A simple, accurate, precise, sensitive, selective, and stability-indicating high-performance thin-layer chromatographic method was developed and validated for determination of duloxetine hydrochloride both in bulk drug as well as in tablet formulation. The stationary phase used in our method consisted of HPTLC aluminum plates precoated with silica gel 60F-254, while, chloroform : methanol (8 : 2, v/v) was used as binary mobile phase. These chromatographic conditions eluted the drug effectively, and distinct compact spots were seen, (Rf, retardation factor, value (0.42 ± 0.20). Densitometric determination of duloxetine hydrochloride was carried out in the absorbance mode at a wavelength of 217 nm. The mean value of corelation coefficient; slope and intercept were 0.9962 ± 0.0015, 121.54 ± 0.61, and 987.3 ± 6.17, in the amount range of 600–2000 ng (nanogram) per spot, respectively. Stress testing validation were performed as per the guidelines of International Conference on Harmonization (ICH) and drug was subjected to stress conditions like acid-hydrolysis, alkali-hydrolysis, oxidation, and thermal degradation. As the method effectively separated the active drug from its degradation products, it can be employed as a stability-indicating assay method (SIAM) for identification and quantitative determination of duloxetine HCl in bulk drug and tablet dosage formulation.
Duloxetine hydrochloride designated in IUPAC as (+)-(S)-N-methyl-gamma-(1-naphthyloxy)-2- thiophenepropylamine hydrochloride (Figure
Structure of Duloxetine HCl.
It is also indicated for the management of neuropathic pain associated with diabetic peripheral neuropathy [
Literature survey revealed many analytical methods for the estimation of duloxetine hydrochloride [
HPTLC method is becoming a routine analysis technique due to its advantages. The major advantage is that, several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thereby minimizing the analysis cost and time. The aim of this work was to develop an accurate, specific, repeatable, and stability-indicating method for the determination of duloxetine HCl in the presence of its degradation products and related impurities as per ICH guidelines [
Duloxetine HCl was received as a gift from Zydus Cadila, Ahmedabad, India. The other chemicals and reagents used were of analytical grade and were purchased from Rankem and Qualigens, India.
HPTLC instrumentation consisted of Camag Linomat IV, and samples were spotted in the form of bands of width 5 mm using Camag microlitre syringe (constant application rate of 1
A stock solution of duloxetine HCl (100
Six replicates of same concentration (600 ng per spot of Duloxetine HCl) were checked for repeatability of the sample application and determination of peak area. The results qualify the requirements of %RSD. of peak area of Duloxetine HCl. The intraday and interday variation for the determination of Duloxetine HCl was carried out at three different concentration levels of 600, 800, and 1000 ng per spot.
The mobile phase volume and temperature were varied, and effect on the quality of chromatograms was recorded. The mobile phase ratios of chloroform:methanol was deliberately changed (2 : 8, 4 : 6, and 6 : 4 v/v), and its effect on results was seen at different concentrations of 600, 800, and 1000 ng per spot.
The background noise of blank sample was determined by analyzing pure methanol, and then signal-to-noise ratio, limit of detection (LOD) and limit of quantification (LOQ) were determined by spotting a minimum concentration of duloxetine HCl (starting from 20 ng per spot, and then concentration was gradually increased).
The preanalysed samples were spiked with additional 80, 100, and 120% of the standard Duloxetine HCl, and the mixtures were reanalyzed by this method. The experiment was repeated in triplicate, and percentage recoveries in formulations were determined.
The drug content in duloxetine HCl in tablet formulation (Tablet, Symbal, label claim: 30 mg per tablet) was determined. Twenty tablets were powdered, and then a powder equivalent to 20 mg was weighed and extracted with methanol. To ensure complete extraction, it was sonicated for 15 min, and then solution was serially diluted with methanol to get a concentration of 0.1
A stock solution was prepared by dissolving 10 mg of Duloxetine HCl in 100 mL of methanol, and 20 mL of this solution was utilized for forced degradation studies.
To 20 mL of methanolic stock solution, 10 mL each of 2 M NaOH (for 8 hours) and 0.5 M HCl (for 4 hours) were added separately at room temperature. A sample was taken every hour and was neutralized (using standard pH paper). The concentration of drug equivalent to 1000 ng per spot of duloxetine was immediately subjected to HPTLC determination to find out the effect of stress conditions. The forced degradation was performed in the dark to prevent the possible degradation by light.
To 20 mL of methanolic stock solution, 10 mL of hydrogen peroxide (30%, v/v) was added at the room temperature. This solution was then heated in boiling water bath for 8 hours to remove the excess of hydrogen peroxide. The resultant solution (1000 ng per spot) was applied on TLC plate, and the chromatograms were recorded.
