DEVELOPMENT AND VALIDATION BY RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF RELUGOLIX, ESTRADIOL HEMIHYDRATE AND NORETHINDRONE ACETATE IN SYNTHETIC MIXTURE
Riya Vora
, Dr. Nachiketa Pandya , Dr. Vanita Marvaniya , Dr. Dhaval Patel
Relugolix, Estradiol hemihydrate, Norethindrone acetate, RP-HPLC, Method validation, Simultaneous estimation, Synthetic mixture, ICH guidelines, Gradient elution, Chromatographic separation
Objective :To develop a simple and reliable RP-HPLC method for the simultaneous determination of Relugolix, Estradiol Hemihydrate, and Norethindrone Acetate in a Synthetic Mixture. To optimize chromatographic conditions using a gradient mobile phase for separation of the three analytes. To validate the developed method according to analytical validation parameters like linearity, precision, LOD, LOQ, and assay. To demonstrate the suitability of the developed method for routine analytical and quality control use.
Experimental Work :The RP-HPLC method was developed with the stationary phase being an Inertsil C18 column (150 × 4.6 mm, 5 μm). A gradient mobile phase (buffer and acetonitrile) was used for chromatographic separation. The optimized gradient program was 0 min (62:38 Buffer:CAN), 5 min (35:65), 11 min (35:65), 11.5 min (62:38), and 15 min (62:38). The flow rate was fixed at 1.0 mL/min, injection volume was 10 μL, and the detection wavelength was 240 nm. Standard solutions of Relugolix, Estradiol hemihydrate, and Norethindrone acetate were prepared and injected one at a time and confirmed the peaks according to retention time. The developed method was validated against the standard parameters for analytical validation.
Results and Discussion :The optimized chromatographic conditions provided for a good separation for all the three analytes with theoretical plates being greater than 2000, tailing factor being less than 2, and the resolution being greater than 2. The method has shown good linearity for Relugolix in concentrations ranging between 200 and 600 μg/mL, 5 and 15 μg/mL for Estradiol hemihydrate, and 2.5 and 7.5 μg/mL for Norethindrone acetate. The LOD values came to 12.9μg/mL for Relugolix, 1.28 μg/mL for Estradiol hemihydrate, and 0.64 μg/mL for Norethindrone acetate, while LOQ values were 39.3μg/mL, 3.89μg/mL, and 1.94μg/mL. The method showed repeatability in a repetitiveness study with %RSD values of 0.12%, 0.16%, and 0.51% for Relugolix, Estradiol, and Norethindrone acetate, respectively. The assay results stood at 98.5±1.73%, 101±0.72%, and 98±0.49% for Relugolix, Estradiol, and Norethindrone acetate, respectively.
Conclusion :Finally, a gradient RP-HPLC method was successively developed and validated for the simultaneous determination of Relugolix, Estradiol hemihydrate, and Norethindrone acetate in a synthetic mixture. The method showed good chromatographic separation with excellent linearity and precision, alongside an acceptable level of sensitivity. All the validation parameters are well within the satisfactory limits proving the reliability and reproducibility of the method. The proposed RP-HPLC method is simple, accurate, and can be used as a routine quality control analysis of these drugs in.
"DEVELOPMENT AND VALIDATION BY RP-HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF RELUGOLIX, ESTRADIOL HEMIHYDRATE AND NORETHINDRONE ACETATE IN SYNTHETIC MIXTURE", JETNR - JOURNAL OF EMERGING TRENDS AND NOVEL RESEARCH (www.JETNR.org), ISSN:2984-9276, Vol.4, Issue 4, page no.712-743, April-2026, Available :https://rjpn.org/JETNR/papers/JETNRTH00015.pdf
Volume 4
Issue 4,
April-2026
Pages : 712-743
Paper Reg. ID: JETNR_233500
Published Paper Id: JETNRTH00015
Downloads: 00062
Research Area: Pharmacy All
Country: Ahmedabad, Gujarat, India
ISSN: 2984-9276 | IMPACT FACTOR: 9.87 Calculated By Google Scholar | ESTD YEAR: 2023
An International Scholarly Open Access Journal, Peer-Reviewed, Refereed Journal Impact Factor 9.87 Calculate by Google Scholar and Semantic Scholar | AI-Powered Research Tool, Multidisciplinary, Monthly, Multilanguage Journal Indexing in All Major Database & Metadata, Citation Generator
Publisher: RJPN (IJPublication) Janvi Wave
