Abstract

STABILITY-INDICATING RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF TRIMETHOPRIM: AN ANALYTICAL QBD APPROACH

Gowtham S.*, Kanmani R., Gokulan P.D. and Senthil Kumar K. L.

ABSTRACT

The purpose of this study is to present a novel approach for determining the concentration of trimethoprim (TMP) through the utilization of reverse-phase high-performance liquid chromatography (RP-HPLC) and a quality-by-design (QbD) method. TMP is commonly used to treat and prevent urinary tract infections (UTIs) such as cystitis. Previous research has shown a lack of a proven technique that accurately measures TMP over an extended period using RP-HPLC with analytical quality by design (AQbD) integration. To optimize the method, the analysis employed the Central Composite Design (CCD), allowing for the establishment of a relationship between various factors and responses to improve study accuracy. The separation of trimethoprim was achieved using a C18 column with dimensions of 250 mm in length, 4.6 mm in diameter, and 5 μm particle size. The column temperature was maintained at 35°C. An isocratic mobile phase of methanol with orthophosphoric acid buffer at pH 3.5 in a 60:40%volumetric ratio was used. The injection dose was 10 μL with a flow rate of 1.2 mL/min. Trimethoprim was identified by measuring its absorbance at 230 nm using a photodiode-array detector. The strategy underwent validation following ICH Q14 Guidelines. The retention time of trimethoprim was 3.1 minutes. The linearity ranged from 3-10 μg/mL. The accuracy, reproducibility, and selectivity of the method were confirmed with an R2 value of 0.9993. The approach also showed significant recovery under various conditions of forced degradation, demonstrating its ability to differentiate between trimethoprim and degradation chemicals. Overall, the method met all assessment criteria, confirming its credibility and reliability.

Keywords: Trimethoprim; Analytical quality by design; HPLC; Method Development; Method Validation.