SciELO - Scientific Electronic Library Online

Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados


Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. vol.55 no.4 Concepción dic. 2010 

J. Chil. Chem. Soc., 55, N0 4 (2010)




*Quality Assurance Laboratory, Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, G. H. Patel Building, Donor's Plaza, the Maharaja Sayajirao university of Baroda, Fatehgunj, Vadodara, Gujarat, india — 390 002


Three spectrophotometric methods have been developed for simultaneous estimation of Phenylephrine hydrochloride (PHE) and Tropicamide (TPC) in their combined dosage form. Method-I: zero-crossing derivative spectrophotometry involves amplitudes measurement of the first derivative spectra of the binary mixture at 284.0nm for PHE and 241.2nm for TPC. Method-II: dual wavelength,which uses the difference in absorbance at 260.8 nm and 268.2 nm for estimation of PHE and 245.4nm and 271.8nm for TPC. Method III: Ratio spectra derivative spectrophotometry, in which division of the absorption spectrum of the binary mixture by a standard spectrum of one of the components and then calculating first derivative amplitudes at 270.8nm for PHE and 240.8nm for TPC . The calibration graph follows Beer's law in the range of 25-125 mg/ml for PHE and 4-20 mg/ml for TPC for all the three methods. The accuracy and precision of the methods were validated statistically.

Keywords: Phenylephrine Hydrochloride(PHE), Tropicamide(TPC), zero-crossing derivative spectrophotometry, dual wavelength, Ratio spectra derivative spectrophotometry.



Chemically, Phenylephrine hydrochloride (PHE), is (R)-2-methylamino-1-(3-hydroxyphenyl) ethanol hydrochloride)'1-31. It is an alpha-adrenergic (sympathomimetic) agent which stimulates alpha-adrenergic receptors, producing pronounced vasoconstriction. PHE alone or in combination with other drugs is reported to be estimated by spectrophotometric method'4-71, HPLC'8-131 and voltametry'141.

Similarly, Tropicamide (TPC), is (R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid-4-ylmethyl) propionamide'1-31. It is a tropic acid derivative endowed with short duration of antimuscarinic activity. Tropicamide alone is reported to be estimated by spectrophotometric method'151 and HPLC'16-171 .

Since no spectrophotometric method is reported for simultaneous estimation of phenylephrine hydrochloride and tropicamide in combination therefore, in the present work, a successful attempt has been made to estimate both these drugs simultaneously by three simple UV spectrophotometric methods (zero crossing derivative spectrophotometry, Dual wavelength method, Ratio spectra derivative spectrophotometry).



A Schimadzu 1700PC double beam spectrophotometer with a fixed slit width (1nm) used coupled with schimadzu UVPC software was used for all the absorbance measurements and treatment of all data.

Chemicals used

PHE and TPC were kindly supplied as gift samples by Promed pharmaceuticals (Gurgaon, India) and were used as received. Analytical grade HCl and distilled water were used as a solvent.

Commercial Ophthalmic formulation

The quantitative assay of the commercial pharmaceutical formulations TROPICACYL PLUS eye drops (INTAS) containing 8mg TPC and 50mg PHE (1:6.25) respectively were carried out using the developed methods.

Standard solutions and synthetic mixtures

Stock solution of 1mg/1ml PHE and TPC were prepared separately in 0.1N HCl. The standard series of the solutions containing 4-20mg/ml TPC and 25-125 mg/ml were prepared from the stock solutions in distilled water for obtaining calibration graphs and spectra. Similarly, accurate volumes of each of PHE and TPC stock solutions were transferred in 10ml volumetric flasks and diluted to volume with distilled water to obtain binary mixtures as well as synthetic mixtures. All the solutions were prepared freshly. The zero order overlay spectra of PHE, TPC and synthetic mixture is shown in figure 1.

Zero Crossing Derivative Spectrophotometry (MethodI):

The absorption spectra of the binary mixture solutions of PHE and TPC were recorded in the range of 200 nm to 400 nm and were stored in the memory of the instrument.and transformed to first derivative with AX = 8nm and scaling factor 10. The amplitudes at 284.0nm were plotted against the respective concentrations of PHE.Similarly, the amplitudes at 241.2 nm were plotted against the respective concentrations of TPC 'Figure 2(a)].

Dual wavelength method (Method II):

The zero-order spectra of pure drugs were derivatized in first order with AX = 8nm and scaling factor 20 for both drugs. In this method, the difference between absorbance at 260.8and 268.2 nm (Difference is zero for TPC) of the 1st derivative spectra of the binary mixture containing PHE and TPC were measured for the determination of PHE. Similarly the difference between absorbance at 245.4 nm and 270.8nm (Difference is zero for PHE) were measured for the determination of TPC 'Figure 2(b)1. Standard laboratory mixtures of PHE and TPC in 1:6.25 ratio were prepared and successfully analyzed using the developed method.

Ratio spectra derivative spectrophotometry (MethodIII):

For selecting the standard solution as a divisor, appropriate concentration of PHE and TPC were tested respectively. Thus, a concentration of 125mg/ml of PHE and 28mg/ml of TPC as divisor gave best results in terms of signal to noise ratio and highest correlation coefficient values, being an indication of the quality of fitting of the data to the straight line.Therefore,the absorption spectra of PHE for the solution in distilled water were divided by the spectrum of the standard solution of 28 mg/ml of TPC in the same solvent and the first derivative was plotted with the interval of Dl=8 nm. It was found that the first derivative amplitude at 270.8 nm was suitable for the determination of PHE in binary mixture with TPC. In similar way, the stored spectra of TPC were divided by 125 mg/ml of PHE and the first derivative was plotted with the interval of Dl=8 nm. It was found that the first derivative amplitude at 240.8 nm was suitable for the determination of TPC in binary mixture with PHE 'Figure 2(c)1. Once the optimum working condition was established, calibration graphs were obtained at 270.8 nm for Phenylephrine Hydrochloride and 240.8 nm for Tropicamide in the standard binary mixture and showed that the proposed method is applicable over the ranges 25-125 mg/ml for PHE and 4-20 mg/ml for TPC.

