Ultrafast Screen for Bath Salts in Urine Using the Agilent RapidFire High-Throughput Mass Spectrometry System

Significance of the Topic

The emergence of synthetic cathinones, commonly known as "bath salts," poses a serious challenge for forensic toxicology due to their structural diversity and rapid market introduction. Traditional immunoassay and chromatographic methods often fail to detect newly modified analogs in a timely manner. High-throughput, rapid analytical approaches are needed to support law enforcement, clinical laboratories, and public safety initiatives by delivering fast, sensitive, and specific screening of these designer drugs in biological matrices such as urine.

Objectives and Study Overview

This study aimed to develop and validate an ultrafast screening method for six prevalent bath salts—mephedrone, methylone, methcathinone, fluoromethcathinone, methoxymethcathinone, and MDPV—in human urine. The goals were to achieve accurate quantitation over a broad concentration range, confirm specificity in the presence of common interferences, and maximize throughput for forensic toxicology applications.

Methodology and Instrumentation

The method employs a simple dilute-and-shoot sample preparation: urine samples spiked with analytes are diluted 1:10 in water containing methylone-d3 as an internal standard and transferred to 96-well plates for direct analysis.

Key instrumentation and conditions:

• Agilent RapidFire 360 system with reversed-phase C4 SPE cartridge (A2).
• Agilent 6490 Triple Quadrupole Mass Spectrometer.
• MassHunter Qualitative and Quantitative Analysis software (B.05.00).
• Cycle time: 14 seconds per injection (>240 samples per hour).
• Buffers: A (water with 0.09% formic acid, 0.01% TFA), B (50% methanol/50% isopropanol with 0.09% formic acid, 0.01% TFA).
• MRM transitions optimized for each analyte and internal standard.

Results and Discussion

The method demonstrated excellent linearity (R² > 0.998) over the range of 31–1,000 ng/mL for all six analytes, with a limit of quantitation of 31 ng/mL. Intra- and interday accuracy remained within ±15%, and precision (CV) was below 10% at all concentration levels, even in the presence of high concentrations (100 µg/mL) of potential interferences such as amphetamines, ephedrine, and pseudoephedrine. A repeatability test of 2,000 sequential injections showed stable peak shapes and consistent instrument response (e.g., methylone CV = 2.05%, accuracy = 101.25%).

Benefits and Practical Applications

This ultrafast SPE/MS/MS workflow offers significant advantages for forensic and clinical laboratories by combining minimal sample preparation with high specificity and sensitivity. The rapid cycle times support large-scale screening campaigns, reduce turnaround time, and enable timely decision-making in cases involving suspected designer drug intake.

Future Trends and Potential Applications

Continued expansion of the analyte panel on the RapidFire platform could accommodate emerging designer cathinones and other novel psychoactive substances. Integration with automated sample handling and data processing will further streamline workflows. The approach may also be adapted to other biological matrices—such as blood or oral fluid—to broaden its forensic and clinical utility.

Conclusion

The developed Agilent RapidFire high-throughput MS method provides a robust, precise, and sensitive screening tool for six key bath salts in urine. With throughput exceeding 240 samples per hour and strong analytical performance, it addresses the growing needs of forensic toxicology laboratories for rapid and reliable detection of designer drugs.

Used Instrumentation

• Agilent RapidFire 360 high-throughput SPE system.
• Agilent 6490 Triple Quadrupole Mass Spectrometer.
• MassHunter Qualitative and Quantitative Analysis software B.05.00.