Design and validation of a sustainable and comprehensive analytical method for tracing Zolpidem in water, alcoholic, and non-alcoholic beverages using CPSME-LC-MS/MS for confirmatory analysis

Abstract

Introduction:
Zolpidem, a widely prescribed non-benzodiazepine hypnotic from the imidazopyridine class, is used for the short-term treatment of insomnia due to its sedative and sleep-inducing properties. However, its rapid onset, strong sedative effects, and amnesic properties make it a substance of choice in drug-facilitated crimes (DFCs). Increasing reports of zolpidem misuse emphasize the need for novel detection methods in forensic toxicological investigations.

Method:
To address the need for a robust and eco-friendly analytical approach, a miniaturized Vortex-Assisted Ultrasonication-based Cellulose Paper Sorptive extraction in combination with Liquid chromatography mass spectrometry/mass spectrometry (VAUS-CPSE-LC-MS/MS) method was developed and validated for the quantification of Zolpidem in different beverages, including alcoholic, non-alcoholic, and water samples. Isopropanol alcohol (IPA) was selected as the elution solvent and a “2×2” cm sized filter paper was selected for the experiment. To optimize the method, the statistical tool “Minitab Statistica 22.0” was employed where the Placket Burman Design and Central Composite Design were formed to identify influential parameters.

Results:
Optimal conditions for the extraction were a vortex time of 90 seconds, ultrasonication time of 12.5 minutes, pH of 7.5, extraction time of 6 minutes, back-extraction solvent volume of 1.5 mL, and 6.5 pieces of filter paper. Chromatographic separation was achieved using UHPLC with a binary mobile phase of 0.1% formic acid in Milli-Q water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). The calibration curve for target analytes was linear in the range of 1.325–1000 ng/mL with a regression coefficient (R²) of 0.999. The method demonstrated excellent sensitivity with limits of detection (LODs) and limits of quantification (LOQs).

Conclusion:
The developed VAUS-CPSE-LC-MS/MS method proved to be a sensitive, eco-friendly, and cost-effective approach for the detection and quantification of Zolpidem in complex beverage matrices. This method offers strong potential for application in forensic toxicology investigations involving drug-facilitated crimes, robberies, and suspicious deaths.