A simple, economic, selective, precise, and stability-indicating high-performance thin-layer chromatographic method for analysis of tamsulosin hydrochloride, both as a bulk drug and in formulations, was developed and validated according to ICH guidelines. The method employed HPTLC aluminium plates precoated with silica gel 60F-254 as the stationary phase while the solvent system consisted of toluene : methanol : triethylamine (3.5 : 1.2 : 0.2 v/v). The system was found to give compact spot for drug (
Benign prostate hyperplasia (BPH) is a common condition in ageing men. Chemically, tamsulosin hydrochloride is [(−)-(R)-5-[2-[[2-(
Tamsulosin hydrochloride was kindly gifted from Sun Pharmaceuticals, Vapi (Gujarat, India). All chemicals and reagents used were of analytical grade and purchased from Merck fine chemicals, Mumbai (Maharashtra, India).
The samples were spotted in the form of bands of width 6 mm with a Camag 100 microlitre sample syringe (Hamilton, Bonaduz, Switzerland) using Camag Linomat 5 (Switzerland) sample applicator on precoated silica gel aluminium plate 60F-254 (10 cm × 10 cm with 0.2 mm thickness, E. Merck, Germany). A constant application rate of 200 nL/s was employed, and space between two bands was 8 mm. The slit dimension was kept 5 mm × 0.45 mm micro and 20-mm/s scanning speed was employed. The mobile phase consisted of toluene : methanol : triethylamine (3.5 : 1.2 : 0.2 v/v). Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase. The optimized chamber saturation time for mobile phase was 20 min at room temperature. The length of chromatogram run was approximately 80 mm. Subsequent to the development, TLC plate was dried in a current of air with the help of an air dryer. Densitometric scanning was performed on Camag TLC scanner 3 in the absorbance mode at 280 nm. The source of radiation was deuterium lamp.
A stock solution of tamsulosin hydrochloride (1000
Repeatability of sample application and measurement of peak area were carried out using six replicates of the same spot (800 ng per spot of tamsulosin hydrochloride). The intraday and interday variation for the determination of tamsulosin hydrochloride was carried out at three different concentration levels of 800, 1200, and 1600 ng per spot.
By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different composition of toluene : methanol : triethylamine (3.5 : 1.2 : 0.2 and 3 : 1.2 : 0.2 v/v) were tried and chromatograms were run. The amount of mobile phase, temperature, and relative humidity was varied in the range of ±5%. The plates were prewashed by methanol and activated at 60 ± 5°C for 2, 5, and 7 min prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from 0, 20, 40, and 60 min. Robustness of the method was done at three different concentration levels: 800, 1200, and 1600 ng per spot.
In order to determine detection and quantification limit, tamsulosin hydrochloride concentrations in the lower part of the linear range of the calibration curve were used. Tamsulosin hydrochloride solution of 200
The specificity of the method was ascertained by analyzing standard drug and sample. The spot for tamsulosin hydrochloride in sample was confirmed by comparing the
The analyzed samples were spiked with extra 80, 100, and 120% of the standard tamsulosin hydrochloride, and the mixture was analyzed by the proposed method. At each level of the amount, six determinations were performed. This was done to check the recovery of the drug at different levels in the formulations (Table
To determine the concentration of tamsulosin hydrochloride in tablets (labeled claim: 0.4 mg per tablet), the contents of 25 tablets were weighed, their mean weight determined, and they were finely powdered. The powder equivalent to 10 mg of tamsulosin hydrochloride was weighed. The drug from the powder was extracted with methanol. To ensure complete extraction of the drug, it was sonicated for 30 min, and the volume was made up to 10.0 mL. The resulting solution was centrifuged at 3000 rpm for 5 min, and supernatant was analyzed for drug content. The 4 mL was taken and volume made up to 10.0 mL by methanol, to make a 400-
The 10 mg of tamsulosin hydrochloride was separately dissolved in methanolic solution of hydrogen peroxide (30.0%, v/v). The solution was kept for “16 h” at room temperature in the dark in order to exclude the possible degradative effect of light. The 4.0 mL of the above solution was taken and diluted up to 10.0 mL with methanol. The resultant solution was applied on TLC plate in triplicate (3.0
The powdered drug was stored at 60°C for “2 h” under dry heat condition showed significant degradation. The degraded products were resolved from the standard. In all degradation studies, the average peak areas of tamsulosin hydrochloride after application was 1200 ng per spot of three replicates.
Tamsulosin hydrochloride (10 mg) was separately dissolved in 10 mL methanolic solution of 2 M HCl and 2 M NaOH. This solution was kept for “6 h” at room temperature in the dark in order to exclude the possible degradative effect of light. Further, it was refluxed for “1 h” at 55°C. The 4 mL of the above solution was taken and neutralized, then diluted up to 10 mL with methanol. The resultant solution was applied on TLC plates in triplicates (3
Tamsulosin hydrochloride (10 mg) was dissolved in 10 mL of methanol. The solution was kept in the sun light for “24 h”. 4 mL of the above solution were taken and diluted up to 10 mL with methanol (Figure
TLC procedure was optimized with a view to developing a stability-indicating assay method. Initially, toluene : 1-propanol (3 : 2 v/v) was tried with good resolution with an
A typical HPTLC densitogram of tamsulosin hydrochloride (
The linear regression data for the calibration curves (
Summary of validation parameters.
