Talanta Open (Aug 2025)
A versatile HPLC method collaborated with NaDES-ultrasound-assisted LLE and machine learning for purity testing and trace analysis of 1,4-benzodiazepines in crime scene evidence, hospital effluents, and pharmaceuticals
Abstract
A green, stability-indicating, selective, and multipurpose HPLC approach was introduced for the simultaneous determination of three 1,4-benzodiazepines (BNZs) (chlordiazepoxide (CDP), alprazolam (ALP), and diazepam (DZP)) in the presence of their degradation product, impurity (2-amino-5-chlorobenzophenone). These BNZs are emerging contaminants in environmental water that have recently attracted more attention due to their potential effects on public health. In addition, the common abuse of BNZs in drug-facilitated crimes has increased, with victims often assaulted or robbed under the influence of BNZs. Therefore, the development of a fast, versatile, and high throughput method for the analysis of these compounds in hospital effluent and food samples from crime scenes is of paramount importance. However, efficient extraction from such complex matrices usually requires large volumes of toxic organic solvents, lengthy time, and high cost. For the first time, microwave-driven synthesis of novel natural deep eutectic solvent (NaDES) in just two min was conducted in this study for efficient and green ultrasound-assisted liquid-liquid extraction of BNZs. The selected NaDES is composed of menthol: fructose (3: 1, molar ratio). The HPLC method employed gradient elution mode of acetonitrile/phosphate buffer (pH 3.0) mobile phase, phenyl column coupled to UV detection with time programming. The experimental variables were optimized using full factorial design and the separation was achieved in <14 min. The method showed excellent linearity and a high sensitivity with detection limits of 0.34, 0.18, and 0.04 µg mL−1 for CDP, ALP, and DZP, respectively. The method was also validated in cream biscuits with % recovery from 95.0 % to 106.0 %. The proposed method paves the way for new NaDES synthesis and extended applications in impurity profiling of pharmaceutical products, environmental monitoring, and forensic toxicology. The method’s greenness was confirmed using ComplexMoGAPI and AGREE tools.
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