Ultrafast Analysis of Gabapentin and Pregabalin in Urine Using the Agilent RapidFire High-Throughput Mass Spectrometry System

Importance of the Topic

The quantification of gabapentin and pregabalin in human urine is vital for therapeutic drug monitoring, clinical toxicology, and quality control in pharmaceutical and forensic settings. Traditional chromatographic approaches often involve lengthy sample preparation, high solvent consumption, and limited throughput. An ultrafast, reliable, and solvent-efficient method addresses the growing demand for rapid analysis in high-volume diagnostic and research laboratories.

Study Objectives and Overview

The primary goal was to develop and validate a rapid online SPE/MS/MS method for measuring gabapentin and pregabalin in urine. Key objectives included:

• Minimizing sample cycle time compared to conventional LC/MS/MS protocols.
• Simplifying extraction via a dilute-and-shoot approach.
• Maintaining analytical performance—linearity, accuracy, and precision—comparable to established methods.

Methodology and Instrumentation

Samples, calibrators (0.5–100 µg/mL), and quality controls were prepared by spiking drug-free urine with analytes and a stable-labeled internal standard (gabapentin-d10), followed by 200-fold dilution in water. Analysis employed a 96-well plate dilute-and-shoot workflow integrated with the Agilent RapidFire High-Throughput MS system and an Agilent 6460 Triple Quadrupole mass spectrometer.

• Agilent RapidFire 360 module for ultrafast solid-phase extraction on a C18 cartridge.
• Agilent 6460 Triple Quadrupole Mass Spectrometer controlled by MassHunter software.
• Mobile phases: 0.1% formic acid in water; 50% methanol/25% isopropanol/25% acetonitrile with 0.1% formic acid.
• Optimized MRM transitions for quantifier and qualifier ions for both analytes and internal standard.

Main Results and Discussion

The method achieved:

• Linear calibration curves from 0.5 to 100 µg/mL with R² > 0.995.
• Inter- and intraday accuracy within ±5% and precision (CV) < 5% across quality control levels.
• An injection-to-injection interval of 25 seconds, enabling throughput > 140 samples per hour.
• No significant carryover detected even after the highest calibrator, owing to a blank injection between samples.
• Robust reproducibility demonstrated by > 2 000 sequential injections with CV of 1.67% for gabapentin and 2.65% for pregabalin.

Benefits and Practical Applications

This methodology offers considerable advantages:

• High-throughput analysis for large-scale clinical or toxicological studies.
• Reduced solvent usage and minimal sample handling.
• Rapid turnaround supporting same-day reporting in diagnostic laboratories.
• Scalability for use in pharmacokinetic, compliance, and forensic testing.

Future Trends and Potential Applications

Emerging directions include expanding the platform to other polar pharmaceuticals and biomarkers, further automation of sample handling, integration with laboratory information management systems (LIMS), and potential coupling with high-resolution MS for broader screening panels. Continued miniaturization and optimization may reduce cycle times further, enhancing applicability in point-of-care environments.

Conclusion

The Agilent RapidFire SPE/MS/MS method enables ultrafast, accurate, and precise quantification of gabapentin and pregabalin in urine. With a 25-second cycle time and performance on par with conventional LC/MS/MS, this approach delivers a high-throughput solution that can transform routine drug monitoring workflows without compromising data quality.