Development of a micelle-enhanced high-throughput fluorometric method for determination of niclosamide using a microplate reader.

The present paper describes the development and validation of a simple and sensitive micelle-enhanced high-throughput fluorometric method for the determination of niclosamide (NIC) in 96-microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λem 444 nm after excitation at λex 275 nm. Tween-80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100-150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r2 ≥ 0.9997) over the concentration ranges of 1-5 and 0.5-5 μg/ml with lower detection limits of 0.01 and 0.008 μg/ml and lower quantification limits of 0.04 and 0.03 μg/ml on using Tween-80 and or CMC, respectively. The developed high-throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.