A STABILITY-INDICATING HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF SELINEXOR AND BORTEZOMIB IN BULK AND PHARMACEUTICAL DOSAGE FORM

A STABILITY-INDICATING HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF SELINEXOR AND BORTEZOMIB IN BULK AND PHARMACEUTICAL DOSAGE FORM

Bavita Gaur *, Sandeep Sahu and Mohan Lal Kori

Faculty of Pharmacy, RKDF University, Bhopal, Madhya Pradesh, India.

ABSTRACT: Symmetric peak and effective peak resolution in chromatography were achieved by adjusting the mobile phase's pH and amount of organic Phase. The retention time of 2.127 and 4.145 minutes was observed after the elution of Bortezomib and Selinexor respectively. To accurately measure both Selinexor and Bortezomib at the same time, an approach was developed with an optimal wavelength of 236nm. Selinexor and Bortezomib were successfully separated by chromatography using a mobile phase of Acetonitrile: 0.1% Formic acid (30:70, v/v) and Luna Phenyl Hexyl (250×4.6mm, 5μ) column running at 1.0 ml/min. When looking at the linearity range, statistics correlating 0.999 indicates a very excellent correlation. For intermediate precision intervals, the relative standard deviation of Selinexor and Bortezomib, respectively, was 0.47 and 0.53, indicating the reproducibility of the analytical approach. The suggested analytical approach has a low LOD, which means that it is also extremely sensitive. This indicates that the suggested approach has sample accuracy by ICH requirements, as 99.9 and 100.2 percent of the spiking Selinexor and Bortezomib, respectively, were recovered. According to ICH recommendations, forced degradation of under twenty per cent is acceptable. Selinexor and Bortezomib showed degradation of under 20 percent using the suggested approach, which indicates stability.

Keywords: Selinexor and Bortezomib linearity range, Recovery, Precision

INTRODUCTION: Xpovio, also known as Selinexor, is a medication used to treat cancer by selectively inhibiting nuclear export. This mechanism operates by inhibiting the function of exportin ¹, thereby preventing the movement of various proteins responsible for promoting the growth of cancer cells from the nucleus to the cytoplasm. As a result, the cell cycle is halted, leading to programmed cell death ². This drug has a unique mechanism of action ^{3, 4}.

Bortezomib is a dipeptide boronic acid derivative and proteasome inhibitor that is commonly prescribed for the treatment of multiple myeloma and mantle cell lymphoma ⁵.

This protein complex, known as the 26S proteasome, plays a crucial role in breaking down ubiquitinated proteins through the ubiquitin-proteasome pathway.

It is believed that bortezomib primarily works by reversibly inhibiting the 26S proteasome, which leads to cell cycle arrest and apoptosis in cancer cells. There may be multiple mechanisms at play in the anticancer activity of bortezomib ⁶. Fig. 1 and 2 display the molecular structure of Selinexor and Bortezomib, respectively.

FIG. 1: STRUCTURE OF (A) SELINEXOR (B) BORTEZOMIB

MATERIALS AND METHOD:

Chemicals and Reagents: Samples of Selinexor and Bortezomib were supplied as the reference material by Supriya Labs in Mumbai. All compounds, including HPLC -grade acetonitrile, Formic acid, were procured from the chemical division of Merck located in Mumbai.

Instrumentation: The investigation was carried out utilizing a high-performance liquid chromatography (HPLC) apparatus comprised of a Waters e 2695 Shimadzu column, a photodiode array (PDA) detector, and Empower software version 2.0 as its driver. a UV detector, a Shimadzu UV-visible spectrophotometer, and a Phoenix 4.5 L digital ultrasonic cleaner was utilized.

Selinexor and Bortezomib Stock Solution: Weigh out precisely 100 mg of Selinexor and 10 mg of Bortezomib for the working standard. Then, transfer the contents to a 100 mL volumetric flask that has been diluted with diluent to volume.

Selinexor and Bortezomib Standard Solution: Take 5 ml of the stock solution of Selinexor and Bortezomib and transfer it to a 50 ml volumetric flask. Next, dilute the solution with the appropriate diluent until the flask is filled to the desired volume.

