Late Bhagirathi Yashwantrao Pathrikar College of Pharmacy, Pathri, Chh. Sambhajinagar Maharashtra 431111.
Relugolix is a modern, orally active, non-peptide gonadotropin-releasing hormone (GnRH) receptor antagonist widely used in the treatment of hormone-dependent disorders such as advanced prostate cancer, uterine fibroids, and endometriosis. Chromatographic separation was achieved using an Inertsil ODS-2 C18 column (4.6 × 150 mm, 5 µm) with a mobile phase composed of Acetonitrile and 0.1% Orthophosphoric Acid buffer in the ratio of 60:40 v/v, delivered at a flow rate of 1.0 mL/min. Detection was carried out at 293 nm using a PDA detector. The developed method produced a sharp, symmetric peak at a retention time of approximately 2.0 minutes, indicating excellent resolution and reduced analysis time. The method exhibited remarkable linearity over the concentration range of 20–100 µg/mL, with a correlation coefficient (R²) of 0.999. The LOD and LOQ were 0.05 µg/mL and 0.17 µg/mL, respectively, demonstrating high sensitivity. Accuracy studies indicated percentage recoveries within 98–102%, while precision results confirmed excellent repeatability with %RSD values below 2%. The method was found to be specific, robust, and free from excipient interference. The assay of a marketed formulation yielded 99.12% of the labelled claim[1].
Pharmaceutical analysis plays a crucial role in ensuring the quality, safety, and efficacy of pharmaceutical products. Accurate and precise analytical methods are essential throughout drug development, manufacturing, and quality control processes. Among various analytical techniques, High Performance Liquid Chromatography (HPLC) has gained widespread acceptance due to its high sensitivity, reproducibility, and ability to separate complex mixtures[1].
Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is one of the most commonly employed chromatographic techniques in pharmaceutical industries for the estimation of drugs in bulk and dosage forms. The technique offers advantages such as better resolution, shorter analysis time, and compatibility with a wide range of compounds.[2-7]
Drug Profile of Relugolix
Relugolix is a novel, orally active, non-peptide gonadotropin-releasing hormone (GnRH) receptor antagonist. Unlike GnRH agonists, Relugolix directly inhibits GnRH receptors in the pituitary gland, leading to immediate suppression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This results in reduced testosterone production without the initial hormone surge (flare effect) associated with GnRH agonists.
Relugolix has been approved for the treatment of advanced prostate cancer and is also used in the management of hormone-dependent conditions such as uterine fibroids and endometriosis. Its oral administration provides better patient compliance compared to injectable therapies[4].
Fig.1. Chemical structure of relugolix
Fig.2.Relugolix Tablet
Chemical Properties of Relugolix
Chemically, Relugolix is designated as (C29H27F2N7O5S) with a molecular weight of approximately 623.63 g/mol. It appears as a white to off-white crystalline powder and is practically insoluble in water but soluble in organic solvents such as methanol and acetonitrile. These physicochemical properties make RP-HPLC a suitable technique for its analysis.
The chemical structure of Relugolix contains aromatic rings and heterocyclic moieties, contributing to its lipophilicity and chromatographic behaviour under reversed-phase conditions [4].
Figure .3.Chemical structure of Relugolix
Pharmacological Profile
Relugolix exerts its pharmacological action by competitively binding to GnRH receptors in the anterior pituitary gland. This binding inhibits the release of gonadotropins, thereby decreasing the synthesis of sex hormones. In males, it reduces testosterone levels, while in females it lowers oestrogen levels.
The drug shows rapid onset of action, dose-dependent suppression of testosterone, and reversible effects upon discontinuation. These characteristics make Relugolix a clinically significant advancement in hormone therapy[8].
Figure.4. Mechanism of action of Relugolix as a GnRH receptor antagonist
Pharmacokinetics
Relugolix demonstrates good oral bioavailability with peak plasma concentration achieved within 2 to 3 hours after administration. It is extensively metabolized in the liver, primarily through CYP3A-mediated pathways, and is eliminated via faecal and renal routes. The elimination half-life supports once-daily dosing, improving patient adherence[8,9,19].
Fig.5.Pharmacokinetic Profile of Relugolix
Need for Analytical Method Development
Reliable analytical methods are required for routine quality control, stability testing, and assay of pharmaceutical formulations. Although advanced techniques such as LC-MS/MS provide high sensitivity, they are expensive and not feasible for routine quality control in all laboratories.
Therefore, there is a need to develop a simple, cost-effective, accurate, and precise RP-HPLC method for the estimation of Relugolix in bulk drug and marketed dosage forms. Such a method would be beneficial for pharmaceutical industries and research laboratories for routine analysis.
Aim and Objectives of the Present Study
Aim:
To develop and validate a simple, rapid, and reliable RP-HPLC method for the estimation of Relugolix in bulk drug and pharmaceutical dosage form.
Objectives:
Analytical Method Validation
Method validation is a critical requirement to ensure the reliability of analytical results. According to ICH Q2(R1) guidelines, validation parameters include specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and robustness. Proper validation confirms that the method is suitable for its intended purpose[1].
SCOPE OF PRESENT WORK
The scope of the present work is focused on the systematic development and validation of a Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the quantitative estimation of Relugolix in bulk drug and marketed pharmaceutical dosage form. With the increasing use of Relugolix in hormone-dependent disorders, there is a growing need for a reliable and economical analytical method suitable for routine quality control analysis.
