A Rapid Method for the Ultra-Sensitive Quantification of Fluticasone Propionate and Salmeterol Xinafoate from Human Plasma

Significance of the topic

The accurate measurement of inhaled corticosteroids and long-acting beta-agonists at sub-picogram-per-milliliter levels is critical for pharmacokinetic studies, therapeutic monitoring, and safety assessment in asthma and COPD management. Ultra-sensitive assays enable reliable detection of trough concentrations, support dose optimization, and contribute to a deeper understanding of systemic exposure from inhaled therapies.

Objectives and study overview

This study presents the development and validation of a rapid, high-throughput workflow for simultaneous quantification of fluticasone propionate and salmeterol xinafoate in human plasma. The goal was to achieve lower limits of quantification (LLOQs) of 0.1 pg/mL for fluticasone propionate and 0.05 pg/mL for salmeterol xinafoate, improving upon previously reported assays.

Methodology and instrumentation

Sample preparation

• Plasma (400 µL) was diluted with 400 µL ZnSO₄/ammonium hydroxide solution (40:60 v/v ZnSO₄:10% NH₄OH) to dissociate protein-bound analytes.
• An Oasis PRiME HLB 96-well µElution plate was used for SPE without conditioning steps.
• Wash solvent: 200 µL of 50:50 methanol/water.
• Elution: two aliquots of 25 µL 10:90 isopropanol/methanol, followed by dilution with 50 µL water.

Chromatography and mass spectrometry

• System: ACQUITY UPLC I-Class PLUS with BEH C18 column (1.7 µm, 2.1×50 mm) at 60 °C.
• Mobile phases: A = 0.1% ammonium hydroxide in water; B = 10:90 isopropanol/methanol.
• Gradient: 50:50 A:B for 1 min, ramp to 5:95 at 3 min, hold, then re-equilibrate; flow rate 0.3 mL/min; injection 10 µL.
• Detection: Xevo TQ-XS triple quadrupole, ESI positive mode. MRM transitions: fluticasone propionate 501.3 → 293.3; salmeterol xinafoate 416.4 → 232.2; internal standard fluticasone-d₃ 504.3 → 293.2.
• Optimized source: capillary 1 kV, cone voltage 30 V, desolvation 500 °C, gas flows: cone gas 150 L/h, desolvation gas 1000 L/h, nebulizer 7 bar.

Key results and discussion

The optimized SPE method delivered matrix effects below 1% and recoveries above 90% for both analytes. Flow rate optimization (100–500 µL/min) indicated 300 µL/min provided optimal peak shape and sensitivity. The assay achieved LLOQs of 0.1 pg/mL (fluticasone) and 0.05 pg/mL (salmeterol) with linear calibration ranges of 0.1–10 pg/mL and 0.05–5 pg/mL, respectively (R2 > 0.99, 1/x weighting). Inter- and intra-day precision (%RSD) and accuracy (% bias) met bioanalytical criteria, with all QC levels demonstrating %RSD < 10% and mean accuracy within 85–115%.

Benefits and practical applications

• The µElution SPE format streamlines sample cleanup, eliminates conditioning, and reduces solvent use.
• High sensitivity supports pharmacokinetic and transthorough concentration studies for inhaled drugs.
• The 96-well format and short UPLC run time enable high throughput in routine bioanalysis and clinical trials.

Future trends and opportunities

Advances may include further miniaturization of sample volumes, automation for large-scale studies, expansion to other low-dose inhaled compounds, integration with high-resolution or hybrid mass spectrometers for enhanced selectivity, and real-time monitoring capabilities in therapeutic drug management.

Conclusion

A robust, ultra-sensitive UPLC-MS/MS method for fluticasone propionate and salmeterol xinafoate quantification in human plasma was developed. The workflow combines simplified SPE, rapid chromatography, and a state-of-the-art triple quadrupole detector to deliver reliable detection at sub-pg/mL levels, meeting stringent requirements for PK and clinical bioanalysis.

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