ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 3, с. 302-309


УДК 543


Department of Pharmaceutical Chemistry, University of Medical Sciences, Faculty of Pharmacy str. Grunwaldzka, 6, 60-780Poznan, Poland 1E-mail: imuszals@ump.edu.pl Received 04.04.2013; in final form 20.05.2014

A selective ultraviolet and visible spectrophotometric method (UV-Vis) for the determination of memantine (MM) and rimantadine (RM) hydrochlorides after derivatization with 1,2-naphtoquinone-4-sulfonic acid sodium salt (NQS) and extraction with dichloromethane was developed and optimized. The reaction of the NQS (0.7%, w/v solution) with memantine was carried out for 40 min in a buffer medium of NaOH/KCl (pH 12.0) at 80°C. A mixture of NQS (0.2%) and rimantadine was heated for 20 min at 50°C, in a medium of NaOH/KCl (pH 12.0). The linearity of the determinations of MM (0.05-0.60 mg/mL) and RM (0.05-0.30 mg/mL) was evaluated. LOD (28 mg/mL for MM, 11 mg/mL for RM) and LOQ (84 mg/mL for MM, 32 mg/mL for RM) values and parameters of precision and accuracy were determined. The UV-Vis method for the determination of rimantadine hydrochloride tablets was validated (selectivity, linearity, precision, accuracy).

Keywords: memantine, rimantadine, 1,2-naphthoquinone-4-sulfonic acid sodium salt, derivatization, UV-Vis spectrophotometry.

DOI: 10.7868/S0044450215030123

Rimantadine and memantine (Scheme 1), derivatives of adamantine, demonstrate different pharmacological activities. Rimantadine is used to treat influenza A. It is involved in inhibiting the replication of the virus by blocking the M2 ion channel, interfering with the passage of hydrogen ions required for the release of ribonucleic acid into the cytoplasm of the host [1—3]. Memantine prevents overstimulation of glutaminergic N-methyl-D-aspartate receptor, improving the cognitive performance of the patient with dementia. Pre-clinical studies confirm the protective effect of me-mantine on the structure of the central nervous system, resulting not only from inhibiting an excessive influx of calcium ions but also from affecting the levels of P-amyloid. Thus, memantine may produce a therapeutic effect also in epilepsy, stroke, multiple sclerosis and Parkinson's disease [4—7].

Memantine Rimantadine

Scheme 1. Structural formulas of adamantane derivatives: memantine and rimantadine.

Adamantane derivatives are not detectable in UV-Vis region because they do not have fluorescent or chro-mophoric groups, necessary for the absorption in this range. Therefore, most methods of determining adamantine derivatives involve derivatization. Chloride anthraquinone-2-sulfonic acid [8] (HPLC with ultraviolet or mass detector: HPLC-UV HPLC-MS), N-methyl-N-(i-butylsilyl)trifluoroacetamide [9], pen-tafluorobenzoyl chloride [10] (gas chromatography with mass or electron capture detector: GC—MS, GC-CD), 4-fluoro-7-nitro-2,1,3-benzoxadiazole [11], o-phthalic aldehyde [12], 2-mercaptoethanol [12], fluo-rescamine [13], chloride 2-naphthol acetylene [14], flu-orocarbonyl chloroformate [15] (HPLC with fluorescence detector HPLC—Flu), 6-carboxyfluorescein N-hydroxysuccinimide ester [16] (micellar electroki-netic chromatography with laser induced fluorescence: MEKC—LIF) or 1,2-naphthoquinone-4-sulfonic acid — NQS [17, 18] (capillary zone electrophoresis— CZE, Vis, Flu) are examples of derivatization reagents used in the determination of rimantadine, memantine or amantadine. The use of NQS as a reagent was questioned by Revilla et al. due to the fact that although the resulting precipitate was dissolved by the addition of a surfactant (Brij 35), the colour of the so-obtained solution was not significantly different from the colour of the blank solution containing the reagent (NQS) [17].

However, this reagent was successfully applied in the spectrophotometric determination of such compounds as cefadroxil and ceftriaxone [19], cefotaxime and valaciclovir [20], isoniazid [21], dapsone [22], tri-metazidine [23], amphetamine and methamphet-amine [24], methyl- and dimethylamine [25] and amantadine [18].

The aim of this study was to optimize the derivati-zation of rimantadine and memantine (derivatives of alkylamino- and aminoadamantane) with 1,2-naph-thoquinone-4-sulfonic acid sodium salt by selecting the optimal reaction pH, temperature, reaction run time and the method of extracting the coloured products. Due to the instability of NQS, it was necessary to assess the stability of the extract of the formed products when exposed to light, which was not considered in previous studies. Following optimization, the Vis method was validated and verified regarding the determination of rimantadine and memantine in pharmaceutical preparations.