Parameter | Data |
---|---|
Linearity range (ng per spot) | 400–2400 |
Correlation coefficient | 0.9982 |
Limit of detection (ng per spot) | 20.49 |
Limit of quantitation (ng per spot) | 62.10 |
Recovery ( | 100.74 |
Precision (% R.S.D.) | |
Repeatability of application ( | 1.43 |
Repeatability of measurement ( | |
Interday ( | 1.45 |
Intraday ( | 0.87 |
Robustness | 1.05 |
Specificity | specific |
The repeatability of sample application and measurement of peak area were expressed in the terms of % R.S.D. and results are depicted in Table
Intraday and interday precision of HPTLC methoda.
Amount (ng per spot) | Intraday precision | Interday precision | ||||||
Mean area | SD | % R.S.D. | S.E.b | Mean area | SD | % R.S.D. | S.E.b | |
800 | 2974.6 | 15.00 | 0.50 | 25.08 | 800 | 43.43 | 1.50 | 8.661 |
1200 | 4015.5 | 32.01 | 0.80 | 23.81 | 1200 | 41.24 | 1.04 | 18.48 |
1600 | 5064.6 | 67.00 | 1.32 | 52.93 | 1600 | 91.68 | 1.82 | 38.68 |
The standard deviation of peak areas was calculated for each parameter, and the percentage of R.S.D. was found to be less than 2%. The low values of the percentage of R.S.D. values, shown in Table
Robustness of the methoda.
Parameter | SDb of peak area | % R.S.D.b |
---|---|---|
Mobile phase composition | 31.35 | 1.05 |
Amount of mobile phase | 34.83 | 1.14 |
Temperature | 38.21 | 1.25 |
Relative humidity | 36.26 | 1.19 |
Development distance | 32.28 | 1.07 |
Time from spotting to chromatography | 32.45 | 1.07 |
Time from chromatography to scanning | 20.85 | 0.70 |
Recovery studiesa.
Excess drug added tob the analyte (%) | Amount recovered (mg) | Recovery (%) | % R.S.D | S.E. |
---|---|---|---|---|
0 | 809.23 | 101.15 | 0.40 | 4.99 |
80 | 1447.91 | 99.79 | 0.57 | 12.71 |
100 | 1613.94 | 100.59 | 0.70 | 17.21 |
120 | 1629.84 | 101.49 | 0.19 | 4.80 |
The calibration curve in this study was plotted between the amount of analyte versus average response (peak area), and the regression equation was obtained (
The peak purity of tamsulosin hydrochloride was assessed by comparing the spectra at peak start, peak apex, and peak end positions of the spot which was obtained between standard and sample spectra of tamsulosin hydrochloride (Figure
The proposed method, when used for extraction and subsequent estimation of tamsulosin hydrochloride from pharmaceutical dosage forms after spiking with 80, 100, and 120% of additional drug, afforded recovery of 99.79%–101.59% as listed in Table
A single spot of
The chromatogram of samples degraded with acid, alkaline hydrolysis, hydrogen peroxide, and heat showed well-separated spots of pure tamsulosin hydrochloride as well as some additional peaks at different
Forced degradation of tamsulosin hydrochloride.
Serial number | Sample exposure condition | Number of degradation products ( | Tamsulosin hydrochloride remained (ng/1200 ng) (±S.D, | S.E. | Recovery (%) |
---|---|---|---|---|---|
1 | 2 N HCl “8 h” at room temp. reflux for “1 h” | 1 (0.29) | 1055.8 (7.90) | 1.810 | 87.99 |
2 | 2 N NaOH “8 h” room temp. reflux for “1 h” | 1 (0.26) | 983.76 (5.01) | 2.092 | 81.98 |
3 | 30% H2O2 “16 h” | 1 (0.34) | 1020.66 (14.00) | 2.034 | 85.06 |
4 | Heat for “2 h” at 60°C | 1 (0.31) | 1028.72 (10.10) | 2.244 | 85.73 |
5 | Photo degradation kept for “24 h” | 1 (0.28) | 1069.81 (16.81) | 0.900 | 89.15 |
HPTLC densitogram of tamsulosin hydrochloride after treatment with 30% hydrogen peroxide for “16 h”.
HPTLC densitogram of tamsulosin hydrochloride after photo degradation at “24 h” in sun light.
HPTLC densitogram of tamsulosin hydrochloride after HCl degradation for “8 h” at room temperature and “1 h” reflux.
HPTLC densitogram of tamsulosin hydrochloride after NaOH degradation for “8 h” at room temperature and “1-hour” reflux.
HPTLC densitogram of tamsulosin hydrochloride after “2-hour” dry heat degradation at 60°C.
Absorption spectra of tamsulosin hydrochloride bulk and tablet formulation.
The developed HPTLC technique is precise, specific, accurate, and stability indicating. The developed method was validated based on ICH guidelines [
The method can be used to determine the purity of the drug available from the various sources by detecting the related impurities. It may be extended to study the degradation kinetics of tamsulosin hydrochloride and for its estimation in plasma and other biological fluids. As the method separates the drug from its degradation products, it can be employed as a stability-indicating one.