2.5. Assay Validation

The LC-MS/MS method was developed and validated in accordance with the current FDA guidelines (Guidance for Industry: Bioanalytical Method Validation of USFDA). Food (2018)

Specificity and selectivity in blank mouse plasma (n=6) were evaluated by comparing the chromatograms with that of 20–223, and IS-spiked sample for the assessment of potential interferences with endogenous compounds at retention time of analytes or IS.. Assay sensitivity was quantified using the signal-to-noise ratio (S/N) of each analyte in the CSs, and the LOD and LLOQ were defined peak areas three and ten-fold greater S/N ratio, respectively.

Calibration curves were constructed by plotting the concentrations of 20–223 on the x-axis and the peak area ratio (analyte/IS) of 20–223:IS on the y-axis. A blank sample, a sample containing IS only (termed “zero sample”), and the CSs listed above were used for the generation of each calibration curve. Each CS and QC were required to be ± 15% standard deviation (SD) of their expected value with the exception of the LLOQ which was required to be ± 20%. CSs were run in ascending order followed by two consecutive “zero samples” in order to assess any carry-over, which was required to be less than 20% of the expected S/N for LLOQ.

The inter-day and intra-day accuracy and precision were determined by analyzing variations in the QCs calculated concentration. This was performed in five replicates over three consecutive days, wherein samples were prepared in mouse plasma as previously described. Precision was defined as the percent relative standard deviation (%RSD) and accuracy was defined as the percent bias (% Bias) with acceptance criteria of ± 15% (except ± 20% at LLOQ) for both.