Assay of ophthalmic formulation by Method I, II & III:

An accurate volume of ophthalmic solution equivalent to about 50mg of PHE, and 8.00 mg of TPC was transferred to 100 ml volumetric flask, dissolved in 20 ml 0.1N HCl and sonicated for 10 minutes. The volume was then made up to the mark using the same solvent. The resulting solution was filtered through Whatmann filter paper grade I. Appropriate dilutions were prepared in distilled water taking suitable aliquots of the clear filtrates and was subjected to analysis using all the three methods described above. The result of analysis of ophthalmic formulation is reported in Table 1.



The proposed methods were validated as per ICH guideline (Table 2):

(i) Linearity: The linearity range was optimised with 25-125 mg/ml and 4-20 mg/ml for PHE and TPC for all three developed methods. The linear regression analysis shows good linearity having regression coefficient greater than 0.999 for all three developed methods.

(ii) Precision: The precision of the methods were established by carrying out the analysis of the analytes (n=5) using the proposed developed methods. The intra and inter day precision were found less than 2 for all the developed methods which shows that methods are precise.

(iii) Sensivity: The LOD and LOQ were calculated using the following equations:

LOD=3.3 x 6/s and LOQ= 10 x s/s

Where 6 is standard deviation of 'y' intercept of calibration curve (n=5) and s is slope of regression equation.

(iv) Accuracy: To check the accuracy of all three developed methods, analytical recovery experiments were carried out by using standard addition method at 80 %, 100 % and 120 % levels. From the total amount of drug found, the % recovery was calculated.The results of recovery studies were summerised in Table 3.

Statistical Analysis

Statistical comparison of developed three methods was carried out using Analysis of variance (ANOVA) with Tukey's multiple comparison test (compares all pairs of columns with one another) with the help of software Graph Pad Prism 5. It was found that there is no significant difference between three methods for both PHE and TPC (Table 4).


Application of derivative technique of spectrophotometry offers a powerful tool for quantitative analysis of multicomponent mixtures. Derivative spectrophotometry is useful by means of resolving two overlapping spectra and eliminating matrix interferences in the assay of two or more component mixture. This paper demonstrates the potential of zero crossing derivative spectrophotometry, dual wavelength method and ratio spectra derivative spectrophotometry as an analytical technique and its usefulness to accurately, rapidly, simply and simultaneously quantitative active ingredients in multicomponent pharmaceuticals. The proposed three analytical UV spectrophotometric methods were developed and validated thoroughly for quantitatively determination of PHE and TPC in ophthalmic solution.


The authors are greatful to M/s Promed Pharmaceuticals, Gurgaon (India) for their generous gift of PHE and TPC bulk drugs.


1. "British Pharmacopoeia", 2009, Her Majesty's Stationary Office, London.PHE- 4682-85, TPC - 6219-22.         [ Links ]

2. Government of India "Indian Pharmacopoeia"1996, Vol. I, The Controller of Publications, Delhi, PHE - 587, TPC - 783.         [ Links ]

3."The United State Pharmacopoeia" 2003, 26th Revision, U. S. Pharmacopoeial Convention Inc., Rockville. Vol. 30,PHE - 2931, TPC -3430.         [ Links ]

4. I.Savic, G. Nikolic, V. Bankovic,Maced. J. Chem. Chem. Eng., 2008, 27, 2, 149-156.         [ Links ]

5. M. Kazemipoura and M. Ansarib, Iran. J. Pharm. Res., 2005, 3, 147-153.         [ Links ]

6. N. Erk, J. Pharm. Biomed. Anal., 2000, 23,1023-1031.         [ Links ]

7. S.A. Shama, J. Pharm. Biomed. Anal., 2002, 30, 1385-1392.         [ Links ]

8. K.Gumbhir and William D. Mason, J. Pharm. Biomed. Anal. , 1996, 14. 623-630.         [ Links ]

9. P. Ptácek, J. Chromatogr. B,2007,858,263-68.         [ Links ]

10. A. Marín, E. García, A. García and C. Barbas, J. Pharm. Biomed. Anal., 2002,29,701-714.         [ Links ]

11. R. Pereira-Rosario ; S. El-Gizawy; J. H. Perrin; C. M. Riley, Drug Dev. Ind. Pharm., 1986, 12, 2443-65.         [ Links ]

12. R. Heydari, Anal. Lett., 2008, 41, 965-976.         [ Links ]

13. N. Erk and M. Kartal, Farmaco, 1998, 53, 617-22.         [ Links ]

14. Y. Zhu, Z. Zhang,, W. Zhao and D. Pang, Sens. Actuator B, 2006, 119, 308-14.         [ Links ]

15. M. Amanlou, E. Souri, S. Izady, H. Farsam, Iran. J. Pharm. Sci., 2003, 1, 43-50.         [ Links ]

16. M. Amanlou, G. Asmardi, P. Andalibi, N. Javadi, F. Khodadady and Z. B. Omarny, J .Chromatogr. A, 2005, 1088, 136-39.         [ Links ]

17. Z. Stefanowicz, J. Stefanowicz, K. Mulas, J. Pharm. Biomed. Anal., 2009, 49, 214-220.         [ Links ]

(Received: July 19, 2010 - Accepted: December 30, 2010)


Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons