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Journal of Spectroscopy
Volume 2013, Article ID 783457, 5 pages
Research Article

Simple, Rapid and Sensitive UV-Visible Spectrophotometric Method for Determination of Antidepressant Amitriptyline in Pharmaceutical Dosage Forms

1Department of Chemistry, Government Danveer Tularam College, Utai, Durg, C.G. 490001, India
2Department of Chemistry, Government Nagarjun P.G. Science College, Raipur, C.G. 492001, India
3Department of Chemistry, Government College, Bhanpuri, Bastar, C.G. 494224, India

Received 5 May 2013; Accepted 11 July 2013

Academic Editor: Lahcen Bih

Copyright © 2013 Pankaj Soni 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.


The paper describes a new and simple approach for spectrophotometric determination of tricyclic antidepressant drug amitriptyline. Enhancement of the colour intensity of the Fe(III)-SCN complex on addition of the drug amitriptyline forms the basis of the proposed method. The value of molar absorptivity of the Fe(III)-SCN amitriptyline ion pair complex in terms of the drug lies in the range of (2.82–3.36) × 103 L·mol−1·cm−1 at the absorption maximum 460 nm. The detection limit of the method was 0.3 μg·mL−1. The slope, intercept, and correlation coefficients for the present method were found to be 0.008, 0.002, and +0.998, respectively. The effect of analytical variables on the determination of the drug and the composition of the complex are discussed in the paper. The method is applicable in the determination of amitriptyline in pharmaceutical preparations.

1. Introduction

Amitriptyline [3-(10,11-dihydro-5H-dibenzol[a,d]cyclohept-5-ylidene) propyldimethylamine] constitutes an important class among the neurotherapeutics belonging to first generation of antidepressant drug [1, 2]. Recent studies show proinflammatory cytokine process takes place during clinical depression, mania, and bipolar disorder, and it is possible that symptoms of these conditions are attenuated by the pharmacological effect of antidepressants on the immune system [38]. Amitriptyline has a carboxylic structure with an exocyclic double bond at C-5 which is substituted with an N, N-dimethyl-1-propanamino side chain (Figure 1).

Figure 1: Amitriptyline.

Amitriptyline hydrochloride [AMIYTP]Cl represents a large group of compounds used in treatment of various mental diseases. The chemical structure of this compound is based on the condensed aromatic three-ring system which includes substituents in positions 2 and 10. The difference in chemical structure causes the different pharmaceutical activity of drug. The value of angle of these molecules is important; the more nearly planar the greater the neuroleptic activity. It is believed that drug amitriptyline acts by blocking the receptors of neurotransmitters, noradrenalin, in the synopsis in the central nervous system, which results in an increase of concentration of both molecules with a subsequent enhancement of their antidepressant potency. However, this drug suffers from several drawbacks, such as antiarrhythmic, anticholinergic, cardiovascular, and hyperthermia side effect, which may be reduced if the drugs are suitably vectored to the organism.

Various analytical methods have been reported for determination of amitriptyline including spectrofluorimetric [9], spectrophotometric [1015], flow injection method [16], UV-Visible spectrophotometric methods [17, 18] based on chromogenic reaction with citric acid-acetic anhydride [19], bromothymol blue [20], ammonium reineckate [21, 22], bismuth-hexaiodide [23], and cerium (IV) [24]. Most of these methods however suffer from several interferences by many basic compounds, until several manipulation steps, and can be applied only to relatively high concentration of drugs. Some of these methods are not simple for the routine analysis, and they need sophisticated instruments which are not commonly available in the routine laboratories, and conventional batch process solvent extraction is a tedious and time-consuming procedure. Therefore it seems necessary to develop a simple and fast identification method for determination of amitriptyline. The proposed method is based on the enhancement of the colour intensity of the Fe(III)-SCN complex on addition of the drug amitriptyline. The optimization of analytical variables is discussed in this paper. The method is simple, sensitive, and found to be reproducible. This method has been applied for determination of the concentration of amitriptyline in pharmaceutical preparations.

