Micellar Electrokinetic Capillary Chromatographic Method for Simultaneous Determination of Drugs Used to Treat Advanced Breast Cancer
J. Rodrfguez Flores 1./J.J. Berzas Nevado 1/ G. Castaneda Penalvo ~/ M. I. Rodrfguez Cdceres 2 1 Department of Analytical Chemistry and Foods Technology, University of Castilla-La Mancha, 13071 Ciudad Real, Spain; E-Marl:
[email protected] 2 Department of Analytical Chemistry, University of Extremadura, Badajoz, Spain
KeyWords Column liquid chromatography MEKC Tamoxifen, anastrozole, letrozole, methotrexate Breast cancerdrugs
Summary A micellar electrokinetic capillary chromatography (MEKC) method has been developed for quantification of four drugs - tamoxifen, anastrozole, letrozole, and methotrexate - used to treat advanced breast cancer. Separation was performed at 25 ~ and 25 kV, with 20 mM borate buffer (pH 9.2) containing 40 mM sodium dodecylsulfate as electrolyte solution. Under these conditions analyses were performed in 12 min. The hnearity of the response was investigated for the concentration range 2.0- 20.0 mg L 1. The intra-day residual standard deviation (n = 4 graphs) bel',,veen the slopes of the calibration graphs was acceptable for the four drugs studied. Detection limits (signal-to-noise ratio = 3) were below 1 mg L 1 for all the compounds. The simplicity, precision, and sensitivity of MEKC proved suitable for quahty control of pharmaceutical preparations used to treat advanced breast cancer. Six different pharmaceutical preparations, each containing one of the above-mentioned drugs, were successfully analyzed.
Introduction Despite significant advances in treatment procedures in recent years, advanced breast cancer remains essentially incurable. Many breast cancers have estrogen receptors (ER), and growth of these tumors can be stimulated by estrogen hormones [1]. Approximately 30% ofpostmenopausal women with advanced breast cancer will respond to endocrine therapy. This response rate increases to more than 50% in postmenopausal women whose tu-
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mors express ER [2]. The mechanism of action of most endocrine agents involves either blockage of estrogen activity at the cellular level (anti-estrogens) or inhibition of estrogen production (aromatase inhibitor). Chemotherapy with such agents (anti-metabolites) is, however, necessary when tumors do not respond to hormonal treatment with anti-estrogens or when estrogen levels are very low. The anti-estrogen tamoxifen (TAM, Figure 1) is the most widely used first-line agent for treatment of post-menopausal
breast cancer, because of its once-daily oral administration and because it is well tolerated. Despite early success with this agent, however, most patients with advanced breast cancer will eventually relapse, their tumors becoming resistant to TAM. Approximately 25% of patients respond to second-line endocrine therapy with selective non-steroidal aromatase inhibitors such as anastrozole (ANA) and letrozole (LET), Figure 1. These agents significantly reduce serum estradiol levels and are orally administered and well tolerated. Both compounds have led to significant reductions of plasma estradiol level in postmenopausal women, with tumor regression in some patients [3, 4]. Patients who do not respond to hormonal therapy are sometimes treated with methotrexate (MET, Figure 1), an agent frequently used in the therapy of acute lymphatic leukemia and other malignant diseases [5 7]. MET is an antimetabolite that inhibits the reduction of natural dihydrofolic acid to tetrahydrofolic acid, leading to reduced purine and pyrimidine synthesis and reduction of tumor cell growth. Other factors can influence the effects of MET on tumor cell growth, including amplification of dihydrofolate reductase gene expression after treatment with MET [8], and a decrease in membrane transport [9]. Although it is useful to monitor residual drug levels in patients who have been treated with several lines of therapy, no method for measurement of ANA and LET by capillary electrophoresis (EC) is currently available in the literature, although non-aqueous capillary electro-
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Reagents and Solutions H3C\
~
/O\
H3C FCH2CH3
TAMOXIFEN
N
H3C" I CH3
/~CH 3 H3C
CN
ANASTROZOLE
LETROZOLE
TAM and MET were supplied by Tocris (Bristol, UK), A N A was from AstraZeneca Laboratories (Madrid, Spain), and LET was kindly provided by Novartis Pharma (Basel, Switzerland). Standard solutions (200 mg L 1) of ANA, LET, and TAM were prepared in 50% (v/v) ethanol-water. A standard solution of MET was prepared in 4:96 (v/v) DMSOethanol. The stability of the solutions was evaluated over a period of 7 days by spectrophotometric measurement of diluted solutions (10 mg L 1), by means of a Beckman (Fullerton, Ca, USA) DU-70 spectrophotometer equipped with 1.0 cm quartz cells and controlled by Beckman Data Leader software resident in an IBMPS 2 model 30 computer. Owing to their lower stability, standard and the diluted TAM solutions were prepared frequently. Sodium dodecylsulfate (SDS) was obtained from Sigma (St. Louis, MO, USA). All chemicals and solvents used were of analytical reagent grade.
