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Journal of Chemistry
Volume 2015, Article ID 672183, 7 pages
http://dx.doi.org/10.1155/2015/672183
Research Article

HPLC-Fluorescent Analysis of Memantine: An Investigation on Fluorescent Derivative Formation

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Road, Rajathevee, Bangkok 10400, Thailand

Received 3 December 2014; Revised 14 January 2015; Accepted 16 January 2015

Academic Editor: Ghada M. Hadad

Copyright © 2015 Pornpan Prapatpong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Memantine is an N-methyl-D-aspartate receptor antagonist recommended for treatment of Alzheimer’s disease. Due to the lack of chromophores/fluorophores in memantine molecule, this work focuses on a novel procedure for memantine-fluorescent derivative formation enabling it to be monitored by a fluorescence detector. 4-(N-Chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulphonyl 2,1,3-benzoxadiazole (DBD-COCl) was chosen as a fluorescent probe since the carbonyl chloride of DBD-COCl could easily react with the amine on memantine to form the fluorescent derivative. The derivatization was achieved at 5 : 1 of DBD-COCl and memantine, at 60°C for 50 min. Structure elucidation from mass spectrometry and infrared spectroscopy spectra confirmed the amide bond of memantine-DBD-COCl. The derivative was stable up to 24 h and could be monitored by high performance liquid chromatography (HPLC) coupled with a fluorescence detector using a VertiSep GES C18 column, acetonitrile and water (80 : 20) as the mobile phase with a flow rate of 1 mL/min using the excitation and emission wavelengths at 430 and 520 nm, respectively. The derivative was eluted at 4.50 min and was separated from the DBD-COCl. Preliminary validation data reveals good linearity (), repeatability (%RSD < 0.54) and accuracy ( between 94 and 119%) with a limit of detection of 0.79 μg/mL.

1. Introduction

Alzheimer disease (AD) was firstly identified by a German psychiatrist and neuropathologist, Alois Alzheimer, in 1906 [1]. AD is a common dementia found in elderly that affects more than 4.5 million Americans and this number is estimated to increase to 14 million by the year 2050 [2, 3]. Destruction of central nervous system, specifically cholinergic system, is a major cause of cognitive function impairments as well as behavioral and psychological symptoms in dementia. Neuropathology and neurotransmitter changes such as senile plaques, neurofibrillary tangles, amyloid angiopathy, and decreasing of neurotransmitter acetylcholine and glutamate in neuron cell are major causes of the disease [4, 5]. AD patients suffer from loss of memory, orientation and language skill, mobility, vision, reasoning, and decision making. These symptoms slowly progress but become devastating since they greatly affect daily life activities of patients [6, 7]. Eventually, complete care is needed at the late stage of AD and patients usually die within 6–12 years.

Currently, there is no curative treatment for AD; the available medication only slows down the progress of the disease. The drugs are classified into three major types, which include (1) acetylcholinesterase inhibitors (e.g., donepezil, rivastigmine, and galantamine), (2) N-methyl-D-aspartate receptor antagonists (e.g., memantine), and (3) supportive medications (e.g., vitamin E, Ginkgo biloba extract, etc.) [8]. Among these drugs, memantine (Figure 1) is recently approved, and the United State Pharmacopeia suggests gas chromatography with flame ionization detection for the assay of the drug substance and tablets [9]. However, memantine monograph is not yet available in the British Pharmacopeia [10]. It is evident that memantine does not absorb UV or fluorescence due to lack of chromophore and fluorophore in its molecules. Therefore, derivatization of memantine with some probes (e.g., anthraquinone-2-sulfonyl chloride, 9-fluorenylmethyl chloroformate, dansyl chloride, 2-(napthoxy)acetyl chloride, o-phthaldialdehyde, and 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl) is required to enable the drug to be detected by UV or fluorescence detectors [11]. For example, precolumn derivatization followed by high performance liquid chromatography (HPLC) with UV or fluorescence detection has been demonstrated for analyses of memantine in rat plasma [1214], human plasma [15], human urine [16], and active pharmaceutical ingredients [17]. Furthermore, HPLC coupled with mass spectrometer (MS) has been employed for quantitation of memantine in human plasma [18] and urine [19]. Other methods including gas chromatography [9, 20, 21], spectrofluorometry [22], and spectrophotometry [22, 23] are reported for quality control of memantine in raw material and tablet formulations.

