HPTLC Method Validation for simultaneous determination of Tamsulosin Hydrochloride and Finasteride in Bulk and Pharmaceutical Dosage Form

Benign Prostate Hyperplasia (BPH) is a common condition in aging men. Chemically tamsulosin hydrochloride is [(−)-(R)-5-[2[[2-( -ethoxyphenoxy) ethyl]amino]propyl]-2-methoxybenzenesulfonamide] and is official in Martindale – The Extra Pharmacopoeia and Merck Index [1,2]. The chemical structures are shown in Figure 1, whereas chemically finasteride is N-(1,1-dimethylethyl)-3-oxo(5α,17β)-4-azaandrost-1-ene-17-carboxamide and official in Merck Index [1,2]. The chemical structures are shown in Figure 1.

The purpose of this work is to establish and validate a simple accurate and reproducible procedure for quantitative TLC analysis of tamsulosin hydrochloride and finasteride in bulk and tablet dosage form as per ICH guidelines [14,15].

Chemicals and reagents
Tamsulosin hydrochloride and Finasteride was kindly gifted from Sun Pharmaceuticals, Vapi (Gujarat), India. Urimax-F tablets containing 0.4 mg of tamsulosin hydrochloride and 5 mg of finasteride were obtained from commercial sources within their shelf life period. All the reagents and solvents used were of HPLC grade.

HPTLC instrumentation
The samples were spotted in the form of bands of width 6mm with a Camag 100 µl sample (Hamilton, Bonaduz., Switzerland) syringe on precoated silica gel aluminium plate 60 F 254 (20 cm x 10 cm with 0.2 mm thickness), supplied by Anchrom technologists, (Mumbai) using a Camag Linomat applicator 5 (Switzerland). A constant application rate of 150 nl sec -1 was employed and space between two band was 15 mm. The slit dimension was kept 6 mm x 0.45 mm. The mobile phase consisted of toluene: n-propanol: triethylamine (3.0:1.5:0.2 v/v). The optimized chamber saturation time for mobile phase was 20 min at room temp (25 o C ± 2) and relative humidity 60% ± 5. The length of chromatogram run was approximately 80 mm. Subsequent to the development; TLC plates were dried in current of air with the help of an air dryer. Densitometric scanning was performed using Camag TLC scanner 3 in the absorbance mode at 260 nm and software used was winCATS 4.0.5. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum in the range of 190 -400 nm.

Preparation of standard solutions for linearity studies
An accurately weighed quantity of 10 mg TAM and 50 mg of FIN were transferred to two different 10 ml volumetric flasks, dissolved in methanol and volume was made up to mark with the same solvent to obtain concentration 1000 ng/μl of Tam and 5000 ng/μl of FIN respec-

Summary
A new simple, precise, accurate and selective TLC-densitometry method has been developed for simultaneous determination of tamsulosin hydrochloride and finasteride in tablet dosage form. Chromatographic separation was performed on aluminum plate precoated with silica gel 60 F 254 using toluene: n-propanol: triethylamine (3.0:1.5:0.2 v/v) as mobile phase. Detection was carried out densitometrically at 260 nm. The RF value of tamsulosin hydrochloride and finasteride were 0.32 and 0.54, respectively. The reliability of the method was assessed by evaluation of linearity which was found to be 200 -1200 ng/spot for tamsulosin hydrochloride and 1000 -6000 ng/spot for finasteride. Accuracy of the method was accessed by percentage recovery and found to be 99.77 ± 0.71 % for tamsulosin hydrochloride and 99.75 ± 0.86 % for finasteride. The method can be used for routine analysis of tamsulosin hydrochloride and finasteride in tablet dosage form.

