Table 4. Result Summary for the Validation of HDIT101 Quantification Assay.

validation components	summary
analyte	HDIT101 mAb using its surrogate peptide LC1. MRM transition: 896.06 > 807.55
IS	stable-isotopically-labelled version of LC1 peptide (SIL-LC1)
matrix	human plasma
sample volume	25 μL
analytical method	UPLC–MRM/MS, 10 μL injection, 3.5 min LC gradient
sensitivity	LLOQ at 20 μg/mL, six replicates in every batch.
	precision (% CV)				accuracy (% bias)
within-batch	9.0	7.2	12.3	13.9	–3.0	4.8	–8.9	–2.1
batch-to-batch	11.0				–2.0
specificity	no significant interfering peak for LC1 (<6.9% of lowest LLOQ signal) and SIL-LC1 (<2.3% of mean SIL-LC1 signal) observed in blank human plasma samples
carry-over	no significant interfering peak for LC1 (<12.9% of lowest LLOQ signal) and SIL-LC1 (<0.2% of mean SIL-LC1 signal) observed in eluent samples following CAL I standards
calibration range	20–5000 μg/mL
QC within-batch results	precision (% CV)				accuracy (% bias)
QC A—60 μg/mL	7.2	7.9	9.2	12.0	3.4	6.1	8.6	–0.1
QC B—2000 μg/mL	5.1	7.8	12.6	12.6	8.2	6.1	3.8	–0.3
QC C—4000 μg/mL	5.9	10.1	11.3	8.4	2.7	4.8	0.3	0.1
QC batch-to-batch results	precision (% CV)				accuracy (% bias)
QC A—60 μg/mL	9.1				4.0
QC B—2000 μg/mL	9.0				4.8
QC C—4000 μg/mL	8.4				1.7
freeze-and-thaw stability	demonstrated over three freeze-and-thaw cycles from –80 °C to room temperature on the three QC levels in triplicates.
processed sample stability	demonstrated for 24 h at 10 °C on full calibration range and QC samples with <11% CV between reinjected and original results.
long-term stability	demonstrated over four months at –80 °C for the three QC levels in duplicates.
recovery	average of 98.2% of recovery for HDIT101 for the three QC levels in triplicates using protein A IP (6.9% CV)
matrix effect	normalizeda matrix-effect consistency for each QC level in duplicate.
QC A—60 μg/mL	36% (11.4% CV)
QC B—2000 μg/mL	40% (6.1% CV)
QC C—4000 μg/mL	71% (6.3% CV)

^aLC1 matrix effect was normalized by the SIL-LC1 matrix effect.