Material and Methods
The sample preparation was performed in line with the MassTox® Immunosuppressants protocol (Chromsystems). In brief: 50 µl of sample (EDTA-whole blood), reconstituted 6PLUS1® calibrator (order no 28039/XL) or MassCheck® control (order no 0081-0085, 0089) was pipetted into a 1.5 ml reaction vial. 25 µl of the reconstituted Internal Standard Mix (order no. 93946/RUO) and 100 µl Extraction Buffer (order no 93005/RUO) were added and mixed briefly. 250 µl of Precipitation Reagent (order no. 93003/RUO) was added, vortexed for 1 min and incubated for 2 min at ambient temperature, then centrifuged for 5 min at 15000 g. The supernatant was diluted with Dilution Buffer 1 (92007/RUO; ratio 1:1).
10 – 50 µl were injected at a temperature for the autosampler of 8°C – 15°C. Substances were separated on MasterColumn® A (order. no 92110) with a column temperature of 70°C. Flow rate, position of selection valve and binary gradient can be found in table 1. Detection was performed with electrospray ionisation in positive ion mode with a Sciex 4500 mass spectrometer. Used multiple reaction monitoring (MRM) transitions are found in table 2.