A Sensitive Microflow LC-MS/MS Method for the Analysis of Fluticasone Propionate in Human Plasma

Importance of the Topic

Fluticasone propionate is an inhaled glucocorticoid used for asthma prophylaxis at low doses. Accurate measurement in plasma requires ultra sensitive assays capable of detecting concentrations below 10 pg/mL. Microflow LC-MS/MS methods with trap-and-elute configurations can provide the necessary sensitivity to define pharmacokinetics at sub picogram levels.

Study Objectives and Overview

The study aimed to develop and validate a microflow LC-MS/MS assay for quantifying fluticasone propionate in human plasma at sub pg/mL levels. By integrating a two-dimensional trap-and-elute approach on the ionKey/MS system, the goal was to improve sample cleanup, increase injection volume, and achieve a lower limit of quantification below 1 pg/mL.

Used Instrumentation

• Waters ionKey/MS System
• Xevo TQ-S Tandem Quadrupole Mass Spectrometer
• ACQUITY UPLC M-Class System
• iKey Separation Device 150 µm × 50 mm, 1.8 µm HSS T3
• Sep-Pak C18 Plate for Solid Phase Extraction
• MassLynx Software for Data Acquisition
• TargetLynx Software for Quantification

Methodology and Sample Preparation

Calibration standards (0.244–250 pg/mL) and QC samples (1.5, 10, 100 pg/mL) were prepared by spiking fluticasone propionate into human plasma. Protein precipitation was performed with zinc sulfate followed by solid phase extraction on a Sep-Pak C18 plate. The eluate was evaporated and reconstituted in 25% methanol. Chromatography employed a trap-and-elute configuration on an iKey UPLC HSS T3 column with a gradient from 40 to 95% organic at 3 µL/min and 50°C. MS/MS detection used ESI+, monitoring transitions 501.3>313.2 for fluticasone and 504.3>313.2 for the d3 internal standard.

Main Results and Discussion

The assay achieved an LOD of 0.244 pg/mL and an LLOQ of 0.488 pg/mL. Calibration curves were linear from 0.488 to 250 pg/mL with r2 greater than 0.99 and accuracy within 98–101% (CV ≤8%). Robustness testing over 3000 injections showed stable system pressure and consistent peak area ratios with RSD under 8%.

Benefits and Practical Applications

This microflow LC-MS/MS approach enables ultra-trace quantification of inhaled drugs in clinical pharmacokinetic studies. The trap-and-elute design enhances sensitivity, reduces solvent consumption, automates cleanup, and protects the analytical column, making it suitable for high-throughput bioanalysis.

Future Trends and Opportunities

Advances in microflow chromatography may include further miniaturization, integration with high-resolution mass spectrometry, and automated sampling for real-time monitoring. Expansion toward multiplexed assays can support broader metabolite and biomarker profiling in clinical and environmental studies.

Conclusion

A robust microflow LC-MS/MS method using the ionKey/MS system in trap-and-elute mode has been validated for fluticasone propionate in human plasma. It delivers the required sensitivity, linearity, and reproducibility for sub pg/mL quantification in pharmacokinetic applications.

References

1. Mather J, Rainville P, Graham K, Plumb R. A high sensitivity UPLC-MS/MS method for fluticasone propionate in plasma. Waters Application Note, 2009.
2. Waters Corporation. Using neutral and basic mobile phases in ACQUITY UPLC M-Class and ionKey/MS systems. P/N 715005272.
3. Roman GT, Johnson J, Murphy JP, Alelyunas Y, Wrona M. Achieving sensitivity, throughput, and robustness with ionKey/MS and trapping. Waters White Paper, 2016.