matrix; QC1, QC2, and QC3; three batches	Accuracy ±20% (±25% at LLOQ/ULOQ) for non-zero calibrators and for QC samples (compared to nominal values), total error ≤40%	Passed	Passed	Passed
			Passed	Passed	Passed
			Passed	Passed	Passed
Inter assay precision	Standard dilution series in surrogate matrix; QC1, QC2, and QC3; three batches	Precision ≤20% (≤25% at LLOQ/ULOQ)	Passed	Passed	Passed
			Passed	Passed	Passed
			Passed	Passed	Passed
Intra assay accuracy	QC1, QC2, and QC3; one batch	Accuracy ±20% (±25% at LLOQ/ULOQ) for QC samples (compared to nominal values), total error ≤40%	Passed	Passed	Passed
			Passed	Passed	Passed
Intra assay precision	QC1, QC2, and QC3; one batch	Precision ≤20% (≤25% at LLOQ/ULOQ)	Passed	Passed	Passed
			Passed	Passed	Passed
Parallelism	Eight samples three times serially diluted 1:2 in surrogate matrix	Accuracy ±20% for at least two dilutions in 80% of the samples (e.g. five of six samples)	up to 1:8 dilution	up to 1:8 dilution	up to 1:2 dilution
Reproducibility	Six to ten samples tested in independent two batches	Precision from two or more batches ≤30% in 80% of the samples	Passed	Passed	Passed
Carryover	Standard dilution series in surrogate matrix and blank samples	Blank samples following the calibrators should be below LLOQ	at levels of >391 ng/mL	none	none
Stability	Freeze / thaw stability sample; up to three cycles	Accuracy ±25% of the nominal value	Passed	Passed	Passed
	Short-term stability sample; 2 hours and 24 hours at RT		Passed	Passed	Passed
	Freeze / thaw stability proteolyzed sample; one cycle		Passed	Passed	Passed