Rapid, Sensitive and Direct Quantitation of Tiotropium at sub-pg/mL in Plasma using Shimadzu LCMS-8060NX

Significance of the Topic

Tiotropium bromide is a long-acting anticholinergic agent used in COPD maintenance therapy. Its low systemic bioavailability challenges the quantification of trace plasma levels in pharmacokinetic and bioequivalence studies. A robust ultra-sensitive assay is crucial to accurately monitor drug exposure and support clinical development.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive, and direct quantitative method for measuring tiotropium in human plasma at concentrations down to sub-picogram per milliliter levels using a Shimadzu LCMS-8060NX coupled with Nexera X2 UHPLC. The method targets reliable bioanalytical performance in selectivity, linearity, precision, accuracy, and matrix effect.

Methodology

Human plasma samples were spiked with tiotropium and internal standard, then extracted using solid phase extraction cartridges conditioned with methanol and water. Analytes were eluted with 50% methanol and analyzed under gradient chromatography at 0.2 mL/min on a C18 column. Detection employed multiple reaction monitoring in positive electrospray mode.

Applied Instrumentation

Shimadzu Nexera X2 UHPLC system with a Shim-pack Velox C18 column (100×2.1 mm, 2.7 μm); Shimadzu LCMS-8060NX triple quadrupole mass spectrometer; electrospray ionization source; nebulizing gas at 3 L/min; drying gas at 10 L/min; desolvation line at 200 °C; heat block at 500 °C; interface at 400 °C.

Main Results and Discussion

The assay showed no significant interferences across six plasma lots. Calibration was linear from 0.20 to 200 pg/mL (r² > 0.99) with 1/x² weighting. Intra- and inter-day precision at LLOQ (0.20 pg/mL) and QC levels yielded accuracy within 103–113% and RSD below 18.6%. Matrix factor ranged 0.77–0.93, indicating acceptable ion suppression. Overall recovery averaged 57.6% (RSD 8.7%) and no carry-over was detected.

Benefits and Practical Applications

This high-throughput method combines ultra-sensitivity with minimal sample volume and simple preparation. It is ideal for pharmacokinetic profiling, bioequivalence assessments, and therapeutic drug monitoring of inhaled anticholinergics, ensuring reliable data at trace concentrations.

Future Trends and Potential Applications

Advancements may include microflow LC-MS platforms for enhanced sensitivity, automated sample handling, high-resolution mass spectrometry for metabolite identification, and integration with clinical diagnostics to facilitate real-time pharmacokinetic studies.

Conclusion

The validated LC-MS/MS assay on Shimadzu LCMS-8060NX provides a rapid, sensitive, and reproducible approach for quantifying tiotropium in human plasma at sub-pg/mL levels, supporting demanding bioanalytical applications.

References

• DrugBank. Tiotropium (DB01409). Accessed Jan 02, 2020.
• Wikipedia. Tiotropium bromide. Accessed Jan 02, 2020.