Selinexor and Bortezomib Sample Solution: Accurately weigh 10 milligrams of Bortezomib and 42.5 mg of Selinexor, then transfer to a 10 ml volumetric flask. To dissolve, add around 7 ml of diluents and sonicate for 20 minutes. Then, dilute with diluent to volume.

FIG. 2: BLANK CHROMATOGRAM

FIG. 3: STANDARD SAMPLE CHROMATOGRAM

Method Development: An in-depth analysis of chromatographic settings, including column type and temperature, mobile phase, and flowing velocity, is essential for optimizing chromatographic methodology, achieving symmetrical peak design, and enhancing resolution. The mobile phase was optimized by trying out numerous mixtures of appropriate solvents, and in the end, Acetonitrile: 0.1% Formic acid (30:70, v/v) was chosen as the optimal mobile phase at a rate of 1.0ml/min. Table 1 displays the optimum chromatographic parameters. Fig. 2 & 3 displayed the blank and optimized chromatogram, respectively.

Method Validation: System suitableness, linearity, accuracy, robustness, precision, and selectivity are only few of the factors that were considered while validating the suggested technique in accordance with ICH Q2R1 recommendations ^7-9.

Linearity: According to ICH, linearity is the extent to which the method of analysis yields test findings that scale linearly with the quantity of analyte in the given sample ^{10, 11}. The spectrum of an analyte concentrations, defined as the difference between the highest and lowest concentrations, is an indicator of the analytical method's accuracy, precision, as well as linearity. In order to establish linearity, small portions were prepared and analyzed in threefold throughout an amount range of 25-150μg/ml for Selinexor and 2.50-15.00μg/ml for Bortezomib. By comparing the calibration graph to the linear regression formula, a correlation coefficient was calculated. Fig. 4 and 5 displayed the linearity results.

FIG. 4: CALIBRATION CURVE OF SELINEXOR       

FIG. 5: CALIBRATION CURVE OF BORTEZOMIB

Precision: Analysis precision was calculated using both intra- and inter-day standards. The RSD is a measure of accuracy ¹².

Accuracy: Accuracy is measured by how much data can be recovered. Pre-examined samples are assessed at three concentrations before being spiked with established quantities of standard Selinexor and Bortezomib ¹³.

LOD and LOQ: According to ICH, the minimum detectable concentration of an analyte (although it is not always its quantitative value) is known as the limit of detection. The minimal detectable concentration of a component in a sample using an appropriate analytical technique is called the LOQ. The LOD and LOQ were calculated through data from a calibration graph.

Forced Degradation Studies: Using forced degradation studies (FDS), the stability-indicating characteristic of the proposed approach was evaluated. A range of conditions were used for the experiments, including thermal degradation, oxidative hydrolysis, acid hydrolysis, alkaline hydrolysis, and photolysis (UV energy-days/dark control). The stability of Selinexor and Bortezomib was evaluated by exposing them to FDS under various stress conditions ^{14, 15}.

RESULTS AND DISCUSSION:

Chromatographic Optimization: After carrying out a series of experimental protocols, it was noted that the mobile phase, which comprised 30:70 of acetonitrile and 0.1% Formic acid displayed a peak characterized by desirable theoretical plate count, resolution, and tailing factor. Therefore, this procedure was optimised and validated.

Specificity and Selectivity: The procedure’s specificity and selectivity were assessed by looking for interference peaks in the chromatograms of placebo and blank samples. In the retention time ranges, the HPLC chromatograms for the drug matrix (a mix of the Drug and placebos) revealed almost no interference peaks. As a result, the HPLC approach presented in this study was restricted.

Linearity: Both Selinexor (y = 31387.33x + 21904.04) and Bortezomib (y = 32255.90x + 1300.61), when tested across the dosage ranges of 25-150 μg/ml and 2.50-15.00 μg/ml, correspondingly, were found to have linear concentration-response relationships.

Table 1 displays the linearity information for Selinexor and Bortezomib.

TABLE 1: CALIBRATION CURVES FOR LINEAR REGRESSION WITH A SAMPLE SIZE OF 6

LOD and LOQ: The LOD and LOQ values for Selinexor were found to be 0.30 and 1.0 μg/ml, respectively, while for Bortezomib, they were determined to be 0.300 and 1.0 μg/ml.

