| Overview of Validation Exercises and Criteria |
| Validation Exercise |
Minimum
Experiments |
Performance Criteria |
| Selectivity |
Matrix blanks: 6 lots, n=1 for each lot
Matrix blank fortified with IS: 6 lots, n=1 for each lot
LLOQ Selectivity Sample: 6 lots, fortified with analyte at LLOQ level and IS. n=3 for each lot |
At least 5 out of the 6 lots must meet the following criteria:
Response for the analyte in matrix blanks or matrix blank fortified with IS must be £20% of the mean analyte response in the acceptable LLOQ calibration standards
Response for IS in matrix blanks must be ≤5% of the mean IS response in the acceptable LLOQ calibration standards
At least two-thirds of the selectivity LLOQ replicates for each lot must meet accuracy acceptance limit, and the mean accuracy must be within ±20.0% of the nominal concentration |
| Cross-analyte Interference |
Each analyte at ULOQ evaluated separately. IS at the level of use evaluated separately |
Interference must be £ 20% of the mean analyte peak response or £ 5% of the mean IS response of the acceptable LLOQ calibration standards |
| Carryover |
Minimum of 2 blank samples after the injection of each ULOQ standard |
Analyte response £20% of the mean analyte response in the acceptable LLOQ standards, IS response ≤5% of the mean IS response in the acceptable LLOQ standards |
| Linearity |
Minimum of 6 non-zero calibration standard (CS) levels |
(R2) ≥0.985 |
Calibration
Standards:
Accuracy |
Injected at the beginning and end of the analytical run |
Minimum 6 non-zero (or 75% of total) CS must be within ±15.0% RE of nominal (exception: LLOQ within ±20.0 %RE) |
QC Samples –
Core Validation |
Three concentration levels: Low, Mid, High; n=6 at each level |
Minimum 50% of the QC replicates at each level and 66.7% of all QCs must be within ±15.0% RE of nominal
Mean inter- and intra-assay accuracy within ±15.0% RE of nominal; precision £15.0% RSD |
QC Samples –
Ancillary Runs |
Three concentration levels: Low, Mid, High; n ≥ 2 at each level |
≥66.7% of all QCs and at least 50% at each level must be within ±15.0% RE of nominal |
LLOQ Samples
(Sensitivity) |
n=6, ≥ 1 run |
Mean accuracy within ±20.0%RE of nominal; precision £20.0% RSD |
| Recovery |
Analyte at low, medium and high levels, and IS at the level of use: pre-extraction spiked samples (n=6) are compared with mean response of post-extraction spiked matrix samples (n=6) |
Recovery for analyte and IS must be
relatively consistent across all QC levels |
| Dilution (Parallelism) |
One level (minimum 10 fold dilution); n=6 |
Mean accuracy within ±15.0% RE of nominal; precision £15.0% RSD |
| Matrix Effect |
Post-extraction spiked samples (n=6, at each QC low, mid and high level) are compared with mean response of 6 injections of analyte or IS in solvent |
MF will be calculated and reported for the analyte and for the IS |
| Ruggedness |
Minimum of two variables over the course of validation (e.g. different column, instrument and/or analyst) |
Mean inter- & intra-assay accuracy within ±15.0% RE of nominal; precision ≤15.0% RSD |
