Liquid chromatography-tandem mass spectrometric assay for the light sensitive tyrosine kinase inhibitor axitinib in human plasma

Abstract

A bioanalytical assay for the new tyrosine kinase inhibitor axitinib was developed and validated. In addition, the light mediated trans to cis isomerization of this drug was investigated. For the quantitative assay, human plasma samples were pre-treated under light protection using protein precipitation with acetonitrile containing erlotinib as the internal standard. The extract was diluted with water and injected into the chromatographic system. The system consisted of a trifunctional bonded octadecyl silica column with isocratic elution using formic acid in a water-methanol mixture. The eluate was transferred into an electrospray interface with positive ionization and the analyte was detected and quantified using the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.2–200 ng/ml concentration range, the lowest level of this range being the lower limit of quantification. Within day precisions were 2.5–6%, between day precisions 4–9% and accuracies were between 91 and 106% for the whole calibration range. Light protected axitinib showed no isomerization and was shown to be chemically stable under all relevant conditions. Finally, the assay was successfully applied for a mouse tissue distribution study using mouse samples diluted with human plasma.

Introduction

Axitinib (AG013736, Fig. 1A) is a selective orally active tyrosine kinase inhibitor (TKI) with anti-angiogenic effects. In vitro, the compound has been shown to inhibit angiogenesis, vascular permeability and blood flow [1], [2]. In phase I and II clinical studies, axitinib showed antitumor activity against different advanced tumors [3], including renal cell carcinoma (RCC) [4], [5], pancreatic cancer [6] and thyroid cancer [7]. At the moment, two phase III clinical studies with axitinib are ongoing [8], [9]. In one study axitinib is combined with gemcitabine for the treatment of advanced pancreatic cancer [9], and in the other phase III trial axitinib is tested as a second line therapy for metastatic RCC in comparison to sorafenib [8]. In both studies 5 mg of axitinib twice daily (BID) is used as a starting dose.

Pharmacokinetic evaluations of axitinib, important during initial drug development, are still scarce [10]. The assay in that first pharmacokinetic study was, however, reported very concisely and used liquid chromatography–tandem mass spectrometry (LC/MS/MS) in combination with (laborious) liquid–liquid extraction [10]. Therefore, the development and validation of a simple, fast and sensitive bioanalytical assay for axitinib in human plasma was conducted and reported in this paper before starting a new clinical trial.

Validated bioanalytical assays using LC/MS/MS for the first TKIs have been reported frequently since 2002 [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], with very recently the introduction of assays for multiple components of this class of drugs [29], [31], [32]. Most often, these assays have been validated for plasma of human subjects [11], [12], [13], [14], [15], [17], [18], [19], [20], [22], [24], [25], [26], [29], [30], [31]. For erlotinib [11], [12], imatinib [19], [20], [21], sunitinib [27] and sorafenib [23] a metabolite could be quantified simultaneously with the parent drugs. In most methods the protonated parent molecule is used in the selected reaction monitoring (SRM) mode using a triple quadrupole (QqQ) system, with a quadrupole–linear ion trap combination [15] as the only exception. This assay of Guetens et al. [15] was also the only method using a capillary LC system. Two new methods used a single quadrupole system in the single ion monitoring mode [30], [31] for dasatinib, nilotinib and imatinib.

Apart from an application using a pentafluorophenyl column [22] and one using a hydrophilic interaction chromatography system [32] all investigators used an octa or octadecyl modified silica phase with at least end-capping as prevention of unwanted interaction of the basic TKI drugs with the free silanol groups of the silica. Typically, methanol is used as organic modifier in the eluent for all imatinib single drug assays [16], [17], [18], [19], [20], [21] and the nilotinib assay [30], while acetonitrile is used for other TKI drugs: erlotinib [11], [12], gefitinib [13], [14], [15], lapatinib [22], [29], vandetanib [28], sunitinib [26], [27], [29], sorafenib [23], [24], [25], [29], dasatinib [29], [31], [32] and nilotinib [29], [30], [31], [32]. To control the pH of the eluent and to improve ionization of the analytes, formic acid [12], [14], [19], [22], [23], [24], [25], [26], [29], [30], [31], [32] and ammonium acetate [11], [13], [15], [16], [18], [20], [21], [22], [24], [29], [32] are both frequently used, with ammonium formate [17], [27] and acetic acid [28] being incidental alternatives.

Lower limits of quantification (LLQs) for TKIs in the sub-ng/ml range (0.1–0.5 ng/ml) were obtained using liquid–liquid extraction with methyl-t-butyl ether [13], [15], [26], [27]. Other pre-treatment methods, including protein precipitation (PP) with acetonitrile [12], [14], [16], [18], [19], [20], [23], [24], [25], [29], [30], [32] or acetonitrile–methanol [31], liquid–liquid extraction with hexane-ethyl acetate [11], [17] or pentane–ethyl acetate [11], [17], [28] and solid-phase extraction on C18 [22] or C8 [21] sorbents, resulted in LLQs in the 1–100 ng/ml range.

Recently, axitinib was reported to form the sulfoxide metabolite and a glucuronide conjugate as circulating metabolites and hydroxy-axitinib in excreta in human ADME studies [33], unfortunately no further details were reported. Therefore, and also because no axitinib metabolites were commercially available, we focused on developing and validating a bioanalytical LC/MS/MS assay for the axitinib parent compound. A QqQ system and PP as a simple pre-treatment technique were used to obtain sensitivity in the sub-ng/ml range for this new TKI. The higher sensitivity compared to other TKI drugs using this approach may partially result from the use of a new generation LC system and column with higher pressures and sub-2 μm particles (Ultra Performance LC), not being used earlier for TKI drugs.