Simultaneous quantification of cyclosporine, tacrolimus, sirolimus and everolimus in whole blood by liquid chromatography–electrospray mass spectrometry
Introduction
Cyclosporine A (CsA), tacrolimus, sirolimus and everolimus (Fig. 1) are immunosuppressive drugs used in organ transplantation [1]. The calcineurin inhibitors (CsA and tacrolimus) block interleukin-2 production, leading to a decrease in T lymphocyte proliferation [2]. The mammalian target of rapamycin (sirolimus and everolimus) blocks interleukin-2 induced intracellular signal transduction, leading to an inhibition of T cell cycle progression [2]. Due to complementary mechanisms of action, these two classes of agents are often combined in the clinic, resulting in a synergistic effect [3]. Due to high pharmacokinetic variability, narrow therapeutic indexes and many potential drug–drug interactions, close monitoring of blood concentrations is required to prevent rejection and minimize toxicity [4]. It is currently recommended to quantify these drugs in whole blood, due to a high distribution in erythrocytes, between 40–60% for CsA [5], about 95% for tacrolimus [6] and sirolimus [7] and 75% for everolimus [8].
Traditional monitoring of CsA used trough concentration (C0) as the parameter of choice [9], however, it has been shown that the latter was not a good surrogate of the area under the concentrations time curve (AUC) and that a measurement 2 h after drug intake (C2) correlated better with clinical outcome [10]. For the monitoring of tacrolimus [11], sirolimus [12] and everolimus [13], C0 measurement currently remains the main approach. The usual whole blood therapeutic indexes, which depend on transplantation type, time after graft, association of other immunosuppressive agents and analytical methods, range between 100–400 ng/mL for CsA at C0 [14], 600–1500 ng/mL for CsA at C2 [15], 5–20 ng/mL for tacrolimus [14], 4–20 ng/mL for sirolimus [16] and 3–8 ng/mL for everolimus [17] at C0.
Immunological methods are largely used in routine laboratories for quantification of immunosuppressive drugs. Despite an increase in the selectivity of these techniques, the disadvantage is that cross-reactions can occur with some metabolites, resulting in an overestimation of the measured drug concentrations with unacceptable biases in some clinical situations [18], [19], [20], [21], [22], [23], [24], [25].
The use of liquid chromatography (LC) for the quantification of immunosuppressive drugs is increasing and drastically enhances the selectivity of the method, and allows the quantification of the main drug independently from its metabolites. These methods are now considered to be the gold standard in therapeutic drug monitoring. Several LC methods coupled with ultraviolet (UV), mass spectrometry (MS) or tandem mass spectrometry (MS/MS) detection have been developed in case of immunosuppressants with various sample preparation procedures including liquid–liquid extraction (LLE), solid-phase extraction (SPE), column-switching and direct injection [26], [27], [28], [29], [30], [31], [32].
In this publication, a rapid, sensitive and selective method for the simultaneous quantification of CsA, tacrolimus, sirolimus and everolimus in whole blood is reported. This method used an on-line SPE coupled with liquid chromatography, electrospray ionization-mass spectrometry detection (LC–ESI-MS). The method was validated in terms of selectivity, signal suppression, limit of quantification (LOQ), function response, trueness, repeatability and intermediate precision.
Section snippets
Chemicals and biologicals
CsA, formic acid, sodium formate and zinc sulfate were purchased from Fluka Chemie (Buchs, Switzerland). Tacrolimus was a kind gift from Fujisawa Pharmaceutical (Osaka, Japan), sirolimus and 27-demethoxy-sirolimus from Wyeth-Ayerst Research (Princeton, USA) and everolimus and cyclosporine D (CsD) from Novartis (Basel, Switzerland). All reagents and solvents were of analytical grade. Methanol (MeOH) was purchased from Biosolve LTD (Valkenswaard, Netherlands). Ultra-pure water was supplied by a
Results
The developed method permits the rapid, sensitive, selective and simultaneous quantification of four immunosuppressants (CsA, tacrolimus, sirolimus and everolimus) in whole blood using automated on-line SPE coupled with LC–ESI-MS after a simple protein precipitation step. A similar approach was also developed for the quantification of CsA in peripheral blood mononuclear cells [43].
Discussion
LC–UV methods developed for the quantification of immunosuppressive drugs often require time-consuming multi-steps off-line extractions procedures (SPE or LLE) and relatively long chromatographic separations to overcome the lack of selectivity of the detection device. They are hardly compatible with large sample batches found in most transplant centres and are prone to potential interference with co-administered drugs. LC–MS or LC–MS/MS methods allow a reduction in chromatographic separation
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