A RAPID METHOD FOR THE ULTRA-SENSITIVE QUANTIFICATION OF FLUTICASONE PROPIONATE AND SALMETEROL XINAFOATE FROM HUMAN PLASMA

Significance of the Topic

Fluticasone propionate and salmeterol xinafoate are inhaled drugs widely used to manage asthma and COPD. Their systemic absorption leads to circulating concentrations at sub-picogram levels, necessitating ultra-sensitive quantification methods. Accurate measurement at these low levels supports pharmacokinetic studies, therapeutic monitoring and safety assessments.

Objectives and Overview of the Study

This work aims to establish a rapid, high-throughput LC-MS/MS assay for simultaneous quantification of fluticasone propionate and salmeterol xinafoate in human plasma, achieving detection limits of 0.1 pg/mL and 0.05 pg/mL respectively. The study builds on existing sample preparation protocols and optimizes chromatographic and mass spectrometric parameters to enhance sensitivity and reliability.

Methodology and Instrumentation

Sample Preparation:

• Plasma volume: 400 µL.
• Protein dissociation by mixing with 400 µL of 40:60 ZnSO4 (0.1 M) to 10% NH4OH solution.
• Solid-phase extraction using Oasis PRiME HLB µElution plates without conditioning steps.
• Wash: 200 µL of 50:50 methanol:water.
• Elution: two 25 µL aliquots of 10:90 isopropanol:methanol.

Chromatography and Mass Spectrometry:

• UPLC system: ACQUITY UPLC I-Class.
• Column: ACQUITY UPLC BEH C18, 2.1 × 50 mm, 1.7 µm, 60 °C.
• Mobile phases: A—10% ammonium hydroxide in water; B—10:90 isopropanol:methanol.
• Flow rate: 0.3 mL/min; gradient: 50:50 to 5:95 (A:B).
• Injection volume: 50 µL; sample temperature: 5 °C.
• Mass spectrometer: Waters Xevo TQ-XS tandem quadrupole with ESI+.
• MRM transitions: fluticasone propionate 501.3 → 293.3; salmeterol xinafoate 416.4 → 232.2.

Main Results and Discussion

The method demonstrated linear calibration curves over 0.1–10 pg/mL for fluticasone propionate and 0.05–5 pg/mL for salmeterol xinafoate, with r2 > 0.99. Lower limits of quantification reached 0.1 pg/mL and 0.05 pg/mL respectively. Intra- and inter-day precision (CV < 10%) and accuracy (85–115%) met bioanalytical validation criteria. Matrix effects were below 1% and recoveries exceeded 90%. Optimization of wash and elution solvents, column selection (BEH C18) and flow rate (300 µL/min) yielded sharp peaks (peak widths ≤12 s) and high signal-to-noise ratios.

Benefits and Practical Applications of the Method

• Ultra-sensitive quantification supports detailed pharmacokinetic profiling at trough levels.
• Rapid and simple µElution SPE reduces preparation time and solvent consumption.
• High throughput suitability for clinical and research laboratories.
• Robust performance with minimal matrix interference enhances reliability in QA/QC settings.

Future Trends and Opportunities

Advances may include automation of sample preparation to increase throughput, integration of microflow LC to further reduce solvent use, and application of high-resolution mass spectrometry for broader metabolite coverage. Adaptation of the workflow to other low-abundance inhaled or oral compounds can extend its utility in drug monitoring and pharmacodynamic studies.

Conclusion

A streamlined LC-MS/MS method combining Oasis PRiME HLB µElution and UPLC-MS/MS achieves femtogram-level detection of fluticasone propionate and salmeterol xinafoate in human plasma. The validated assay offers high sensitivity, precision and throughput, making it well suited for clinical pharmacokinetics and routine bioanalysis.

Used Instrumentation

• ACQUITY UPLC I-Class system.
• Oasis PRiME HLB 96-well µElution plates.
• ACQUITY UPLC BEH C18 column, 2.1 × 50 mm, 1.7 µm.
• Waters Xevo TQ-XS triple quadrupole.

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