Spectrophotometric and Stability Indicating RP-HPLC-PDA method for Simultaneous Determination of Finasteride and Tamsulosin in Combined Tablet Dosage form

Atul H Kategaonkar¹, Dhaval M Patel¹, Bhushan P Itkar¹, Vishnu P Choudhari¹*, Bhanudas S Kuchekar¹ and Ana G Nikalje²

¹MAEER’S Maharashtra Institute of Pharmacy, S.No. 124, MIT Campus,  Paud Road, Kothrud, Pune-411038, India.

²Post-graduate Department of Pharmaceutical Chemistry, Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad.431001. MS. India

*Corresponding Author E-mail: viraj1404@rediffmail.com

ABSTRACT

Simple, accurate, precise, and sensitive ultraviolet spectrophotometric and stability indicating reversed phase high performance liquid chromatographic methods for simultaneous estimation of Tamsulosin and Finasteride in combined tablet dosage form have been developed and validated. The spectroscopic method employs an absorbance correction method using 228 and 246 nm as two wavelengths for estimation with methanol as solvent. Beer's law is obeyed in the concentration range of 2-10 and 25-125 μg/mL for Tamsulosin and Finasteride respectively. The reversed phase high performance liquid chromatographic separation was achieved on a Waters Symmetry C₁₈ column (250 mm x 4.6 mm, 5.0 μ particle size) using Methanol: Water: THF (75: 15: 10 v/v/v) mobile phase. Trifluoroacetic was used to adjusted pH to 3.7 with,  flow rate was 0.7 mL/min and column  temperature was maintained at 32⁰C. Quantification was achieved with PDA detector at 275 nm over the concentration range of 50 to 400µg/mL for Finasteride and 4 to 44µg/mL for Tamsulosin. Both methods have been successfully applied for the analysis of the drugs in a pharmaceutical formulation. Results of analysis were validated statistically and by recovery studies.

KEYWORDS: Spectrophotometry, Stability indicating RP-HPLC, Tamsulosin, Finasteride.

INTRODUCTION:

Tamsulosin (TAM) ¹ is chemically known as 5-[(2R)-2-[[2-(2-ethoxyphenoxy) ethyl] amino] propyl]-2-methoxybenzenesulfonamide, which an alpha-adrenoceptor blocker with enhanced specificity for the alpha-adrenoceptors of the prostate, is commonly used to treat benign prostatic hyperplasia (BPH). Finasteride (FINA)² is chemically known as (5α, 17β)-N-(1, 1-Dimethylethyl)-3-oxo-4- azaandrost-1-ene-17-carboxamide. Finasteride is a competitive and specific inhibitor of Type II 5α-reductase, an intracellular enzyme that converts the androgen testosterone into Dihydro testosterone (DHT). For the treatment of symptomatic benign prostatic hyperplasia (BPH) in men with an enlarged prostate to improve symptoms, reduce the risk of acute urinary retention, and reduce the risk of the need for surgery including transurethral resection of the prostate. The chemical structures of TAM and FINA are shown in Figure 1.The literature survey revealed that the many more HPLC and Spectroscopic methods for TAM and FINA are reported as a single drug formulation.

But to my knowledge, no method was reported for determination of combination of FINA and TAM therefore it is felt necessary to develop stability indicating HPLC and simple absorbance correction UV spectrophotometric methods for this combination.

Several analytical methods that have been reported for the determination of TAM^3-6 individually in biological fluids and pharmaceutical formulations which include liquid chromatography coupled with mass spectrometry, HPLC, chiral liquid chromatography and capillary electrophoresis. For FINA several analytical methods have been reported for its individual determination which includes Bromometric assay, spectrophotometry, and liquid chromatography ^6-12. This paper describes simple, accurate, precise and sensitive UV spectrophotometric method and stability indicating RP-HPLC method for simultaneous determination of TAM and FINA in combined tablet dosage form. The proposed methods were optimized and validated as per the International Conference on Harmonization (ICH) guidelines ¹³.

