High-performance liquid chromatographic method for therapeutic drug monitoring of cyclosporine A and its two metabolites in renal transplant patients

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Abstract

A novel fast HPLC method was developed for the determination of cyclosporine A (CyA) and its two metabolites M17 (AM1) and M21 (AM4N) in blood. Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane. Chromatography was carried out using a microbore RP-column under isocratic elution with acetonitrile–methanol–water (200:80:140, v/v/v) at 70°C and a detector set at 205 nm. Linearity for all three compounds was tested in the range of 1–1000 ng/ml. Recovery was 97–109%, and a coefficient of variation was 1.6–8.8% depending on the particular compound and its concentration. The method was used for a group of renal transplant patients having an inadequate response to CyA therapy in order to evaluate the possible role of CyA and its metabolites on the occurrence of hypertension and other toxicological events.

Introduction

Cyclosporine A (CyA) (Fig. 1), is a commonly used immunosuppressive drug with a narrow therapeutic window. Hence, its monitoring is of great importance in order to optimize CyA level within a range that minimizes both the risk of rejection and drug-induced toxicity. Among methods used for the determination of CyA blood levels [1], [2], [3], [4], only HPLC methods can provide levels of the main metabolites. Various consensus panels concluded that routine measurement of metabolites is not necessary [5], [6]. However, all agreed as well that there are some circumstances when the levels of metabolites would be helpful and justified further studies aimed to explore the role of metabolites from a clinical point of view [6]. These situations include, e.g., persistence of CsA levels caused by metabolic disorders [7] or clinical complications associated with high levels of CsA metabolites [8]. The primary metabolites M17 (AM1), M1 (AM9), and M21 (AM4N), seem to be the most important ones from the clinical point of view with regard to their possible high concentration in some patients, to higher risk of nephrotoxicity and to metabolite/parent drug ratio [9], [10]. A novel HPLC method has been developed in this work in order to evaluate whether these values can be individual for each renal transplant patient on a standard immunosuppressive therapy.

Section snippets

Chemicals

Methanol of HPLC gradient grade, acetonitrile for HPLC and hydrochloric acid were obtained from Merck, water, n-hexane and diethyl ether, all HPLC grade, were from Sigma–Aldrich and Fluka Chemie, Cyclosporine A, an official USP 23 standard, and cyclosporine D (98%, HPLC), were from Galena (Czech Republic). Metabolites M17, dihydro-M17, M21, and M1 were obtained from Galena, their sources were semisynthesis [11], producing fungus [12], and dog liver, respectively, and their purity was above 98%

Results

Least square calibration curves for CyA, M17 and M21 were constructed by plotting the peak area ratio of substances versus internal standard to standard’s concentrations in the range of 1–1000 ng/ml. The correlation coefficients were 0.999 for CyA and M17 and 0.998 for M21. A chromatogram of standard sample is shown in Fig. 2B. When blood from an untreated volunteer was analyzed, no interfering peaks occurred, as can be seen from chromatogram 2A. Chromatogram 2C shows retention time and

Discussion

Although there are more than 30 various cyclosporine A metabolites described in the literature [15], most of them lack any immunosuppressive activity and do not exhibit toxicity in in vitro assays. Moreover, in order to distinguish two possible clinical events, i.e., persistence of CsA high levels without metabolism [7] or accumulation of metabolites [8], it is useless to analyze all of them, but some of them can serve as suitable markers. We have chosen primary metabolites M17 (AM1), with

Acknowledgements

This work was supported by grant COST OC –B15.20 (1999).

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