The thermal degradation of the drug was carried out by heating the stock solutions at 70°C for 5 hours. The resultant solutions were appropriately diluted every hour, 1000 ng per spot was applied on TLC plate, and chromatograms were recorded.
The main purpose of our HPTLC method development and validation was to devise a stability-indicating assay method. The analysis of both pure drug as well as of degraded products were tried in different solvent systems. Initially combinations of butanol, hexane, toluene, and acetic acid with varying proportions were tried, but finally, the mobile phase consisting of chloroform : methanol (8 : 2, v/v) gave well-defined spots having good resolutions. The optimum development time was found to be 40 min at room temperature.
The linear regression data for the calibration curves (
Linear regression data (ng per spot) for the calibration curvesa.
Linearity range | Slope ± SD | Confidence limit of slopeb | Intercept ± S.D. | Confidence limit of interceptb | |
---|---|---|---|---|---|
600–2000 | 120.98–122.08 | 982.14– 992.92 |
a
The repeatability of sample application and measurement of peak area were expressed in terms of relative standard deviation (%RSD) and was found to be less than 0.1%. The results depicted in Table
Intraday and interday precision of HPTLC method.
Amount | Intraday precision ( | Interday precision ( | ||||||
Mean area | SD | %RSD | SE | Mean area | SD | %RSD | SE | |
600 | 5403.23 | 24.43 | 0.45 | 9.97 | 5356.38 | 25.23 | 0.47 | 10.30 |
800 | 7209.17 | 22.69 | 0.31 | 9.26 | 7154.16 | 24.29 | 0.34 | 9.91 |
1000 | 9015.28 | 21.79 | 0.24 | 8.89 | 8928.81 | 22.16 | 0.25 | 9.04 |
SD: standard deviation; SE: standard error.
Our method was robust, since %RSD was found to be less than 2.0% after introducing deliberate small changes in temperatures and mobile phase ratio.
As per the ICH guidelines, signal-to-noise ratio having values of 3 and 10 were considered as LOD and LOQ, respectively. The LOD and LOQ were found to be 100 and 350 ng per spot, respectively.
The proposed method was utilized for extraction studies, and subsequent estimation of duloxetine hydrochloride in pharmaceutical dosage forms after spiking a preanalysed sample with 80, 100, and 120% of additional drug. The recoveries were found to be between 99–101% as listed in Table
Recovery studies of duloxetine hydrochloride.
Excess drug spiked to Preanalyzed drug (%)* | Drug content (ng per spot) | Recovery (%) | %RSD | SE |
---|---|---|---|---|
0 | 600 | 99.36 | 0.55 | 0.45 |
80 | 1080 | 100.49 | 0.78 | 0.64 |
100 | 1200 | 100.45 | 0.65 | 0.53 |
120 | 1420 | 100.41 | 0.62 | 0.51 |
*(
A single peak was observed in the chromatogram obtained in the analysis of drug samples extracted from tablets, and no additional interfering peak(s) were observed due to the presence of excipients in the tablets. The drug content was found to be 99.28% with a %RSD of 0.48%.
The method distinctively separated the degraded products of duloxetine HCl due to acid, base, oxidative, and thermal treatments. The chromatograms of degraded products were well resolved from the chromatograms of drug as shown in Figures
Stress degradation of duloxetine hydrochloride.
Stress conditions* | Degradation products | Recovery |
---|---|---|
0.5 M HCl RT (4 h) | 2, (0.12, 0.21) | 83.13% |
2.0 N NaOH RT (8 h) | 2, (0.19, 0.28) | 92.28% |
30% H2O2 RT (8 h) | 2,(0.19, 0.31) | 91.11% |
Heat (70°C) (5 h) | 1, (0.24) | 94.45% |
*(
Summary of validation parameters.
Parameter | Data |
---|---|
Linearity range | 600–2000 (ng per spot) |
Limit of detection | 100 (ng per spot) |
Limit of quantification | 350 (ng per spot) |
Correlation coefficient | |
Recovery (%) | |
Precision (%RSD) | |
Interday | 0.35 |
Intraday | 0.33 |
A typical HPTLC chromatogram of duloxetine hydrochloride
A HPTLC chromatogram of alkali-treated duloxetine hydrochloride.
A HPTLC chromatogram of hydrogen peroxide-treated duloxetine hydrochloride.
A HPTLC chromatogram of acid-treated duloxetine hydrochloride.
A HPTLC chromatogram of thermally degraded duloxetine hydrochloride.
The proposed HPTLC method was developed and validated for precision, specificity, and accuracy. The method was applicable both for determination of bulk drug as well as the tablet formulation. The method separates the drug from its degradation products, and as such it can be described as a stability-indicating assay method (SIAM).
The authors thank Zydus Cadila, Ahmedabad, India, for providing gift sample of duloxetine.