Table 1: Regression Analysis of Calibration Curves for Method I and Method II

Parameter	METHOD I		METHOD II
	TAM	FINA	TAM	FINA
λ max(nm)	228	246	275^1
Beer’s law limit (µg/mL)	2-10	25-125	1- 44	12.5- 550
Correlation coefficient (r)	0.9998	0.9992	0.9997	0.9998
Molar absorptivity (L mol^-1 cm^-1)	1955.62	4911.23	_	_
Linear regression Equation^2 (y= mx+c)
Intercept (c)	0.0013	- 0.0181	7089.8	-320.04
Slope  ±  S.D.^3	0.04756±0.50	0.00518±0.48	6847.5± 0.30	876.78±0.53
Detection limit (µg/mL)	0.72	0.86	1.38	13.17
Quantitation limit (µg/mL)	2.19	2.62	4.20	39.93

¹ is the Detection Wavelength for HPLC method, ²With respect to y = mx + c, where y is the absorbance and x is the concentration (μg/mL).

³Standard deviation of slope.

Figure1:  Chemical structures of a) FINA and b) TAM

a)    FINA                                                          b) TAM

MATERIALS AND METHODS:

Drugs and Chemicals:

Methanol, Water and Tetrahydrofuran(THF)  used for HPLC method were of HPLC grade and analytical reagent grade Trifluoro Acetic Acid (TFA) was used.

Spectroscopic grade Methanol was used for specrtrophotometric method. All the solvents and reagents used were purchased from LOBA Chemie Pvt. Ltd., Mumbai. Tablet used for analysis were VELTAM – F form two batches (Batch No. DH 1189 and DH 1338) manufactured by Intas Pharmaceuticals, Dehradhun, India containing FINA USP 5mg and TAM 0.4 mg per tablet. Pure drug sample of FINA % purity 99.78 was kindly supplied as a gift sample by Ranbaxy Laboratories Ltd. and pure drug sample of TAM % purity 99.92 was gifted by Aarti drugs Pvt. Ltd. Mumbai. These samples were used without further purification.

Instruments:

An UV-Vis double beam spectrophotometer of makes Varian Cary 100 Australia with 10 mm matched quartz cells were used for spectrophotometric method. Waters HPLC system, Milford USA consisted of binary Pump (Waters 515), with Auto sampler (model Waters; 717) having injection capacity of 5-200 μL.  Photodiode array detector (PDA) (Waters 2998) was used. Data was integrated using Waters Empower 2 system and Waters Symmetry C₁₈ column (250 mm x 4.6 mm, 5.0 μ particle size) and and Kromasil C₁₈ (250 mm × 4.6 mm, 5.0 µ) were used and maintained at 32⁰C using column oven. The mobile phase

consisted of methanol: water: THF (75:15:10, v/v/v) with pH adjusted in between 3.60 to 3.80 by TFA and filtered through 0.45μm nylon filter and degassed in ultrasonic bath prior to use. Measurements were made with injection volume 10µl and ultraviolet (UV) detection at 275 nm. For analysis of forced degradation samples, the PDA was used in scan mode with a scan range of 220–400 nm.

Procedure:

Method I: Absorbance Correction Method¹⁴:

Stock solution of drugs having concentration 1000µg/mL was prepared by dissolving FINA and TAM separately in methanol. Aliquots of stock solutions were further diluted in methanol and were scanned in the wavelength range of 300–200 nm. Zero order-overlain spectrums are represented in Figure 2. Two wavelengths selected for determination were 228 nm and 246 nm, λ_max of TAM and FINA respectively. TAM and FINA show significant absorption at 228 nm but at the λ_max of FINA (246) TAM shows practically nil absorption. The Beer’s law was obeyed over the concentration range 2-10 μg/mL at 228 nm by TAM and over the concentration range 25-125 μg/mL at 228 nm and 246 nm by FINA. The absorptivity values at 228 nm and 246 nm for both the drugs were determined by checking absorbance values for working standards of TAM and FINA. The determination of FINA has been done at 246 nm using its absorptivity value, since there is no interference of TAM. An accurate determination of TAM has been achieved after correction for absorbance by FINA at 228 nm.

Table 2: System Suitability Parameters for RP-HPLC Method

Parameter	TAM	FINA
Theoretical plates	3593.86	7275.75
Tailing Factor	1.295	1.189
Capacity factor	2.0903	4.1983
Resolution^*	-	9.188
Selectivity factor	-	1.97

^* For FINA calculated with respect to TAM

Table 3: Result of analysis of commercial formulation.

Method	Label Claim (mg/tablet)		% of Label claim Estimated* ±SD		% R.S.D.
	TAM	FINA	TAM	FINA	TAM	FINA
Method I	0.4	5	99.59±0.59	101.629±0.48	0.581	0.488
Method II	0.4	5	101.91±0.30	100.62±0.53	0.294	0.53

*Average of six Determinations

Table 4: Robustness evaluation of HPLC method (n=3).

