Factorial design optimization of a novel NBD-Cl derivatized spectrofluorimetric method for sensitive determination of vilazodone in pharmaceutical formulations and human plasma

Abstract

A sensitive, selective, and systematic spectrofluorimetric method has been developed and validated for the determination of the antidepressant drug vilazodone (VLZ) in pharmaceutical formulations and human plasma. The method is based on the nucleophilic substitution reaction between VLZ and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) under optimized alkaline conditions (pH 8.8) to form a highly fluorescent derivative measured at λ_em 548 nm after excitation at λ_ex 470 nm. The reaction mechanism was confirmed through comprehensive LC-MS/MS analysis, identifying the specific site of derivatization and key fragmentation patterns. A 2^6-2 fractional factorial design was employed to systematically optimize six critical variables affecting the derivatization reaction. Statistical analysis revealed that buffer pH, NBD-Cl volume, temperature, and buffer volume significantly influenced the fluorescence response, with notable interaction effects between pH and both NBD-Cl and HCl volumes. The mathematical model demonstrated excellent fit (R² = 0.994) and predictive capability, enabling precise determination of optimal reaction conditions: buffer pH 8.8, NBD-Cl concentration 0.1 %, reaction temperature 60 °C, and reaction time 12 min. The method was validated according to ICH guidelines, demonstrating excellent linearity (10–500 ng/mL), precision (RSD < 1.35 %), and accuracy (recovery 99.36–100.27 %). The limits of detection and quantification were 3.22 ng/mL and 9.75 ng/mL, respectively, with a correlation coefficient (r²) of 0.9997. Selectivity studies confirmed negligible interference from common pharmaceutical excipients, plasma components, and inorganic ions. The optimized method was successfully applied to the determination of VLZ in pharmaceutical tablets (99.45 % recovery) and spiked human plasma (98.55 % recovery) with excellent stability under various storage and handling conditions. This derivatization-based spectrofluorimetric approach overcomes the photoinduced electron-transfer quenching effect inherent to VLZ’s molecular structure and offers a simpler, more accessible alternative to chromatographic techniques for routine quality control and potential pharmacokinetic studies.