Table of Contents
ISRN Spectroscopy
Volume 2014, Article ID 541970, 7 pages
http://dx.doi.org/10.1155/2014/541970
Research Article

Novel Spectrophotometric Methods for the Determination of Selegiline Hydrochloride in Bulk and Its Pharmaceutical Preparation

Department of Post-Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangothri 574199, India

Received 27 November 2013; Accepted 18 January 2014; Published 23 February 2014

Academic Editors: Y. C. Ling and T. Strunskus

Copyright © 2014 Kumble Divya and Badiadka Narayana. 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

A simple and highly selective spectrophotometric method has been developed for the determination of selegiline hydrochloride in bulk and formulations. Method A is based on the oxidation of 3-methyl-2-benzothiazolinone hydrazone in the presence of ceric ammonium sulphate, followed by its coupling reaction with drug to form a colored product having of 629 nm. Method B is based on the coupling reaction of drug with 4-aminoantipyrine to give a new ligand that reacts with copper(II) to give intense bluish red colored chelate which is measured at 539 nm. Beer’s law is obeyed in the range of 10.00–85.00 μg mL−1 with molar absorptivity of for method A and 20.00–120.00 μg mL−1 with molar absorptivity of for method B. The optimum reaction condition and the analytical parameters are evaluated. The results obtained indicate that the methods are free from interference of the ingredients; thus they are successfully applied to pharmaceutical formulations.

1. Introduction

Selegiline hydrochloride (SGE) (Figure 1) (deprenyl), [(R)-(2)-N-methyl-(1-phenyl-2-propyl)-N-propinylamine] hydrochloride [1], is a levomethamphetamine derivative which belongs to a class of drugs called phenethylamines [2]. SEG is a selective, irreversible inhibitor of monoamine oxidase (MAO-A) [3]. It is used for the treatment of early-stage Parkinson’s disease, depression, and senile dementia [4]. It is useful adjunct in the treatment of cocaine addiction [5]. SEG (brand name Anipryl) is also used (at extremely high dosages relative to humans) in veterinary medicine to treat the symptoms of Cushing’s disease and cognitive dysfunction (canine cognitive dysfunction) in dogs [6]. Recommended dosage of SEG is about 10 mg/day; further increase in the dosage will lead to the nonselective inhibition of MAO [7]. Therefore, it is essential to develop a standard analytical method for monitoring residual drug in bulk and in pharmaceutical formulations. The pharmaceutical importance of drug has prompted us to devise methods for the rapid determination of SEG. A very few methods are reported in the literature for the determination of SEG in pharmaceutical formulations which include high performance liquid chromatography [812], gas chromatography [13, 14], fluorescence polarization immunoassay (FPIA) and gas chromatography-mass spectrometry (GC/MS) [15], spectrofluorometry [16], and stereoselective analyses [17]. The literature survey reveals that so far there is no visible spectrophotometric method for the analysis of SEG.

541970.fig.001
Figure 1: Chemical structure of SEG.

Unlike gas chromatograph and high performance liquid chromatograph the UV-Visible spectrophotometer is a simple, low-cost instrument and on the other hand, in terms of simplicity and expense, the method could be considered superior in comparison with the previously reported methods. Thus, the present work reports new spectrophotometric methods for the determination of SEG in pure and in pharmaceutical formulations.

2. Materials and Methods

2.1. Apparatus

A UV-Visible spectrophotometer (SHIMADZU, UV 2550, Japan) with 1 cm quartz cells was used for the absorbance measurements.

2.2. Chemicals and Reagents

All the reagents were of analytical grade and used without further purification. All the solutions were prepared in distilled water. SEG bulk drug was obtained as gift sample from CAD Pharma Inc., Bangalore, India. Pharmaceutical formulations of SEG were obtained commercially.

2.3. Standard Drug Solution

Stock solution of SEG (1000 μg/mL) was prepared by dissolving 100 mg of SEG in distilled water and making the volume 100 mL in a standard volumetric flask. The stock solution was diluted approximately to get working concentration.

3-Methyl-2-benzothiazolinone Hydrazone (MBTH, 0.2% w/v). 0.2 g of MBTH was accurately weighed and dissolved in 100 mL of distilled water.

Ceric Ammonium Sulphate (CAS, 1% w/v). 1 g gm of CAS was dissolved in 20 mL of 0.1 N sulphuric acid and the volume became 100 mL.