Chromatographic   parameters	Retention time		Capacity factor		Tailing Factor
A: Flow Rate: -	TAM	FINA	TAM	FINA	TAM	FINA
0.665 (-5%)	3.261	5.413	2.259	4.413	1.275	1.153
0.7 (normal)	3.004	5.26	2.002	4.238	1.295	1.189
0.735 (+5%)	2.974	4.945	1.974	3.944	1.232	1.148
Mean ± SD (n=3)	3.07±0.15	5.26±0.23	2.07±0.15	4.19±0.65	1.26±0.03	1.16±0.02
B: Percentage of methanol in mobile phase:
70% (-5%)	3.195	5.420	2.195	4.422	1.237	1.148
75 (normal)	3.004	5.26	2.002	4.238	1.295	1.189
80 (+5%)	2.972	4.913	1.972	3.913	1.242	1.147
Mean ± SD (n=3)	3.05±0.12	5.19±0.25	2.05±0.12	4.19±0.66	1.25±0.03	1.16±0.02
C: Temperature:
30.4 (-5%)	3.058	5.091	2.058	4.091	1.248	1.152
32 (normal)	3.004	5.26	2.002	4.238	1.295	1.189
31.6 (+5%)	3.062	5.100	2.062	4.100	1.239	1.149
Mean ±SD (n=3)	3.04±0.03	5.15±0.09	2.04±0.03	4.14±0.65	1.26±0.02	1.16±0.02
D: Columns form different manufacturers:
Symmetry	3.004	5.26	2.002	4.238	1.295	1.189
Kromasil	3.580	5.503	2.580	4.503	0.982	1.170
Mean ±SD (n=3)	3.29±0.40	5.38±0.17	2.29±0.40	4.37±0.51	1.13±0.22	1.17±0.01

Table 5: Recovery studies of TAM and FINA by method I and method II.

Drug	Excess drug added to the analyte %.	Theoretical   content (µg).	% Recovery ^n		% RSD
			Method		Method
			I	II	I	II
FINA	80	270	100.57	101.46	0.67	0.34
	100	300	98.90	99.69	0.59	0.65
	120	330	99.46	100.95	0.51	0.85
TAM	80	21.6	98.67	99.62	1.30	0.99
	100	24	99.77	99.62	0.69	0.57
	120	26.4	100.89	99.78	0.57	0.26

n = Average of six Determinations

Procedure for Analysis of Tablet Formulation:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 5 mg of FINA and 0.4 mg of TAM was transferred to 25 mL volumetric flask, 20 mL of methanol was added and sonicated for 30 min and diluted to 25 mL with methanol. The sample solution was centrifuged for 5 minutes at 8000 rpm. The solution was further diluted with methanol to obtain the dilution within the Beer’s law range. Then absorbance of sample was measured at 228 nm and 246 nm. The content of TAM and FINA were calculated using the following formula

                                C_FINA = A1/ax1

                                C_TAM = A2 – ax2 C_FINA / ay2

Where, C_FINA and C_TAM are concentrations of standard FINA and TAM respectively; A1 and A2 are absorbance’s of sample solution at 228 nm and 246 nm respectively; ax1 (183.95) is absorptivity of standard FINA at 246 nm; ax2 (823.94) and ay2 (273.55) are absorptivity values of standard FINA and TAM at 228 nm respectively.

Method II: A stability indicating RP-HPLC method

Standard stock solution:

Standard stock solution of FINA and TAM were prepared by dissolving 50 mg of FINA and 4 mg of TAM in 100 mL of mobile phase separately to get concentration of 500 and 40µg/mL respectively. 1 mL of stock solution was further diluted to 10 mL with mobile phase to get a working standard solution of concentration 50 and 4 µg/mL. Further dilutions for the calibration graph were also prepared from stock solution of 500 and 40 µg/mL.

Selection of analytical wavelength:

From the standard stock solution further dilutions were prepared using mobile phase and scanned by PDA detector over the range of 200-350 nm and the spectras were overlaid. It was observed that TAM, which was in low concentration in tablet formulation as compared to FINA showed considerable absorbance at 275 nm and the area of both the drugs was nearly same at this point. Therefore 275 nm was selected as wavelength of analysis.

