ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 2, с. 179-182
ОРИГИНАЛЬНЫЕ СТАТЬИ =
CAPILLARY ELECTROPHORESIS COUPLED WITH MICRODIALYSIS
FOR CONTINUOUS MONITORING OF FREE METOPROLOL IN RABBIT BLOOD © 2015 Li Wang
Department of Pharmaceutics, School of Pharmacy, Jiangsu University Zhenjiang 212013, P.R. China E-mail: email@example.com Received 03.09.2013; in final form 30.01.2014
A microdialysis method followed by a capillary electrophoresis (CE) procedure has been performed for the determination of metoprolol in rabbit blood. A microdialysis probe was inserted into the ear vein for blood sampling. The electrophoresis conditions were: 47 (40) cm x 50 p.m silica capillary, 50 mM acetate buffer solution (pH 4.0), 10 s hydrodynamic load, detection wavelength 214 nm, run voltage 18 kV, temperature 25°C. The calibration was linear within the range of 1.0—100 p.g/mL (r > 0.999). The limit of detection (LOD) was 0.5 p.g/mL. Intra-day and inter-day repeatabilities with relative standard deviations (RSDs) < 5.1% were obtained. The method was then applied to pharmacokinetics profiling of metoprolol in the blood following oral administration of metoprolol to rabbits.
Keywords: metoprolol, microdialysis, capillary electrophoresis, pharmacokinetic.
Metoprolol is a relatively selective beta(1)-adreno ceptor antagonist that has been used extensively to treat such cardiovascular disorders as hypertension, arrhythmia, and heart failure. Several methods have been reported for determination of metoprolol including GC—MS [1, 2], HPLC [3-5], LC-MS [6, 7] and LC-MS-MS [8-10], CE-flame ionization detection , CE-elec-trochemiluminescence detection  in biological fluids. The biological samples of metoprolol were prepared mostly by the liquid-liquid extraction method, which is somewhat complicated.
In this work, microdialysis probe for blood sampling and fast and accurate CE method for determination of metoprolol in rabbits were used. Microdialysis provides the advantage of clean samples that do not require cleanup prior to analysis. The technique can substantially reduce the consumption of animals without withdrawal of biological fluids and involve minimal disturbance of physiological function. Moreover, multiple sampling sites provide detailed pharmacoki-netic information . The method was fully validated and subsequently applied to the pharmacokinetics study of metoprolol tartrate tablets to verify its availability in the non-clinical pharmacokinetics study of metoprolol formulations.
Chemicals and reagents. Metoprolol and propranolol were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Metoprolol tartrate tablets were purchased from AstraZeneca Medicine Co. Ltd (Jiangsu, China). Heparin sodium was obtained from Beijing AoBoXing Universeen Bio-Tech Co. Ltd (Beijing, China). The other chemicals were of analytical grade and the water was deionized and double distilled.
Solutions. Stock solution of 1 mg/mL metoprolol and 0.5 mg/mL propranolol were prepared in water. On the day of analysis, various metoprolol working solutions were prepared through sequential dilution, and 5 ^g/mL propranolol working solution was obtained by dilution of propranolol stock solution. Ringer's solution had the following composition: NaCl 145.0 mM, KCl 2.7 mM, MgCl2 1.0 mM, CaCl2 1.2 mM (pH 7.4). Running electrolytes were filtered through a 0.45 ^m nylon membrane and degassed before use.
Apparatus. CE was performed on a Beckman P/ACE 5000 equipped with UV-absorbance detector using a filter of 214 nm. Data were acquired with the P/ACE Station software system (version 1.21). Both of these were purchased from Beckman (Beckman Coulter, Fullerton, CA, USA).
Separation procedure. Separations were carried out with a 47 cm x 50 ^m i.d. fused-silica capillary (Beck-
for 20 min with 0.1 M NaOH, then ultra-pure water (7 min) and finally running buffer (7 min). The temperature was held at 25°C.
Microdialysis. The experiments were performed in four female, rabbits weighing 2.5—3.5 kg. On the day of the experiment the animals were fixed on a wooden chassis. At 20 min prior to the start of the experiment, the animals were given an intravenous injection of 5000 IU of heparin sodium injection via the edge vein of the rabbit's ear to avoid clogging by blood coagulation during the experiments. The animals were treated with a single per oral dose of 60 mg/kg metoprolol. A "shunt" type microdialysis probe (MP, molecular weight cutoff 10000 Da, Xi'an Kangpei New Technology Co., China) (membrane length 10 mm) was used for examining the time course of MP plasma concentrations. The entrance of the heparinized probe was inserted in the left edge vein of rabbits' ear (outward the heart). The microdialysis probe was perfused with water using a perfusion pump (Beijing Silugao High Technology Development Co. Ltd, China). The flow rate was 3 ^L/min and samples were collected at 15 min intervals. Briefly, 5 ^L of propranolol were added as the internal standard (IS) to each 20 ^L sample. The MP levels were determined by capillary electrophoresis and UV detection.
