ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 6, с. 613-616
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
SENSITIVE SPECTROFLUORIMETRIC DETERMINATION OF ALFUZOSIN IN PHARMACEUTICAL PREPARATIONS AND HUMAN URINE USING DANSYL CHLORIDE © 2015 Ayga Karasakal*, Sevgi Tatar Ulu**, 1
*Department of Chemistry, Faculty of Science and Letters, Namik Kemal University
Tekirdag, Turkey
**Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University
34416 Istanbul, Turkey 1E-mail: sevgitatar@yahoo.com Received 02.12.2013; in final form 25.09.2014
A sensitive and selective spectrofluorimetric method was developed for the determination of alfuzosin in pharmaceutical preparations and urine. The method is based on reaction of alfuzosin with 1-dimethylami-nonaphthalene-5-sulphonyl (dansyl) chloride in carbonate buffer of pH 9.7 to yield a highly fluorescent derivative that is measured at 516 nm after excitation at 405 nm. The developed method was validated according to the criteria for analytical method, showing good performances in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The fluorescence intensity versus concentration plot was rectilinear over the range of 25 to 500 ng/mL. The LOD and LOQ were found to be 8 and 25 ng/mL, respectively. The method was successfully applied to the determination of alfu-zosin in pharmaceutical preparations and urine.
Keywords: alfuzosin, dansyl chloride, derivatization, urine, pharmaceutical preparations.
DOI: 10.7868/S0044450215060055
Alfuzosin (AZ, Scheme) is an uroselective and competitive adrenergic-receptor antagonist, used to treat arterial hypertension and symptomatic benign prostatic hyperplasia, a common disorder in older men [1].
Salt of alfuzosin hydrochloride (AZH) is chemically known as N-[3-[(4-amino-6,7-dimethoxy—quinazolin-2-yl)-methylamino]propyl]tetrahydrofuran-2-carboxa-mide hydrochloride [2].
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Chemical structures of alfuzosin and dansyl chloride
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Several methods have been reported for the determination of AZH in pharmaceutical preparations and biological fluids. These are classified as spectrophotome-try [3], HPLC with fluorimetric and luminescence detection [4—8] and liquid chromatography with electro-spray ionization—mass spectrometry (LC—ESI—MS-MS) [9]. Dansyl chloride is fluorogenic reagent for the determination of primary and secondary aliphatic
amines. According to the screening of the literature, there was no use of dansyl chloride as fluorescence probe for the complexion ofAZH.
That is, this study would be the first for the determination of AZH with dansyl chloride. Therefore, it was aimed to study on the determination of the drug by its derivative employing dansyl chloride measuring the
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Fig. 1. Emission spectra ofblank (a), 500 ng/mL AZ—dan-syl derivative (b), and urine spiked with 500 ng/mL AZ— dansyl derivative (c).
purchased from Fluka (Buchs, Switzerland). All solvents used were of analytical grade (Merck Darmstadt, Germany). Water was purified by aquaMAXTM ultra (Young instrument, Korea) — ultra water purification system. From a healthy volunteer urine samples were collected into a glass tube and kept in a refrigerator at —20°C until analysis.
Preparation of the solutions. Stock solution ofAZH was prepared in the concentration of 0.1 mg/mL in methanol. Then, the standard solution (100 ^g/mL ) was prepared by diluting from the stock solution with methanol. The solution of dansyl chloride and its 0.02% dilution were prepared in acetone. All solutions and reagent were kept in a pyrex tubes covering them with aluminum folia. It was always kept in dark and refrigerator. Sodium bicarbonate (0.1 M) solution was prepared in water and adjusted to pH 9.7 with 0.1 M NaOH.
Preparation of sample tablets. Ten tablets were weighed, finely powdered and portions equivalent to 100 mg AZH were transferred into a 100 mL volumetric flask; 50 mL methanol was added, shaken thoroughly to dissolve. Then, it was made up to volume with methanol, mixed well and filtered. An aliquot of 10 mL of the filtrate was diluted to 100 mL to prepare of sample tablet solutions (100 ^g/mL).
Spiked human urine. The proposed method was applied to the determination of AZH in spiked urine provided from one healthy volunteer. Spiked urine was 100-fold diluted with distilled water. Increasing amount of each AZH was quantitatively transferred into a series of 12 mL volumetric flasks, in the concentration range 25—500 ng/mL, in the following order: 1 mL urine, 198 ^L dansyl chloride, 100 ^L buffer (pH 9.7) and mixed well. The mixture was kept for 30 min at room temperature. The content of tube was extracted three times with 3 mL of dichloromethane. The combined organic phases were adjusted to 10 mL with the dichloromethane. The fluorescence intensity was measured in a 10 mm path-length quartz cell with excitation and emission wavelengths of 405 and 516 nm, respectively.
intensities using spectrofluorimetry and to develop a sensitive, simple, rapid and accurate method.
