A stability-indicating RP-TLC/Densitometry method for analysis of Raloxifene hydrochloride both in bulk material and in tablets was developed and validated. Densitometric analysis of Raloxifene hydrochloride was carried out at 311 nm on TLC aluminium plates precoated with silica gel 60RP-18
Raloxifene hydrochloride (RLX), [6-Hydroxy-2-(4-hydroxy-phenyl) benzo [b] thien-3-yl] [4-[2-(1-piperidinyl)-ethoxy] phenyl]-methanone-, hydrochloride (Figure
Chemical structure of Raloxifene hydrochloride (RLX).
Literature survey revealed that RLX was analyzed by HPLC [
In view of the above factors, an HPTLC method was well thought-out, to be cheaper, faster, and sometimes more efficient than RP-HPLC and UPLC. From the literature survey, it is revealed that no stability-indicating RP-TLC/Densitometry method has been reported in the literature for analysis of RLX as bulk material or in pharmaceutical formulations.
Hence, the objective of the present investigation was to develop a simple stability-indicating RP-TLC/Densitometry method offering lower analysis time and less cost per analysis for estimation of RLX in bulk material and in tablets and to validate the method according to the ICH guidelines [
Raloxifene hydrochloride (RLX) was procured from Cipla India Ltd, Mumbai, India. All chemicals and reagents used were of Analytical grade and were purchased from Merck Chemicals, India.
Chromatography was performed on aluminium plates precoated with Silica gel 60 RP-18 F254 S (20 × 10 cm, E. Merck, Germany). The plates were prewashed with methanol and activated at 100°C for 10 min prior to chromatography. The samples were spotted in the form of bands of 6 mm width with a Camag microlitre syringe using a Camag Linomat 5 applicator with a constant rate of application, 150 nL per second. Linear ascending development with methanol : water : ammonia (95 : 05 : 0.1 v/v) as mobile phase was performed in a 20 × 10 cm twin-trough glass chamber (Camag), with tightly fitting lid, previously saturated with mobile phase vapour for 25 min at room temperature (25 ± 2°C). The development distance was 8 cm. After development, the plates were dried in current of air by an air dryer. Densitometric scanning was then performed at 311 nm with a Camag TLC Scanner 3 in absorbance mode operated by winCATS software. The source of radiation was a deuterium lamp. Slit dimensions were 5 mm × 0.45 mm and the scanning speed 20 mm per second.
Stock standard solution was prepared by dissolving 10 mg of RLX in 10 mL methanol. From it, appropriate volume 0.2–1.2 mL was transferred into six separate 10 mL volumetric flask and volume was made up to the mark with methanol. With the help of linomat 5 applicator, 5
Twenty tablets (RALISTA, label claim: 60 mg of RLX per tablet) were weighed and crushed into fine powder. The quantity of powdered drug equivalent to 50 mg of RLX was weighed and transferred in 100 mL volumetric flask containing 50 mL methanol, sonicated for 10 min, volume was adjusted to mark and filtered using 0.45
Repeatability of sample application and measurement of peak area were performed using six replicates of the same spot (400 ng per band of RLX). The intra, and interday variation for the estimation of RLX was carried out at three different concentration levels of 200, 300, and 500 ng per band.
In order to determine detection and quantification limit, RLX concentrations in the lower part of the linear range of the calibration curve were used. From the stock standard solution RLX 100, 120, 140, 160, 180, and 200 ng per band was applied in triplicate on RP-TLC plate. The LOD and LOQ were calculated using equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where “N” is standard deviation of the peak areas of the drugs
The specificity of the method was checked by analyzing drug standard and sample. The band for RLX in sample was confirmed by comparing the
Ruggedness of the method was performed by spotting 400 ng of RLX by two different analysts keeping same experimental and environmental conditions.
The preanalysed samples (200 ng per band) were spiked with extra 80, 100, and 120% of the standard RLX, and the mixture was then reanalysed by the proposed method. At each level of the amount, three determinations were performed. This was done to check the recovery of the drug at different levels in the formulations.
