INTRODUCTION:

Multi-drug administration is often associated with clinically significant interaction, especially of narrow therapeutic index drugs, either at pre-absorption or post-absorption Chemically, Phenylephrine hydrochloride (PHE), is (R)-2-methylamino-1-(3-hydroxyphenyl) ethanol hydrochloride)'. 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'.Similarly, Tropicamide (TPC), is (R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid-4-ylmethyl) propionamide^'. 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^' .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 simple UV spectrophotometric methods (simultaneous equation method).^5,6

INSTRUMENT

A double-beam Jasco, V630 Visible spectrophotometer, spectral bandwidth of 2nm, wavelength accuracy ±0.5nm and a pair of 1-cm matched quartz cells was used to measure absorbance of the resulting solution.

CHEMICALS AND REAGENTS

The gift sample of Tropicamide and phenylepherin hydrochloride was obtained from local market. Eye drops of brand TROPICACYL PLUS eye drops (INTAS) containing 8mg TPC and 50mg PHE (1:6.25) respectively were carried out using the developed methods.

PREPARATION OF SOLUTIONS

Stock solution of 1µg/ml PHE and TPC were prepared separately in distilled water. The standard series of the solutions containing 4-20 mg/ml TPC and 25-125 mg/ml PHE were prepared from the stock solutions in distilled water for obtaining calibration graphs and spectra as shown in figure (1) and figure (2). 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. Shown in Table (1)^1,2,3,14

Simultaneous equation,

Cx = (A2ay1-A1ay2)/ (ax2ay1-ax1ay2) eq. (1)

Cy = (A1ax2-A2ax1)/ (ax2ay1-ax1ay2) eq. (2)

APPLICATION OF PROPOSED METHOD FOR ANALYSIS OF MARKETED FORMULATION:

An accurately weighed sample equivalent to TPC (8mg) and PHE (50mg) was taken in a volumetric flask (100mL), distilled water (40mL) was added and sonicated for 10 min. Volume was made up to the mark with distilled water and filter through whatmann filter paper. The aliquot portions of above solutions were further diluted with solvent to get final concentrations of TPC (8µg/ml) and PHE (50µg/ml) ,respectively. The above solution was analyzed for the content of TPC and PHE using the method described above, and as shown in table (2)^1,2,13

VALIDATION OF PROPOSED METHOD

The proposed analytical method was validated as per recommendations ICH guidelines for the Parameters like Linearity, recovery, precision, ruggedness and repeatability.

Linearity: The linearity range was optimized with 25-125 µg/ml and 4-20 µg/ml for PHE and TPC for all three developed methods. The linear regression analysis shows good linearity having regression coefficient greater than 0.99 for all three developed methods. Table (2)^4,5,8

Recovery study:

The accuracy of an analytical method is closeness of test results obtained by that method to the true value. The accuracy of an analytical method should be established across its range. A known amount of standard solution of pure drugs TPC and PHE were added to pre analyzed sample solution TPC 8µg/ml and PHE 50µg/ml. These solutions were subjected for analysis. The lower the values of standard deviation (SD) indicate the method is accurate, shown in Table (3)^5,6,9

Precision:

The precision of an analytical method is the degree of agreement among the individual test results when the method is applied repeatedly to multiple samplings of a homogeneous sample. Variation of results within the same day (intra day), variation of results between days (inter day) were analyzed. 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. Table (4)^4,7,8

Ruggedness:

The ruggedness of analytical method is the degree of reproducibility of test results obtained by the analysis of the same sample under a variety of conditions, such as different laboratories, different analysts, different instruments, and different lots of reagent. Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slot by two analyst using same operational and environmental conditions and the results are reported in Table(5)^7,8

Repeatability:

Repeatability was determined by analyzing TPC (8µg/ml) and PHE (50µg/ml) concentration of drug solutions for six times and results are reported in Table (6)^9,10,14

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 summarized in Table (7)^11,12

