Memantine hydrochloride, low-to-moderate affinity noncompetitive N-methyl-D-aspartate receptor antagonist, is used in the treatment of patients with moderate to severe Alzheimer’s disease. Chemically, Memantine is an aliphatic tricyclic primary amine compound which lacks any UV or fluorescence properties. Previous studies have reported the spectrophotometric quantification of memantine after derivatization reactions with different reagents. In this paper, we reported the formation of a fluorescent product by reacting memantine hydrochloride and o-phthalaldehyde in the absence of a thiol. Experimental design methodologies were used in the optimization step. The variables under investigation were: pH; the ratio between memantine hydrochloride: o-phthalaldehyde and heating time. The results of statistical analysis of two sequential full factorial design were used for quantification of memantine hydrochloride at optimized reaction condition. Optimal conditions for derivatization were: aqueous NaOH, pH 12.2, memantine hydrochloride:o-phthalaldehyde ratio of 1:5, and heating at 70oC for 45 min. A new eco-friendly spectrofluorimetric derivatization procedure has been developed and validated for analysis of memantine hydrochloride. The linear range was 0.25–10 µg/mL with a quantification limit of 1.97 µg/mL, detection limit of 0.65 µg/mLwith maximum fluorescence intensity development at λex 350 nm and λem 427 nm
Keywords: Memantine; o-phthalaldehyde; Spectrofluorimetry; Factorial design
The rapid chemical reaction of o-phthalaldehyde (OPA), a very common flourogenic agent, with primary amines at room temperature in the presence of thiol originate a fluorescent compound, isoindole derivative, which is used for their spectrophotometric analysis [1-3]. In this reaction, schematically shown in Scheme 1, the primary amine and a mercaptan react with OPA under basic condition to form highly fluorescent 1-alkylthio-2-alkyl substituted isoindoles [3, 4].
O 2 R S 1 R NH H 2 1 R N 2 H R SH O
This reaction was used for the spectrophotometric or chromatographic quantification of amino acids [1,5,6] and different drugs having primary amino groups [7-9] after derivatization with OPA/thiol mixture. Different mercaptans were used in literatures such as 2- mercaptoethanol [9, 10], thioglycolic acid [3], 3- mercaptopropionic acid [1, 2], N-acetyl-L-cysteine [11, 12] and other chiral thiols [13]. The influence of different experimental variables, was studied by several authors. [2, 9, 12].
Primary amines [14], amino acids [15, 16] and urea [17, 18] were reported toreact with OPA in the absence of thiol under alkaline condition to yield l,3- dihydroxyisoindoline derivatives, which upon heating eliminate one molecule of water to form afluorescent product, isoindolin-1-onederivatives, as shown in the following Scheme 2.
O HO O R NH2 - H2O H R N R N H HO O CH 3 In case of memantine, R = CH3
Memantine hydrochloride (MTH), low-to-moderate affinity noncompetitive N-methyl-D-aspartate receptor antagonist, is used in treatment of patients with moderate to severe Alzheimer’s disease, Parkinsonism, movement disorders [19], dementia syndromes [20], and it has recently been described as a potential treatment for glaucoma [21]. MTH was registered in Europe in 2002 with the indication of moderately severe to severe Alzheimer’s disease and was first marketed in the USA in 2003 [22].
Chemically, MTH, 1-amino-3,5-dimethyladamantane hydrochloride, is an aliphatic tricyclic primary amine compound which lacks any UV or fluorescence properties as shown in figure 1 [10, 11]. MTH has a primary amino group and the above-described reactions can be used for its identification and quantification. Previous studies have been reported the spectrophotometric quantification of MTH with the reaction of this compound with OPA and a thiol, [11, 12, 23, 24] but none, to our knowledge, was reported for spectrofluorimetric analysis of MTH after derivatization with OPA in the absence of thiol. There have been some reports about the analysis of the MTH determination by HPLC [25, 26], GC-MS [27, 28], LC- MS [29, 30], and capillary electrophoresis [31]. However, these methods are too much complex, expensive instrumentation, time-consuming sample preparation and long chromatographic elution time for analysis of MTH [10, 25, 26]. Few spectrophotometric methods were developed for the assay of MTH either by UV, colorimety
The aim of this paper was to develop an eco-friendly, simple spectrofluorimetric based analytical method for determination of MTH in bulk and pharmaceutical preparations. Special attention was put on optimization of the reaction of MTH with OPA in the absence of a thiol, forming a fluorescent isoindolin-1-one derivative, to avoid the unpleasant odor and toxicity of free thiols [33]. The optimal conditions of this reaction were systematically optimized by employing full factorial experimental design methodologies [34-36].
® Memexa Tablets are manufactured by Copad Egypt for trade and pharmaceutical industries (Copad Pharma): Al Obour City-Cairo, Egypt and each tablet claimed to contain 10 mg MTH. MTH (99.9%) was kindly provided by Sigma for Pharmaceutical Industries Co. Mubarak Industrial Zone, 1st Zone; Quesna; Elmenoufia; Egypt. OPA and sodium hydroxide werepurchased from Fluka Chemical Industries, Ltd. (Buchs, Switzerland).Water was obtained by double distillation.
Aqueous solutions of sodium hydroxide (pH = 8.5, 10.5 and 12.2) were prepared. Three stock solutions of MTH containing 0.1 mg/mL at three different pH values (pH = 8.5, 10.5 and 12.2) were prepared by dissolving 25 mg of the MTH in (100 mL) NaOH solutions (pH = 8.5, 10.5 and 12.2) and volumes were adjusted to 250 mL with the same solution. Three stock solutions of OPA (2.0 mg/mL) were also prepared by dissolving50 mg OPA in 25mL of NaOH solutions (pH = 8.5, 10.5 and 12.2), and were o protected from light and kept at 4 C. In the experimental design optimization, the MTH and OPA standard stock solutions were prepared in NaOH solutions (pH = 8.5, 10.5 and 12.2) to concentrations of 1.0 mg/mL.
Spectrofluorimetric measurements were made inFP- 6300 Jasco, (Japan) spectrofluorimeter equipped with a 150 W xenon lamp and connected to personal computer loaded with {Jasco} –{spectra manager} software. The pH measurements were made with HANNA pH 211 Microprocessor, pH Meter with double Junction glass combination electrode. Statistical analysis of data including factorial design was made using Minitab ® 18 software [37].
Aliquots of MTH containing (0.25–20 μg/mL) MTH were transferred to a set of light-protected screw capped test tubes, appropriate volume of OPA was added and mixed o well. Test tubes were warmed in a water bath at 70 C then, the contents were allowed to cool. The solutions were transferred to 5.0 mL volumetric flasks and the volume was adjusted to the mark with NaOH solutions. The fluorescence intensity of the resulting solutions was measured at emission 427 nm after excitation at 350 nm. The blank determination without the addition of the analyte was also prepared simultaneously and was used to adjust the background fluorescence intensity. The fluorescence intensity was plotted versus the final drug concentration (μg/mL) to get the calibration graphs and the corresponding regression equations were derived.
Twenty tablets of Memexa® with labeled amount of 10 mg/tablet was weighed and crushed to fine powder. The powder corresponding to one tablet was weighed, dissolved in 50 mL aqueous NaOH and sonicated for 10 min. The volume was adjusted to 100mL with NaOH. The solution was filtered and solution (0.5mL) was transferred to 10mL volumetric flask. The analytical procedure was followed and the quantitation was made from the external calibration using the corresponding regression equation.
Different authors have described several derivatization reagents used for analysis of MTH, such as FMOC-Cl [5], (2-naphthoxy) acetylchloride, dansyl chloride [38], bromothymol blue [39], sulphonphthalein acid dyes [40] and ninhydrine [34]. In this research, an eco-friendly, simple, one step derivatization reaction for quantification of MTH was achieved by forming a fluorescent product resulting from the reaction of OPA with MTH under alkaline condition which showed maximum absorbance at 427 nm after excitation at 350 nm.
The general strategy was based on a preliminary evaluation of factors affecting the derivatization reaction between MTH and OPA. The possible interactions between factors will then be investigated by statistical analysis of full factorial experimental design, followed by optimization of the significant factors [34, 36]. Three main factors were appeared to be affecting the derivatization reaction of memantine with OPA; the reaction heating time, pH, and the volume ratio between the MTH and OPA. An initial evaluation of these factors was performed in a univariate way under alkaline condition and it was found that: i) The minimum volume ratio needed for the reaction fluorescence development is (1:1, MTH:OPA). ii) The reaction needs strong alkaline condition to liberate the free amino group as a direct relationship between pH and the fluorescence intensity is reported. iii) Higher absorbance is observed with elevating the temperature and heating time. iv) The formed fluorescent product is stable. (v) Higher fluoresces are observed with NaOH aqueous solutions than with borate or acetate buffers. The results obtained confirmed the formation of stable fluorescent product up to 120 min which has a wavelength of maximum absorbance (427 nm) after excitation at (350 nm).
For optimization of derivatization conditions, a 3 multilevel, 2 full factorial design was carried out and each experiment was repeated in triplicate using Minitab ® 18 Software [37]. A set of 24 experiments was performed where two levels for each factor were used as following: heating time (15 and 30 min), pH (5.5 and The reported response for each experiment was the fluorescence intensity of isoindolin-1-one derivative products as reported in Table 1. The effect of each factor was tested using a Student (t) test with a corresponding pvalue. All studied factors with p-values less than 0.05 and consequently, are considered statistically significant [36, 41].