By introducing small deliberate changes in the mobile-phase composition, the effects on the results were examined. Mobile phases having different composition of methanol : water : ammonia (96 : 4 : 0.1 v/v) and (94 : 6 : 0.1 v/v) were tried and chromatograms were run. The amount of mobile phase was varied in the range of ±2 mL. The plates were prewashed by methanol and activated at 100 ± 5°C for 5 and 15 min prior to chromatography. Time from spotting to chromatography and from chromatography to scanning was varied from 0, 20,40 min.
To assess the stability of RLX in methanol and mobile phase; the sample solutions were separately prepared in methanol and mobile phase and stored at room temperature for 24 (h). The sample solutions were assayed at an interval of 6 (h) for 24 (h).
The sample solution was applied on RP-TLC plate, kept for 72 (h), and scanned at an interval of 12 (h) as described above.
Accurately weighed quantity 10 mg of RLX was separately dissolved in 10 mL methanolic solution of 0.5 M HCl and 0.5 M NaOH and 3% (v/v) hydrogen peroxide, respectively; solutions were kept for period of 12 (h) at room temperature in dark to avoid likely degradative effect of light. An appropriate volume 1.0 mL of above solution was taken, neutralized, and diluted up to 10 mL with methanol. The resultant solution was applied on RP-TLC plates in triplicates (5
Accurately weighed quantity 10 mg of RLX stored at 80°C for 24 (h) in an oven. It was transferred to 10 mL volumetric flask containing methanol and volume was made up to the mark. The 1.0 mL of above solution was taken and diluted up to 10 mL with methanol. The resultant solution was applied on RP-TLC plate in triplicate (5
Accurately weighed quantity 10 mg of RLX was dissolved in 10 mL methanol and solutions was kept for period of 24 (h) in light. An appropriate volume 1.0 mL of above solution was taken and diluted up to 10 mL with methanol. The resultant solution was applied on RP-TLC plate in triplicate (5
For the selection of appropriate mobile phase for RLX, several runs were exercised using mobile phases containing solvents of varying polarity, at different concentration levels. Among the different mobile-phase combinations employed, the mobile phase consisting of methanol : water : ammonia (95 : 05 : 0.1 v/v) gave a sharp and well-defined peak at
Chromatogram of RLX standard (
The linear regression data for the calibration curves showed good linear relationship over the concentration range 100–600 ng per band. Linear regression equation was found to be
Three dimensional chromatograms of RLX sample (100–600 ng per band).
The precision of the developed method was represented in terms of % relative standard deviation (% RSD) of the peak area. The results depicted indicated high precision of the method are presented in Table
Repeatability and Intraday, Interday precision.
Parameters | Concentration ng per band | % Amount found | % RSD |
---|---|---|---|
Repeatability | 400 | 100.94 | 1.67 |
Intraday | 200 | 99.98 | 1.27 |
300 | 101.44 | 0.94 | |
400 | 99.70 | 0.41 | |
Interday | 200 | 99.50 | 1.37 |
300 | 99.68 | 1.33 | |
400 | 101.97 | 1.35 |
The LOD and LOQ were determined from the slope of the lowest part of the calibration plot. The LOD and LOQ were found to be 9.27 ng and 27.10 ng, respectively, which indicates the sensitivity of the method is adequate (Figure
Calibration curve containing LOQ value as the lowest point.
The recovery studies were executed out at 80%, 100%, and 120% of the test concentration as per ICH guidelines. The % recovery of RLX at all the three levels was found to be satisfactory. The amounts of drug added and determined and the % recovery are listed in Table
Recovery studies.
Drug | Initial amount (ng per band) | Amount of drug standard added (%) | % Drug recovered | % RSD |
---|---|---|---|---|
RLX | 200 | 0 | 100.20 | 1.2 |
200 | 80 | 99.78 | 1.2 | |
200 | 100 | 99.74 | 1.4 | |
200 | 120 | 100.57 | 1.5 |
The peak purity of RLX was assessed by comparing the spectra at peak-start, peak-apex, and peak-end positions of the band, that is,
Peak purity spectra of RLX standard (a) and RLX extracted from tablets (b) scanned at peak-start, peak-apex and peak-end position.
