ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2012, том 67, № 6, с. 632-636
ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
PHARMACOKINETIC STUDY OF METOPROLOL IN RABBIT PLASMA BY CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION © 2012 Yuyun Chen", Weiping Yang4
aCollege of Environmental Science and Engineering, Chang'an University Xi'an, Shannxi 710054, China bEducation School of Nationalities, Shaanxi Normal University Xi'an 710062, China Received 17.08.2010; in final form 22.08.2011
This work described a sensitive method for determination of metoprolol in rabbit plasma. The method involved purification by ultrafiltration, derivatization with Fluorescein isothiocyanate, separation by capillary electrophoresis and determination by laser-induced fluorescence detector. Other components in plasma including a variety of amino acids and proteins did not interfere with the determination of metoprolol under experimental conditions. The assay had a wide range (2.0—500 ng/mL) of linearity and a detection limit of 0.8 ng/mL. The intra- and inter-day precisions of the QC samples were satisfactory with RSD less than 10% and accuracy within 10%. This method was successfully applied to pharmacokinetic study of metoprolol in rabbit blood.
Keywords: capillary electrophoresis, metoprolol, laser-induced fluorescence, ultrafiltration, pharmacokinetic.
Metoprolol, 1-(isopropylamino)-3- [p-(2-methoxy-ethyl)phenoxy]-2-propanol, is a kind of P-adrenaline receptor blocker. It is widely used for the treatment of hypertension, angina, myocardial infarction, arrhythmia, hyperthyroidism and other related diseases [1, 2]. It is so sensitive that even a small oral dose of the drug gives sufficient blockade. The drug has been added to the list of forbidden drugs by the International Olympic Committee [3]. Therefore, the development of sensitive and selective analytical methods for the determination of the P-blockers is of great importance.
Up to now, several methods have been reported for determination of metoprolol in plasma and other biological fluids, which include gas chromatography-mass spectrometry (GC—MS) [4], high-performance liquid chromatography (HPLC) [5], LC-MS [6, 7] and LC-MS-MS [8].
Capillary electrophoresis (CE) represents an alternative to LC or GC, allowing fast separation and requiring very limited amounts of samples and reagents. Moreover, the principle that enables the separation in CE is based on migration of the analytes under the influence of an electric field and not on the partition between two phases as in LC or GC. Until now, CE has been applied in chiral/achiral separation and determination of metoprolol in some studies [9-12]. Park et al. [13] determined the stability constants of the inclusion complexes of metoprolol and carboxymethyl-P-cyclodextrin (CMCD) using HPLC and CE. Zhang
et al. [14] developed a method based on on-line cation-exchange preconcentration and capillary electrophoresis coupled by tee joint interface for determination of metoprolol, which method enhanced the detection sensitivity of CE-UV about 5000 times for metoprolol and propranolol. There were also a few CE methods reported for chiral/achiral separation or determination of metoprolol in different biological samples [15, 16]. A CE method [15] was evaluated for enantioselective analysis of metoprolol and its deme-thylated and carboxylated metabolites, and the concentration of enantiomers in human urine was determined. Wang et al. [16] developed a CE method for determination of P-blockers including clenbuterol, metoprolol, ractopamine, isoxsuprine and salbutamol in pig feed, urine and liver.
However, few CE methods were used for the determination of metoprolol in plasma. As far as we know, only one paper described a CE method for the enanti-oseparation and determination of metoprolol in serum sample [17]. The UV detector was used and the detection limit for metoprolol was 50 ng/mL [17]. Most probably, this is due to the low sensitivity of this technique that limits its applicability to drug analysis in plasma samples, if not enhanced by suitable detection equipment. CE in conjunction with laser-induced fluorescence (LIF) detection can overcome the drawback of low sensitivity. However, there are not many ana-lytes of interest applicable by CE that naturally possess
a high fluorescence intensity. Also, determination of native fluorescent compounds is limited by their different excitation wavelengths, and their compatibility with available laser sources. To overcome this drawback, derivatization of analytes with a fluorophore is an important technique for improving sensitivity in CE. Therefore, derivatization reaction plays an important role in CE-LIF method. Some derivatizing reagents have been applied in CE-LIF [18, 19]. Fluorescein isothiocyanate (FITC) is an excellent derivatizing reagent, for its derivatives have good electrophoretic property.
A method based on the use of CE-LIF detection was developed for determination of metoprolol in rabbit blood in this paper. The advantages of this method include high sensitivity, inexpensive chemicals used and small sample volume required. The detection limit was 0.8 ng/mL, which was lower than those for traditional methods. In addition, the pharmacokinetics of metoprolol in rabbit blood was investigated.
