LC-MS/MS Analysis of Urinary Benzodiazepines and Z-drugs via a Simplified, Mixed-Mode Sample Preparation Strategy

Importance of Topic

Benzodiazepines and related Z-drugs are among the most prescribed sedatives and anxiolytics worldwide, but their misuse and overdose incidents have increased dramatically. Accurate, high-throughput urinary screening is critical in forensic toxicology, clinical diagnostics, and workplace drug testing to monitor compliance, detect impairment, and guide treatment.

Study Objectives and Overview

This work aimed to develop a simplified, mixed-mode solid-phase extraction (SPE) protocol combined with LC-MS/MS for the simultaneous quantification of 21 benzodiazepines, their metabolites, and two Z-drugs in human urine. Key goals included reducing sample-prep time, minimizing solvent use, and maintaining robust analytical performance.

Methodology and Instrumentation

A 200 µL urine aliquot was hydrolyzed in-well with β-glucuronidase and buffered at pH 5.0. Acidification followed, then direct loading onto Oasis MCX µElution Plates without conditioning or equilibration. Two wash steps (0.02 N HCl and 20% methanol) and a rapid high-vacuum dry preceded elution with 60:40 ACN:MeOH containing 5% ammonia. Final extracts were diluted prior to injection.

Used Instrumentation

• UPLC System: ACQUITY UPLC I-Class (FL) with CORTECS UPLC C18+ 1.6 µm, 2.1×100 mm column
• Alternative HPLC Scale: ACQUITY UPLC H-Class with CORTECS UPLC C18+ 2.7 µm, 3.0×100 mm column
• Mass Spectrometer: Xevo TQ-S micro with ESI+ and MRM detection
• Software: MassLynx and TargetLynx Application Manager

Main Results and Discussion

Chromatographic separation achieved baseline resolution of all analytes within 5.5 minutes (5.2 minutes on UPLC); HPLC scale retention increased by ~30% but maintained critical separations under 4000 psi. Average SPE recoveries were 91% (range 76–102%), with intra- and inter-batch CVs of 5–15%. Mixed-mode SPE reduced absolute matrix effects to 17.7% versus 25.3% for reversed-phase HLB. Calibration curves (0.5–500 ng/mL) exhibited R² ≥ 0.997, mean deviations < 10%, and limits of quantitation of 0.5 ng/mL. QC accuracy ranged 96–108% with precision ≤ 10%.

Benefits and Practical Applications

This approach condenses traditional six-step SPE into four steps, cuts solvent consumption, eliminates sample transfers, and integrates hydrolysis in-well. The workflow is scalable to 96-well format, supports both UPLC and HPLC platforms, and delivers reliable data for routine forensic, clinical, and pharmaceutical laboratories.

Future Trends and Possibilities

Further automation of in-well SPE, coupling with high-resolution MS, and expansion to multiresidue panels (e.g., opioids, stimulants) may enhance throughput. Miniaturization and online SPE–MS integration could drive on-site testing, while novel mixed-mode sorbents may broaden matrix compatibility.

Conclusion

The simplified in-well mixed-mode SPE combined with rapid LC-MS/MS provides accurate, precise, and high-throughput analysis of urinary benzodiazepines and Z-drugs. The method reduces hands-on time, solvent use, and matrix interferences, making it ideal for forensic and clinical toxicology applications.

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