Introduction

Raloxifene hydrochloride belongs to the benzothiophene class of compounds and used for the osteoporosis in postmenopausal women by its SERM activity(selective estrogen receptor modulator) (Salazar, Codevilla, Meneghini, & Bergold, 2015). Raloxifene HCl tablet is given as a dosage form for the administration of oral. 55.7 mg of freebase molar equivalent contains in each 60mg of raloxifene HCl tablet. The excipients used in tablet dose forms are crospovidone, lactose, carnauba wax, hypromellose, magnesium stearate, lactose monohydrate, modified pharmaceutical glaze, magnesium stearate, polysorbate 80, propylene glycol, polyethylene glycol, povidone, and titanium dioxide (Salazar et al., 2015). Literature view report that different spectrophotometric methods are available for raloxifene determination (Basavaiah, Kumar, Tharpa, & Vinay, 2008; Kalyanaramu & Raghubabu, 2011; Sivasubramanian & Pavithra, 2006) and through Reversed-Phase HPLC in dosage and bulk drug form (Kumar, Kumar, Suresh, & Apsar, 2011; Salazar et al., 2015; Suneetha & Rao, 2010). Plackett–Burman design is used for the evaluation of raloxifene hydrochloride and its impurities by a new LC Validation method (Stojanović, Rakić, Slavković, & Kostić, 2013). UPLC method to analysis raloxifene and its corresponding impurities in drug and dosage form reported (Saini, Baboota, Ali, & Patel, 2012). Identification and description of possible impurities through the synthesis of the raloxifene hydrochloride drug in bulk (Reddy, 2012). Structural explanation of viable impurities of raloxifene HCL by LC/ESI-MS (Jagadeesh, Kumara, Jayashreeb, & Mohantya, 2014). Confirmation of raloxifene and glucurovides in urine samples by LC-MS/MS method (Trdan, Roškar, Trontelj, & Ravnikar, 2011).

The main object of this work is an advanced, fast, accurate, precise and simple method for the decide and quantification of 5 related substances in raloxifene. The structures of the drug substances and related substances are presented in Figure 1. The specification limits of all impurities are tabulated on the support of ICH thresholds and official monographs (USP, Ph.Eur and BP). According to the guidelines of FDA (Food and Drug Administration) and ICH (International Conference on Harmonization), this method was demonstrated with respect to the limit of detection, the limit of quantification, linearity, precision, accuracy, specificity and stability studies.

Materials and Methods

Reagents and Standards

Reference standards and related compounds of drug substances are obtained by GSN Pharmaceuticals Pvt., Ltd. Hyderabad. Raloxifene tablets dosage forms were brought from the local market. Acetonitrile at HPLC class was acquired from Qualigens, India. Water is purified through Milli-Q Millipore for HPLC analysis. 0.45 µm filter nylon used to filter the mobile phase all the solutions.

Instrumentation

The HPLC system used in the current method was Waters 2695 separation module includes of binary pump plus autosampler, degasser, column oven and 2996 photodiode array detector. The Empower software of Waters Corporation was used to detect and process the output signals. Inertsil C8 150 x 4.6 mm ID, 3.5μm column was used for LC studies. The flow rate of the mobile phase is 1.0 mL/ minute. The column temperature was continued at 300C and the detection observed at a wavelength 280nm. 10 µL was used for injection.

Impurity stock solutions and Standard preparation

Finely powdered after weighing the tablets, with the mortar and pestle. 100mg equivalent of raloxifene was taken into a volumetric flask of 50ml. Extract all the active compounds and their related impurities add 35ml of diluent and fill up after sonication. By using Nylon 0.45 µm syringe, discard the first 2ml of the filtrate and transfer the remaining into 2ml HPLC vial.

Results and Discussion

Analytical method development

As raloxifene hydrochloride shows a basic character, it was hypothesized that acidic pH could improve the ionization capacity of the drug. Therefore, a pH of 4.5 was maintained and consequently, good separation of peaks in the column was obtained. The mobile phase composition was finalized after examines with different solvent systems. It was found that the use of methanol with water or pH 4.5 phosphate buffer caused peak broadening with substantial loss of peak area. Mobile phase A (0.05 mM KH2PO4 buffer at pH 4.5) and mobile phase B (acetonitrile) are optimal for good separation and for obtaining a sharp peak of raloxifene HCL and its five impurities. Peak tailing was a major criterion for selection of the stationary phase. Initial trials with C18 columns of two different makes, (Phenomenex® C18, 250 mm long and 4.6 mm internal diameter, particle size 5 μm and Kromasil C18, 250 mm long and 4.6 mm internal diameter, particle size 5 μm) showed peak tailing less than 1.5. Finally, good separation and taling was found in Inertsil C8 column. Based on the trials, an optimum column temperature of 30 ºC was selected to avoid a shift in retention time and the best peak shape. Wavelength depends on λmax, which was selected as 280 nm, based on the below given UV-Vis spectra of raloxifene.