Figure 1: Structures of memantine and DBD-COCl.

This work aimed to develop a new and convenient derivatization procedure for memantine analysis using 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulphonyl 2,1,3-benzoxadiazole (DBD-COCl) as a fluorescent probe (Figure 1) [24]. The amine functional group of memantine could rapidly react with the carbonyl chloride of DBD-COCl under mild conditions to form a fluorescent derivative, which could be monitored by HPLC coupled with a fluorescence detector. Ratios of DBD-COCl and memantine, temperature, reaction time, and pH were investigated since they are key factors for the formation of the memantine fluorescent derivative. Formation of the derivative was achieved and it was characterized by HPLC, mass spectrometry, and infrared spectroscopy. Preliminary method validation supports the potential of the method for the analysis of memantine by HPLC coupled with fluorescence detection.

2. Materials and Method

2.1. Reagents and Equipment

Memantine hydrochloride was from Sigma-Aldrich (Munich, Germany). DBD-COCl was obtained from Santa Cruz Biotechnology (California, USA). Acetonitrile was of HPLC grade from RCI Labscan (Bangkok, Thailand). Pyridine was of reagent grade. Solid phase extraction (SPE) cartridges were Empore C18-SD 4 mm/1 mL (Minnesota, USA). HPLC instrument was a Flexar-UHPLC from Perkin Elmer equipped with a UHPLC pump, column oven, and UV/Vis and FL (fluorescent) detectors (Waltham, Massachusetts, USA). HPLC column was a VertiSep GES C18 (4.6 × 150 mm, 5 μm) (Vertical, Bangkok, Thailand). Analytical balance was from Sartorius and pH meter was from Consort (Turnhout, Belgium). Mass spectrometer is LCQ Fleet ion trap from Thermo Scientific Waltham (Massachusetts, USA). Fourier transform infrared spectrometer was Nicolet 6700 from Thermo Scientific Waltham (Massachusetts, USA).

2.2. Solution Preparation

Memantine stock solution (1 mM) was prepared by dissolving 5.39 mg memantine with 25 mL acetonitrile. The working solution was prepared by diluting the stock solution with acetonitrile to obtain the concentration of 20 μM. DBD-COCl stock solution (1 mM) was prepared by dissolving 8.32 mg DBD-COCl with 25 mL acetonitrile. DBD-COCl solution at concentrations of 20, 40, 60, 80, and 100 μM was prepared by transferring appropriate amounts of DBD-COCl stock solution to 50 mL volumetric flasks and adjusting to volume with acetonitrile.

2.3. Memantine Fluorescent Derivative Formation

Memantine fluorescent derivative formation was studied by varying several factors such as ratios of DBD-COCl and memantine, reaction time, and temperature. One milliliter of the memantine working solution (20 μM) was mixed with 1 mL of different concentrations of DBD-COCl (20–100 μM) in amber glass vials, heated on a water bath at various temperature (60–80°C) and reaction time (0–90 min). After every 10 min, 1 mL of water was added to the vials to stop the reaction and left to cool down at room temperature. One milliliter of the solution was filtered through a 0.22 μm membrane filter prior to HPLC analyses.

HPLC analysis was performed on a C18 reversed phase column using acetonitrile and water (80 : 20) as the mobile phase at the flow rate of 1 mL/min. The injection volume was 20 μL and the fluorescence detection was performed at the excitation and emission wavelengths of 430 and 520 nm, respectively.

Extraction of the memantine fluorescent derivative was carried out by SPE to ensure the purity of the derivative. The cartridge was preconditioned with 150 μL of acetonitrile and 300 μL of water. After loading the reaction solution to the cartridge, the excess DBD-COCl was washed with 100 μL of 40% acetonitrile three times and 100 μL of 90% acetonitrile two times, respectively. The derivative was eluted with 100 μL of methanol two times and left to dryness and the process was repeated five times. The residues were reconstituted with 1 mL methanol and injected into the HPLC and the LC fractions were collected and checked by mass spectrometry. The pure fractions were evaporated and subjected to infrared spectroscopy for structure elucidation.

