As with the original method, a fast and inexpensive on-line solid-phase extraction (SPE) is performed through a twodimensional liquid chromatography configuration. Using this configuration, sample clean up effectively minimizes any ion suppression interference during the MRM measurements. This permits the use of Cyclosporin D (CsD) as an internal standard for quantitation of all 5 immunosuppressant drugs included in the extended panel.
Samples were prepared for MPA measurements by adding 100 μL of a diluting solution* to either 50 μL of either plasma samples, calibrators or controls, This mixture was then vortex mixed for 30 seconds. After centrifugation at 13000 rpm x 5 min., 100 μL of supernatant were transferred to either an autosampler vial or a microtiter plate.
The hardware configuration included an Applied Biosystems/MDS SCIEX API 3200TM Triple Quadrupole Mass Spectrometer equipped with a Turbo VTM source. This source operates in positive ion mode at a voltage of +5500 volts and with a Turbo V gas heated at 650 C.
Multiple Reaction Monitoring (MRM) measurements were made using declustering potential (DP) and collision energy (CE) values, which were automatically optimized by the Analyst software for each of the analytes. DP values ranged between 40 and 140 V; CE values were between 30 and 65 eV.
Two-dimension chromatography was performed through a split arrangement of the modules of the LC pumping system (either a Perkin Elmer Series 200 Micro Pump, an Agilent 1100 Binary Pump with the pump heads disconnected from the mixing tee or a Shimadzu Prominence or 10Dvp pumps) and a computer-controlled Valco Valve (10-port, 2 positions) plumbed as illustrated in Figure 2. www.appliedbios