ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2014, том 69, № 7, с. 735-742
ОРИГИНАЛЬНЫЕ СТАТЬИ
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ADSORPTIVE AND AFFINITY LINEAR SWEEP VOLTAMMETRY FOR THE DETERMINATION OF SINGULAIR IN TABLETS AND ITS INTERACTION WITH HUMAN SERUM ALBUMIN
© 2014 Deia Abd El-Hady*, **, Ahmed K. Youssef**
*Chemistry Department, Faculty of Science-North Jeddah, King Abdulaziz University
Saudi Arabia E-mail: deiaabdelhady@yahoo.com **Chemistry Department, Faculty of Science, Assiut University 71516-Assiut, Egypt Received 27.04.2012; in final form 14.01.2013
A new simple, sensitive and reliable adsorptive and affinity linear sweep voltammetric method at in situ mercury film electrode for the determination of singulair in tablets and its interaction with human serum albumin (HSA) was investigated. The solubility of singulair in aqueous media was enhanced by inclusion with hydrox-ypropyl-P-cyclodextrin (HPpCD). The nature of the electrochemical process of singulair was studied by affinity cyclic voltammetry (ACV). Reproducibility of the proposed method was checked giving interday precision of 0.073 standard deviation. The limit of detection and limit of quantification were 0.05 and 0.16 fg/L, respectively. In the present work, singulair was interacted with HSA by 1 : 1 stoichiometry to form electroin-active supramolecular complex. The binding constant was precisely estimated by coupling affinity linear sweep voltammetry (ALSV) and ACV with non-linear regression analysis based on the shifting of analyte peak potentials. The proposed experiments and data analysis could be used to investigate the binding constant of drug with protein within one hour.
Keywords: singulair, adsorptive voltammetry, affinity voltammetry, human serum albumin, binding constant, tablet.
DOI: 10.7868/S0044450214070056
Singulair is a registered trademark by Merck of a prescription medicine which is used to thwart asthma and other nasal allergies that are caused due to runny or stuffy nose and also due to exercising. Singulair is obtainable in the form of granules and tablets that is rapidly absorbed following oral administration [1]. It is chemically named montelukast sodium:
Cl
COO-Na+
H3C
It is a class II drug so it shows very low aqueous solubility and freely soluble in ethanol and methanol [1]. The difficulty of singulair solubility in aqueous media leads to limit the study of its interaction with macro-biomolecules such as protein under the physiological conditions. Cyclodextrins (CDs), the natural oligosaccharides possessing a hydrophilic exterior and hydrophobic cavity, are well-known safe encapsulating ma-
terials [2]. In fact, CDs are continually being examined for their ability to improve complexity in order to obtain both higher solubility of drugs, and stabilizing or solubilizing agents [3]. The hydroxypropyl derivatives of CD are very often used in pharmacy due to their better complexation ability than that of natural CD and they increase the solubility and bioavailability more than natural CD [4]. Therefore, in the present work, HPpCD was used to enhance the solubility power of singulair in aqueous media.
Reviewing the literature revealed that some separation methods have been developed to determine singu-lair using chromatographic assays in dosage formulations [5—12] and human plasma samples [13—19]. It was possible to separate singulair and several impurities using capillary electrophoresis [20, 21]. Spectrophotometry methods for the estimation of singulair in tablet dosage form have been developed and validated [22—24]. Voltammetry can be considered as a convenient alternative to those techniques. The choice of working electrode is a crucial point that should have favorable electrochemical behavior of the analyte of interest, friendly environment and a reproducible sur-
face area with low background current. In situ plated mercury film electrode [25—27] is the best one that could achieve a large surface area with low volume ratio, high plating efficiency, high sensitivity and good selectivity due to the fast diffusion of analyte through the film. To the best of our knowledge, there is only one report for the voltammetric determination of sin-gulair in tablets and spiked plasma [28] at hanging mercury electrode, pH 2.00. This method was limited for further applications because mercury electrode was internationally forbidden, analyte was completely soluble in alcohol and the determination was performed in strong acidic medium.