2. Experimental

2.1. Apparatus

Systronics (India) make UV-Visible double beam spectrophotometer—model 2201 with quartz cell having path length of 1 cm was employed to determine the absorbance. Source and sample positions are software-optimized to keep the unit always at its peak performance.

2.2. Reagents

All chemical used were of analytical grade reagent (Merck). Deionized double distilled water was used throughout the experiment. Glass wares were cleaned with acidified solutions of KMnO4 or K2Cr2O7, followed by washing with concentrated HNO3 and rinsed several times with deionized double distilled water. All solutions used were prior filtered and degassed.

2.2.1. Standard Solution of Amitriptyline (1 mg·mL−1 or 1000 ppm)

The standard solution (1 mg·mL−1 or 1000 ppm) of amitriptyline (Mol. Wt. 319) was prepared by dissolving 1.0 gm amitriptyline in 1 litre deionized double distilled water. The working solutions of amitriptyline were prepared by the appropriate dilution of the stock solution.

2.2.2. Reagent Solution Ammonium Thiocyanate (1.8 M)

It was prepared fresh by dissolving 14 gm ammonium thiocyanate in 100 mL deionized double distilled water.

2.2.3. Reagent Solution of Iron(III) 1.25 × 10−3 M

It is prepared by dissolving 0.0506 gm of Fe(NO3)3·9H2O (Mol. Wt. 404) in 0.01 M nitric acid and made up the solution to 100 mL with 0.01 M nitric acid solution.

2.3. Procedure

2.5 mL aliquots of the standard solutions of amitriptyline having varying concentrations from 1.0 to 10.0 μg mL−1 were taken in the 25 mL volumetric flasks. In each volumetric flask 2.5 mL of the iron solution and 5 mL of NH4SCN solution were added, then made up the solutions upto the mark on volumetric flask with double distilled water, then measured the absorbance at 460 nm against the reagent blank, and prepared the calibration graph by plotting absorbance versus concentration of amitriptyline (Figure 2). The similar procedure was repeated with the sample solution, which was prepared from the pharmaceutical product. The concentration of amitriptyline in the sample solution was computed by using the calibration curve prepared under similar condition.

Figure 2: Calibration graph for determination of amitriptyline, 1.0 to 10.0 μg mL−1.

3. Results and Discussion

3.1. Reaction Mechanism and Composition of Complex

Ferric ions react with SCN ions to give a variety of red-orange complexes, but the presence of amitriptyline activates the formation of a higher thiocyanato species, which enhances the colour intensity of the complex. The proposed reaction for the formation of higher thiocyanato complex in presence of the drug cation, that is, [AMIYTP]+ can be expressed as where the value of may vary from 2 to 6.

This reaction has been used for the determination of cationic antidepressant drug in the present work.

The mole ratios of Fe(III) to SCN and [AMIYTP]+ ions involved in formation of the ion-associated complex were determined on the basis of the spectrophotometric analysis of these constituents at their different concentrations. The values of (the absorbance of the complex when the reagent was in equilibrium) and (the absorbance when the reagent was in constant excess) were determined spectrophotometrically, and the values of were calculated for different concentrations of Fe(III), SCN and [AMIYTP]+. Their molar ratio was determined using curve-fitting method by plotting () versus (where = molar concentration) values of SCN, and [AMIYTP]+. The values of slope for SCN and [AMIYTP]+ were found to be 4.2 and 0.7 close to integers 4 and 1, respectively. For each mole of Fe(III), the involvement of SCN and [AMIYTP]+ was assumed to be 4 : 1. Hence, the curve-fitting method suggested the molar ratio of Fe(III), SCN, and drug cation in the complex to be in 1 : 4 : 1 ratio, respectively (Figures 3 and 4).

Figure 3: Curve-fitting method for the determination of mole ratio of the Fe(III) to SCN in the Fe(III)-SCN-[AMIYTP]+ complex.
Figure 4: Curve-fitting method for the determination of mole ratio of the Fe(III) to [AMIYTP]+ in the Fe(III)-SCN-[AMIYTP]+ complex.

