Samples were prepared by adding 200 μL of a diluting solution to 100 μL of either blood serum, calibrators, or controls, and vortex mixing the sample for 30 seconds. After centrifugation at 13,000 g x 10 min., 200 μL of supernatant were transferred to either an autosampler vial or a microtiter plate.
The hardware configuration included an Applied Biosystems/MDS Sciex API 3000 Triple Quadrupole Mass Spectrometer equipped with TurboIonSpray source. The source operates in positive ion mode at a voltage of + 4500 volts and with a turbo gas flow of 8 L/min of air heated at 350C (nominal heating-gun temperature).
MRM measurements were made using declustering potential (DP) and collision energy (CE) values, which were automatically optimized by the software for each of the analytes. (DP values ranged between 40 and 70 V; CE values were between 19 and 45 eV).
Two-dimension chromatography was performed through a split arrangement of the single heads of an Agilent 1100 Binary Pump and a computer-controlled Valco Valve (10-port, 2 positions), plumbed as depicted in Figure 1. First dimension chromatography was accomplished via an Applied Biosystems POROS R2/20, 2.1 x 30 mm perfusion-column. Second dimension chromatography was performed by a Phenomenex Luna 5 μm Phenyl- Hexyl, 2 x 50 mm column (p.n. 00BA 4257-B0) housed in an Agilent Column Oven kept at 60oC. 10 μL samples were injected into the column via an Agilent 1100 Wellplate Autosampler, plumbed as shown in Figure 1.
All hardware set-up details, solution and reagent requirements, and operating parameters were stored on CD for quick transfer of the proposed methodology.
* Diluting solution consisted of acetonitrile containing 50 ng/mL of DESRapamycin as an internal standa