Precision: Intermediate precision findings for Selinexor and Bortezomib, respectively, reveal a %RSD of 0.47 and 0.53, respectively, demonstrating the approach's accuracy.

Table 2 respectively highlight the outcomes of accuracy.

TABLE 2: INTERMEDIATE PRECISION OF SELINEXOR AND BORTEZOMIB

Accuracy: The reliability of the procedure was tested by analysing recoveries after 50%, 100%, and 150% spiking. Using %RSD, we calculated that the mean recovery rates for Selinexor were 99.6, 99.9, and 100.2, while those for Bortezomib were 100.4, 100.2, and 100.1. The results were 0.20, 0.17, 0.14 for Selinexor and 0.37, 0.46, 0.17 for Bortezomib. Table 3 shows the results of the Selinexor and Bortezomib accuracy values.

TABLE 3: ACCURACY FOR SELINEXOR AND BORTEZOMIB

Robustness: In terms of robustness, there is not a significant variance in peak area or resolution comparing Selinexor and Bortezomib after making minor and intentional modifications to mobile phase proportion, column temperatures, and rate of flow.  Information on robustness may be found in Table 4.

TABLE 4: ROBUSTNESS OF SELINEXOR AND BORTEZOMIB

Assay: With % relative standard deviations (RSDs) of 0.221 for Selinexor and 0.276 for Bortezomib, the analysis of the drug product showed that there was not any peak interference by degradants, additives, or impurities, at retention duration of Selinexor and Bortezomib. Fig. 6 shows chromatogram of Marketed formulation.

FIG. 6: CHROMATOGRAM OF MARKETED FORMULATION SAMPLE

Forced Degradation Studies: Sample solutions were subjected to a variety of stresses in order to conduct accelerated degradation investigations. Selinexor and Bortezomib were found to degrade under acidic, alkaline and peroxide, according to the results of degradation experiments. Chromatograms depicting degradation as a function of pH, acidity, peroxide concentration, temperature, and light intensity are presented in Fig. 7, 8, 9, 10, and 11. Fig. 12 displays that neither Selinexor and Bortezomib showed any degradation peaks under, Reduction, Thermal, Photolytic hydrolytic conditions. Table 5 is a summary of the findings we collected on degradation.

FIG. 7: CHROMATOGRAM OF ACID DEGRADATION

FIG. 8: CHROMATOGRAM OF ALKALI DEGRADATION

FIG. 9: CHROMATOGRAM OF PEROXIDE DEGRADATION

FIG. 10: CHROMATOGRAM OF REDUCTION DEGRADATION

FIG. 11: CHROMATOGRAM OF THERMAL DEGRADATION (105°C FOR 72HRS)

FIG. 12: CHROMATOGRAM OF PHOTO DEGRADATION (1.2 MILLXH AND 200WH/M2 LIGHT)

FIG. 13: CHROMATOGRAM OF HYDROLYSIS DEGRADATION (HEAT ON WATER BATH 60°C FOR 30MIN)

TABLE 5: SUMMARY OF DEGRADATION DATA

CONCLUSION: The current HPLC technique used for the measurement of bulk and dosage forms adopts great sensitivity and reliability.  The present procedure was validated using the ICH Q2R1 recommendations. When compared to other approaches, the suggested approach stood out with regard to validation criteria and stability-indicating investigations. Findings for limits of detection, limits of quantification, and accuracy were all within the permitted limits during validation, demonstrating that the instrument can provide reliable findings down to very low concentrations. Peaks in degradation may be identified in research.

ACKNOWLEDGEMENT: The authors are thankful to Faculty of Pharmacy, RKDF University, Bhopal, Madhya Pradesh, India for providing the necessary facilities to carry out this work.

CONFLICT OF INTEREST: The authors confirm that this article content has no conflict of interest.

