Purpose: An HPLC-MS/MS method for the determination of Compound-A in human plasma after inhaled administration was developed and successfully applied to regulated analysis of human plasma samples. Challenges encountered during method development will be presented
Method: Plasma samples were prepared for analysis using automated salt assisted liquid -liquid extraction (SALLE) in 96 well plates. A stable isotope labeled internal standard was used. HPLC-MS/MS analysis was performed on a Waters HSS T3 column (50 x 2.1 mm, 1.8 µm) and a 3.5 minute gradient elution using 0.1% propionic acid and 0.1% propionic acid in acetonitrile. The eluent was introduced to the electrospray source of the triple quadrupole mass spectrometer instrument at 0.75 ml/min flow rate.
Results: Salt assisted liquid-liquid extraction and solid phase extraction (SPE) were assessed with respect to sample preparation. SALLE resulted high recoveries and favorable matrix effects comparable to SPE. A variety of organic acid additives were evaluated for impact on sensitivity. The addition of propionic acid to the HPLC mobile phase resulted in a signal response 3x higher as compared to formic acid. Using 0.25 mL human plasma sample aliquot, the assay was validated over the concentration range of 4-2000 pg/mL. Inter-day sample accuracy was between 93.6-104.7%. Compound-A was stable in human plasma after three freeze-thaw cycles and 4.0 hours at room temperature.
Conclusion: An HPLC–MS/MS method for the determination of Compound-A in human plasma was developed and validated. The absence of a relative matrix effect and interferences was demonstrated. The method was successfully applied to human clinical samples, and incurred sample analysis results indicated that the method was reliable and reproducible.