Materials and reagents. Memantine hydrochloride, rimantadine hydrochloride and 1,2-naphthoquinone-4-sulfonic acid sodium salt were purchased from Sig-ma-Aldrich Co., USA. Memantine (Ebixa®, H.Lund-bech A/S, Denmark) and rimantadine (Rimantin®, Genexo Sp. z o.o., Poland) tablets were labeled to contain 10 and 50 mg of active substance, respectively. The excipients of memantine tablets core were: lactose monohydrate, microcrystalline cellulose, colloidal anhydrous silica, talc, and magnesium stearate, and the components of the coating were: methacrylic acid-ethyl acrylate copolymer 1 : 1, and polysorbate 80. The excipients of rimantadine tablets were: lactose, potato starch, and stearic acid. Average weights of the tablets were: 0.2516 ± 0.0006 g (RSD 0.51%, n = 20) and 0.1499 ± 0.0010 g (RSD 1.40%, n = 20) for Ebixa® and Rimantin®, respectively. All other chemicals and solvents were of analytical grade. For filtration the filter papers with grade 390, diameter 125 mm and density 84 g/m2 from Munktell & Filtrak GmbH (Germany) were used.

Apparatus. A UV-Vis spectrophotometer (Lambda 20, Perkin Elmer, UK) with 1 cm matched quartz cells was used for all absorbance measurements. The absor-bance of orange extracts was measured in closed cuvettes in the range from 320 to 570 nm against blank samples and the absorbance was read at the analytical wavelength ^max (453 nm for the MM product and 452 nm for the RM product). The pH measurements were performed with an HI 110 pH-meter (Hanna Instruments, Hungary).

Preparation of buffer solutions. Borate buffer pH 8.0-10.8: boric acid solution (0.1 M) was adjusted to the appropriate pH with 0.1 M NaOH. Buffer

pH 11.5-13.0: 25.0 mL of 0.2 M KCl were transferred to calibrated flasks, then 2.5-66.0 mL of 0.2 M NaOH were added and diluted to 100.0 mL with deionized water.

Preparation of stock solutions. MM or RM solutions (1 mg/mL) in HCl (0.005 M): 0.1 g of sample (MM or RM) was dissolved in 5.0 mL of 0.1 M HCl by shaking for about 5 min. The so-obtained solutions were diluted to 100.0 mL with deionized water. Reagent solution (NQS, 1%, w/v): 0.5 g of NQS was transferred to a 50.0 mL calibrated flask, 30 mL of water was added and the mixture was sonicated in an ultrasonic bath for 3 min. Then, the solution was diluted with deionized water. The solution was protected from light.

Optimization of the conditions of MM and RM reactions with a solution of NQS. The reaction mixture containing 2.0 mL of the tested substance (MM 0.6 mg/mL or RM 0.3 mg/mL in 0.005 M HCl), 3.0 mL of buffer and 1.0 mL of reagent solution (NQS 0.6%, w/v) was heated for 45 min in a water bath (80°C). The solution was then cooled for 2 min in ice water, and 5.0 mL ofthe mixture was transferred to a 50 mL separating funnel. The solution was extracted for 5 min with 10.0 mL of dichloromethane and allowed to separate. After 15 min, the lower (dichloromethane) layer was filtered through filter paper containing 0.2 g of anhydrous sodium sulphate.

The optimization was aimed at following conditions: the reaction medium (pH 8.0-10.8, borate buffer; pH 11.5-13.0, NaOH/KCl buffer), the concentration of NQS reagent (1.92-38.4 mM, 0.05-1.00%, w/v), the temperature and heating time of the reaction mixture (25-90°C) and the type of extracting solvent (CH2Cl2, CHCl3, CCl4). Solutions of MM, RM, and NQS of desired concentrations were prepared by diluting the stock solutions.

Stability of the extract. 2.0 mL of the test solution in 0.005 M HCl (MM 0.4 mg/mL, RM 0.25 mg/mL), 3.0 mL of buffer pH 12.0 and 1.0 mL of NQS (0.7 and 0.2%, w/v for MM and RM, respectively) were placed in a test tube. The mixtures containing MM were heated for 40 min at 80°C, and those containing RM for 20 min at 50°C. After heating the solutions were cooled for 2 min in ice water, mixed and transferred (5.0 mL) into separating funnels. Then, the so-formed products were extracted for 5 min with 10.0 mL of dichloromethane and allowed to separate. After 15 min, the dichlo-romethane layers were filtered to flasks through filter paper containing 0.2 g of anhydrous sodium sulphate and stored protected and unprotected from light. Absorbance was measured at ^max at specified intervals.

Calibration curve, precision and accuracy of the determination of MM and RM in solutions. A series of dilutions of the stock solutions of memantine and rimantadine hydrochlorides in hydrochloric acid (0.005 M)

was prepared in the range from 0.05 to 0.60 mg/mL for MM and from 0.05 to 0.30 mg/mL for RM. As described in the assessment of the stability of extracts, de-rivatization of the test substances was made and the products were extracted. The extracts were protected from light. Determination of the tested substances in each solution was repeated three times and the values of Amax were used to determine the parameters of calibration curves.

The accuracy and precision of the method were estimated by determining the content of the active substances in solutions of different concentrations in 0.005 M

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