EXPERIMENTAL
Metoprolol was purchased from the National Institute for Drugs and Bioproducts Inspection. FITC was purchased from Sigma. The commercial metoprolol tablets were purchased from Jiufu Medicine Co. Ltd. containing 50 mg of metoprolol per tablet. Acetone, hydrochloric acid, sodium hydroxide and sodium borate were of analytical reagent grade.
The experiments were performed on a Beckman P/ACE 5000 system equipped with a laser-induced fluorescence detector. The detector was carried out with excitation at 488 nm and emission at 520 nm. All separations was carried out with a 75-^m I.D. uncoat-ed fused-silica capillary (length 57 cm, 50 cm to detector). Z383 high-speed centrifuge was purchased from Hermle Labortechnik Gmbh Company. The ultrafiltration tubes (Centriprep centrifugal filter devices) were from Millipore Co. The eluting peaks were processed with P/ACE Station Version 1.21 software (Beckman Instrument, Fullerton, CA, USA).
The capillary temperature was maintained at 20°C. Samples were loaded by pressure injection at 3.45 kPa for 5 s for a total volume of about 4.5 nL and were separated at an applied voltage of 18 kV. In all experiments samples were introduced at the anodic end of the capillary and detected by laser-induced fluorescence detector. The capillary was pre-rinsed with water (2 min) and separation buffer (2 min). Before each elec-trophoretic run, the capillary was flushed (5 x 2 min) in sequence with water, 0.1 M sodium hydroxide, water, 0.1 M hydrochloric acid and water.
Several variables were investigated, including the pH and composition of the run buffer and applied voltage. Of these, pH and composition of run buffer
were found to have the greatest effects on the separation. Sodium borate solution was used as buffer in present work because FITC-metoprolol derivative can emit strong fluorescence when excited in alkaline environment. The effect of buffer concentration was studied. The results showed that the use of higher concentrations of run buffer and lower applied voltages improved resolution. However, these conditions resulted in longer analysis times. In addition, the use of high concentrations of buffer resulted in high separation currents and increased Joule heating. Considering all of these factors, the best separation was obtained under the following conditions: 50 mM buffer, pH 9.0 and 18 kV applied voltage.
As metoprolol does not exhibit native fluorescence when the present Ar-ion laser with 488 nm excitation wavelength is employed, a derivatization step is required for LIF detection. For this purpose, fluoresce-in-based derivatives are the most frequently used labelling agents. FITC is well known for reacting with primary and secondary amino groups in a basic environment. The maximum absorption and excitation wavelengths are 490—495 and 520—530 nm, respectively. So FITC was used as derivative reagent in this experiment. The solution of FITC was prepared by dissolving 5 mg of FITC in 25 mL of acetone (5.0 x 10-4 M) and stored at —20°C in the dark. The sodium borate solution was prepared by double-distilled water at 100 mM.
Four rabbits (2.5—3.0 kg) were used in this experiment. After oral administration of 2 mg/kg of meto-prolol to rabbits, 300 ^L blood samples were drawn from veins of rabbit ears twelve times in 0, 0.5, 1, 1.5, 2, 3, 5, 8, 10, 16, 20 and 24 h. After the blood samples were collected in polypropylene micro-centrifuge tubes, they were centrifuged at 900 rpm for 10 min at 5°C. After centrifugation, 100-^L aliquots of plasma were ultrafiltered. Then 25 ^L of plasma ultrafiltrate were treated with 5 x 10-4 M FITC in dark at 25°C for 12 h. The reaction equation of metoprolol and FITC is as follows (Fig. 1). The ultrafiltration technique has been applied to plasma processing for drug and biogenic amine monitoring assay [20]. The ultrafiltration procedure offers the advantages of simplicity and high reproducibility, eliminating problems associated with the precipitation procedures (e.g., sample dilution, incomplete protein precipitation, drug coprecipitation, acid-catalyzed degradation etc.).
RESULTS AND DISCUSSION
The effect of temperature of derivatization was studied. An increase in temperature produced an increase in the rate of derivatization. However, a high temperature would result in a high volatilization of organic solvent which would lower the derivatization yield. Taking into account the rate of derivatization and reaction time, the temperature was selected at
N=C=S
O—„
O FITS
СИ(СИз)2
OH
CH2CH2OCH3
NH CH2°
25°C dark
OCH2CHCH2NHCH(CH3)2
OH
Metoprolol
O=
CH2CH2OCH3
O
Fig. 1. The reaction of FITC and metoprolol.
+
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.