3. Results and Discussion

Obviously, Figure 1 shows that memantine does not contain any chromophore or fluorophore in its molecule. Therefore, derivatization is necessary to enable it to be detected by UV or fluorescence detection. Presently, an approach for the formation of memantine fluorescent derivative was proposed (Figure 2). Reactions of DBD-COCl and memantine occurred via unimolecular nucleophilic substitution due to the nucleophilic property of amine group on memantine that can easily react with –COCl on DBD-COCl to form amide bond. The reaction was considered to be stoichiometric and straightforward. However, factors affecting the derivative formation needed to be optimized to establish the derivatization protocol.

Figure 2: Proposed mechanisms for the memantine fluorescent derivative formation.

Effects of DBD-COCl and memantine ratios, reaction time, and temperature on the formation of memantine fluorescent derivative were optimized. DBD-COCl and memantine at ratios of 1 : 1, 2 : 1, 3 : 1, 4 : 1, and 5 : 1 were reacted for 10–90 min on a water bath at 60°C and the peak areas of the fluorescent derivative were monitored. At all ratios, peak area of the derivative increased as the reaction times lengthened and the area remained unchanged after 50 min (Figure 3). Except for the ratio 4 : 1, the peak area started to decrease dramatically after 50 min. After 80 min, the area declined independently on the DBD-COCl and memantine ratios. The maximum peak area was obtained at the ratio of 5 : 1 at the reaction time of 50 min. Next experiments were investigated by reacting DBD-COCl at the ratio of 5 : 1 for 10–50 min and varying the reaction temperature from 60 to 80°C. Elevating the temperature did not improve the peak area of the derivative. The maximum peak area was observed at 60°C after 50 min (Figure 4). This result was in good agreement with that obtained from the effects of DBD-COCl and memantine ratios. Thus, the optimal condition for memantine fluorescent derivative formation was by reacting DBD-COCl with memantine at the ratio of 5 : 1 for 50 min at 60°C.

Figure 3: Effects of DBD-COCl and memantine ratios and time on the memantine fluorescent derivative formation.
Figure 4: Effects of temperature on the memantine fluorescent derivative formation.

The memantine fluorescent derivative was cleaned up by SPE using the condition described in Section 2. This procedure would be essential for the future work since the method will be applied to biological samples (e.g., serum or urine). The eluate was analyzed by HPLC and the pure fraction was preliminarily confirmed with the mass of 476.85 by MS. Structure elucidation of the derivative from the pure fraction was performed by IR and results are in Table 1 and Figure 5. The main difference among memantine, DBD-COCl, and the fluorescent derivative was the occurrence of the carbonyl amide at a wavenumber of 1644 cm−1 indicating the amide bond of the derivative.

Table 1: Wavenumbers for the functional groups of memantine, DBD-COCl and the fluorescent derivative.
Figure 5: Infrared spectra of (a) memantine, (b) DBD-COCl, and (c) memantine fluorescent derivative.

Figure 6 compares chromatograms of the memantine fluorescent derivative, underivatized memantine, mobile phase, and DBD-COCl. Under the reversed phase-HPLC, the derivative was eluted as a symmetric peak at 4.50 min and was well separated from DBD-COCl, which co-eluted as a solvent front. No interferences from the excess DBD-COCl, underivatized memantine, or mobile phase were observed. The method is selective for the fluorescent derivative.

Figure 6: Chromatograms of (a) memantine, (b) mobile phase, (c) DBD-COCl, and (d) memantine fluorescent derivative. HPLC condition: see Section 2.3.

Stability of the memantine fluorescent derivative was evaluated at 0, 12, and 24 h after the formation, using the HPLC condition described in Materials and Method. The derivative was stable during the investigated period and no significant changes in peak shape, resolution, retention time, and peak area were observed. The derivative was eluted at the same retention time of about 4.50 min (Figure 7) with the %RSDs for the peak area of 0.29%.

Figure 7: Stability of memantine fluorescent derivative in 4 h. HPLC condition: see Section 2.3.