4-Aminoantipyrine (AAP, 0.1% w/v). 0.1 g of AAP was dissolved in 10 mL of ethanol and made up to be 100 mL with distilled water.

Copper(II) Chloride (1% w/v). 1 g of Cu(II) chloride was accurately weighed and dissolved in 100 mL of distilled water.

2.4. Recommended Procedures

Method A. Aliquots containing 10.00–100.00 μg mL−1 of SEG were transferred into a series of 10 mL volumetric flasks. Then 1.0 mL of 0.2% MBTH and 1.0 mL of 1% CAS solutions were added and the flasks were kept aside for 15 min at room temperature. The solutions in each flask were made up to the mark with distilled water and the absorbance was measured at 629 nm against reagent blank.

Method B. Aliquots containing 20.00–120.00 μg mL−1 of SEG were transferred into a series of 10 mL volumetric flasks, followed by the addition of 2 mL of 1% AAP and 1 mL of 0.1% Copper chloride.The resulting reddish violet colored mixtures were kept aside for 10 min and make up the solution to be 10 mL with acetone; the absorbance values were measured at 539 nm against the reagent blank solution.

2.5. Preparation of Pharmaceutical Formulation

Twenty tablets of SEG (5 mg) were crushed thoroughly in a mortar, dissolved in 30 mL of ethanol, and diluted to 100 mL by using water. The solution was filtered through Whatman filter paper number 41 and diluted quantitatively with water to obtain a suitable concentration for the analysis. A convenient aliquot was then subjected to the analysis by using proposed method.

3. Results and Discussion

Method A. In this method MBTH undergoes oxidation with Ce(IV), by losing two electrons and one proton to form electrophilic intermediate, which act as the active coupling species [18, 19]. This intermediate undergoes electrophilic substitution reaction with SEG (Scheme 1) to give bluish green colored product that can be measured at 629 nm (Figure 2).

541970.sch.001
Scheme 1: Probable reaction of SEG with MBTH.
541970.fig.002
Figure 2: Absorption maximum for method A.

Method B. In this method AAP reacts with SEG to form a new ligand (Scheme 2) having low sensitivity at 329 nm. This sensitivity has been increased by complexation with Cu(II) [20] to give intense bluish red colored chelate which can be measured at 539 nm (Figure 3).

541970.sch.002
Scheme 2: Probable reaction of SEG with AAP and Cu(II) chloride.
541970.fig.003
Figure 3: Absorption maximum for method B.

3.1. Determination of Effective Reagents Concentration

Experiments were carried out to optimize the reaction condition for complete color formation. It was found that 1.0 mL of 0.2% MBTH and 1.0 mL of 1% Ce(IV) solutions for method A and 2 mL of 1% 4-AAP and 1 mL of 0.1% Cu(II) solutions for method II were found to be optimum to get the stable and maximum color intensity.

3.2. Quantification

In order to analyze the linearity of the developed methods, absorbance was measured for a series of solutions containing increasing amounts of SEG under optimum condition. Regression analysis of Beer’s law plots (Figures 4 and 5) at their respective values revealed good correlation. The validity of Beer’s law, molar absorptivity, and Sandell’s sensitivity values were evaluated and were given in Table 1. The proposed methods showed excellent linearity for the determination of SEG drugs with a good correlation coefficient. Limit of detection (LOD) and limit of quantification (LOQ) were calculated as per the ICH guidelines [21].

tab1
Table 1: Spectral and statistical data for the determination of SEG.
541970.fig.004
Figure 4: Calibration curve for method A.
541970.fig.005
Figure 5: Calibration curve for method B.
3.3. Accuracy and Precision

Accuracy and precision of the proposed methods were tested by carrying out determinations of five replicates of bulk and commercial samples for both the methods, whose concentrations lie within Beer’s law range. The relative standard deviation (RSD) and relative error results indicated that the methods were precise and accurate. The relative error (%) which is a measure of accuracy and RSD (%) which is a measure of precision were summarized in Table 2, revealing the high accuracy and precision of the methods.

tab2
Table 2: Evaluation of accuracy and precision.
3.4. Stoichiometry of the Reaction Product

The stoichiometric ratio of the colored product of method A and method B was investigated by applying the continuous variation (Job’s) and mole ratio methods [22] using equimolar solutions of the reagents. As shown in Figures 6 and 7, the molar ratio which gave maximum absorbance is found to be 1 : 1 (drug : reagent) for SEG-MBTH system and 1 : 2 for Cu-ligand system. In view of this result, a reaction mechanism is proposed for the developed method.