Table 6: Summary of stress degradation study of TAM and FINA.

Exposure conditions	FINA		TAM
	% degredation	tr of degredant(s)	% degredation	tr  of degredant(s)
Acid hydrolysis	24.22	2.88,3.89,7.89	74.42	4.10
Base Hydrolysis	21.39	3.57,3.83,4.78	93.73	3.49,4.87,6.15
Hydrogen peroxide 30 %w/v	73.72	4.39,6.97	50.05	3.53
Neutral Hydrolysis	37.11	3.85,6.61	66.27	3.9,6.09

Figure 2: Zero order overlain Spectrum of TAM (6μg/mL) and FINA (75μg/mL) in methanol.

Checking the resolution of drugs:

The column was saturated with the mobile phase (approximately, 10 column volumes and indicated by constant back pressure at desired flow rate). Mixed standard solution of FINA and TAM was injected to get the chromatogram. The retention times of the drugs were found to be 3.006 min and 5.108 min for TAM and FINA rescpectively (Figure 3).

Procedure for Analysis of Tablet Formulation:

Sample preparation:

Twenty tablets were weighed accurately powdered and a quantity of tablet powder equivalent to 50  mg of FINA and 4 mg of TAM was weighed and transferred to a 100 mL volumetric flask containing about 70 mL of methanol, ultrasonicated for 30 min and volume was made up to the mark with the mobile phase. The resulting solution was centrifuged at 8000 rpm for 5 min. Clear supernatant was suitably diluted to get solutions of concentrations of 150 µg/mL of FINA and 12 µg/mL of TAM. The sample solution was then filtered through 0.45 µ nylon filter and 10 µL of the sample solution was injected in triplicate by using autosampler 717 under the conditions described above.  The chromatogram was obtained and the peak areas were recorded and amount of each drug present per tablet was estimated from the respective calibration curves. Procedure was repeated three times for analysis of homogenous sample. The results of formulation analysis are given in Table 3.

In peak purity analysis with photo diode array detector, purity angle was always less than purity threshold for all the analytes. This shows that the peak of analytes was pure and excipients in the formulation did not interfere the analytes.

Robustness:

In the robustness study, the influence of small, deliberate variations of the analytical parameters on retention time of the drugs was examined. The following factors were selected for change, proportion of methanol in mobile phase (75% ± 5%), flow rate of the mobile phase (0.7 ± 5% mL/min), operating temperature (32 ± 5%), and Column from different manufacturer. One factor at the time was changed to estimate the effect. The solutions containing 150 mg/mL of FINA and 12 mg/mL of TAM were injected in the column. A number of replicate analyses (n = 3) were conducted at 3 levels of the factor (-, 0, +). Results of robustness study are presented in Table 4.

Recovery Studies:

To study the accuracy of the above methods, recovery studies were carried out by addition of standard drug solution to pre analyzed sample at three different levels 80 %, 100 % and 120 %. Results of recovery study are presented in Table 5.

Procedure for forced degradation study ¹⁵:

Two different stock solutions of FINA and TAM containing 250 mg of drugs in 250 mL methanol were prepared. These solutions were used for forced degradation to provide an indication of the stability indicating property of proposed method. In all degradation studies the average peak area of   FINA and TAM standards after injections were recorded in order to study the degradation products of both the drugs.

Acid and Base induced degradation:

To 20 mL of methanolic stock solution of both drugs, 5 mL of each of 1 N H₂SO₄ and 1 N NaOH were added separately. These mixtures were refluxed for 3 hours at 70⁰ C. The forced degradation in acid and base was performed in dark conditions in order to exclude the possible degradative effect of light.

Oxidative degradation:

To 20 mL of methanolic stock solutions 5 mL of 30% v/v of hydrogen peroxide was added separately. The solutions were refluxed for 4 h at 70⁰ C and then heated on boiling water bath for 10 min to remove the excess of hydrogen peroxide, and then volume was made up with methanol.

Neutral hydrolysis:

To 20 mL of methanolic solutions of both drugs, 5 mL of double distilled water was added and the mixture was refluxed for 4 h at 70⁰ C to study the degradation under neutral conditions.

