2.4. Sample Preparation and LC-MS/MS Quantification of MMAE

A detailed tissue homogenization procedure has been reported previously [14]. Briefly, different volumes of RIPA buffer containing protease inhibitor were added into the weighted tissue samples to obtain different dilution factors in each tissue. The dilution factor was 5 for heart, liver, lung, spleen, pancreas, kidney, skin, and fat, 8 for bone and muscle, and 4 for brain samples. Tissue samples were homogenized using a BeadBug™ microtube homogenizer (Benchmark, USA) at the maximum speed for 15 s followed by a 30-s ice cool down, and repeated three times. Blood samples without dilution were directly homogenized for 30 s at maximum speed and were treated the same way as tissue samples.

Then, 500 µL acetonitrile containing 0.1% formic acid was added into 100 µL of plasma, tissue, or tumor homogenate samples and then spiked with 20 µL of IS solution (D8-MMAE 150 ng/mL). After vortexing and centrifugation at 15,000× g for 15 min at 4 °C, the supernatants were transferred to glass tubes and dried under nitrogen flow at 32 °C. The dried residuals were reconstituted with 60 µL of acetonitrile/water (95:5 v/v) containing 0.1% formic acid, gently vortexed, and immediately transferred into HPLC vials.

Standards and quality control samples (QCs) were prepared for plasma and each tissue matrix. Then, 250 µL of the control plasma or matrices were spiked with 20 µL of IS solution (D8-MMAE 150 ng/mL) and 10 µL of MMAE stock solution diluted with acetonitrile, and then, 500 µL of acetonitrile containing 0.1% formic acid was added. The final concentrations of standard were 0.5, 1, 10, 100, 200, and 500 ng/mL. QCs were prepared similarly for the final concentrations of 5, 50, and 250 ng/µL.

A Waters Acquity LC-MS/MS system was used with electrospray interphase and triple quadrupole mass spectrometer. An ACQUITY UPLC BEH Amide column (Waters, Milford, MA, USA) was used with 5 mM ammonium formate and 0.1% formic acid as the aqueous phase, and 0.1% formic acid and 1 mM ammonium formate were used as the organic phase. The gradient flow rate was 0.25 mL/min. The lower limit of quantification of MMAE was 0.2 ng/mL (or ng/g) for plasma, blood, tissues, and tumor.