541970.fig.006
Figure 6: Application of Job’s method to method A.
541970.fig.007
Figure 7: Application of Job’s method to method B.
3.5. Interference Studies

In pharmaceutical analysis, it is important to test the selectivity towards the excipients added to the pharmaceutical preparations. The effects of the excipients associated with formulations of SEG in its pure form and its formulations were investigated using the developed methods. This method does not suffer any interference from commonly associated excipients such as sucrose, lactose, dextrose, starch, and sodium chloride in the preparation of tablets. Data of the interference studies are given in Table 3.

tab3
Table 3: Effect of excipients on assay of SEG.
3.6. Analytical Application

Commercial formulations (tablets) containing 5 mg of SEG were successfully analyzed by the proposed methods. The results obtained for the Student’s -test at 95% confidence level are less than the theoretical values, which confirm the good accuracy of the methods. Obtained values were listed in Table 4. However, there is no method described in the literature for the assay of SEG in pharmaceutical preparations. Thus the present work reports an elegant method for the determination of SEG in pure and pharmaceutical formulations.

tab4
Table 4: Result of assay of formulation by the proposed method.

4. Conclusions

The developed spectrophotometric methods are quite simple and do not require any pretreatment of the drug and tedious extraction procedure. The in both the methods was considerably higher which is a decisive advantage and interferences by common excipients are generally very less. The described procedure is reliable, very simple, and conveniently applicable in most laboratories due to the accessibility of the reagents employed and reasonably low time of analysis. Thus, these methods can be used for routine analysis of SEG in pharmaceutical industries, hospitals, and research laboratories.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors are thankful to CAD Pharma Inc., Bangalore, India, for providing the drug sample. Badiadka Narayana thanks UGC for financial assistance through BSR one-time grant for the purchase of chemicals. Kumble Divya thanks DST-PURSE project for the financial support.