The probable reaction which occurred in the following solution in HNO3 medium is expressed as where value of “” may vary from 1 to 3 and (AMIYTP)+ = cation of the drug amitriptyline.

3.2. Absorption Spectra

The Fe(III)-SCN-[AMIYTP]+ complex exhibits the absorption maximum at 460 nm (Figure 5). The position of did not change when the drug amitriptyline is added, but the absorbance was increased. In presence of the drug, a hyperchromic shift was observed due to formation of a higher thiocyanate complex.

Figure 5: Absorption spectra of the Fe(III)-SCN and Fe(III)-SCN-[AMIYTP]+ complexes (concentration of amitriptyline = 5 μg mL−1).
3.3. Optimization of Analytical Variables

Among the acids (HCl, H2SO4, and HNO3) tested during the investigation, HNO3 was found most suitable because of the better performance of the complex in the nitric acid medium. Another reason for the selection of nitric acid is its oxidizing nature because of which several reductants, that is, CN, S2−, , oxalic acid, ascorbic acid, and so forth did not interfere in the reaction. Use of 0.01 M nitric acid in the iron solution was found adequate during the detailed investigation. During the optimization of analytical variables, the minimum concentrations of iron(III) and ammonium thiocyanate solutions (required to obtain the maximum and stable absorbance) were found to be 1.25 × 10−3 M and 1.8 M, respectively. Their further addition decreased the absorbance. As the solution of ammonium thiocyanate seemed to be slightly viscous in comparison with the iron(III) solution, the SCN solution was prepared using double volume of distilled water. Thus volume of 2.5 mL of 1.25 × 10−3 M iron(III) and 5.0 mL of 1.8 M ammonium thiocyanate solutions was applied in order to avoid the matrix interference during analysis.

3.4. Optimum Concentration Range, Detection Limit and Statistics

The absorbances for the different concentrations of amitriptyline are shown in Figure 2. The method followed linearity in the range 1.0 to 10.0 μg mL−1 of amitriptyline, with the slope, intercept, and correlation coefficient of 0.008, 0.002, and +0.998, respectively. The value of molar absorptivity (calculated by taking the molar concentration of amitriptyline and path length of the cell to be 1 cm) with amitriptyline in terms of the drug was found to be in the range (2.82–3.36) × 103 L mol−1 cm−1 at the absorption maximum 460 nm. The detection limit (absorbance >3 × SD) of the method was 0.3 μg mL−1. The relative standard deviation for the analysis of six different solutions containing 5.0 μg mL−1 amitriptyline was found to be ±1.7%.

4. Application of the Method

The proposed method was applied for the determination of amitriptyline in commercial pharmaceutical tablets Amil-25 (Mano Pharmacy, India) and Amitriptyline hydrochloride tablet USP/IP-25 mg (From (Iron) drugs and pharmaceuticals Pvt. Ltd., India). Sample solutions were prepared from the tablets. Five tablets of each product were weighed and powdered. The powder equivalent to their single tablet was dissolved in methanol, and the obtained solution was filtered and evaporated to dryness. Then the residue was dissolved in distilled water and made up the volume up to 100 mL in a volumetric flask. These stock solutions were further diluted to contain the requisite concentrations. Then the procedure described earlier for the determination of amitriptyline followed, and it was compared with the results obtained from the official method as shown in Tables 1 and 2. The amitriptyline content determined by the proposed method in the drug samples was in excellent agreement with the already reported extractive spectrophotometric ammonium reineckate method (Table 1). The performance of the proposed method was compared with the official method (Table 2).

Table 1: The results of analysis of “Amitriptyline” tablets by proposed and official methods.
Table 2: Performance data for spectrophotometric determination of amitriptyline.

5. Conclusion

The method was successfully applied for the determination of amitriptyline in the pharmaceutical preparation. The method is very simple as there is no requirement of prior separation or extraction of the complex, and the reagents are low cost and commonly available in routine laboratories. The results obtained from the proposed method were comparable with the established methods. The method has good potential in simplicity, sensitivity, and reproducibility, and the reaction used in the proposed work is expected to give better results with flow injection analysis.