EXPERIMENTAL
Apparatus. Fluorescence spectrum and measurements were performed by employing ofModel RF-1501 Shimadzu spectrofluorimeter (Kyoto, Japan) equipped with a 150 W Xenon lamp and a 1-cm quartz cell used throughout the study.
Materials and reagents. Pure powder of AZH (all >98% purity) was obtained from Sigma (Steinheim, Germany). Pharmaceutical preparation (Xatral tablets, 10 mg) supplied from Sanofi Aventis was purchased from a local pharmacy. Dansyl chloride was
RESULTS AND DISCUSSION
Derivatization. Dansyl chloride is a reagent that reacts with some primary, secondary amines, carboxyl and phenols to yield a highly fluorescent derivatives [10—12]. AZH was determined spectrofluorimetrical-ly by derivatization with dansyl chloride. The fluorescence intensities of the products were measured at 516 nm while exciting at 405 nm (Fig. 1).
Optimization of the derivatization conditions. The optimum derivatization reaction conditions between AZH and dansyl chloride was determined. Experimental parameters affecting the reaction were determined, such as pH, reaction temperature and time, dansyl chloride concentration and volume, amount of the dansyl chloride, extraction solvent and stability.
SENSITIVE SPECTROFLUORIMETRIC DETERMINATION
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Fig. 2. Effect of pH on the derivatization ofAZH with dan-syl chloride.
Effect of pH. At the beginning of this study the effect of carbonate buffer pH on the fluorescence intensity of AZ—dansyl derivative was examined in the range 9.0— 11.0. Maximum fluorescence intensity was obtained at pH 9.7 (0.1 M carbonate buffer, Fig. 2).
Effect of reaction temperature and time. The effect of temperature on the reaction was also investigated from room temperature to 60°C. Maximum fluorescence intensity develops within 30 min at room temperature at a concentration of 250 ng/mL (Fig. 3).
Effect of dansyl chloride concentration and volume. The influence of the concentration of dansyl chloride was studied using different volumes (66—462 ^L) of 0.02% of the reagent solution. The maximum fluorescence intensity was obtained with 198 ^L (Fig. 4). The reagent amount required was examined by changing the mole ratio of dansyl chloride to AZH. A 6-fold molar excess of reagent was found to be necessary to complete the reaction.
Effect of extraction solvent. The effect of different extraction solvents on the fluorescence intensity of AZ— dansyl derivative was investigated using ethyl acetate, chloroform and dichloromethane. It was found that dichloromethane was the best solvent for extraction since it gave the highest fluorescence intensity (Fig. 5).
Stability of the fluorigenicproduct. The stability of the AZ—dansyl derivative in dichloromethane as a function of time was also studied. It was found that the derivative remained stable for about 3 h at room temperature (25 ± ± 2°C) in daylight and 3 days at 4°C in the dark.
Method validation. The validity of the proposed method was tested regarding linearity, limit of detection, limit of quantification, precision, accuracy, recovery and robustness according to International Conference on Harmonization Q2B recommendations [13].
Linearity. Calibration curve of AZ—dansyl derivative was linear over the concentration range of 25— 500 ng/mL for urine. The calibration curve equation is F = (1.85 ± 0.01)c + (42 ± 5) (mean ± SD is given).
LOD and LOQ were calculated according to the following equation: LOD = 3.3 a/a and LOQ = 10 a/a, where is the standard deviation of the intercept of regression equation, a is the slope of the calibration curve.
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Fig. 3. Effect of temperature and time on the derivatization of AZH with dansyl chloride. Temperature, °C: 1 — room temperature, 2 - 40, 3 - 50, 4 - 60.
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Fig. 4. Effect of added volume of 0.02% dansyl chloride solution.
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Fig. 5. Effect of organic solvents on the derivatization of AZH with dansyl chloride.
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AYgA KARASAKAL, SEVGI TATAR ULU
LOD and LOQ values for AZ—dansyl derivative were found to be 8 and 25 ng/mL for urine.
Precision and accuracy. Accuracy and precision of the assay was determined by intra- and inter-day for 3 consecutive days. Three different concentrations of AZH were determined in six independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision). The accuracy and pre
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