Application of a design of experiment approach in the development of a sensitive bioanalytical assay in human plasma

To support a first-in-human (FIH) clinical study in healthy volunteers, a human plasma assay, a 20-fold more sensitive method than the validated non-clinical LC-MS/MS assays, was requested. For the clinical assay, a LLOQ of 0.050 ng/mL for Compound A and 0.100 ng/mL for Compound B was desired to accurately determine the analyte concentrations in human plasma samples across all treatment groups. A design of experiment (DOE) investigation was performed in an effort to optimize the extraction procedure of the bioanalytical assay used to support the first in human study and future clinical studies. Three factors, extraction buffer pH (two pHs), volume ratio of organic solvent to plasma (two ratios), and extraction shake time (three times), were selected for the DOE. Both analytes were analyzed at a low concentration, 0.150 ng/mL, and a stable isotope label internal standard was used for each analyte. To estimate the recovery of each analyte from the extraction, the response ratio of each analyte over the respective internal standard was used, and to estimate matrix effects, the absolute response (peak area) of each analyte was used. The results of the DOE indicated that the three factors tested had a more significant effect on the extraction of the metabolite, Compound B, compared to that of the parent, Compound A. The extraction buffer pH had the greatest influence on Compound B and the volume of extraction solvent had an influence on both analytes. Unexpectedly, a longer extraction time caused an apparent decrease in the overall recovery for both analytes. This was presumably due to an increased extraction of interfering matrix components. Optimal conditions were achieved for the combined analysis of both compounds using the DOE approach.