Preliminary experiments on method validation showed that the method was linear over a range of 1–5 μg/mL with of 0.99 and the %RSDs for retention times and peak areas were less than 0.54% (). Accuracy of the method was represented by percent recovery (), which was between 94.8 and 119.4% (%RSD = 7.71%). Limits of detection and quantitations (calculated from the 3.3 SD/S and 10 SD/S, respectively, where SD was standard deviation of blank and S was slope from the calibration curve) were 0.79 and 2.42 μg/mL, respectively. The data indicates the potential of the method for analysis of memantine, and sensitivity improvement is in progress for determination of the drug in biological samples.

4. Conclusion

This work reports the first fluorescent derivative formation of memantine using DBD-COCl as the derivatizing reagent. The reaction was based on the amide bonding between carbonyl chloride of DBD-COCl and the amine group of memantine. The amide bond formation was confirmed by IR spectra. The fluorescent derivative was stable for 24 h and could be detected by reversed phase-HPLC-fluorescent using the excitation and emission wavelengths at 430 and 520 nm, respectively. In addition, preliminary results on the method validity show that the developed method could potentially be employed for memantine analysis because of its simplicity and reliability. Sensitivity enhancement and application of the method to real samples are under investigation.

Conflict of Interests

The authors confirm no conflict of interests to the paper contents.

Acknowledgment

Financial supports from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program, the Higher Education Commission, and Mahidol University (Grant no. PHD/0306/2551) to Pornpan Prapatpong and Leena Suntornsuk and the Nation Research Council of Thailand through Mahidol University (NRC) (NG P 0812555) and the Office of the High Education Commission and Mahidol University under the National Research Universities Initiative are acknowledged. Leena also thanks Thailand-United States Educational Foundation (Fulbright) for encouragement and time on the paper preparation and revision.