Method validation

The specificity of the method verified by well resolution of both the impurities and any degradants (by forced degradation study) from raloxifene peak. The method sensitivity was established by the limit of quantification (LOQ) and limit of detection (LOD). The LOQ values were investigated and confirmed by six injections of the impurities at the estimated concentrations at 4.0 ppm. Linearity was assessed by making and injecting the standards in between of LOQ (0.4 ppm) to 150% (6 ppm) of raloxifene working concentration (2000 ppm). Method Accuracy was carry out by having the impurities at a known concentration at LOQ, 2, 4 and 6 ppm levels in triplicate preparations. The precision was checked by method precision and ruggedness. Method precision was checked by injecting six freshly prepared spiked raloxifene impurities at specification level (4 ppm w.r.t 2000 ppm raloxifene working concentration) on the same day. The same experimental procedure was take on for the studies of ruggedness in different days with different columns and reagents. The robustness process was investigated with a slight adjustment in the mobile phase flow rate, Column temperature. The actual flow rate of the mobile phase was 1.0 mL. This was changed by 10%, i.e. from 0.9 to 1.1 mL/min. The column temperature studied on the resolution at 350C and 250C (±50C) was effective, mobile phase pH altered ±0.2 to the actual mobile phase pH of 4.5. At the room temperature and different time gaps as well as refrigerator condition (2-8°C) through standard and spiked sample solution stability of impurities in the raloxifene sample solution was investigated. System suitability was evaluated by injecting the system suitability solution and raloxifene by a series of six injections of the standard solution. The corresponding standard chromatograms are shown in Figure 4; Figure 3; Figure 2 and system suitability results are shown in Table 1.

Forced degradation

Degradation samples were injected into the HPLC system and % degradation of the drug sample, and the purity of peaks obtained from stress samples were checked using a PDA detector.

Results in Table 2 shows that the purity angle is less than the purity threshold and demonstrated that the peak homogeneity of the analytes is confirmed. Assay of stressed samples was tested and check the performance by the comparison with standard references and the mass balance (%impurities + % assay) for stressed samples.

Conditions of the forced degradation studies are included in Table 3, and degradation chromatograms were shown inFigure 10; Figure 9; Figure 8; Figure 7; Figure 6; Figure 5.

Limit of detection and quantification

The limit of detection (LOD) and limit of quantitation (LOQ) were checked by injecting diluted solutions having known concentration of known impurities and raloxifene to obtain a signal- to- noise ratio close to 3 for LOD and close to 10 for LOQ. List of Table 4 contains results of analyte peak and each impurity.

Linearity

The linearity of the method in multiple reaction monitoring modes was satisfactorily investigated by injecting dissimilar concentrations i.e., LOQ (0.4 ppm), 50% (2 ppm), 100% (4 ppm), 120% (5 ppm) and 150% (6 ppm) w.r.t 0.2% of specification level (specification level 4 ppm of sample concentration 2000 ppm ). The calibration curve was obtained by drawing the graph with concentration and peak areas. The slope, intercept, correlation coefficient values were presented in Table 5, which represents an excellent correlation between peak areas and concentrations.

Accuracy

Accuracy was conducted by the standard addition method. Therefore, the accuracy of the method was determined by spiking three-levels, i.e. LOQ (0.4 ppm), 50% (2.0 ppm), 100% (4.0 ppm) and 150% (6.0 ppm) of the specification limit (0.2% w.r.t sample concentration 2000 ppm). Recoveries are calculated the content of all impurities in spiked sample preparation and determined the % recovery in raloxifene sample.

Precision

The present method was verified through method precision and intermediate precision. Method precision was checked by raloxifene spiked sample by injecting six individual preparations (2000 ppm of raloxifene HCl, 4.0 ppm of RLF IMP-1 to RLF IMP-5 in diluent). Precision results are given in Table 6, and %RSD was found to below 10%. Results of intermediate at separate columns and reagents on separate days included in Table 7 and %RSD was found to below 10%

Robustness

Table 8 report the parameters of the method that were altered to test the robustness of the method. Prepared a standard solution (4.0 ppm of raloxifene hydrochloride) and spiked sample solution (RLF IMP-1 to RLF IMP-5 in diluent), these solutions were run to assess the robustness method whether these changes had a significant effect on the chromatography of the method or not. All system suitability results provide proof of its positive during normal usage.

As per the ICH guidelines, robustness parameters are performed at 10% of the actual conditions. Of course, during the method development, the flow rate was altered up to 20% variation of the actual flow rate, i.e. at 0.8 mL/min, and 1.2 mL/min and not much variation observed. If the flow rates go beyond the 20% of the actual flow, peaks were not eluted properly.

Solution stability

The solution stability investigations were executed systematically to assess the stability of raloxifene standard solution and spiked solution at room temperature (25oC) as well as cooler temperature (2-8oC). Each sample was injected and studies the % recovery of the impurities with respect to the fresh standard solutions. Table 9 reports the results. The results demonstrated that the raloxifene standard solution and spiked solution were stable both at 250C and refrigerator conditions for 24 hrs, which was evidenced by its good recovery values in the between of 97.6% to 99.9%. The difference between the recoveries at 0th hr and 24th hr was 95% to 105%, which indicates that the standard and spiked solutions prepared in diluent were stable for ‘24' hrs.

Application of Method

The results obtained in this study demonstrated that the present HPLC method is selective, linear, precise, rugged, robust and stability specify for the confirmation of related in raloxifene HCl tablet dosage forms. Therefore, By using the below-mentioned equation, it is suitable for the proposed method. Local market samples analyzed as per proposed impurity method results found within in the criteria

Conclusion

A basic, selective, highly sensitive, more accurate and rapid analytical method was enhanced for the determination and quantification of 5 related substances in Raloxifene. Raloxifene is a non-polar molecule, which is slightly soluble in water. Satisfactory extraction of tablet dosage forms found in the mixture of pH 4.5 buffer and acetonitrile is 50:50 (% v/v). Good separation and quantification of 5 related substances in raloxifene was achieved by selecting KH2PO4 buffer with pH 4.5 and acetonitrile as mobile phase B, stationary phase used is Inertsil C8 with the dimensions 150 x 4.6 mm ID, 3.5μm. 1.0 mL/min was the flow rate in liquid chromatography. The more accurate value of the method was proved by the recovery by the range 80.0% to 120.% with %RSD not more than 5.0, with a correlation of 0.999. The method is validated for specificity, the limit of quantification, the limit of detection, linearity, accuracy, method precision, intermediate precision, robustness and stability this method is validated.