PharmaceuticalTreatment H2N\ /N... / N . .
CH3
Tablets Y
NIH2
-N
CH2N---{ (
) )---CONH
HOOC-CHz-CH2---C---COOH
I
H METHOTREXATE Figure 1. The chemical structures of the drugs.
Fourteen tablets were finely powdered and an amount equivalent to the mean mass of one tablet was dissolved in 25 mL ethanol by sonication for 15 min. The solution was then filtered. For analytical purposes, however, complete solubility of MET was achieved with 20:80 (v/v) DMSO-ethanol only.
Inlection phoresis [10 12] has been applied to the determination of TAM and metabolites and/or acid hydrolysis products. MET has been widely studied by isotachophoresis [13], capillary zone electrophoresis with UV detection [14, 15], and micellar electrophoresis with UV detection [16] or laser-induced fluorescence detection [17]. In this paper we describe a micellar electrokinetic capillary chromatographic (MEKC) method for resolution of four drugs often used to treat advanced breast cancer. The MEKC method was suitable for determination of the drugs in six pharmaceutical products at very different levels (from 1.0 mg A N A in Arimidex tablets to 50.0 mg MET in Metotrexato (A1mirall) injection).
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Experimental Micellar ElectrokineticCapillary Chromatography M E K C was performed with Beckman (Fullerton, CA, USA) P/ACE System MDQ equipment equipped with a diodearray detector and a Beckman capillary electrophoresis software system controller. Separation was performed in a 57 cm (50 cm to detector) x 75 ixm i. d. fused-silica capillary housed in a cartridge maintained at 25 ~ with a 800 Ixm x 100 Ixm detector window. The wavelengths selected for monitoring of the electropherograms were 215 nm for A N A and LET, 257 nm for TAM, and 300 nm for MET.
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The injection volume was diluted to 25 mL with 4:96 (v/v) DMSO-ethanol.
Lyophilized Preparations The total sample was dissolved in 4:20:76 (v/v) DMSO-water-ethanol (25 mL).
Operating Conditions and Procedure Before first use the capillary was conditioned by flushing with 0.1 M N a O H for 20 min, then with water for 10 min, and finally with 40 mM SDS in borate buffer (20 mM, pH 9.2, measured by means of a Crison (Barcelona, Spain) MicropH 2002 pH meter) for 10 min. The rinse step was Original
performed by use of vials different from the separation vials, to keep the level of buffer constant in the anodic separation vial. At the start of each sequence the capillary was washed with 0.1 M NaOH for 2 min then with electrolyte separation buffer for 2 min, to restore the capillary wall surface and re-equilibrate the capillary between sample injections. All separations were conducted using 2 mL vials for rinsing and washing and 200 ixL polypropylene vials as sample vials. All vials were refrigerated at 10 ~ inside the equipment. Capillary electrophoresis was performed at 25 ~ The first two injections of a sequence were used for system-equilibration purposes only; the results from these were not included in the data. The capillary was filled with separation buffer for 2 min, followed by 7-s hydrodynamic injection of the samples. Separation was performed at 25 kV for 12 min (with 31.25 kV min 1 ramp voltage). Under these conditions the current was 641xA. Sample measurements were performed in triplicate and average corrected peak areas (CPA) (area/migration time) were used for quantitative analysis.
Results and Discussion
Preliminary Experiments and Optimization of the Separation Conditionsfar CapillaryZane Electrophoresis(CZE) The pH of the running electrolyte has a significant impact on ionization of the acidic silanols of the capillary wall and on the electrophoretic mobilities of the compounds studied. Because of the structures of the analytes (Figure 1), basic or acidic buffers could be used to promote their ionization. Experiments were performed at pH 2.5 12 to evaluate the effect ofpH on the separation of MET, TAM, ANA, and LET. No references were found to the pK~ values of ANA, LET, and TAM; MET has three pKvalues 3.8, 4.8 and 5.6. ANA, LET, and TAM were in the unionized forms at each pH studied; for this reason their migration times were the same as that of the electroosmotic flow (EOF). MET was in the cationic form at pH < 3.8 (the first pK~) and in anionic form at pH > 5.6 (the third pK~). Because it was impossible to achieve electrophoretic resolution of ANA, LET, and TAM by CZE, we decided to investigate MEKC. Original
6000
-
EOF
215 n m
MET
