Reversed-phase high-performance liquid chromatographic method for the simultaneous quantitation of the carboxylate and lactone forms of the camptothecin derivative irinotecan, CPT-11, and its metabolite SN-38 in plasma

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Abstract

Irinotecan, also known as CPT-11 (I), is a potent semi-synthetic derivative of 20(S)-camptothecin (CPT). Like all known active derivatives of CPT, the lactone forms of I and its active metabolite SN-38 (II) are reversibly hydrolysed to inactive carboxylate forms. We describe a sensitive and selective HPLC method using the ion-pairing reagent tetrabutylammonium phosphate (TBAP) which allows the simultaneous determination of the carboxylate and lactone forms of I and II in human plasma samples following the precipitation of plasma proteins with an ice-cold mixture of acetonitrile and methanol. The mobile phase was 0.075 M ammonium acetate buffer (pH 6.4)—acetonitrile (78:22, v/v) containing 5 mM TBAP. Separation of the compounds was performed on a radially-compressed C18 column. The limits of quantitation in human plasma were 2 and 10 ng/ml for the two forms of II and I, respectively. In addition, we propose the use of CPT lactone both as an internal standard and as a “watchdog” for sample status. Under unsuitable storage conditions, CPT elutes increasingly in its carboxylate form thereby alerting the operator of possibly erroneous determinations of the concentrations of both forms of I and II. The retention times of the peaks of interest are well separated from the solvent front. This enables the detection of early eluting metabolites. The method was successfully used for monitoring of the two forms of I and II in patients treated with I.

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    This hydrolysis depends mainly on pH: at pH less than 5 the lactone form is favored, while at pH > 9 the carboxylate form is formed [2,13,14]. Only the lactone form can bind the topoisomerase I, however, it has been previously described that the quantification of the lactone and carboxylate forms (total irinotecan and SN-38), is as clinically relevant as the quantification of the lactone form, since the pharmacokinetics of both forms is correlated [2,15,16]. The typical chromatograms obtained after spiking the analytes and internal standard in human plasma at the lower and upper limit of quantification are shown in Fig. 2 (A and B, respectively).

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    Moreover, these compounds can also undergo other metabolic transformations (apart from lactone hydrolysis) or be present within complex background; thus, the method used to monitor hydrolysis should be general enough to differentiate between the lactone and carboxylate forms of the drug, even if other metabolic changes or complex background interferences occur. To date, several reports have described the hydrolysis and lactonization reactions of CPTs measured by ultraviolet spectrometry (UV–vis) and spectrofluorimetry [11–13], capillary zone electrophoresis [14], high-performance liquid chromatography (HPLC) with ultraviolet [15], fluorescence detection [16, 17], and mass spectrometry [5]. The above methods are indeed useful for measuring the rates of hydrolysis and lactonization.

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    Although it is uncertain why these values significantly deviated only in the High QC sample of irinotecan, we presumed that the pH of the mobile phase and stock solutions might have been affected. In solution, the lactone ring of irinotecan is in equilibrium with the open-ring carboxylate form, and mobile phase pH and solution concentration can significantly distort its analysis [23–25]. The deviation of the High QC irinotecan sample was unfortunate, however, an analysis in the low concentration range is potentially more relevant to occupational exposure studies [26].

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