References

  1. W. Birkmayer, J. Knoll, P. Riederer, and M. B. Youdim, “(-)-Deprenyl leads to prolongation of L-dopa efficacy in Parkinson's disease,” Modern problems of pharmacopsychiatry, vol. 19, pp. 170–176, 1983. View at Google Scholar · View at Scopus
  2. A. Clarke, F. Brewer, E. S. Johnson et al., “A new formulation of selegiline: improved bioavailability and selectivity for MAO-B inhibition,” Journal of Neural Transmission, vol. 110, no. 11, pp. 1241–1255, 2003. View at Publisher · View at Google Scholar · View at Scopus
  3. J. Birks and L. Flicker, “Selegiline for Alzheimer's disease,” Cochrane Database of Systematic Reviews, no. 1, Article ID CD00044, 2003. View at Google Scholar · View at Scopus
  4. H. Allain, J. Cougnard, and H.-C. Neukirch, “Selegiline in de novo parkinsonian patients: the French selegiline multicenter trial (FSMT),” Acta Neurologica Scandinavica, Supplement, vol. 84, no. 136, pp. 73–78, 1991. View at Google Scholar · View at Scopus
  5. J. D. Amsterdam, “A double-blind, placebo-controlled trial of the safety and efficacy of selegiline transdermal system without dietary restrictions in patients with major depressive disorder,” Journal of Clinical Psychiatry, vol. 64, no. 2, pp. 208–214, 2003. View at Google Scholar · View at Scopus
  6. B. G. Katzung, Basic & Clinical Pharmacology, Lange Medical Books, McGraw-Hill, New York, NY, USA, 9th edition, 2004.
  7. J. A. Braddock, D. B. Church, I. D. Robertson, and A. D. J. Watson, “Inefficacy of selegiline in treatment of canine pituitary-dependent hyper-adrenocorticism,” Australian Veterinary Journal, vol. 82, no. 5, pp. 272–277, 2004. View at Publisher · View at Google Scholar · View at Scopus
  8. S. Pichini, R. Pacifici, M. Pellegrini et al., “Development and validation of a high-performance liquid chromatography—mass spectrometry assay for determination of amphetamine, methamphetamine, and methylenedioxy derivatives in meconium,” Analytical Chemistry, vol. 76, no. 7, pp. 2124–2132, 2004. View at Publisher · View at Google Scholar · View at Scopus
  9. K. Nishida, S. Itoh, N. Inoue, K. Kudo, and N. Ikeda, “High-performance liquid chromatographic-mass spectrometric determination of methamphetamine and amphetamine enantiomers, desmethylselegiline and selegiline, in hair samples of long-term methamphetamine abusers or selegiline users,” Journal of Analytical Toxicology, vol. 30, no. 4, pp. 232–237, 2006. View at Google Scholar · View at Scopus
  10. M. H. Slawson, J. L. Taccogno, R. L. Foltz, and D. E. Moody, “Quantitative analysis of selegiline and three metabolites (N-desmethylselegiline, methamphetamine, and amphetamine) in human plasma by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry,” Journal of Analytical Toxicology, vol. 26, no. 7, pp. 430–437, 2002. View at Google Scholar · View at Scopus
  11. R. La Croix, E. Pianezzola, and M. S. Benedetti, “Sensitive high-performance liquid chromatographic method for the determination of the three main metabolites of selegiline (L-deprenyl) in human plasma,” Journal of Chromatography B, vol. 656, no. 1, pp. 251–258, 1994. View at Publisher · View at Google Scholar · View at Scopus
  12. M. Katagi, M. Tatsuno, A. Miki, M. Nishikawa, K. Nakajima, and H. Tsuchihashi, “Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry,” Journal of Chromatography B, vol. 759, no. 1, pp. 125–133, 2001. View at Publisher · View at Google Scholar · View at Scopus
  13. G. Szebeni, J. Lengyel, G. Szekacs, K. Magyar, J. Gaal, and I. Szatmari, “Gas chromatographic procedure for simultaneous determination of selegiline metabolites, amphetamine, methamphetamine and demethyl-deprenyl in pig plasma,” Acta Physiologica Hungarica, vol. 83, no. 2, pp. 135–141, 1995. View at Google Scholar · View at Scopus
  14. K. S. Patrick, B. Lan Nguyen, and J. D. McCallister, “Gas chromatographic-mass spectrometric determination of plasma selegiline using a deuterated internal standard,” Journal of Chromatography, vol. 583, no. 2, pp. 254–258, 1992. View at Publisher · View at Google Scholar · View at Scopus
  15. H. H. Maurer and T. Kraemer, “Toxicological detection of selegiline and its metabolites in urine using fluorescence polarization immunoassay (FPIA) and gas chromatography-mass spectrometry (GC-MS) and differentiation by enantioselective GC-MS of the intake of selegiline from abuse of methamphetamine or amphetamine,” Archives of Toxicology, vol. 66, no. 9, pp. 675–678, 1992. View at Publisher · View at Google Scholar · View at Scopus
  16. J. Netriová, J. Sádecká, and I. Skačáni, “Spectrofluorimetric determination of selegiline,” Farmaceuticky Obzor, vol. 74, no. 7, pp. 181–186, 2005. View at Google Scholar · View at Scopus
  17. M. Hasegawa, K. Matsubara, S. Fukushima, C. Maseda, T. Uezono, and K. Kimura, “Stereoselective analyses of selegiline metabolites: possible urinary markers for selegiline therapy,” Forensic Science International, vol. 101, no. 2, pp. 95–106, 1999. View at Publisher · View at Google Scholar · View at Scopus
  18. R. G. Vijaya, Y. Triveni, K. Divya, N. K. Lakshmi, and G. G. Venu, “Application of MBTH and sulphanilic acid for the quantitative spectrophotometric determination of diacerein in bulk and formulations,” Der Pharma Chemica, vol. 1, no. 2, pp. 285–229, 2009. View at Google Scholar
  19. E. Sawicki, T. W. Stanley, T. R. Hauser, W. Elbert, and J. L. Noe, “Spot test detection and colorimetric determination of aromatic amines and imino heteroaromatic compounds with 3-methyl-2-benzothiazolone hydrazone,” Analytical Chemistry, vol. 33, no. 6, pp. 722–725, 1961. View at Google Scholar · View at Scopus
  20. N. Theia Al-Sabha, “Spectrophotometric assay of phenylephrine hydrochloride using 4-aminoantipyrine and copper (II),” Pakistan Journal of Analytical and Environmental Chemistry, vol. 11, no. 1, pp. 01–07, 2010. View at Google Scholar
  21. International Conference on Harmonization (ICH) Guidelines, http://www.ICH.org/.
  22. G. D. Christian and J. E. O'Rilly, “Jobs method,” in Instrumental Analysis, Prentice Hall, Upper Saddle River, NJ, USA, 2nd edition, 1980. View at Google Scholar