In vitro dialysis experiments. Microdialysis probes were inserted in the test tube filled with the following concentrations of metoprolol in Ringer's solution: 1, 5, and 10 ^g/mL; and the probe was perfused with water. A circulator (SHH ■ W21 ■ 420, HeBei, China) maintained the temperature of the circulating water at 37°C to mimic the blood temperature. The microdialysis flow rate was 3 ^L/min and sampling was carried out at 15 min interval for 64 min. Relative recovery in vitro (%) was calculated from the concentration in the dialysate (cout) divided by the concentration of the spiked drug in the test tube (cin) multiplied by 100, that is
= [Cout/Cin] X 100.
Fig. 1. Typical electropherograms of a blank blood dialysate from the microdialysis probe prior to drug administration (a), a blood dialysate sample spiked with IS (5 ^g/mL) (b) and a blood dialysate sample containing metoprolol (4.3 ^g/mL) and IS (5 ^g/mL) (c).
man Coulter, Fullerton, CA, USA) having an effective length of 40 cm. On-column detection was carried out through a 3 mm wide window opened by removing the polyimide cover of the capillary. An 18 kV voltage was applied, with currents typically less than 35 A. A 10 s hydrodynamic injection was made from the anodic side of the capillary by applying a 3.45 kPa (0.5 psi) pressure at the outlet of the capillary. Each day, before analyses began, the capillary was sequentially flushed
Method validation. Calibration samples were obtained by spiking 19 parts of blank microdialysate with one part ofworking solutions of metoprolol (Section 2.2) to yield seven concentrations in the range of 1.0 to 100 ^g/mL. Quality control samples were prepared in the same way. The intra- and inter-assay variabilities of metoprolol were assayed (six replicates) at concentrations of1.0, 5.0, 10, 25, 50 and 100 ^g/mL on the same day and on 6 sequential days, respectively. The accuracy (bias, %) was calculated from the nominal concentrations (cnom) and the mean value of observed concentrations (c0bS) as follows: bias (%) = [(c0bS - cnom)/cnom] x x 100. The precision coefficient of variation (RSD) was calculated from the observed concentrations as follows: RSD (%) = (SD/cobs) x 100. Accuracy (bias, %) and precision (RSD, %) values within 15% covering the range of actual experimental concentrations were considered acceptable. The LOD was defined as the
CAPILLARY ELECTROPHORESIS COUPLED WITH MICRODIALYSIS
analyte concentration resulting in a S/N ratio of 3 : 1. The limit of quantification (LOQ) was defined as the analyte concentration that could be analyzed with acceptable precision and accuracy.
Data analysis. The dialysate metoprolol concentrations were calculated from the calibration curves. Protein unbound metoprolol concentration data were obtained by correcting the microdialytic data for in vitro recovery of the respective microdialysis probes using the following equation.
cin cout/^in vitro'
where cin is the substance concentration in blood, and cout is the concentration of the dialysate. Pharmacoki-netic calculations were done by entering the data into the computer program NDST-21 (China Pharmaceutical University, China), which subsequently processed all data for the calculation of pharmacokinetic parameters following the no-compartmental model.
RESULTS AND DISCUSSION
Analytical developments. In order to optimize the separation conditions, different acetate buffers were tested between pH 3.0 and 5.0 with buffer concentrations of 25 and 60 mM. The best separation was obtained with 50 mM sodium acetate-acetic acid buffer (pH 4.0). A 10 s hydrodynamic injection of sample, made by applying a 3.45 kPa (0.5 psi) pressure at the outlet of the capillary, was chosen as a good compromise between separation and sensitivity. Indeed, if the time of injection was increased, the width of all peaks increased and the analytes were less separated. Finally an adjustment of the applied voltage was needed. Voltages between 17 and 30 kV were tested and the best separation was obtained at 18 kV (data not shown). In summary, the separations were carried out using a running voltage of 18 kV (25°C). To improve the concentration sensitivity, water was used as perfusion medium.
Under the optimal electrophoresis conditions, me-toprolol in blood dialysate was adequately resolved at a relatively short retention time of 8.0 min and the migration time IS was about 7.7 min (Fig. 1). Figure 1a shows a typical electropherogram of blank blood di-alysate, Fig. 1b shows a blood dialysate sample spiked with IS and Fig. 1c shows the electropherogram of a blood dialysate sample containing metoprolol and IS. None of the observed peaks interfered with the determination of metoprolol.
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