A Highly Sensitive RP HPLC‐PDA Analytical Method for Detection and Quantification of a Newly Synthesized (E‐2‐((E)‐4‐(5‐Ethoxy‐3‐Methyl‐1‐Phenyl‐1H‐Pyrazole‐4‐yl)but‐3‐en‐2‐Ylidene)) Hydrazine‐1‐Carbothioamide in Nanosuspension

ABSTRACT Analytical methods development and validation are vital for the precise detection, quantification, and characterization of novel therapeutic compounds, especially those with poor aqueous solubility, such as pyrazolone derivatives. This study aimed to develop and validate a sensitive, accurate, and efficient RP HPLC‐PDA method for the detection and quantification of novel (E‐2‐((E)‐4‐(5‐ethoxy‐3‐methyl‐1‐phenyl‐1H‐pyrazole‐4‐yl)but‐3‐en‐2‐ylidene) hydrazine‐1‐carbothioamide in nanosuspension. The method was optimized for high sensitivity and specificity using a Shim‐pack GIST C18 (5 µm, 150 × 4.6 mm) column, with an isocratic mobile phase of ACN and 0.1% TFA in water (75:25 v/v). It employed a 0.5 mL/min flow rate, a 20 µL injection volume, and detected the compound at 333 nm. The method showed excellent linearity ( R 2 = 0.9994) over a concentration range of 2.5–50 µg/mL, with high precision, accuracy, and reproducibility, in compliance with ICH Q2 (R1) guidelines. The LOD and LOQ were 2.43 and 7.38 µg/mL, respectively. Recovery rates ranged from 110% to 112%, with RSD below 2%. The validated RP HPLC‐PDA method was effectively applied to detect, characterize, and quantify the novel compound in its nanosuspension form. This method offers a reliable analytical tool for the quality control of this novel compound, both in raw material and finished product forms, as well as for impurity profiling, drug release, and stability testing, which will, in turn, facilitate new drug development.