LC-MS/MS Method for the Quantification of Budesonide in Human Plasma

Significance of the Topic

Glucocorticoids such as budesonide play a critical role in anti-inflammatory therapy for asthma, allergic rhinitis, and dermatological conditions. Due to its low oral bioavailability, achieving reliable quantitation of budesonide at low picogram-per-milliliter levels in plasma is essential for accurate pharmacokinetic and bioequivalence assessments.

Objectives and Study Overview

This application note describes the development and validation of a sensitive LC-MS/MS assay using the SCIEX Triple Quad™ 5500+ – QTRAP® Ready system to quantify budesonide in human plasma across a concentration range of 2 to 1024 pg/mL.

Methodology and Experimental Procedures

• Sample Preparation
  • Human plasma samples (200 µL) spiked with budesonide standards and deuterated internal standard (budesonide-D8).
  • SPE cleanup on Phenomenex Strata-X RP cartridges: conditioning with methanol and water, sample load, wash with 5% methanol, and elution with 100% methanol.
  • Drying under vacuum and reconstitution in 40% acetonitrile for LC injection.
• Chromatography
  • Column: C18 (100 × 2.1 mm, 1.7 µm) at 50 °C.
  • Mobile phase A: 5 mM ammonium bicarbonate; B: acetonitrile; flow rate 0.5 mL/min.
  • Gradient: 40% B at 0 min to 95% B at 2 min, held to 3 min, returned to 40% B by 4 min.
  • Injection volume: 10 µL.
• Mass Spectrometry
  • SCIEX Triple Quad™ 5500+ – QTRAP® Ready in positive electrospray ionization.
  • MRM transitions: m/z 431.3→323.2 and 431.3→147.1 for budesonide epimers; 439.3→323.2 for the internal standard.
  • Source settings: curtain gas 30, temperature 600 °C, GS1 = 60, GS2 = 80, ion voltage 3000 V, dwell time 75 ms.
• Data Processing
  • Acquisition in Analyst® 1.7.1; quantitation via SCIEX OS-Q with 1/x²-weighted linear regression.

Main Results and Discussion

• Sensitivity and Linearity
  • LLOQ of 2 pg/mL achieved without significant matrix interference.
  • Calibration curve linear from 2 to 1024 pg/mL (r = 0.99).
• Precision and Accuracy
  • %CV ranged from 1.9% to 12.2%; accuracy between 95% and 106% across the calibration range.
• Robustness
  • SPE sample cleanup effectively minimized matrix effects.
  • Fast run time (<4 min) and stable performance with Turbo V™ source.

Benefits and Practical Applications

• High sensitivity supports low-level pharmacokinetic profiling.
• Wide dynamic range accommodates both therapeutic and trace-level measurements.
• Rapid sample preparation and chromatographic run increase laboratory throughput.

Future Trends and Opportunities

• Adoption of high-resolution mass spectrometry for enhanced metabolite confirmation.
• Automation of SPE workflows to improve consistency and throughput.
• Expansion toward multiplexed assays for respiratory biomarkers.

Conclusion

This validated LC-MS/MS method delivers selective, sensitive, and high-throughput quantitation of budesonide in human plasma, fulfilling stringent bioanalytical requirements for pharmacokinetic and clinical studies.

Reference

1. Collins BA, Dima MA, Ivosev G, Zhong F (2013) A high dynamic range pulse counting detection system for mass spectrometry. Rapid Commun Mass Spectrom. 28(2):209–216.