The authors are thankful to the Director, Shri Shankaracharya College of Engineering and Technology, Bhilai (C.G.), for providing laboratory facilities for this work. They are also thankful to the All India Council for Technical Education (AICTE), New Delhi, for financial support during the present investigation.


  1. R. J. Baldessarini, “Drugs and the treatment of psychiatric disorders, depression and mania,” in Goodman & Gilman's: The Pharmacological Basis of Therapeutics, J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon, and A. G. Gilman, Eds., chapter 19, pp. 431–459, McGraw Hill, New York, NY, USA, 9th edition, 1996. View at Google Scholar
  2. C. D. Las Cuevas, W. Penate, and E. J. Sanz, “Psychiatric outpatients' self-reported adherence versus psychiatrists' impressions on adherence in affective disorders,” Human Psychopharmacology: Clinical and Experimental, vol. 28, no. 2, pp. 142–150, 2013. View at Publisher · View at Google Scholar
  3. A. Remlinger-Molenda, P. Wojciak, M. Michalak, and J. Rybakowski, “Activity of selected cytokines in bipolar patients during manic and depressive episodes,” Psychiatria Polska, vol. 46, no. 4, pp. 599–611, 2012. View at Google Scholar
  4. S. M. O'Brien, P. Scully, L. V. Scott, and T. G. Dinan, “Cytokine profiles in bipolar affective disorder: focus on acutely ill patients,” Journal of Affective Disorders, vol. 90, no. 2-3, pp. 263–267, 2006. View at Publisher · View at Google Scholar · View at Scopus
  5. E. Obuchowicz, A. Marcinowska, and Z. S. Herman, “Antidepressants and cytokines—Clinical and experimental studies,” Psychiatria Polska, vol. 39, no. 5, pp. 921–936, 2005. View at Google Scholar · View at Scopus
  6. C.-J. Hong, Y. W.-Y. Yu, T.-J. Chen, and S.-J. Tsai, “Interleukin-6 genetic polymorphism and Chinese major depression,” Neuropsychobiology, vol. 52, no. 4, pp. 202–205, 2005. View at Publisher · View at Google Scholar · View at Scopus
  7. I. J. Elenkov, D. G. Iezzoni, A. Daly, A. G. Harris, and G. P. Chrousos, “Cytokine dysregulation, inflammation and well-being,” NeuroImmunoModulation, vol. 12, no. 5, pp. 255–269, 2005. View at Publisher · View at Google Scholar · View at Scopus
  8. M. Kubera, M. Maes, G. Kenis, Y.-K. Kim, and W. Lasoń, “Effects of serotonin and serotonergic agonists and antagonists on the production of tumor necrosis factor α and interleukin-6,” Psychiatry Research, vol. 134, no. 3, pp. 251–258, 2005. View at Publisher · View at Google Scholar · View at Scopus
  9. F. A. Mohamed, H. A. Mohamed, S. A. Hussein, and S. A. Ahmed, “A validated spectrofluorimetric method for determination of some psychoactive drugs,” Journal of Pharmaceutical and Biomedical Analysis, vol. 39, no. 1-2, pp. 139–146, 2005. View at Publisher · View at Google Scholar
  10. J. Karpinska and J. Suszynska, “The spectrophotometric simultaneous determination of amitryptyline and chlorpromazine hydrochlorides in their binary mixtures,” Journal of Trace and Microprobe Techniques, vol. 19, no. 3, pp. 355–364, 2001. View at Publisher · View at Google Scholar · View at Scopus
  11. F. A. F. Nour El-Dien, G. G. Mohamed, and N. A. Mohamed, “Spectrophotometric determination of trazodone, amineptine and amitriptyline hydrochlorides through ion-pair formation using methyl orange and bromocresol green reagents,” Spectrochimica Acta Part A, vol. 65, no. 1, pp. 20–26, 2006. View at Publisher · View at Google Scholar · View at Scopus
