Low level quantitation of Fluticasone and Salmeterol from plasma using LC/MS/MS

Significance of the Topic

Combination therapies of fluticasone propionate and salmeterol are widely used to manage asthma and chronic obstructive pulmonary disease (COPD). Accurate measurement of these compounds at ultra-trace levels in plasma supports pharmacokinetic studies, therapeutic drug monitoring, and quality control. Implementing a robust LC/MS/MS assay with high sensitivity and minimal sample preparation addresses challenges posed by complex biological matrices and low endogenous concentrations.

Objectives and Study Overview

This study aimed to develop and validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of fluticasone and salmeterol in human plasma. Using the Shimadzu LCMS-8050 triple quadrupole system coupled to a UHPLC platform, the method targets lower limits of quantitation (LOQ) in the sub–picogram per milliliter range while ensuring accuracy, precision, and absence of matrix interference.

Methodology and Instrumentation

Sample Preparation:

• Aqueous calibration standards (0.5–200 pg/mL) were prepared in water–acetonitrile (1:1, v/v).
• Matrix-matched standards were generated by protein precipitation of plasma (1:1 with acetonitrile), followed by C18 solid‐phase extraction, evaporation, and reconstitution in water–acetonitrile (1:1, v/v).

Chromatographic and Mass Spectrometric Conditions:

• Column: Shim-pack XR-ODS (50×3 mm, 2.2 μm) with guard cartridge.
• Mobile Phase: 10 mM ammonium formate in water (A) and acetonitrile (B) with a gradient from 50% to 90% B over 3.5 minutes, total run time 7.5 minutes.
• Flow Rate: 0.3 mL/min; Column Temperature: 40 °C; Injection Volume: 50 μL.
• Mass Spectrometer: Shimadzu LCMS-8050 triple quadrupole with heated electrospray ionization (ESI) source.
• Heated ESI Probe: High-temperature gas merging with nebulizer spray for enhanced desolvation and ionization efficiency.
• MRM Transitions: Fluticasone m/z 501.00→293.05/313.10; Salmeterol m/z 416.00→232.20.

Main Results and Discussion

Linearity and Sensitivity:

• Excellent linearity was achieved over the calibration range for both compounds (r² > 0.996 for aqueous; r² > 0.998 for matrix‐matched).
• LOQ for fluticasone: 2 pg/mL (aqueous) and 5 pg/mL (plasma).
• LOQ for salmeterol: 0.5 pg/mL (aqueous) and 1 pg/mL (plasma).

Accuracy and Precision:

• Accuracy ranged from 87.7% to 117.2% for fluticasone and 94.3% to 109.5% for salmeterol across quality‐control levels.
• Precision (%RSD) remained below 14% for low concentrations and below 8% at higher levels.

Matrix Effects and Selectivity:

• No interfering peaks were observed in blank plasma extracts at the retention times of fluticasone (3.64 min) or salmeterol (2.44 min).

Benefits and Practical Applications

• The heated ESI probe significantly reduces chemical noise and boosts signal intensity, enabling ultra-trace quantitation.
• Minimal sample cleanup accelerates throughput and reduces potential analyte loss.
• The method supports pharmacokinetic profiling, bioequivalence studies, and clinical monitoring of inhaled corticosteroids and β2-agonists.

Future Trends and Potential Applications

• Integration of automated on‐line sample preparation to further increase throughput.
• Expansion to include additional inhaled therapies for comprehensive respiratory drug panels.
• Application of higher-resolution mass analyzers or ion mobility to enhance selectivity.
• Use in personalized medicine to optimize dosing regimens based on individual pharmacokinetics.

Conclusion

A highly sensitive and reproducible LC/MS/MS method for fluticasone and salmeterol in plasma was established using the Shimadzu LCMS-8050 system with heated ESI. The assay achieves sub–picogram LOQs, robust linearity, and minimal matrix interference, making it well suited for advanced pharmacokinetic and clinical research applications.

References

1. Shah RR et al. International Journal of PharmTech Research. 2011;3(3):1801–1806.
2. Krishnaswami S et al. Journal of Pharmaceutical and Biomedical Analysis. 2000;22:123–129.