Extraction of Benzodiazepines in Urine with Polymeric SPE Cation Exchange, Agilent Bond Elut Plexa PCX

Importance of the Topic

Accurate measurement of benzodiazepines in human urine is critical in forensic toxicology, clinical monitoring and therapeutic drug management. Complex urine matrices containing salts, pigments and proteins can suppress ionization in LC/MS analysis, necessitating robust sample cleanup to achieve sensitivity and reproducibility.

Aims and Study Overview

This study evaluates the performance of Agilent Bond Elut Plexa PCX polymeric cation exchange SPE in extracting 14 benzodiazepines and metabolites from urine. A streamlined generic protocol was developed to remove neutral and acidic interferents while concentrating basic analytes prior to LC/MS/MS quantification.

Methodology and Instrumentation

• Sample Pretreatment: Hydrolysis of glucuronide conjugates with 5,000 U β-glucuronidase in 1 M acetic acid (pH 3.8) at 60 °C for 2 h.
• SPE Workflow: Condition (1 mL MeOH, 1 mL H₂O), load diluted urine (1 mL urine with 2% formic acid), wash (2 mL 2% formic acid, 2 mL 50% MeOH), elute (1 mL 5% NH₃ in MeOH), dry and reconstitute in 200 µL 50:50 0.1% formic acid/MeOH.
• LC/MS/MS: Agilent Pursuit XRsUltra C18 (2.0×100 mm, 2.8 µm), gradient from 40:60 to 20:80 aqueous 0.1% formic acid/methanol, flow 0.2 mL/min. ESI source (dry gas 350 °C, 30 psi, capillary 70 V), negative ion mode, CID in argon.

Main Results and Discussion

The combined SPE and LC/MS/MS method achieved a detection limit of 1 ng/mL. Recoveries ranged from 95% to 120% with RSDs below 15% at both 1 and 100 ng/mL levels. Calibration curves exhibited linearity over three orders of magnitude (1–1,000 ng/mL) with correlation coefficients > 0.995. Minimal matrix-induced ion suppression was observed, and chromatographic separation allowed unambiguous peak identification.

Benefits and Practical Applications

The generic Plexa PCX protocol offers rapid, reproducible sample preparation for basic analytes in complex matrices. Its high throughput capability, minimal method development time and enhanced sensitivity suit forensic, clinical and pharmaceutical laboratories.

Future Trends and Opportunities

Emerging developments include integration of online SPE, miniaturized and automated platforms, advanced sorbent chemistries for broader analyte scope, and high-resolution MS to further improve throughput and analytical depth.

Conclusion

Agilent Bond Elut Plexa PCX provides a robust SPE solution for sensitive and reproducible quantification of benzodiazepines in urine, combining simplified workflow with high analytical performance.