Main effect plots, interaction plots and contour plots shown in figures (2 and 3), revealed that the fluorescence intensity, the response, is optimizing at the maximum level of pH, heating time and minimum level of the ratio between MTH and OPA while an interaction between pH and heating time is also observed. A graphical display of the ordered standardized effect of each factor, Pareto chart (Figure 4 (a)), showed that pH is the most statistically significant factor. Optimum conditions for derivatization reaction was obtained from the response optimizer plot by using the input variable combinations as shown in figure 4 (b). It involves the use of 0.2 (1:5) ratio between (MTH-OPA) at pH 10.5 and heating the reaction o mixture at 70 C for 30 min to get the best response.
The optimized spectrofluorimetry conditions were successfully applied to analyze MTH after derivatization with OPA. The linearity of the method was established by
preparing a calibration curve in the range 0.5-20 μg/mL.
Triplicates of each experiment were performed and the fluorescence intensity was recorded. The mean fluorescence intensity was plotted against concentration (μg/mL) to construct the calibration curve-1. The correlation coefficient and regression equation were determined as shown in Table 2.
A final univariate evaluation was done by increasing, the ratio between MTH-OPA from 0.1 (1:10) to 1.0 (1:1) at o pH12.2, and heating the reaction mixture at 70 C for 45 min. The results found are presented in Figure 8. An increase in the fluorescence intensity was observed until a ratio of (1:5) followed by a posterior decrease. With these optimized conditions, the calibration curve was obtained using a series of MTH standard concentrations and the merit of the calibration curve are reported in Table 4. Under the final optimized conditions, the regression data of the calibration curve-2are superior compared to the same calculated data after the first factorial design-1 as observed in Tables 2 and 4. Our developed method showed a comparable better results regarding linearity range (0.25-10 μg/mL), LOD (0.6512 μg/mL), LOQ (1.9735 μg/mL) at (λex = 350, λem = 427 nm) relative to a reported method [11] using OPA in the presence of a thiol which showed the linearity range (5-50 μg/mL), LOD (0.858), and LOQ (2.600) at (λex = 340, λem = 476 nm).A preliminary factorial design-1 of experiment followed by another optimizing factorial design-2 resulted in a more sensitive and accurate procedure approved by the remarkable lower values of the intercept, LOD and LOQ than other reported method using OPA and thiol.
The accuracy and precision of the proposed method was assessed by determining the concentration of MTH at three different concentration levels within the linearity range in one day and on three consecutive days. The standard deviation, relative standard deviation, percentage of error and mean recovery obtained by intra-day and inter-day assay were calculated and are summarized in Table 5. The results of the assays are acceptable and reasonable according to ICH guidelines [42].
The specificity of the method was investigated by performing recovery experiments through standard addition technique. For this purpose, a known amount of MTH was added to the pre-analyzed dosage form, Memexa® tablet (10mg), at three different percentage levels (50%, 100% and 150% of that in tablet) and then determined by the proposed methods. The results, in Table 6, showed that no interference from the common dosage form excipients was observed and establishes some degree of specificity of the proposed methods. For the assessment of method robustness, some experimental parameters were interchanged; volume ratio of MTH:OPA (1:5 ±0.1 mL) and pH of NaOH (12.20 ±0.10). The analysis was performed at two different concentrations of MTH (5 and 10μg/mL). The ability remained unaffected by small deliberate variations. The results, presented in Table 7, indicate the acceptable robustness of the proposed methods.
The proposed methods were applied to the quantification of MTH in tablet dosage forms purchased from a local pharmacy store. The results, shown in Table 8, suggest that the method is suitable for the determination of MTH with good accuracy and precision. The excipients in the dosage forms do not interfere in the assay procedure and three replicates were determined.
Copad Pharma, Egypt for trade and pharmaceutical industries: Cairo, Egypt a b The average of three separate determinations.
An eco-friendly, new spectrofluorimetric method was developed for the quantification of MTH using OPA in the absence of thiol. Two sequential factorial design experiments were developed to optimize the factors affecting the derivatization reaction between MTH and OPA. The developed method is validated as per ICH guidelines. It was observed that all validation parameters such as linearity, precision, accuracy, selectivity, and robustness convene the predetermined acceptance criteria.
Thus, it has been concluded that the proposed method is validated for the routine analysis of the drug in pure and tablet dosage form.The proposed spectrofluorimetric method has the advantages of simplicity, precision, accuracy and convenience for the quantitation of MTH.
Consequently, the proposed method can be used for the quality control of the cited drug and can be extended for their routine analysis in dosage forms.