Measurement of intracellular accumulation of anthracyclines in cancerous cells by direct injection of cell lysate in MEKC/LIF detection

Abstract

Anthracyclines are chemotherapeutic drugs that are broadly used in the treatment of various types of solid cancers and leukemia. Herein, we report on a novel analytical method for intracellular accumulation of anthracyclines using MEKC/LIF detection. An aqueous separation system permitted the injection of cell lysates directly into the capillary. The MEKC migrating solution was made up of borate buffer at pH 9.22 containing sodium taurodeoxycholate, (2-hydroxypropyl)-gamma-CD, and SDS. The anthracyclines, Doxorubicin (DOX) and epirubicin (EPI) were detected by LIF using a Nd:YAG laser (532 nm) or an argon ion laser (488 nm) for excitation. Two cell lines, human humerus tumor cells (RDES) and human lung tumor cells (A549), were treated with a mixture of the two anthracyclines for fixed periods of time, and then intracellular concentrations were determined by injecting cell lysates directly. Recovery values of 96.0-100.8% were obtained for DOX and EPI. Reproducibility quantified by RSD was less than 3.9% intraday and 6.7% interday at concentrations ranging between 50 and 500 nM. The uptake of EPI was found to be slightly less than that of DOX for A549, but higher levels of EPI were observed in RDES. Intracellular accumulation of anthracyclines was greater in RDES than in A549, but both types of cells excreted anthracyclines after 12 h. These results indicate that MEKC with an aqueous medium is useful for investigating intracellular uptake and accumulation of drugs, since cell lysates can be used directly with no pretreatment such as deproteination or solvent extraction of analytes.

Capillary Electrophoresis with Laser-induced Fluorescence Detection for Application in Intracellular Investigation of Anthracyclines and Multidrug Resistance Proteins

“…This MEKC-LIF method has been shown to be useful in the base-line separation of DOX and EPI (Figs. 2(A) and 2(B)) 26 or DOX, EPI, IDA and DAU (Fig. 2(C)) 43 contained in cancerous cells.…”

“…While MEKC determination of DOX in the presence of other anthracyclines like IDA and DNR 39 is less demanding because of some basic structural differences, the separation of DOX from EPI 26 is challenging because the two are epimers, only differing in the axial and equatorial orientations of the OH group at C-4 in daunosamine sugar moiety (Fig. 1).…”

“…For direct MEKC-LIF determination, a lab assembled setup, equipped with an argon-ion laser emitting at 488 nm, was employed. 26 The migration solution used to achieve the separation of anthracyclines is made up of 20 mM sodium tetraborate buffer containing 35 mM sodium taurodeoxycholate (STDC), 3.5% (wt/v) (2-hydroxypropyl)-γ-CD (HP-CD), and 20 mM sodium dodecyl sulfate (SDS). Two chiral selectors, HP-CD and STDC, enhance the separation selectivity between DOX and EPI, whereas SDS was needed to separate an internal standard from the analytes.…”

“…However, the quantitation range of DOX determined by LC-ESI-MS/MS 33 (10 -100 ng mL -1 ) is narrow compared to that determined by LIF-CE. By the CE-LIF method employing aqueous buffer, 26,43 the influx of anthracyclines in cancerous cells as well as accumulation can be monitored continuously over a wide time range.…”

“…[26][27][28] This review examines the use of MEKC-LIF in the determination of anthracyclines, which are known anticancer agents, and one group of cell proteins (multidrug resistance associated protein, MRP1) that is responsible for efflux xenobiotics including anticancer agents, covering research work done in Kyushu University's Applied Chemistry Department over a period of three years (2008 -2011).…”