REFERENCES:

1. Xpovio- selinexor tablet, film coated. Daily Med. 19 August 2019. Archived from the original on 29 August 2021. Retrieved 20 November 2019.
2. Nexpovio Product information. Union Register of medicinal products. Retrieved 3 March 2023.
3. Fung HY and Chook YM: Atomic basis of CRM1-cargo recognition, release and inhibition. Seminars in Cancer Biology. 2014; 27: 52–61.
4. Gandhi UH, Senapedis W, Baloglu E, Unger TJ, Chari A, Vogl D and Cornell RF: Clinical Implications of Targeting XPO1-mediated Nuclear Export in Multiple Myeloma. Clinical Lymphoma, Myeloma & Leukemia. 2018; 18(5): 335–345.
5. Chen D, Frezza M, Schmitt S, Kanwar J and Dou QP: Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives. Curr Cancer Drug Targets. 2011 Mar;11(3):239-53.
6. Schwartz R and Davidson T: Pharmacology, pharmacokinetics, and practical applications of bortezomib. Oncology (Williston Park). 2004 Dec;18(14 Suppl 11):14-21.
7. Reddy KTK, Gandla K, Suthakaran R, Parija S, Lakshmi Surekha M and Gandu S: High Performance Liquid Chromatography for The Simultaneous Estimation of Anti-Ulcer Drugs in Pharmaceutical Dosage Form. Journal of Positive School Psychology2022; 6(9: 4524-4528.
8. Rao PV, Reddy CS, Marram RK and Rao DD: Simultaneous Determination of Omeprazole and Domperidone in Capsules and in Vitro Dissolution Studies by Using Stability Indicating UPLC. Journal of Liquid Chromatography & Related Technologies 2012; 35 (16): 2322-2332.
9. Vadagam N, Haridasyam SB, Venkatanarayana M, Lakka NS and Chinnakadoori SR: Separation and quantitative estimation of stereo-selective enantiomers of montelukast in pharmaceutical drug substance and tablets dosage forms by using stability-indicating normal phase-HPLC method. Chirality 2023; 35(12): 952-965.
10. Vadagam N, Haridasyam SB, Venkatanarayana M, Lakka NS and Chinnakadoori SR: Separation and quantitation of valacyclovir enantiomers using stability-indicating chiral liquid chromatography method with a chiral stationary phase of amylose tris-(3,5-dimethylphenylcarbamate). Separation Science Plus 2023; 6(12): 2300145.
11. Lakka NS, Kuppan C, Vadagam N, Ravinathan P, Chepuri K and Chinnakadoori SR: Molecular docking, in-vitro anticancer evaluation and ADME profiling of 7-Oxo Midostaurin. Journal of Molecular Structure 2023; 1293:136159.
12. Vadagam N, Haridasyam SB, Venkatanarayana M, Lakka NS and Chinnakadoori SR: Separation and simultaneous estimation of enantiomers and Diastereomers of muscarinic receptor antagonist Solifenacin using stability-indicating Normal-phase HPLC technique with chiral stationary phase amylose tris-(3,5-dimethylphenylcarbamate). Chirality 2024; 36(2): e23632.
13. Pasham M, Haridasyam SB, Vadagam N, Praveen Boppy NVVD, Chinnakadoori SR and Lakka NS: Separation and quantification of organic-related impurities of beta-adrenergic receptor blocking agent propranolol in pharmaceutical solid dosage forms: Impurity profiling using stability-indicating HPLC method. Separation Science Plus 2023;2300159.
14. Praveen Boppy NVVD, Haridasyam SB, Vadagam N, Venkatanarayana M, Chinnakadoori SR and Lakka NS: Separation and quantification of organic-related impurities of anti-histamine drug hydroxyzine in pharmaceutical dosage forms using stability-indicating high-performance liquid chromatography, liquid chromatography-mass spectrometry, and high-resolution mass spectrometry techniques, Separation Science Plus 2024; 2300157.
15. Konatham TKR and Anuradha M: A stability indicating method development and validation of Telmisartan and Nifedipine in pure form using RP-HPLC. International Journal of Pharmaceutical, Biological and Chemical Sciences 2020; 9(3): 36-44.
    

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    How to cite this article:

    Gaur B, Sahu S and Kori ML: A stability-indicating HPLC method for simultaneous estimation of selinexor and bortezomib in bulk and pharmaceutical dosage form. Int J Pharm Sci & Res 2024; 15(7): 2183-90. doi: 10.13040/IJPSR.0975-8232.15(7).2183-90.

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