The standard deviation of peak areas was calculated for each parameter and % R.S.D. was found to be less than 2%. The low values of % RSD values indicate robustness of the method; results are shown in Table
Robustness of the method.
Parameter | ±SD of peak area | % RSD |
---|---|---|
Mobile-phase composition: methanol : water : ammonia (96 : 04 : 0.1 v/v) | 50.89 | 1.5 |
Mobile-phase composition: methanol : water : ammonia (94 : 06 : 0.1 v/v) | 52.74 | 1.6 |
Mobile-phase volume (±2 mL) | 47.91 | 1.4 |
Development distance (±0.5 cm) | 44.03 | 1.3 |
Activation of TLC plate (±5 min) | 48.84 | 0.9 |
Duration of saturation (±5min) | 44.97 | 1.4 |
Time from spotting to chromatography (±10 min) | 40.16 | 0.9 |
Time from chromatography to scanning (±10 min) | 49.34 | 1.2 |
The stability study of RLX in methanol demonstrates no significant change in the chromatogram obtained.
Similarly, the stability study of RLX in mobile phase also does not show any noticeable change in the chromatogram.
No major changes were observed in chromagram when plate’s were scanned at 0, 12, 24, 36, 48, 72 (h) interval.
The validation of the method is summarized in Table
Summery of validation parameter.
Parameter data | RLX |
---|---|
Linearity range (ng per band) | 100–600 |
Correlation coefficient ( | 0.9969 |
Limit of detection (ng) | 9.27 |
Limit of quantification (ng) | 27.10 |
Recovery | 99.74–100.57 |
Analyst-I | 1.53 |
Analyst-II | 1.33 |
Repeatability of application | 0.96–1.49 |
Interday | 1.33–1.37 |
Intraday | 0.41–1.27 |
Robustness | Robust |
Specificity | Specific |
A single spot at
The results of the forced degradation study of RLX are summarized in Table
Summary of forced degradation studies.
Stress conditions | Time (h) | Recovery (%) | |
---|---|---|---|
0.5 M HCl | 12 | 99.5 | No degradants formed |
0.5 M NaOH | 12 | 80.2 | 0.32,0.44,0.70 |
3% (v/v | 12 | 91.45 | 0.64 |
Day light (8 (h)/day) | 24 | 99.9 | No degradants formed |
Heat (80°C) | 24 | 99.6 | No degradants formed |
RP-TLC chromatogram obtained from forced degradation studies. (a) Base degradation (0.5 M NaOH, 12 (h), RT) showing three degradants at
Peak purity spectra of RLX recovered after degradation in 0.5 M NaOH, 3% (v/v) H2O2, degradants, and RLX standard scanned at peak-start, peak-apex, and peak-end positions.
No additional peaks were found in acid, dry heat, and photodegradation. Therefore, RLX is stable in acidic, dry heat, and photoconditions.
The developed method was found to be simple, rapid, selective, sensitive, and suitable for determination of Raloxifene hydrochloride in bulk material and pharmaceutical dosage forms without any interference from excipients. As the method is stability-indicating one, it can be used to determine the purity of the drug available from various sources by detecting the related impurities. Furthermore, it can be concluded that the impurities present in the drug could be due to hydrolysis or oxidation during processing and storage of the drug.
The proposed procedure fits precision and accuracy usually requested by official methods and can be used as a convenient alternative to HPLC analysis for quantitation of Raloxifene hydrochloride in both bulk and tablet dosage forms. Therefore, the proposed RP-TLC/Densitometry method can be used as an alternative tool in the drug quality control laboratories for quantitative determination of Raloxifene hydrochloride.
The authors are thankful to R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (MS), India, for providing the required facilities to carry out this research work.