Original Article A NEW RELATED SUBSTANCES METHOD DEVELOPMENT AND VALIDATION OF METRONIDAZOLE AND PREDNISOLONE BY USING EFFECTIVE LIQUID CHROMATOGRAPHIC METHOD M. MANORANJANI *1 , D. ANITHA 2 , NANDURI GAYATRI DEVI 3 *1 Department of Chemistry, P B Siddhartha College of Arts and Science, Vijayawada, AP-520010, India. 2 Chemistry Department (B Sandamp; H), Dhanekula Institute of Engineering and Technology, Ganguru, Vijayawada, India. 3 Department of Chemistry, Ch. S. D. St. Theresa’s College for Women (A), Eluru-534003, Andhra Pradesh, India * Corresponding author: M. Manoranjani; * Email: ranjani.20ranjani@gmail.com Received: 31 Mar 2025, Revised and Accepted: 21 Jul 2025 ABSTRACT Objective: The present study was aimed at developing and successively validating novel, simple, responsive and stable RP-UPLC (Reverse Phase Ultra Performance Liquid Chromatography) method for the estimation of active pharmaceutical ingredients of Metronidazole and Prednisolone and their related substances. Methods: The method of chromatography was fine-tuned with the use of the solution containing the contaminant. A phenyl column with dimensions of 50x2.1 mm and a particle size of 1.7 μm was used in the chromatographic procedure. The mobile phase consisted of acetonirile and 0.1 percent ortho-phosphoric acid (OPA) in a 50:50 v/v ratio, and isocratic elution was employed. The experimental parameters called for a PDA (Photo Diode Array) detector operating at 271 nm and a flow rate of 0.2 ml/min. Results: The resolution of Metronidazole, Prednisolone and their four related substances were greater than 2.0 for all pairs of components. The high correlation coefficient (R 2 >0.999) values indicated clear correlations between the investigated compound concentrations and their peak areas within the test ranges. The Repeatability and intermediate precision expressed by the Relative Standard Deviation (RSD) were less than 2. The accuracy evaluated by performing recovery studies via a spike method was 50-150%. The performance of the method was validated according to the present ICH (International Council for Harmonization) guidelines. Conclusion: We used the created technique to measure Metronidazole and Prednisolone with their associated contaminants and it was determined to be appropriate to routine analysis. Keywords: Metronidazole, Prednisolone, RP-UPLC, Development, Validation © 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2025v17i5.54959 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION The antiprotozoal [1] and antibiotic [2, 3] drug known as metronidazole and goes by many brand names, including Flagyl. It treats bacterial vaginosis, pelvic inflammatory disease [4, 5], endocarditis [6, 7] and bacterial vaginosis [8, 9]. Several parasitic diseases, including dracunculiasis [10], giardiasis [11], trichomoniasis [12] and amebiasis [13], respond well to it. When other antibiotics, such as vancomycin or fidaxomicin, are not available, it may be used to treat mild to severe Clostridioides difficile [14, 15] colitis [16]. We may take metronidazole by mouth, topically as a gel or lotion, or intravenously as a gradual infusion. Nausea, headaches, a metallic taste, and decreased appetite are common adverse effects. Seizures or drug allergies might happen from time to time. While some sources say it's not safe to take metronidazole in early pregnancy, others say it's fine for trichomoniasis dosages. In most cases, nursing may safely continue while using metronidazole. As a steroid hormone known as a corticosteroid [17, 18], prednisolone is used to treat a variety of illnesses, including allergies, inflammation [19], autoimmune disorders [20, 21], cancer, electrolyte imbalances [22], and skin issues. Adrenocortical insufficiency, hypercalcemia [23], rheumatoid arthritis [24], inflammation of the eyes, asthma, dermatitis [25], multiple sclerosis [26], and phimosis [27] are among these disorders. It has several administration options, including oral, intravenous, topical, and ocular drops. Having a hydroxyl instead of a ketone at the 11th carbon is what distinguishes it from the similarly called prednisone. Symptoms such as nausea, dizziness, trouble sleeping [28], increased hunger, and exhaustion are common with short-term usage. Mental health issues are among the most serious side effects, and they may affect around 5% of the population. Weakness, yeast infections, easy bruising [29], bone loss, and other common adverse effects are reported with long-term usage. Using it for a short period in the third trimester is generally safe, but using it for a long period or in the first trimester might sometimes cause damage to the baby. Created from hydrocortisone, it is known as cortisol, and it is a glucocorticoid [30]. Fig. 1 shows the chemical structures of Metronidazole, Prednisolone and their related substances. The development of a new and dependable UPLC technique for the detection of Metronidazole, Prednisolone, and their associated related substances has become more important due to the lack of existing methods in the literature. MATERIALS AND METHODS Chemicals The following materials were procured from Merck India Ltd, Mumbai, India: acetonitrile, water, and ortho-phosphoric acid of HPLC (High Performance Liquid Chromatography) grade. The reference standards for Metronidazole and Prednisolone, as well as their related substances, were obtained from Glenmark Pharmaceuticals Pvt Ltd in Hyderabad. Instrumentation A Phenyl column (50x2.1 mm, 1.7 μ) and a detector of light diode array (model 2998) from Waters Acquity liquid chromatographic system were used in this study. The system was monitored using an Empower 2.0 data processing system. International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 17, Issue 5, 2025