  12. T. Aman, A. A. Kazi, M. I. Hussain, S. Firdous, and I. U. Khan, “Spectrophotometric determination of amitriptyline-HCl in pure and pharmaceutical preparations,” Analytical Letters, vol. 33, no. 12, pp. 2477–2490, 2000. View at Publisher · View at Google Scholar · View at Scopus
  13. J. O. Onah, “Spectrophotometric determination of amitriptyline by the method of charge-transfer complexation with chloranilic acid,” Global Journal of Pure and Applied Sciences, vol. 11, no. 2, pp. 237–240, 2005. View at Google Scholar
  14. D. J. Patel and V. Patel, “Simultaneous estimation of amitriptyline HCl and perphenazine in tablets by UV-Visible spectrophotometric and HPTLC,” International Journal of Pharmaceutical Sciences and Research, vol. 1, no. 2, pp. 20–23, 2010. View at Google Scholar
  15. P. Venkatesan, P. V. R. S. Subrahmanyam, and D. Raghu Pratap, “Spectrophotometric determination of pure amitriptyline hydrochloride through ligand exchange on mercuric ion,” International Journal of ChemTech Research, vol. 2, no. 1, pp. 54–56, 2010. View at Google Scholar · View at Scopus
  16. R. M. El-Nashar, N. T. Abdel Ghani, and A. A. Bioumy, “Flow injection potentiometric determination of amitriptyline hydrochloride,” Microchemical Journal, vol. 78, no. 2, pp. 107–113, 2004. View at Publisher · View at Google Scholar · View at Scopus
  17. D. J. Patel and V. Patel, “simultaneous estimation of amitriptyline hydrochloride and perphenazine by absorption ratio (Q-analysis) UV spectrophotometric method in combined tablet dosage form,” International Journal of Pharmaceutical Sciences and Research, vol. 1, no. 12, pp. 133–137, 2010. View at Google Scholar
  18. B. Starczewska and A. Jasińska, “Analytical application of the reactions of amitriptyline with eriochrome cyanine R and pyrocatechol violet,” Acta Poloniae Pharmaceutica, vol. 60, no. 6, pp. 417–423, 2003. View at Google Scholar · View at Scopus
  19. Y. A. Beltagg, “Colorimetric determination of tricyclic antidepressant and dibenzazepine and dibenz—cycloheptadine with citric acid—acetic anhydride,” Pharmazie, vol. 31, pp. 483–488, 1976. View at Google Scholar
  20. W. N. French, F. Matsui, and J. F. Truelove, “Flow-injection spectrophotometric determination of amitriptyline hydrochloride,” Canadian Journal of Pharmaceutical Sciences, vol. 3, no. 3, pp. 33–37, 1968. View at Google Scholar
  21. E. Domagalina and L. Przyborowski, “Flow injection extractive spectrophotometric determination of antidepressant amitriptyline hydrochloride,” Chemia Analityczna, vol. 7, pp. 1153–1159, 1962. View at Google Scholar
  22. E. M. Elnemma, F. M. El Zawawy, and S. S. M. Hassan, “Determination of amitriptyline, imipramine and orphenadrine in antidepressant drugs by potentiometry, spectrophotometry and atomic absorption spectrometry,” Mikrochimica Acta, vol. 110, no. 1-3, pp. 79–88, 1993. View at Publisher · View at Google Scholar · View at Scopus
  23. B. Dembinski, “Extractive colorimetric determination of amitriptyline and imipramine hydrochloride with bismuth hex iodide,” Acta Poloniae Pharmaceutica, vol. 34, pp. 509–514, 1977. View at Google Scholar
  24. H. E. Hamilton, J. E. Wallace, and K. Blumk, “Spectrophotometric and gas-liquid chromatographic determination of amitriptyline,” Analytical Chemistry, vol. 47, no. 7, pp. 1139–1144, 1975. View at Publisher · View at Google Scholar