Dutasteride belongs to a class of drugs called 5-alphareductase inhibitors, which block the action of the 5-alpha-reductase enzymes that convert testosterone into dihydrotestosterone (DHT). Finasteride also belongs to this group, but while Dutasteride inhibits both isoforms of 5-alpha reductase, finasteride inhibits only one. Even so, a clinical study done by GlaxoSmithKline, the EPICS trial, did not find Dutasteride to be more effective than finasteride in treating BPH [5].

Mechanism of action: Dutasteride inhibits the conversion of testosterone to 5 alpha-dihydrotestosterone (DHT), which is the androgen primarily responsible for the initial development and subsequent enlargement of the prostate gland. Testosterone is converted to DHT by the enzyme 5 alpha-reductase, which exists as 2 isoforms, type 1 and type 2 [6]. Dutasteride is a competitive and specific inhibitor of both type 1 and type 2, 5-alpha-reductase isoenzymes, with which it forms a stable enzyme complex. Dissociation from this complex has been evaluated under in vitro and in vivo conditions and is extremely slow. Dutasteride does not bind to the human androgen receptor [7,8].

To develop new RP HPLC method for the simultaneous estimation of Tamsulosin Hydrochloride and Dutasteride in pharmaceutical dosage form.

• Solubility determination of Tamsulosin Hydrochloride and Dutasteride in various solvents and buffers.

• Determine the absorption maxima of both the drugs in UV– Visible region in different solvents/buffers and selecting the solvents for HPLC method development.

• Optimize the mobile phase and flow rates for proper resolution and retention times.

• Validate the developed method as per ICH guidelines.

Tables 1 and 2

A mixture of 40 volumes of 20mM Ammonium acetate buffer pH 3.5:30 volumes of Acetonitrile: 30 volumes of Methanol. The mobile phase was sonicated for 10 min to remove gases.

In simultaneous estimation of two drugs isobestic wavelength is used. Isobestic point is the wavelength where the molar absorptivity is the same for two substances that are interconvertible. So this wavelength is used in simultaneous estimation to estimate both drugs accurately.

25 mg of Tamsulosin Hydrochloride was weighed and transferred in to 250ml volumetric flask and dissolved in methanol and then make up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

25mg of Dutasteride was weighed in to 250ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

Were soluble it was used as solvent for λ max determination by UV-Visible Spectroscopy.

Preparation of mixed standard solution: Standard stock solutions of Tamsulosin Hydrochlorideand Dutasteride (microgram/ml) were prepared by dissolving 40 mg of Tamsulosin Hydrochloride and 32 mg of Dutasteride dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min and dilute to 100 ml with mobile phase. Further dilutions are prepared in 5 replicates of 40 μg/ml of Tamsulosin Hydrochlorideand 32 μg/ml of Dutasteride was made by adding 1 ml of stock solution to 10 ml of mobile phase.

Sample preparation: 10 tablets (each tablet contains 0.5 mg of Tamsulosin Hydrochloride and 0.4 mg of Dutasteride) were weighed and taken into a mortar uniformly mixed. Test stock solutions of Tamsulosin Hydrochloride (40 μg/ml) and Dutasteride (32 μg/ml) were prepared by dissolving weight equivalent to 40 mg of Tamsulosin Hydrochloride and 32 mg of Dutasteride and dissolved in sufficient mobile phase. After that filtered the solution using 0.45-micron syringe filter and Sonicated for 5 min and dilute to 100ml with mobile phase. Further dilutions are prepared in 5 replicates of 40 μg/ml of Tamsulosin Hydrochloride and 32 μg/ml of Dutasteride was made by adding 1 ml of stock solution to 10 ml of mobile phase.

The amount of TOLCAPONE and QUINAPRILpresent in the formulation by using the formula given below, and results shown in table 3.

$$\%Assay=\frac{AT}{AS}\times \frac{WS}{DS}\times \frac{DT}{WT}\times \frac{P}{100}\times \frac{AW}{LC}\times 100$$

Where,

AT = Peak area of sample preparation,

AS = Average Peak area of standard preparation,

WS = Weight of drug in mg,

DS & DT = Dilution of standard and sample preparation,

WT = Weight of Sample in Assay preparation,

P = Percentage purity of working standard,

LC = Label Claim of drug.

Observation: The amount of Tamsulosine Hydrochloride and Dutasteride present in the taken dosage form was found to be 98.93 % and 99.16% respectively.

Validation: Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, quality, purity and potency of the drug substances and drug products.

a) Specificity / Selectivity

b) Accuracy

c) Precision

d) Linearity & Range

e) Limit of Detection

f) Limit of Quantitation

g) Robustness

h) Ruggedness

i) System Suitability

Figure 1

Table 4

System suitability: Standard solutions were prepared as per the test method and injected into the chromatographic system. The system suitability parameters like theoretical plates, resolution and asymmetric factor were evaluated.

Tables 5 and 6

Tables 7,8 and 9

Figures 2 and 3

Observation: The correlation coefficient for linear curve obtained between concentration vs. Area for standard preparations of Tamsulosin Hydrochloride and Dutasteride is 0.9961 and 0.9981.

Tables 10 and 11

Observation: The percentage mean recovery of Tamsulosin Hydrochloride and Dutasteride is 100.23% and 99.47% respectively.

Observation: Test results for Tamsulosin Hydrochloride and Dutasteride are showing that the % RSD of Assay results are within limits.

$$LOD=\frac{3.3\sigma }{S}$$

Where, σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte.

LOD of Tamsulosin Hydrochloride = 0.74 µg/ml

LOD of Dutasteride = 1.29 µg/ml

Observation: The LOD for this method was found to be 0.74 µg/ml & area 33.46 for Tamsulosin Hydrochloride and 1.29 µg/ml & area 41.79 for Dutasteride.

$$LOQ=\frac{10\sigma }{S}$$

Where,

σ = the standard deviation of the response

S = the slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte.

LOQ of Tamsulosin Hydrochloride = 2.24µg/ml

LOQ of Dutasteride =3.91 µg/ml

Observation: The LOQ for this method was found to be 2.24 µg/ml & area 101.40 for Tamsulosin Hydrochloride and 3.91 µg/ml & area 126.64 for Dutasteride.

Observation: From the observation it was found that the system suitability parameters were within limit at all variable conditions.

Observation: From the observation the between two analysts Assay values not greater than 2.0%, hence the method was rugged.

A simple and selective LC method is described for the determination of Tamsulosin Hydrochloride and Dutasteride tablet dosage forms. Chromatographic separation was achieved on a C₁₈ column using mobile phase consisting of a mixture of Phosphate buffer (KH_{2PO4) pH: 3.5:30 Acetonitrile: Methanol (40:30:30v/v), with detection of 223 nm. Linearity was observed in the range 19.2-44.8 µg/ml for Tamsulosin Hydrochloride (r² = 0.9961) and 24-56µg/ml for Dutasteride (r² = 0.9981) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.}

The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing % RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.

From the above experimental results and parameters it was concluded that, this newly developed method for the simultaneous estimation Tamsulosin Hydrochloride and Dutasteride was found to be simple, precise, accurate and high resolution and shorter retention time makes this method more acceptable and cost effective and it can be effectively applied for routine analysis in research institutions, quality control department in meant in industries, approved testing laboratories studies in near future.