Differential Mobility Separation with SCIEX SelexION®- A Novel Technique for the Bioanalysis of Poorly Fragmenting Molecules like Valproic Acid

Significance of the Topic

Valproic acid (VPA) is a widely used antiepileptic agent whose straightforward structure yields poor mass spectrometric fragmentation. This limitation poses challenges for sensitive and selective quantitation in biological matrices. Developing a robust bioanalytical approach for VPA can support pharmacokinetic investigations and therapeutic monitoring.

Objectives and Overview of the Study

This work demonstrates the application of differential mobility separation, using the SCIEX SelexION® device, to improve selectivity, reduce background noise, and enhance the limit of quantitation for VPA in human plasma. The method covers a calibration range of 50–8000 ng/mL and aims to streamline sample preparation and chromatographic requirements.

Methodology

• Sample preparation: protein precipitation with methanol in spiked human plasma, centrifugation, and direct injection of the supernatant.
• Chromatography: reversed-phase C18 column (Atlantis dC18 100×2.1 mm, 3 µm) at 50 °C; gradient elution with ammonium acetate and methanol at 0.5 mL/min.
• Mass spectrometry: Q1 multiple ion monitoring of the [M–H]– ion (m/z 143.1) without conventional fragmentation, augmented by differential mobility separation in infusion mode to maximize analyte signal and remove interferences.

Used Instrumentation

• SCIEX Triple Quad™ 5500 LC–MS/MS system
• SCIEX SelexION® differential mobility separation device
• Turbo V™ ion source

Main Results and Discussion

Differential mobility separation provided an orthogonal separation step before the mass analyzer, leading to:

• A reduction of baseline chemical noise from approximately 7.5×10⁵ to 2×10³ counts per second.
• Complete removal of an isobaric interference coeluting at 1.68 minutes in blank plasma.
• A fourfold improvement in signal-to-noise at the lower limit of quantitation (50 ng/mL).
• Linear calibration (r > 0.99) over 50–8000 ng/mL, with accuracy within ±10% and precision below 15% across quality control levels.

Benefits and Practical Applications

• Enhanced selectivity in the absence of stable product ions, enabling reliable quantitation of poorly fragmenting analytes.
• Reduced reliance on long chromatographic gradients or derivatization steps.
• Simplified protein precipitation workflow compatible with high-throughput bioanalysis.
• Robust performance suitable for regulated environments.

Future Trends and Potential Applications

The integration of differential mobility separation is expected to expand in bioanalytical assays for small molecules with limited fragmentation. Future developments may include:

• Automation of mobility optimization for multi-analyte panels.
• Combination with microflow or nanoLC formats to further reduce sample volume and enhance sensitivity.
• Broader adoption in clinical and veterinary pharmacokinetics to streamline method development.

Conclusion

SelexION-enhanced LC–MS/MS provides a powerful solution for quantifying valproic acid in plasma, overcoming intrinsic mass spectrometric limitations. The orthogonal mobility separation step delivers improved sensitivity, selectivity, and throughput, making it an attractive approach for complex bioanalytical challenges.

References

• Bhaskar Rao G, et al. Liquid chromatography–tandem mass spectrometry method for simultaneous determination of valproic acid and its ene-metabolites in epilepsy patient plasma. Journal of Pharmaceutical Analysis. 2016;6:112–116.