Highly Sensitive and Selective Method for Estimation of Formoterol at Sub-pg/mL in Human Plasma Using Shimadzu LCMS-8060NX

Significance of the Topic

The accurate quantification of formoterol, a potent long-acting β2-agonist used in asthma and COPD management, is critical due to its low bioavailability and rapid pharmacokinetics. Ultra-sensitive measurement of plasma levels supports pharmacokinetic studies, dose optimization, and safety assessments.

Objectives and Overview of the Study

This work aimed to develop and partially validate a highly sensitive, selective, and high-throughput LC–MS/MS method for measuring formoterol at sub-pg/mL concentrations in human plasma, following international bioanalytical guidelines.

Methodology and Used Instrumentation

A one-step solid-phase extraction was performed on 200 µL plasma after protein precipitation with 20% methanol. Cartridges were conditioned with methanol and water, washed with dilute ammonia and water, then eluted with 10% acetonitrile. Chromatography employed a Shimadzu Shim-pack Velox C18 column (2.1×100 mm, 2.7 µm) with a gradient of 0.1% formic acid in 5 mM ammonium acetate (A) and 0.1% formic acid in acetonitrile (B), at 0.3 mL/min, 50 °C, 20 µL injection.
The MS detection used a Shimadzu LCMS-8060NX with positive ESI and MRM transitions m/z 345.35→149.10 for formoterol and 351→152 for the D6 internal standard. Source parameters (interface voltage 1 kV, temperatures 100–300 °C, gas flows) and collision energies were optimized for maximum sensitivity.

Main Results and Discussion

The method exhibited a linear calibration range of 0.2–100 pg/mL (r2 = 0.9967). Intra-day precision (%RSD) was ≤4.8% and accuracy 95.9–108.8%; inter-day precision ≤11.6% and accuracy 98.8–101.4%. Mean analyte recovery was ~62% with consistent performance across LQC, MQC, HQC levels. Matrix effects were minimal (IS-normalized factor ~1.03), confirming selectivity and robustness.

Benefits and Practical Applications of the Method

• Exceptionally low LLOQ (0.2 pg/mL) for micro-dose and PK studies.
• Short run time and streamlined sample preparation enable high throughput.
• Minimal plasma volume requirement reduces patient burden.
• Highly selective SPE cleanup reduces matrix interferences.

Future Trends and Applications

Emerging directions include integration of microflow LC to further lower sample consumption, automated on-line sample cleanup, and coupling with high-resolution MS for increased specificity. The method may be adapted for therapeutic drug monitoring, microdosing trials, and biomarker studies.

Conclusion

A rapid, sensitive, and selective LC–MS/MS assay for formoterol in human plasma was developed and partially validated. Its low LLOQ, strong precision, accuracy, and robustness make it well suited for supporting pharmacokinetic and bioequivalence studies.

References

• Gaikwad A, Atmakuri C, Arote Y, Kelkar J, Rasam P. Highly Sensitive and Selective Method for Estimation of Formoterol at Sub-pg/mL in Human Plasma Using Shimadzu LCMS-8060NX. Shimadzu Analytical (India) Application Note, Jul 2024.
• ChemSpider. Formoterol Structure. https://www.chemspider.com/Chemical-Structure.2340731.html (accessed Feb 13, 2024).