Fast, Reproducible LC-MS/MS Analysis of Dextromethorphan and Dextrorphan

Significance of the Topic

Quantitative analysis of dextromethorphan and its primary metabolite dextrorphan in plasma is crucial for pharmacokinetic profiling and metabolic phenotyping. Accurate measurement at low concentrations provides insights into enzyme activity, drug–drug interactions, and individual variability in drug metabolism. This information supports the development of safer and more effective therapeutics.

Objectives and Study Overview

This study aimed to establish a rapid, sensitive, and reproducible LC-MS/MS workflow for measuring dextromethorphan and dextrorphan in rat plasma. Key goals included achieving a lower limit of quantification (LLOQ) of 0.1 ng/mL, minimizing matrix effects, and ensuring high throughput through optimized sample preparation and chromatographic separation.

Methodology

The analytical workflow combined solid-phase extraction (SPE) with high-performance liquid chromatography and tandem mass spectrometry.

Sample Preparation:

• Matrix: Rat plasma spiked with calibrants (0.1–100 ng/mL) and internal standard (dextromethorphan-d3).
• SPE cartridge: SOLA™ CX (Thermo Fisher Scientific).
• Conditioning: Methanol followed by 0.1% formic acid in water.
• Loading: Plasma supernatant applied at 0.5 mL/min.
• Washing: 40:60 methanol/water to remove interferences.
• Elution: 5% ammonia in methanol (4 × 250 µL), drying under nitrogen, reconstitution in 50:50 acetonitrile/water.

Chromatographic Conditions:

• Instrument: Dionex UltiMate 3000 RSLC system.
• Column: Accucore® C18, 2.6 µm, 50 × 2.1 mm.
• Mobile phases: A) Water + 0.1% formic acid; B) Acetonitrile + 0.1% formic acid.
• Gradient: 5% B (0 min) to 95% B (1 min), return to 5% B by 1.01 min; total cycle 2 min.
• Flow rate: 1.4 mL/min; column temperature: 40 °C; injection volume: 2 µL.

Used Instrumentation

• SPE: SOLA CX cartridges/plates.
• Evaporation: UltraVap™ nitrogen manifold.
• LC: Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC.
• Column: Accucore™ C18 2.6 µm, 50 × 2.1 mm.
• MS: Thermo Scientific™ TSQ Vantage™ with HESI, positive ion mode.

Main Results and Discussion

Chromatography achieved sharp, symmetrical peaks for both analytes within a 2-minute runtime. Calibration was linear from 0.1 to 100 ng/mL (r2 > 0.998). Accuracy of back-calculated concentrations remained within ±14%, and precision across six replicates at three QC levels yielded CV ≤ 8%. Recovery was consistent (95.4% for dextromethorphan, 101.6% for dextrorphan). Matrix suppression was minimal (−0.5% and +7.4%, respectively), confirming effective clean-up and reliable quantification in complex plasma samples.

Benefits and Practical Applications

• High sensitivity (LLOQ 0.1 ng/mL) suitable for low-level exposure studies.
• Fast analytical cycle (2 min) supports high throughput.
• Robust SPE deliver reproducible extraction and low solvent consumption.
• Minimal matrix effects enhance confidence in bioanalytical results.
• Applicable to pharmacokinetic, toxicology, and metabolic phenotyping investigations.

Future Trends and Potential Applications

Emerging directions include automated SPE integration, expansion to multiplexed assays for additional metabolites or co-administered drugs, and coupling with high-resolution mass spectrometry for structural elucidation. Integration into digital laboratory workflows and miniaturized sample preparation platforms could further increase throughput and reduce costs.

Conclusion

The described LC-MS/MS method combining SOLA CX SPE and Accucore C18 chromatography provides a rapid, reproducible, and highly sensitive approach for quantifying dextromethorphan and dextrorphan in plasma. Its robust performance, minimal matrix interference, and short cycle time make it well-suited for both routine bioanalysis and research applications.

References

• Phipps K. Fast, Reproducible LC-MS/MS Analysis of Dextromethorphan and Dextrorphan. Application Note 20685. Thermo Fisher Scientific; 2013.