The present study aims to optimize various chromatographic parameters such as selection of column, mobile phase composition, flow rate, detection wavelength, and run time to achieve adequate resolution, sharp peak shape, and reproducible retention time for Relugolix. Special emphasis is given to developing a method that is simple, rapid, and cost-effective, making it suitable for use in quality control laboratories.
The scope of the work also includes validation of the developed RP-HPLC method in accordance with International Council for Harmonisation (ICH) Q2(R1) guidelines. Validation parameters such as specificity, linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), and robustness are evaluated to confirm the reliability and suitability of the method for its intended purpose.
Furthermore, the validated method is applied for the assay of Relugolix in a marketed tablet formulation to demonstrate its practical applicability in routine pharmaceutical analysis. The study also aims to ensure that the developed method can be effectively used for quality control testing, batch release analysis, and stability studies of Relugolix-containing formulations.
Overall, the scope of the present work is to provide a validated, efficient, and reproducible RP-HPLC method that can serve as a valuable analytical tool in pharmaceutical industries and research laboratories.
METHOD DEVELOPMENT AND VALIDATION
Introduction
Analytical method development and validation are essential to ensure accurate and reliable estimation of pharmaceutical drugs. In the present study, a simple, rapid, and economical RP-HPLC method was developed for the estimation of Relugolix in bulk drug and marketed dosage form. The developed method was validated according to ICH Q2(R1) guidelines to confirm its suitability for routine quality control analysis.
Selection of Analytical Technique
RP-HPLC was selected for the analysis of Relugolix due to its high precision, accuracy, reproducibility, and suitability for compounds with moderate lipophilicity. Compared to advanced techniques such as LC-MS, RP-HPLC is cost-effective and widely used in pharmaceutical quality control laboratories.
Materials and Reagents
Relugolix bulk drug was obtained as a gift sample. Marketed Relugolix tablets (Orgovyx®, 120 mg) were purchased from a local pharmacy. Acetonitrile and methanol of HPLC grade and orthophosphoric acid of analytical grade were used. Distilled water was used throughout the study.
Instrumentation
Chromatographic analysis was carried out using a Shimadzu RP-HPLC system equipped with a quaternary pump, autosampler, and PDA detector. Data acquisition was done using LabSolutions software. Separation was achieved using an Inertsil ODS-2 C18 column (250 × 4.6 mm, 5 µm).
Selection of Detection Wavelength
The detection wavelength was selected by scanning the standard solution of Relugolix in the UV range of 200-400 nm. Relugolix showed maximum absorbance at 293 nm, which was selected for HPLC analysis.
Method Development
Different mobile phase compositions were tried to obtain a sharp and symmetric peak with acceptable retention time. The mobile phase consisting of Acetonitrile and 0.1% OPA (60:40 v/v) provided good resolution and peak symmetry. A flow rate of 1.0 mL/min and run time of 6 minutes were selected.
Optimized Chromatographic Conditions
Preparation of Standard Solution
An accurately weighed 10 mg of Relugolix was dissolved in methanol and diluted to obtain a standard stock solution of 1000 µg/mL. Further dilutions were made with mobile phase to prepare working standard solutions.
Preparation of Sample Solution
Twenty tablets were weighed and powdered. Powder equivalent to 10 mg of Relugolix was dissolved in methanol, sonicated, filtered, and appropriately diluted with mobile phase for analysis.
Method Validation
The developed method was validated as per ICH Q2(R1) guidelines.
Specificity
No interference was observed at the retention time of Relugolix, indicating specificity.
Fig.6.HPLC Chromatogram of sample Solution of marketed Relugolix tab formulation
Fig.7.Overlay of Chromatogram for specificity study
4.10.2 Linearity
The method was linear in the concentration range of 20–100 µg/mL with a correlation coefficient close to 1.
Fig.8.HPLC Chromatogram of standard Relugolix solution
Fig.9.HPLC Chromatogram of sample solution linearity
4.10.3 Accuracy
Recovery studies at 80%, 100%, and 120% levels showed acceptable recovery, confirming accuracy.
Fig.10.System suitability Chromatogram of Relugolix showing retention ,time and tailing
Fig.11.Recovery study chromatogram of Relugolix at 80%,100% and 120%.
4.10.4 Precision
Intra-day and inter-day precision studies showed %RSD values less than 2%.
4.10.5 LOD and LOQ
LOD and LOQ values indicated good sensitivity of the method.
4.10.6 Robustness
Small deliberate changes in flow rate and wavelength did not significantly affect results, confirming robustness.
Fig.12.Effect of minor variation in flow Rate
CONCLUSION
The present study focused on the development and validation of a Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the estimation of Relugolix in bulk drug and marketed pharmaceutical formulations. The method was systematically optimized to ensure accuracy, precision, and reproducibility, following the ICH Q2(R1) guidelines.
The developed RP-HPLC method demonstrated the following:
REFERENCES
Jyoti Darade, Komal Bankar, Maya Dudhare, Mamata Chavan, Gaytri Fuke, Priya Jadhav, Vaishnavi Pakhare, Overall Review on Development and Validation of RP-HPLC Method for Relugolix in Bulk and Marketed Formulations, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 1, 629-638. https://doi.org/10.5281/zenodo.18173767
10.5281/zenodo.18173767
