Introduction
Cyclosporine A, tacrolimus, sirolimus, and everolimus are four of the most commonly administered immunosuppressant drugs and play a central role in the success of tissue and organ transplants. These drugs are most typically analyzed from whole blood using LC/MS/MS. However, because of the analytical challenges posed when working with whole blood, many of the published methods rely upon complex and/or expensive extraction steps utilizing offline solid phase extraction, on-line solid phase extraction, or the use of pre-columns prior to the actual analytical column. In this current work, we present a rapid and effective method for the analysis of these four immunosuppressants from whole blood that use a simple protein precipitation step followed by direct injection onto a wide-pore core-shell HPLC column (Aeris WIDEPORE 3.6 μm XB-C18). The method displays excellent accuracy and is sensitive down to the low μg/L (ng/mL) range.
Immunosuppressants are a class of drugs that inhibit the body’s immune response and are typically administered to prevent the rejection of transplanted organs (e.g. kidney) or tissue (e.g. bone marrow), and may also be used to treat various autoimmune disorders such as Crohn’s Disease or rheumatoid arthritis. The first effective immunosuppressant drug was cyclosporine A (or CsA), an undecapeptide, initially discovered by researchers at the pharmaceutical company Sandoz.1 Since the development of CsA, many other immunoppressant drugs have been developed, including the macrolides tacrolimus (FK506), serolimus (also known as rapamycin), and everolimus. While all of these drugs ultimately act to suppress the immune response, they each exert their effects through different mechanisms. Cyclosporine A binds to the protein cyclophillin, and the resulting CsA-cyclophillin complex blocks the calcineurin-mediated transcription of the interleukin 2 (IL-2) gene in antigen activated T cells, thus preventing the growth, differentiation, and proliferation of T cells that mediate the immune response.2,3 Tacrolimus binds to the protein FKBP12 (FK506 binding protein), and the resulting complex prevents the cascade of reactions that ultimately lead to a reduction in IL-2 transcription.3 Unlike CsA and tacrolimus, which block synthesis of IL-2, sirolimus and everolimus exert their activity by blocking the response of T-cells to IL-2.4Because of their potent immunosuppressant effects and relatively narrow therapeutic index, therapeutic drug monitoring of patients is required in order to insure the efficacy of the treatment, and also to minimize toxic side effects.5, 6 Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) has become the analytical method of choice for the analysis of immunosuppressants. These drugs must be monitored from whole blood, which poses a sample preparation challenge as matrix effects can confound analyses through ion suppression and/or enhancement, and can also affect the reproducibility and accuracy of analytical methods. To overcome the challenges posed when working with whole blood, many methods that have been developed for immunosuppressant analysis involve off-line solid-phase extraction7, which can be time-consuming and expensive, or complex on-line extraction methods that not all labs are equipped to operate.8, 9, 10 Herein, we present a simple and rapid method for the analysis of immunosuppressants from whole blood that utilizes a simple protein precipitation step followed directly by LC/MS/MS analysis using a wide-pore coreshell HPLC column. This fast, simple method shows excellent precision and accuracy down to the μg/L concentration range.
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