Quantification of the anticancer agent STI-571 in erythrocytes and plasma by measurement of sediment technology and liquid chromatography–tandem mass spectrometry

doi:10.1016/S0021-9673(03)00775-1

Journal of Chromatography A

Volume 1020, Issue 1, 5 December 2003, Pages 27-34

https://doi.org/10.1016/S0021-9673(03)00775-1 Get rights and content

Abstract

An isocratic high-performance liquid chromatographic method coupled to tandem mass spectrometry for the quantification of the revolutionary and promising anticancer agent STI-571 (tradenames Gleevec, Glivec, Imatinib) in blood plasma and red blood cells (RBCs) is described. The method involves measurement of sediment technology for RBCs and a subsequent single protein precipitation step by the addition of acetonitrile to both the RBC isolate and plasma. The sample mixture was centrifuged (10 min, 3600 g), and the supernatant filtered through a HPLC filter (0.45 μm). The analytes of interest, STI-571 and the internal standard [²H₈]STI-571 were eluted on a Waters Symmetry C₁₈ column (50×2.1 mm I.D., 3.5 μm particle size) using a methanol–0.05% ammonium acetate (72:28, v/v) mixture. STI-571 and [²H₈]STI-571 were detected by electrospray tandem mass spectrometry in the positive mode, and monitored in the multiple reaction monitoring transitions 494>394 and 502<394, respectively. The lower limit of quantitation of STI-571 was 2.1 ng/ml in RBCs and 1.8 ng/ml in plasma. The recovery from both plasma and RBCs was between 65 and 70%. The method proved to be robust, allowing simultaneous quantification of STI-571 in RBCs and plasma with sufficient precision, accuracy and sensitivity and is useful in monitoring the fate of this signal transduction inhibitor in whole blood of cancer patients.

Introduction

The pathological activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukaemia (CML). A hallmark of CML is a reciprocal chromosomal translocation involving the long arms of chromosomes 9 and 22. This somatic mutation fuses a segment of the bcr gene, from chromosome 9, to a region upstream of the second exon of the c-abl gene from chromosome 22. c-abl encodes a non receptor tyrosine kinase that is strictly controlled in normal cells. However, Bcr-Abl fusion proteins have a constitutive catalytic activity in the transformation and further malignant development of cells. A series of inhibitors, based on the 2-phenylaminopyrimidine class of pharmacophores, exhibits intrinsic affinity and specificity for Abl. The small molecule inhibitor STI-571 (Fig. 1) is one of the most potent, and has proved effective in the treatment of CML, and also several solid tumors [1], particularly gastrointestinal stromal tumors (GISTs). In the latter type of tumors, STI-571 anticancer activity is based on interaction with c-kit, which is amongst key regulators in cellular transduction.

In order to support pharmacokinetic studies with sufficient speed, suitable analytical procedures are required. Methods of quantification in plasma have been described very recently [2], [3]. A semi-automated solid-phase extraction procedure with liquid chromatography–tandem mass spectrometry (LC–MS–MS) was an initial development [2], but with the extension of clinical studies of this promising agent, a high-throughput system is needed. The assay was adapted to meet the demand, and has proved robust, allowing the simultaneous quantification of STI-571 and its main metabolite (CGP 74588) in human plasma at high rates of throughput [3].

However, it has been demonstrated during the past decade that plasma monitoring alone misses important information since blood cells [red blood cells (RBCs), erythrocytes], and sometimes other blood cells are not analyzed [4], [5]. We therefore developed a device, based on measurement of sediment (MESED) technology (Fig. 2), to quantify substances of interest in sediments, such as RBCs [5].

In the case of blood, RBCs are by far the most important transport cells, as they have the largest theoretical volume and surface. It has been shown that RBCs can carry high quantities of anticancer moieties, such as the mustard species originating from the oxazaphosphorines (i.e., cyclophosphamide and ifosfamide), as well as other anticancer agents [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. MESED is of particular interest in RBC analysis: it not only provides an opportunity for simultaneous RBC and plasma analysis, but also an opportunity for easy and fast mass analysis. A direct relation between mass and volume can than be obtained. In this report, the data of MESED technology coupled to LC–MS–MS analysis of STI-571 and possibly its metabolite(s) is presented for the first time. MESED–LC–MS–MS is routinely applied to monitor STI-571 and calculate the RBC versus plasma partition of the substance in patients blood.

Section snippets

Reagents and materials

High-purity solvents were obtained from Acros Organics (Geel, Belgium). All other chemicals were commercially available and of analytical grade. Blank RBCs and plasma samples were obtained from volunteers and, as an individual control, from patients immediately before STI-571 treatment. All water used in the analyses was HPLC grade and obtained from Merck (Darmstadt, Germany). STI-571 and the internal standard [²H₈]STI-571 (STI-571-d₈) were synthesized at Novartis (Basel, Switzerland).

Results and discussion

The recently available MESED device, enables the collection of an exact volume and mass of RBCs with collection of the associated solvent, i.e., blood plasma. The procedure only requires two centrifugation steps of 3000 rpm for 10 min and allows RBC and plasma analysis in a routine manner [4], [5], [6], [7], [8], [9], [10], [11], [12], [14], [15], [16]. Guidelines for assay validation such as those of the US Food and Drug Administration (FDA), are only available for blood plasma. The assay of

Acknowledgements

G.G. thanks the Flemish Cancer League for a grant to support a research assistant.

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Quantification of the anticancer agent STI-571 in erythrocytes and plasma by measurement of sediment technology and liquid chromatography–tandem mass spectrometry

Abstract

Introduction

Section snippets

Reagents and materials

Results and discussion

Acknowledgements

Lancet

J. Pharm. Biomed. Anal

J. Chromatogr. B

Clin. Biochem

J. Pharm. Sci

Ann. Oncol

J. Chromatogr. B

Semin. Oncol