References

  1. H. S. Anderson, “Alzheimer Disease,” Medscape reference, 2012, http://emedicinemedscape.com/ article/1134817-overview.
  2. Alzheimer's Association, “2013 Alzheimer's disease facts and figures,” Alzheimer's & Dementia, vol. 9, no. 2, pp. 208–245, 2013. View at Publisher · View at Google Scholar
  3. Alzheimer's Association, “Alzheimer's disease fact and figure,” Alzheimer's Disease and Dementia, vol. 8, no. 2, pp. 131–168, 2012. View at Publisher · View at Google Scholar
  4. L. Bertram and R. E. Tanzi, Progress in Molecular Biology and Translational Science, vol. 167, 2012.
  5. D. M. Walsh and D. B. Teplow, “Alzheimer's disease and the amyloid β-protein,” Progress in Molecular Biology and Translational Science, vol. 107, pp. 101–124, 2012. View at Publisher · View at Google Scholar · View at Scopus
  6. Y. Gazes, A. Soldan, and Y. Stern, Alzheimer's Disease Encyclopedia of Human Behavior, 2nd edition, 2012.
  7. J. Koppel, S. Sunday, T. E. Goldberg, P. Davies, E. Christen, and B. S. Greenwald, “Psychosis in Alzheimer's disease is associated with frontal metabolic impairment and accelerated decline in working memory: findings from the Alzheimer's disease neuroimaging initiative,” The American Journal of Geriatric Psychiatry, vol. 22, no. 7, pp. 698–707, 2014. View at Publisher · View at Google Scholar · View at Scopus
  8. D. G. Standaert and A. B. Young, “Treatment of central nervous system degenerative disorders,” in Goodman & Gilman's The Pharmacological Basis of Therapeutics, chapter 20, McGraw-Hill, New York, NY, USA, 11th edition, 2006. View at Google Scholar
  9. The United States Pharmacopeia Convention, The United States Pharmacopeia 37: The National Formula 32, The United States Pharmacopeia Convention, Rockville, Md, USA, 2014.
  10. British Pharmacopoeia, The Stationary Office, London, UK, 2015.
  11. C. Poole, Liquid Chromatography, Elsevier, Amsterdam, The Netherlands, 2013.
  12. A. Zarghi, A. Shafaati, S. M. Foroutan, A. Khoddam, and B. Madadian, “Sensitive and rapid HPLC method for determination of memantine in human plasma using OPA derivatization and fluorescence detection: application to pharmacokinetic studies,” Scientia Pharmaceutica, vol. 78, no. 4, pp. 847–856, 2010. View at Publisher · View at Google Scholar · View at Scopus
  13. C. Shuangjin, F. Fang, L. Han, and M. Ming, “New method for high-performance liquid chromatographic determination of amantadine and its analogues in rat plasma,” Journal of Pharmaceutical and Biomedical Analysis, vol. 44, no. 5, pp. 1100–1105, 2007. View at Publisher · View at Google Scholar · View at Scopus
  14. M. G. Hassan, K. M. Emara, H. A. Mohamed et al., “Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide,” Biomedical Chromatography, vol. 26, no. 2, pp. 214–219, 2012. View at Publisher · View at Google Scholar · View at Scopus
  15. R. F. Suckow, M. F. Zhang, E. D. Collins, M. W. Fischman, and T. B. Cooper, “Sensitive and selective liquid chromatographic assay of memantine in plasma with fluorescence detection after pre-column derivatization,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 729, no. 1-2, pp. 217–224, 1999. View at Publisher · View at Google Scholar · View at Scopus
  16. T.-H. Duh, H.-L. Wu, C.-W. Pan, and H.-S. Kou, “Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation,” Journal of Chromatography A, vol. 1088, no. 1-2, pp. 175–181, 2005. View at Publisher · View at Google Scholar · View at Scopus
  17. B. Narola, A. S. Singh, P. R. Santhakumar, and T. G. Chandrashekhar, “A Validated stability-indicating reverse phase hplc assay method for the determination of memantine hydrochloride drug substance with UV-detection using precolumn derivatization technique,” Analytical Chemistry Insights, vol. 2010, no. 5, pp. 37–45, 2010. View at Google Scholar · View at Scopus
  18. M.-Y. Liu, S.-N. Meng, H.-Z. Wu, S. Wang, and M.-J. Wei, “Pharmacokinetics of single-dose and multiple-dose memantine in healthy chinese volunteers using an analytic method of liquid chromatography-tandem mass spectrometry,” Clinical Therapeutics, vol. 30, no. 4, pp. 641–653, 2008. View at Publisher · View at Google Scholar · View at Scopus
  19. R. K. Konda, B. R. Challa, B. R. Chandu, and K. B. Chandrasekhar, “Bioanalytical method development and validation of memantine in human plasma by high performance liquid chromatography with tandem mass spectrometry: application to bioequivalence study,” Journal of Analytical Methods in Chemistry, vol. 2012, Article ID 101249, 8 pages, 2012. View at Publisher · View at Google Scholar · View at Scopus
  20. K. Siddappa, M. Mallikarjun, T. Mahesh, K. Mallikarjun, and R. Chandrakanth, “Development and validation of a gas chromatographic method for the assay of memantine hydrochloride in pure and tablet dosage forms,” Facta Universitatis—Series: Physics, Chemistry and Technology, vol. 9, no. 1, pp. 1–8, 2011. View at Publisher · View at Google Scholar
  21. S. A. Jadhav, S. B. Landge, N. C. Niphade, S. R. Bembalkar, and V. T. Mathad, “Development and validation of stability-indicating GC-FID method for the quantitation of memantine hydrochloride and its nonchromophoric impurities in bulk and pharmaceutical dosages,” Chromatography Research International, vol. 2012, Article ID 806068, 10 pages, 2012. View at Publisher · View at Google Scholar
  22. K. Michail, H. Daabees, Y. Beltagy, M. Abdel-Khalek, and M. Khamis, “Spectrophotometric and spectrofluorimetric determination of memantine hydrochloride in bulk and pharmaceutical preparations,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 3, no. 3, pp. 180–185, 2011. View at Google Scholar · View at Scopus
  23. A. P. Rani, S. Bhawani, C. Nagalakshmi, and C. B. Sekaran, “Determination of memantine hydrochloride by spectrophotometry using anionic dyes, bromothymol blue and solochrome black Tin bulk and tablet dosage forms,” Journal of Chemical Sciences, vol. 2012, p. CSJ-60, 2012. View at Google Scholar
  24. K. Imai, T. Fukushima, and H. Yokosu, “A novel electrophilic reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulphonyl-2, 1,3-benzoxadiazole (DBD-COCI) for fluorometric detection of alcohols, phenols, amines and thiols,” Biomedical Chromatography, vol. 8, no. 3, pp. 107–113, 1994. View at Publisher · View at Google Scholar · View at Scopus