Development of an LC/MS/MS Method for Bupropion in Human Plasma Using a 6460 Triple Quadrupole LC/MS System

Significance of the Topic

Liquid chromatography tandem mass spectrometry (LC/MS/MS) is a cornerstone technique in bioanalysis, offering high sensitivity and specificity for drug quantitation in biological matrices. Successful transfer of validated methods between laboratories and instrument platforms ensures consistent data quality and regulatory compliance in pharmacokinetic and clinical research.

Objectives and Study Overview

The primary aim was to demonstrate the seamless transfer of a bupropion assay from a Shimadzu LC-20A/ABI-SCIEX API 4000 system to an Agilent 1290 Infinity/6460 Triple Quadrupole LC/MS system. Key performance metrics—linearity, precision, and accuracy—were assessed across five independent batches to verify equivalent analytical performance with minimal downtime.

Methodology and Instrumentation

Sample preparation involved protein precipitation of human plasma with methanol following addition of an internal standard (risperidone). Calibration curves ranged from 1.5 to 456 ng/mL with three QC levels (LQC, MQC, HQC). Data acquisition and quantitation were performed using Agilent MassHunter software.

• Liquid Chromatography: Agilent 1290 Infinity LC with Gemini C18 (50 × 3 mm, 5 µm) column at 40 °C; mobile phase 10 mM ammonium formate:acetonitrile (20:80 v/v); isocratic flow 0.8 mL/min (70% to MS), 2 min run.
• Mass Spectrometry: Agilent 6460 Triple Quadrupole with Jet Stream ESI in positive mode; MRM transitions m/z 239.8→183 (bupropion) and 411.1→190 (risperidone); optimized source parameters (dry gas 13 L/min, 350 °C; sheath gas 12 L/min, 350 °C; capillary 3000 V; fragmentor 135 V for bupropion, 90 V for IS; collision energy 7 eV for bupropion, 25 eV for IS).

Main Results and Discussion

The method exhibited excellent linearity (R2 ≥ 0.998) across ten calibration levels. The LLOQ (1.502 ng/mL) displayed a signal-to-noise ratio > 1600 and CV of 3.8%. Precision (%CV) for calibration standards ranged from 2.22% to 11.90%, and accuracy from 94.2% to 108.3%. QC samples across all levels showed precision of 6.72%–7.69% and mean accuracy of 98.2%–103.9%. These metrics meet established bioanalytical validation criteria, confirming robust transfer without modification of LC conditions.

Benefits and Practical Applications

Transferring the bupropion assay with minimal adjustments demonstrates the flexibility of modern LC/MS platforms and streamlines method implementation in multi-site studies. Labs can leverage existing protocols to reduce development time, ensure data consistency, and support regulatory submissions in drug development and therapeutic monitoring.

Future Trends and Applications

• Automation of method transfer workflows to further reduce instrument downtime and human error.
• Expansion to multiplex assays for simultaneous quantitation of drug metabolites.
• Integration with high-throughput sample preparation to support larger clinical trials.
• Standardization initiatives across vendors to facilitate cross-platform data comparability.

Conclusion

A bioanalytical LC/MS/MS method for bupropion quantitation was successfully transferred from one vendor’s platform to another with equivalent sensitivity, precision, and accuracy. Optimizing source parameters on the Agilent 6460 system enabled rapid adoption while preserving the original chromatographic and MRM settings, underscoring the method’s robustness and adaptability.

References

1. Zhou S et al. Critical Review of Development, Validation, and Transfer for High Throughput Bioanalytical LC-MS/MS Methods. Current Pharmaceutical Analysis. 2005;1:3–14.
2. Bansal S, DeStefano A. Key Elements of Bioanalytical Method Validation for Small Molecules. The AAPS Journal. 2007;9(1):Article 11.