Detection and quantitation of benzodiazepines in less than 3 min using PESI-MS and isotope dilution approach

Significance of Topic

Benzodiazepines remain widely prescribed and misused sedative drugs with important roles in clinical toxicology and forensic analysis. Fast and reliable quantitation is critical for emergency screening, drug-facilitated crime investigations and therapeutic monitoring.

Objectives and Study Overview

This study aimed to develop and validate an ultrafast method for measurement of 18 benzodiazepines, including classical and designer compounds, in human serum using Probe Electrospray Ionization tandem mass spectrometry combined with an isotope dilution approach.

Methodology

Chemical reagents and standards were prepared in methanol or buffer. Sample preparation involved:

• Mixing 10 µL serum with 500 µL ethanol/ammonium formate buffer (10 mM)
• Adding 10 µL internal standard mix (50 µg/L)
• Applying 10 µL of the mixture onto the PESI sample plate

Used Instrumentation

The analysis was performed using a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer equipped with a DPiMS-8060 Probe Electrospray Ionization source. Key settings:

• PESI probe movement frequency 3.1 Hz (184 actions/min)
• Run time 2.56 min per sample
• Positive ionization mode with multiple reaction monitoring transitions optimized for each analyte and corresponding isotopically labeled internal standard

Key Results and Discussion

Validation on eight classical benzodiazepines across seven concentration levels (5–2000 µg/L) demonstrated inter- and intra-day precision and bias below 20% for all analytes. Application to 40 routine samples yielded 100% agreement with conventional LC-MS/MS (r2=0.93) and highlighted lower false negatives compared to immunoassay (three cases missed by immunoassay). No designer benzodiazepines were detected in these samples.

Benefits and Practical Applications

The developed method delivers:

• Minimal sample volume requirement (10 µL serum)
• Rapid turnaround (~2.5 minutes per analysis)
• High specificity and sensitivity through isotope dilution and PESI ionization
• Improved throughput for clinical and forensic laboratories

Future Trends and Applications

Advancements may include extension to additional drug classes, integration of PESI-MS with portable mass spectrometers for point-of-care testing, and automation of sample handling for high-throughput toxicology screening.

Conclusion

An ultrafast PESI-MS/MS method combined with isotope dilution has been established and validated for quantitation of 18 benzodiazepines in serum. It offers rapid, accurate and low-volume analysis suitable for clinical toxicology, emergency screening and forensic applications.