3.3. Samples Preparation

Blank lung tissue and pharmacokinetic experimental lung tissue samples were homogenized using the KT 30 homogenizer (Korea Process Technology, Seoul, Korea). Lung tissues were accurately weighed, and four-fold (w/w) cold phosphate-buffered saline (PBS) was added to process the homogenization. Tissues were kept in the ice-bath during the homogenization process, and tissue homogenates were stored at −20 °C until use.

The stock solution of sirolimus (1 mg/mL, in acetonitrile) was appropriately diluted with acetonitrile to obtain sets of working standard solutions (5–500 ng/mL). All the stock and working standard solutions were stored at −80 °C until experimental analysis. Standard samples were prepared in whole blood by spiking 90 μL of blank porcine blood with 10 μL of each working standard solution. Similarly, standard samples in porcine lung tissues were prepared by spiking 15 μL of each working standard solution into 135 μL of blank lung tissue homogenate. The final sirolimus concentrations in blood and lung tissue homogenate were 0.5, 1, 2, 5, 10, 20, and 50 ng/mL. Extraction of the drug and IS was performed using a protein precipitation method as previously described [38,39]. To each standard sample, a 2-fold volume (200 μL for blood and 300 μL for tissue homogenate samples) of IS solution in acetonitrile (1 ng/mL) was added, followed by vortexing for 1 min for deproteinization. After centrifugation at 14,000 rpm for 15 min, 100 μL of the supernatant was collected for analysis using the devised LC-MS/MS methods. QC samples were separately prepared in blood and lung tissue homogenate using similar procedures. The QC sample included LLOQ (0.5 ng/mL), LQ (1.5 ng/mL), MQ (15 ng/mL), and HQ (40 ng/mL).