Method validation
Accurately weighed 10 mg of TMS and 50 mg of FIN were transferred to 10 ml volumetric flask, dissolved in methanol and volume was adjusted to mark. This mix standard solution was used for validation study.
Precision: Repeatability of measurement of peak area was determined by spotting 400 ng/spot of TMS and 2000 ng/spot of FIN. Precision of the method was assessed by intra-day and inter-day variations. Intra-day variations were assessed by spotting 400, 600, 800 ng/spot of TMS and 2000, 3000, 4000 ng/spot of TMS on TLC plate on three different times within the same day. Inter-day variations were performed by analyzing same concentrations described above for TMS and FIN in three different days over a period of week.
Specificity: Specificity of the method was ascertained by analyzing standard drug and sample. The mobile phase resolved both the drugs very efficiently, as shown in Figure 2. The spot for TMS and FIN was confirmed by comparing the RF and spectra of the spot with that of standard. A typical absorption overlain spectrum of TMS and FIN shown in Figure 3; wavelength 260 nm was selected for densitometric scanning. Peak purity of TMS and FIN was assessed by comparing the spectra of sample with that of standard at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions.

Accuracy:
The pre-analyzed samples were over spotted with extra 80, 100 and 120 % of the standard drug solution of TMS and FIN on TLC plate. The total concentrations of the drugs were determined. The experiment was conducted in triplicate. This was done to check for the recovery of the drug at different levels in formulation.

Robustness:
Robustness of the method was performed by spotting 400 ng of TMS and 2000 ng of FIN on TLC plate by making small deliberate changes in chromatographic conditions. Mobile phases having different composition like toluene: n-propane: triethylamine (3.5: 1: 0.2 v/v/v) and toluene: n-propane: triethylamine (3.0: 1.5: 0.2 v/v/v) were tried and chromatograms were run. The development distance was varied from 7, 7.5 and 8 cm. The amount of mobile phase (4.7 and 9.4) was tried and chromatograms were run. Temperature and relative humidity was varied in the range of ± 5%. The plates were prewashed by methanol and activated at 60 ± 5 o C for 2, 5 and 7 min prior to chromatography. Duration of saturation time of chamber was varied as 15, 20 and 25 min. Time from spotting to chromatography and time from chromatography to scanning was varied from 0, 20, 40 and 60 min. Robustness of the method was done at three different concentration levels.
Ruggedness: Ruggedness of the method was performed by spotting 400 ng of TMS and 2000 ng of FIN, respectively by two different analyst keeping same experimental and environmental conditions.

Limit of detection (LOD) and limit of quantification (LOQ):
In order to determine detection and quantification limit, concentrations in the lower part of the linear range of the calibration curve were used. Stock solutions of TMS (1000 µg/ml) and FIN (5000 µg/ml) were prepared separately and different concentrations 200, 240, 280, 320, 360 and 400 ng of TMS and 1000,1200, 1400, 1600, 1800 and 2000 of FIN were separately spotted on TLC plates in triplicate. The LOQ and LOD were calculated using equation LOD = 3.3 x N/B and LOQ = 10 x N/B, where, N is standard deviation of the peak areas of the drugs (n=3), taken as a measure of noise, and B is the slope of the corresponding calibration curve.

Application of proposed method to tablet formulation
Twenty tablets were weighed; average weight determined and crushed in to fine powder. An accurately weighed tablet powder equivalent to 4 mg of tamsulosin hydrochloride and 50 mg of finasteride was transferred into 25 ml volumetric flask containing 15 ml methanol, an accurately weighed quantity of standard tamsulosin hydrochloride (6mg) was added to obtain the proportion of 1:5 for TMS and Fin re-

HPTLC method development and validation
The TLC procedure was optimized with a view to develop method for simultaneous determination of TMS and FIN. The mobile phase toluene: n-propanol: triethylamine 3.0: 1.5: 0.2 (v/v) gave good resolution, sharp and symmetrical peak with R F value of 0.35 for TMS and 0.60 for FIN. It was observed that prewashing of TLC plates with methanol (followed by drying and activation) and pre-saturation of TLC chamber with mobile phase for 20 min ensure good reproducibility and peak shape of both the drugs.