Interaction of drugs with protein has aroused great interest in supra-molecular chemistry due to understanding their structure and function [29]. A drug's efficiency may be affected by the degree to which it binds to proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Notably, it is the unbound fraction which exhibits pharmacological effects. It is also the fraction that may be metabolized and/or excreted. The bound portion may act as a reservoir or depot from which the drug is slowly released as the unbound form in order to maintain equilibrium [27]. Many methods have been proposed for the study of the binding reactions of proteins such as spectrophotometry [30], fluorometry [31] and light scattering techniques [32]. Compared with spectroscopic methods, affinity voltammetry is simple, reliable and practical with low detection limits and wide dynamic ranges. Because the electrochemical reaction occurs on the electrode/solution interface, it is especially suitable for small amounts of sample. Affinity voltammetry is a useful technique for the study of the interaction of small molecules with bio-molecules, and have been used to investigate the binding reaction of proteins with drugs [33, 34].
In the present study, a new simple, sensitive and reliable adsorptive linear sweep stripping voltammetric method at in situ mercury film electrode for the determination of singulair in tablets was developed in aqueous medium. As well, for the first time the precise binding constant of singular with HSA was calculated by coupling the ACV or ALSV with non linear regression analysis based on the changes of electrochemical responses.
EXPERIMENTAL
Apparatus. All voltammetric investigations were performed in a 10.00 mL glass voltammetric cell using commercially available electrode stand (Metrohm, Switzerland). The electrode was connected via IME-663 module (Netherlands). Potentials were controlled using a 3-electrode configuration comprising a rotating glassy carbon disc working electrode (3 mm diameter, Metrohm), a Ag/AgCl (3.00 M KCl) reference electrode and a platinum wire counter electrode. Turbidity was obtained by using turbidity and chlorine benchtop meter,
LaMotte LTC-3000we, 0-4000 NTU & 0-10 ^g/mL. The pH values were measured using the Fischer Scientific pH meter model 810 equipped with a combined glass electrode, which was calibrated regularly with buffer solutions (pH 4.0 and 7.0) at 22 ± 2°C.
Chemicals and reagents. Singulair (reference material) was obtained by Global Nabi Pharmaceuticals, Egypt. It was used without any further purification. A fresh solution of singulair was prepared daily in 10.0 mL of 0.25 mM HPßCD and diluted as required for quantitative analysis. Human serum albumin (HSA, 99%, Sigma) was used as received. The 1.0 g/L stock solution of HSA was prepared in twice distilled water and stored at 4°C. The working solutions were obtained by diluting the stock solution with phosphate buffer. A concentration of 67.0 mM phosphate buer solution was used to control the pH of the solutions tested. It was prepared by mixing definite weights of Na2HPO4 and NaH2PO4 (Sigma) at each desired pH value. Stock solution of mercuric ion (0.01 M) was prepared by dissolving the required weight of basic nitrate (May & Baker LTD., Dagenham, UK) in twice distilled water. All other reagents were of analytical reagent grade.
Procedure. Voltammetric determination of singulair at in situ-MFE. The glassy carbon electrode (GCE) was prepared as described in our previous works [2527] with little modifications. The electrode was polished at the beginning of the experiments with 0.05 ^m aluminum oxide (particle size 0.1 ^m, Metrohm, Switzerland) and rinsed thoroughly with water to obtain a clean and renewed electrode surface. The electrode was connected to the potentiostat and placed in the buffer solution; the potential was cycled 50x between -800.0 to +800.0 mV using cyclic voltammetry at a scanning rate of 100.0 mV/s. The electrochemical pretreatment was repeated daily, and the polishing only when damage of the electrode surface was suspected. The in situ mercury film electrode was prepared by pippeting 10.0 mL of the phosphate buffer at a definite pH into the cell followed by the simultaneous depositing of singulair and 30.0 ^M mercury(II) on GCE. Oxygen was removed by purging 5 min with nitrogen and then deposition was carried out for 30150 s at -600.0 mV, whilst the electrode was rotated at 1100 rpm. Subsequently after 10 s quiescent time the stripping step was cathodically performed from —600.0 to -1300.0 mV using a linear sweep scan with pulse height 50.0 mV. Each scan was preceded by an electrochemical cleaning step to remove the previous film using a conditioning potential of 600.0 mV for a period of 60 s. All measurements were performed under ambient conditions.
Voltammetric study of singulairHSA interaction. Into a 10.00 mL volumetric cell, 0.5 mL of 100.0 mM singulair and an appropriate amount of HSA solution were added. The mixture was diluted with 67.0 mM phosphate buffer, pH 7.4, and mixed homogeneously
by rotation for 5 s. Subsequently, the linear sweep or cyclic voltammetric curves were recorded to show the electrochemical changes of the reaction system in the potential range from —600.0 to +1300.0 mV. As well, such interaction was studied by titration of different singulair concentrations with fixed concentration of HSA under the same experimental co
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