High throughput online solid-phase extraction tandem mass spectrometry for immunosuppressive drugs determination in whole blood

Abstract

High throughput mass spectrometry-based clinical assay technologies have emerged in response to the increase in the number of patients who require fast, accurate, and robust analysis methods. Typical runtimes for quantitation based on liquid chromatography – tandem mass spectrometry (LC–MS/MS) are in the order of a few minutes per sample. The use of tandem mass spectrometry with online solid phase extraction (SPE-MS/MS) instead of chromatography columns can offer high-throughput determination without compromising sensitivity, precision, and accuracy. In this communication, we describe a newly developed fast, accurate, and robust method on the Agilent RapidFire-MS/MS System (RF-MS/MS) for the simultaneous determination of Sirolimus, Everolimus, Tacrolimus and Cyclosporine in whole blood. The commercial multilevel calibrator and quality control (from Chromsystems) were precipitated using a zinc sulfate and methanol mixture solution containing the deuterated internal standards. Precipitated samples were centrifuged and transferred to a 96-well plate for analysis. Immunosuppressive drugs analysis was performed online with C18 extraction small cartridge onto an Agilent RapidFire 400 and 6495 Triple Quadrupole Mass Spectrometer by MRM mode in positive electrospray ionization. Imprecision and accuracy were investigated analyzing 10 Asqualab External Quality Controls. We compared the results obtained with both techniques. The new method showed good linearity 2.35–24.90 μg/L for Sirolimus, 2–21.80 μg/L for Everolimus, 1.43–23.20 μg/L for Tacrolimus and 22–757 μg/L for Cyclosporine with an r 2 > 0.995. RF-MS/MS showed good intraday and interday precision (< 10%) for all immunosuppressive drugs. High accuracy was achieved with 10 samples from the Asqualab program (< 10%). No significant carryover was measured for any of the drug analytes and high endurance of the solid phase extraction cartridge (> 15,000 injections). The RapidFire – MS/MS system provided excellent agreement with our routine LC–MS/MS method. Its main advantage is the high throughput analysis of 13 s per sample, the low consumption of toxic organic solvents, and the high robustness. It could represent an alternative to LC–MS/MS instruments in therapeutic drug monitoring in human biological fluids.