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A rapid HPLC-MS/MS method for the simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human blood samples

Nature Protocols volume 4, pages 526–534 (2009)Cite this article

Abstract

In the past few years, high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has matured to a true alternative to antibody-based immunoassays in routine therapeutic drug monitoring. In transplantation medicine, mass spectrometry-based assessment of immunosuppressant drug levels is considered a gold standard diagnostic procedure. We describe a fast state-of-the-art routine online solid-phase extraction (SPE) HPLC-MS/MS analysis platform that allows monitoring of cyclosporine A, tacrolimus, sirolimus and everolimus from 50-μl aliquots of EDTA whole blood specimens within 3.4 min total analysis time. Sample purification is done by offline protein precipitation followed by two automated chromatographic separation steps. Mass spectrometry-based analyte quantification relies on selected reaction monitoring experiments. The assay underwent complete validation and performance evaluation studies and performs very well in several international proficiency testing schemes. In daily routine, it allows reporting of about 75 patient sample results per work shift with a typical total individual sample turnaround time of less than 3 h.

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Figure 1: Plumbing of the six-port switching valve located in the column compartment and connecting the SPE and HPLC chromatography step.
Figure 2: HPLC-MS/MS-derived analyte chromatograms for the lowest calibrator level (LLOQ) used.
Figure 3: Relative Bland Altman plot for the comparison of HPLC-MS/MS and immunoassay (FPIA)-derived cyclosporine A through level measurements.
Figure 4: Longitudinal cyclosporine A trough level profile of a male (m) heart transplant (HTX) recipient.

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Authors and Affiliations

  1. Division of Mass Spectrometry and Chromatography, Institute of Medical and Chemical Laboratory Diagnostics (ZIMCL), University Hospital Innsbruck, Anichstraße 35, Innsbruck, A-6020, Austria

    Christoph Seger, Karin Tentschert & Andrea Griesmacher

  2. Biocrates Life Sciences AG, Innrain 66, Innsbruck, A-6020, Austria

    Wolfgang Stöggl & Steven L Ramsay

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  1. Christoph Seger
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  4. Andrea Griesmacher
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  5. Steven L Ramsay
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Correspondence to Christoph Seger.

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Seger, C., Tentschert, K., Stöggl, W. et al. A rapid HPLC-MS/MS method for the simultaneous quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human blood samples. Nat Protoc 4, 526–534 (2009). https://doi.org/10.1038/nprot.2009.25

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