Ultrafast Analysis of Barbiturates in Urine by the Agilent RapidFire High-Throughput Triple Quadrupole Mass Spectrometry System

Importance of the Topic

Barbiturates remain important targets in forensic toxicology due to their therapeutic use, abuse potential, and risk of addiction. Rapid and reliable screening methods are essential in clinical and forensic laboratories to handle growing sample volumes and provide timely results.

Objectives and Study Overview

• Develop an ultrafast screening method for qualitative detection of common barbiturates in human urine.
• Compare analytical performance and throughput with conventional LC/MS/MS workflows.

Methodology and Instrumentation

• Sample Preparation: Simple dilute-and-shoot approach. Urine samples spiked with analytes and butalbital-d5 internal standard, then diluted 50-fold in ultrapure water.
• Analysis: Agilent RapidFire high-throughput SPE coupled to a 6460 triple quadrupole mass spectrometer. Cycle time of 16 seconds per sample for >225 samples per hour.
• Chromatography: Phenyl reversed-phase SPE cartridge (G9208A) and aqueous/organic buffers for sample loading and elution.
• Detection: Negative electrospray MRM transitions for phenobarbital, secobarbital, butalbital, amobarbital/pentobarbital (isobars), and internal standard.
• Calibration: Linear range 50–10 000 ng/mL with 1/X² weighted regression.

Instrumentation Used

• Agilent RapidFire 360 system
• Agilent 6460 Triple Quadrupole Mass Spectrometer
• Agilent MassHunter software for acquisition, qualitative, and quantitative analysis
• RapidFire phenyl SPE cartridge (G9208A)

Main Results and Discussion

• Throughput: Achieved >225 injections per hour with 16 s cycle times.
• Linearity: All analytes showed R²>0.995 over 50–10 000 ng/mL. Amobarbital and pentobarbital coelute and cannot be distinguished qualitatively.
• Accuracy and Precision: Interday and intraday accuracy within ±10% and CVs below 10% across the calibration range.
• Matrix Effects and Carryover: Negligible matrix suppression (<10%) and no significant carryover under standard operating conditions.

Benefits and Practical Applications

• Speed: Over 15-fold faster than traditional LC/MS/MS, enabling rapid forensic screening.
• Efficiency: Minimal sample preparation reduces labor, solvent use, and overall cost.
• Flexibility: Method can be extended to additional analytes with minor adjustments.
• Robustness: Consistent qualitative performance suitable for high-throughput toxicology workflows.

Future Trends and Applications

• Expand panels to include other classes of drugs and metabolites for comprehensive screenings.
• Integrate automated data processing and reporting for real-time decision support.
• Adapt workflows for quantitative assays and alternate biological matrices (blood, oral fluid).
• Develop new SPE cartridge chemistries to resolve isobaric interferences and improve selectivity.

Conclusion

The ultrafast RapidFire/MS/MS method offers a sensitive, accurate, and high-throughput solution for qualitative screening of five key barbiturates in urine. By combining simple sample prep with 16-second cycle times, this approach meets the demands of modern forensic laboratories while maintaining analytical performance comparable to LC/MS/MS.

References

• June Feng, Lanqing Wang, Ingrid Dai, Tia Harmon, John T. Bernert. Simultaneous Determination of Multiple Drugs of Abuse and Relevant Metabolites in Urine by LC/MS/MS. Journal of Analytical Toxicology, 31(9):359–368, 2007.