Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection

Abstract

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 μM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635±27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61±13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid–liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid–liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.

Introduction

Doxorubicin (DOX) is a widely used anthracycline that has proven to be effective against a variety of human malignancies [1], such as leukemia and breast cancer [2]. When this drug reaches the targeted organ as free drug administered intravenously or encapsulated in liposomes [3], [4] it enters the malignant cells, reaches their nuclei, and stabilizes the topoisomerase IIα–DNA complex, halting cell proliferation [5]. This process is believed to be the main mechanism of action. Unfortunately, the DOX treatment is accompanied by the appearance of cardiac and liver toxicity [6], [7], [8], [9] and drug resistance [6], [10], [11] which may result from cellular processes involving the parent compound or drug metabolites [12], [13]. Furthermore, transformation of DOX into metabolites, which may not be pharmacologically active, decreases the concentration of the parent compound affecting the efficacy of the treatment. Therefore, it is important to design sensitive analytical procedures that can detect the various metabolites that may be present in the cell after DOX treatment.

DOX metabolism has been studied previously [12], [13], [14]. Based on the DOX metabolic scheme proposed by Licata et al. [13] the expected metabolites are shown in Fig. 1. This work was based on the in vitro incubation of the parent compound with human cardiac cytosol. Andersen et al. reported five metabolites [14]. Since most of the cytotoxic activity of DOX is derived from the aglycone ring system of the molecule [1], [12], those metabolites that retain this molecular feature are expected to be toxic. In order to fully explain the role that DOX metabolites play in its mechanism of action, unwanted cytotoxicity, and appearance of drug resistance it is necessary to develop direct methods that are capable of extracting, separating, and detecting doxorubicin metabolites. Some of these methods are discussed below.

The most common methods for extraction of DOX are solid-phase extraction [15], [16], [17], enzymatic digestion of cellular material [14], and liquid–liquid extraction (LLE) [6], [18], [19]. In the liquid–liquid extraction procedures reported in the literature, a biological sample is treated with chloroform or a chloroform/alcohol mixture in order to lyse the cells and extract the DOX into the organic phase. The phases are separated and the organic phase is dried under a steady stream of nitrogen.

Following the extraction by any of the procedures mentioned above, anthracyclines, including DOX and their metabolites are separated and commonly characterized by reversed-phase liquid chromatography [6], [18], [20], [21], [22], [23]. Because anthracyclines, including DOX, and their metabolites exhibit native fluorescence due to the conjugated aglycone ring system present in the molecule, fluorescence is the preferred detection scheme. Using excitation at either 460 or 480 nm and detection at 550 nm, DOX limits of quantification in the fmol range have been reported [18], [23].

Another separation technique that may be used for the analysis of anthracyclines is capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The determination of anthracyclines including DOX by CE–LIF was done first by Reinhoud et al. [24]. The limit of detection for the CE–LIF method was 24 amol, a limit of detection three orders of magnitude lower than those obtained by reversed-phase liquid chromatography analyses. Recently the CE separation of a mixture of anthracyclines containing DOX, daunorubicin, and idarubicin was reported [25]. Hempel et al. have used CE–LIF for separation and quantification of parent anthracyclines and their main metabolite: idarubicin from idarubicinol and DOX from doxorubicinol in samples from plasma of patients treated with the respective drug [19], [26]. Furthermore, Siméon et al. have reported the CE–LIF analysis of an anthracycline, daunorubicine, extracted from tumor biopsies [27]. However, they did not report the separation of anthracycline metabolites produced by the cells of the biopsy.

One of the unique applications of CE has been its application to the analysis of the contents of single mammalian cells [28], [29], [30], [31], [32]. In single cell analysis a whole cell is rapidly disrupted inside a capillary resulting in the release of all its metabolites which then can be separated by CE. This procedure eliminates the risk of metabolite degradation during a lengthy extraction procedure and guarantees complete recovery [33]. So far, metabolic studies based on single cell analysis by CE–LIF have been limited to synthetic fluorescent substrates that resemble the natural substrates [29], [33], [34]. Because of these structural modifications it is expected that the analysis will result in a slightly altered description of metabolism. Single-cell analysis by CE may provide an accurate description of doxorubicin metabolism because doxorubicin is an unmodified substrate.

Here we demonstrate a sensitive CE–LIF method for the separation and detection of doxorubicin and at least five of its metabolites in bulk preparations and in single cells. Using this method, we present the most complete description of low-abundance DOX metabolites produced in vivo by cultured cells to date.