Plasma samples were thawed and mixed well by vortexing. The plasma samples were extracted by protein precipitation (PPT) with acetonitrile. A 50 μL of rat plasma was spiked with 10 µL of the mixed IS solution (100 ng/mL in 50% methanol). After adding 200 μL of acetonitrile, the sample was vortexed for 1 min and centrifuged at 10,000 × g for 5 min. The supernatant of 100 µL was diluted with distilled water of 100 μL and briefly vortexed and then 3 µL of aliquots were injected into the UPLC-MS/MS system. The thawed tissue samples were weighed and homogenized (Ultra-Turrax®, IKA® T10 basic) in five volumes of distilled water. The homogenized tissue samples (100 μL) was spiked with 10 µL of the mixed IS solution (100 ng/mL in 50% aqueous methanol). After adding 400 μL of acetonitrile, the sample was vortexed for 1 min and centrifuged at 10,000 × g for 5 min. The next step was analyzed in the same manner as in plasma. The measured sample concentrations above the upper limit of the standard curve should be diluted and reanalyzed.
Method validation A simultaneous determination method of the three PFCs in rat plasma and tissues was validated in accordance with the Guidance for Industry: Bioanalytical Method Validation by the US Food and Drug Administration . The verification of method was estimated by specificity, linearity, precision, accuracy, and recovery.
RESULTS AND DISCUSSION
Analytical method development and validation
The mass spectrometric parameters were optimized…