Figure 3: Chromatogram of standard mixture solution of FINA (400µg/ml) and TAM (32µg/ml)

RESULTS AND DISCUSSION:

The HPLC method was optimized with a view to develop a stability indicating assay method. Pure drug was injected in different mobile phase compositions. Initially acetonitrile and water in different ratios were tried. But in that, both drugs did not showed development. Hence, Acetonitrile was replaced by methanol and different ratios were tried. In this mobile phase TAM showed the development but peak shape was not proper and the elution time was too short, and that for FINA was too long (around 18 min). Therefore, to decrease the polarity of mobile phase, various proportions of THF were tried. 10% of THF gave desirable elution times for both the drugs, but still the peak shape of TAM was poor. So, to improve the quality of peak, TFA was used in the Water-THF mixture, and pH was adjusted to around 3.60 - 3.80 and column maintained at 32⁰ C. Now, mobile phase Methanol: Water: THF (75: 15:10), pH 3.7 and column temperature 32⁰ C shown good resolution, peak shape and desired elution. Flow rate was set to 0.7 mL / min and UV detection was carried out at 275 nm. Chromatogram showed symmetrical peaks with good shapes; tailing factor for TAM and FINA was within range and the resolution of the standard drugs was satisfactory.  Retention time of TAM was 3.004 min and that of FINA was 5.198. Number of theoretical plates was also greater than 2000 at 0.7 mL/min. The system suitability parameters observed by using this mobile phase were reported in Table 2.  Ultimately mobile phase consisting of Methanol: Water: THF (75: 15:10) pH 3.7and 0.7 mL/min flow rate was selected for validation and stability studies. The mobile phase and sample solutions was filtered using 0.45 µm membrane filter and was degassed by ultrasonication for 10 min prior to use. All determinations were performed at 32⁰C. RP-HPLC method found to be more sensitive and accurate compared to spectrophotometric method. (Table1).

Stability indicating studies (Specificity):

Stability indicating studies of FINA:

The chromatograms of acid degraded sample showed three additional peaks at t_r 2.88, 3.89, 7.89 min and base degraded samples showed three additional peaks at t_r 3.57,3.83and 4.78 min respectively. The sample degraded under neutral conditions showed two additional peaks at t_r 4.39 and 6.97 min. The sample degraded with 30% v/v hydrogen peroxide showed two additional peaks at 3.85 and 6.61 min. This indicates that the drug is susceptible to acid-base hydrolysis, oxidation, and neutral hydrolytic degradation. (Figure 4).

Stability indicating studies of TAM:

The chromatograms of acid degraded sample showed one additional peak at t_r 4.10 min and base degraded samples showed three additional peaks at t_r 3.49, 4.87 and 6.15min. The sample degraded under neutral conditions showed additional peak at t_r 3.53 min along with peak of standard drug. The sample degraded with 30% v/v hydrogen peroxide showed two additional peaks at 3.90 and 6.09 min. This indicates that the drug is susceptible to acid, base hydrolysis, oxidation and neutral hydrolytic degradation.  (Figure5).

Figure 4: Typical HPLC Chromatogram of: (A) Acid hydrolysis-degraded FINA; (B) Base hydrolysis-degraded FINA; (C) Neutral hydrolysis-degraded FINA; (D)  Oxidative degraded FINA.

CONCLUSION:

The validated Spectrophotometric and stability indicating RP-HPLC methods employed here proved to be simple, fast, accurate, precise and sensitive, thus can be used for routine analysis of Tamsulosin and Finasteride in combined tablet dosage form without prior separation. RP-HPLC method   may further be extended to study the degradation kinetics of Tamsulosin and Finsateride and also for its determination in plasma and other biological fluids. As the method separated the drug from its degradation products, it can be employed as stability -indicating one.

Figure5: Typical HPLC Chromatogram of: (A) Acid hydrolysis-degraded TAM ; (B) Base hydrolysis-degraded TAM; (C) Neutral hydrolysis-degraded TAM; (D) Oxidative degraded TAM.

ACKNOWLEDGEMENTS:

The authors are thankful to Pune University for financial assistance. The authors wish to express their gratitude to M/s Ranbaxy Laboratories Ltd., India, for the sample of pure Finasteride and M/s Aarti Drugs Pvt. Ltd. Mumbai, India, for the sample of pure Tamsulosin. The authors are also thankful to the management of MAEER’s Maharashtra Institute of Pharmacy for providing necessary facilities.

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Received on 25.04.2009        Modified on 21.06.2009

Accepted on 27.07.2009        © AJRC All right reserved

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 414-419