Rapid, Highly Sensitive and Direct Quantification of Fluticasone Propionate at Sub-pg/mL in Plasma Using LCMS-8060

Importance of the Topic

Fluticasone propionate is a widely used synthetic corticosteroid for treating respiratory conditions such as allergic rhinitis and asthma. Due to its low systemic exposure following therapeutic administration, plasma concentrations often fall below traditional detection limits. Developing a rapid, sensitive, and robust bioanalytical method is critical for accurate pharmacokinetic profiling, supporting drug development, dose optimization, and safety monitoring in clinical studies.

Study Objectives and Overview

This work describes the development and partial validation of an ultra-sensitive LC–MS/MS assay using the Shimadzu LCMS-8060 system. The primary aim was to achieve a lower limit of quantification (LLOQ) of 0.2 pg/mL for fluticasone propionate in human plasma, while streamlining sample preparation and maximizing throughput for high-throughput pharmacokinetic investigations.

Methodology and Instrumentation

• Sample Preparation: Single-step solid-phase extraction (SPE) from 500 µL of K2EDTA plasma using a reversed-phase cartridge, washed with 20% acetonitrile, eluted with 50% methanol, and direct injection into the LC–MS/MS system.
• Chromatography: Shim-pack GIST C18 column (2.1×50 mm, 3 μm) at 50 °C; gradient elution with 0.1% ammonia in water (A) and acetonitrile (B) at 0.5 mL/min.
• Mass Spectrometry: ESI in positive mode on LCMS-8060; multiple reaction monitoring (MRM) transitions m/z 501.0→293.05 for fluticasone propionate and m/z 506.0→313.10 for internal standard (fluticasone propionate-D5); optimized collision energy at –20 V.
• Validation Criteria: Partial validation following US FDA guidelines for selectivity, linearity, precision, accuracy, recovery, matrix effect, and stability (bench-top, autosampler, freeze–thaw).

Key Results and Discussion

• Linearity: 0.20–120.00 pg/mL with r² > 0.99 (weighted 1/x²), demonstrating consistent response across the range.
• Precision and Accuracy: Intra-day RSD ≤ 16.33% at LLOQ QC and ≤ 7.41% at higher QC levels; inter-run RSD ≤ 18.33% at LLOQ QC and ≤ 11.14% for LQC, MQC, HQC; accuracy within ±20% at LLOQ and ±15% at other levels.
• Recovery: Mean absolute recovery ~64% across QC levels with RSD 7.26%, confirming reproducible extraction efficiency.
• Matrix Effect: Factor range 0.95–1.06 for analyte and internal standard, indicating negligible suppression or enhancement.
• Selectivity and Carry-Over: Interference in blank plasma <10% of LLOQ signal; no detectable carry-over following highest calibrator injection.

Benefits and Practical Applications

• Exceptional Sensitivity: Achieves LLOQ of 0.2 pg/mL, outperforming previous assays (1 pg/mL).
• High Throughput: Simplified SPE and direct injection reduce sample preparation time and increase laboratory productivity.
• Robust Performance: Compliance with regulatory guidelines ensures reliability for clinical and preclinical pharmacokinetic studies.

Future Trends and Applications

• Broader Bioanalysis: Adaptation of this approach to other low-dose pharmacological agents requiring ultra-trace quantification.
• Automation and Miniaturization: Integration with robotic platforms and microextraction techniques to further enhance throughput and reduce sample volumes.
• Data-Driven Optimization: Leveraging machine learning for predictive method development and real-time quality control monitoring.

Conclusion

The described LC–MS/MS method on the Shimadzu LCMS-8060 platform provides a rapid, highly sensitive, and streamlined workflow for quantifying fluticasone propionate at sub-picogram levels in human plasma. Its validated performance makes it an excellent tool for supporting high-throughput pharmacokinetic and bioequivalence studies in pharmaceutical research.

References

1. FDA. Bioanalytical Method Validation Guidance for Industry, 2018.
2. Ji A.J. et al. Ultrasensitive and automated 1 pg/mL fluticasone propionate assay in human plasma using LC–MS/MS. Bioanalysis. 2013;5(4):423–435.