Fig (1)-overlain spectra of TPC and PHE

Fig (2)-Calibration curve of TPC and PHE

Table-1 Analysis of TPC and PHE in laboratory mixture

Sr. No	Amount Taken	Amount Found* (µg/ml)	%Amount Found±SD
1	TPC(8µg/ml)	7.88	98.5±0.02
2	PHE(50 µg/ml)	49.52	99.04±0.5

*Mean of five readings

Table-2 Application of Proposed Method for Analysis of marketed preparation

Sr. No	Lable claimed	% amount Found*±SD
1	TPC(8µg/ml)	98.25±0.12
2	PHE(50µg/ml)	100.08±0.14

Table-3: Recovery Study Data

Sr.no	Sample solution	Amount of standard drug added (µg/ml)	%Recovery* ± SD
1	TPC(8µg/ml)	8.00	99.02±0.35
2	PHE(50µg/ml)	50.00	99.46±0.44

Table-4: Precision Data

Sr.no	drug	Conc.	Intra-day	±SD	Inter-day	±SD
1	TPC	5µg/ml	4.84	0.7	5.02	0.09
		10µg/ml	9.91	1.2	9.63	0.3
		15µg/ml	14.23	0.8	14.68	0.6
2	TPC	5µg/ml	4.95	0.3	4.55	0.7
		10µg/ml	9.69	0.5	9.78	1.2
		15µg/ml	14.87	0.4	14.79	0.7

Table-5: Ruggedness Data

Drug	Amount taken (μg/ml)	Analyst I*	%SD	Analyst II*	%SD
TPC	8	8.03	0.95	7.96	1.3
PHE	50	49.66	0.23	49.67	1.5

Table-6: Repeatability Data

Sr.no

Drug

Amount

Taken(µg/ml)

Amount Found(µg/ml)

TPC

8.01

1.3

PHE

50.08

1.6

ACKNOWLEDGEMENT:

The authors are greatly thankful to P.D.V.V.P.F’s College of Pharmacy, Ahmednagar, India for providing access to facilities and necessary infrastructure to carry out research work.

RESULTS AND DISCUSSION:

The market survey revealed that the above combination is recently introduced in the market and literature survey also revealed that no methods are reported for the simultaneous estimation of TPC and PHE in their combined dosage form. Hence, an attempt has been made to develop the spectrophotometric method for simultaneous estimation of TPC and PHE in ophthalmic dosage form. The overlain spectra of both drugs showed good absorbances at 253 and 235 nm, hence these wavelengths were selected for estimation of TPC and PHE. Linearity of both TPC and PHE were obeyed in the concentration range of 4-20 µg/ml and 5-125 µg/ml with the correlation coefficient 0.996 and 0.994, respectively. The absorptivities was then calculated and substituted in equation 1 and 2 to obtained concentration of both drugs.

REFERENCES:

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

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

3. I. Savic, G. Nikolic, V. Bankovic, Maced. J. Chem. Chem. Eng., 2008, 27, 2, 149-156

4. M. Kazemipoura and M. Ansarib, Iran. J. Pharm. Res., 2005, 3, 147-153

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10. R. Heydari, Anal. Lett., 2008, 41, 965-97612. N. Erk and M. Kartal, Farmaco, 1998, 53, 617-22.

11. Y. Zhu, Z. Zhang^,, W. Zhao and D. Pang, Sens. Actuator B, 2006, 119, 308-14

12. M. Amanlou, E. Souri, S. Izady, H. Farsam, Iran. J. Pharm. Sci., 2003, 1, 43-50

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Received on 14.12.2011 Modified on 15.01.2012

Asian J. Research Chem. 5(5): May 2012; Page 678-680

Sample	Label Claim	Amount Found(mg/tab)	Amount Taken		% Recovery	LOD	LOQ	C.V
			%	mg/ml
TPC	8mg/ml	7.51	120	10	98.76	0.07	0.128	1.272
			100	8	98.73
			80	6	98.76
PHE	50mg/ml	49.78	120	60	99.01	0.33	0.204	1.08
			100	50	99.12
			80	40	99.12

Simultaneous Spectrophotometric Estimation of Tropicamide and Phenylepherin Hydrochloride in Ophthalmic Dosage Form.

INTRODUCTION: