Highly sensitive quantitative analysis of budesonide from plasma using LC/MS/MS

Significance of the Topic

Budesonide is a potent inhaled glucocorticoid used in asthma, allergic rhinitis and dermatological conditions. Its low oral bioavailability (≈10 %) and the presence of two non-interconverting epimers (22R more active than 22S) necessitate highly sensitive quantitative assays in plasma for pharmacokinetic and bioequivalence studies.

Objectives and Study Overview

This work aimed to develop and validate an ultra-sensitive LC/MS/MS method for quantifying budesonide in human plasma. Using Shimadzu’s LCMS-8060 triple quadrupole mass spectrometer and UHPLC separation, the study targeted a lower limit of quantitation (LLOQ) of 2 pg/mL to support bioanalytical applications.

Methodology

Sample Preparation

• Calibration standards: 2–200 pg/mL in plasma; QC levels: LQC 7.5 pg/mL, MQC 75 pg/mL, HQC 175 pg/mL.
• Internal standard: budesonide-D8 added to all samples except blanks.
• Centrifugation: 4 mL plasma aliquots spun at 4 500 rpm for 5 min.
• Solid phase extraction (SPE): conditioning (1 mL MeOH, 1 mL H2O), loading, washing (1 mL H2O, 1 mL 5 % MeOH), elution (1 mL MeOH).
• Evaporation at 50 °C under nitrogen; reconstitution in 200 µL mobile phase.

Instrumentation Used

• UHPLC: Shimadzu Nexera X2 with C18 column (100 × 3 mm, 2.1 µm).
• Mass spectrometer: Shimadzu LCMS-8060 triple quadrupole with heated ESI probe.
• Mobile phase: aqueous buffer (A) and acetonitrile (B); gradient from 40 %–90 % B over 4 min; flow 0.4 mL/min; injection 20 µL; column 40 °C.
• ESI settings: nebulizing gas 3 L/min, drying gas 10 L/min, heating gas 10 L/min; DL 250 °C, heat block 400 °C, interface 300 °C.
• MRM transitions: budesonide m/z 431.10 > 323.20; budesonide-D8 m/z 439.10 > 323.20.

Main Results and Discussion

The assay achieved an LLOQ of 2 pg/mL for budesonide in plasma with no blank interference. Calibration was linear from 2 to 200 pg/mL (r = 0.9992). Accuracy for calibration standards ranged 94.5–104.0 %, and QC precision (n=6) showed %RSD ≤ 6.4 and accuracy 96.9–101.0 %. The heated ESI probe significantly enhanced desolvation, ionization efficiency, and reduced background noise.

Benefits and Practical Application

• Enables reliable quantitation at low picogram levels, suitable for pharmacokinetic, bioequivalence, and therapeutic drug monitoring studies.
• High throughput: UHPLC run time ~4 min supports large sample batches.
• Robust accuracy and precision meet regulatory bioanalytical criteria.

Future Trends and Possibilities

Advances may include microflow LC-MS for reduced solvent use and increased sensitivity, incorporation of automated sample preparation and on-line SPE, and application of high-resolution mass spectrometry for simultaneous multi-analyte profiling. Emerging ionization sources and AI-driven data analysis could further enhance assay performance and throughput.

Conclusion

A highly sensitive, rapid and robust LC/MS/MS method for budesonide quantitation in plasma was validated on the Shimadzu LCMS-8060 platform. The developed assay meets stringent bioanalytical requirements and is suitable for detailed pharmacokinetic investigations.

References

1. Deventer K., Mikulcikova P., Van Hoecke H., Van Eenoo P., Delbeke F.T. Detection of budesonide in human urine after inhalation by liquid chromatography–mass spectrometry. J. Pharm. Biomed. Anal. 2006;42:474–479.
2. Streel B., Cahay B., Klinkenberg R. Using total error concept for the validation of liquid chromatography-tandem mass spectrometry method for the determination of budesonide epimers in human plasma. J. Chromatogr. B.