Method Development, Validation, and Sample Analysis for Regulated Quantitative Bioanalysis Using LC-MS/MS - LC-MS in Drug Bioanalysis - page 57

Experiment #1 . For interference compounds which are not quantified within the
assay, spike the interference compound at the C max or minimum at ULOQ level into
Low QC.

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

Experiment #2 . For interference compounds which are quantified within the assay,
each analyte need to be spiked individually, and the contribution of the single ana-
lyte to all other analytes are evaluated.

Acceptance . Same as above for carryover assessment.

5.2.6 Relative Extraction Recovery

Purpose . To obtain the relative extraction recovery from matrix for all analyte and
internal standard. This experiment is required for stable isotope labeled IS.

Experiment . Three analyte levels generally near the low, medium, and high QC
concentrations and the internal standard at the working spiking concentration were
tested. The response of extracted samples (Preprocess) is compared to the response
of samples representing 100 % recovery (Postprocess).

Acceptance . There is no requirement for overall recovery as long as the RSDs for
replicates at each concentration (preprocess and postprocess) are £ 20.0 %

5.2.7 Short- and Long-Term Solution Stability

Purpose . To evaluate the stability of the analyte and internal standard in stock and
working solvent if they differ. It contains two sets of experiments:

Experiment #1 . Short-term analyte and IS bench top (i.e., room temperature or wet
ice bath) solution stability. A minimum of one concentration such as ULOQ spiking
solution must be tested. Some laboratories require solution stability at standard 2
(which has concentration typically twice of the LLOQ) or LLOQ spiking solution
level. Test for minimum 6 h.

Experiment #2 . Long-term analyte and IS storage solution stability.

Acceptance . The stability of solution is acceptable for the period of storage if the % dif-
ference between the stability of the bench top solution and control solution is £ 10.0 %.

5.2.8 Short and Long-Term Matrix Stability

Purpose . To evaluate the stability of the analyte in matrix. There are three different
experiments: