Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS - LC-MS in Drug Bioanalysis - page 55

centration as lowest standard; Low = 3× the concentration of the lowest standard;
Medium: ~40-60 % of the ULOQ; High: ~75-85 % of the ULOQ.

5.2 Method Validation Experiments

5.2.1 Intra- and Interday Precision and Accuracy Evaluation

Purpose . To evaluate assay accuracy and precision based on the overall performance
of analytical QCs as well as STDs.

Experiment . Three test batches which are prepared, extracted, and analyzed in three
separate days. It contains replicate of two sets of STDs and six replicates QC at
LLOQ, Low, Medium, and High QC. The STDs must be prepared fresh at the
day of extraction. QCs can be from previously prepared and qualified pools.

Acceptance . For STDs, the back-calculated concentration must not deviate £ 15.0 %
(£20.0 % for the LLOQ) from their individual target concentrations. At least three-
fourths of the individual STDs must be acceptable. For QC, the mean of the repli-
cates at each QC concentration and Relative Standard Deviation (RSD) must be
£15.0 % (£20.0 % for the LLOQ) from their target concentrations. At least two-
thirds of the individual QCs at each level must be acceptable.

5.2.2 Dilution Integrity and Linearity

Purpose . to evaluate accuracy and precision of the QC with an analyte concentra-
tion originally above the ULOQ.

Experiment . Two levels of Dilution QC are required. The Dilution QC for dilution
integrity is typically at 10× of the High QC. The Dilution QC for dilution linearity
should be at a concentration above projected maximum concentration ( C max ). If the
projected C max is not available, the recommended concentration would be 50× or
100× of High QC. To test dilution integrity or linearity, the Dilution QCs need to be
diluted at dilution factor (DF) of 10, 50 or 100 to bring the final concentration
within the range. It may require multiple steps of dilution. This experiment only
needs to be conducted once per each validation.

Acceptance . Same as described in intra- and interday precision and accuracy
evaluation.

5.2.3 Carryover of Analyte and Internal Standard

Purpose . To evaluate the impact of carryover on the quantitation of the analyte and
internal standard.