Sensitive Quantitation of Glucagon-like peptide-1 (GLP-1) Analog Tirzepatidein Plasma Using Automated LC-MS/MS workflow

Significance of the Topic

Glucagon-like peptide-1 receptor (GLP-1R) agonists such as tirzepatide have transformed obesity and type 2 diabetes management. Accurate quantitation of these peptides in plasma is essential for pharmacokinetic and toxicokinetic evaluation during drug development.

Objectives and Study Overview

This study aimed to develop an automated LC-MS/MS workflow for sensitive and reproducible quantitation of tirzepatide in human plasma. It compared traditional organic solvent precipitation with AssayMAP RP-S cartridge cleanup to enhance assay performance.

Methodology

A 100 µL plasma aliquot fortified with tirzepatide and semaglutide (internal standard) underwent protein precipitation using a 1:1 acetonitrile:methanol mixture. After centrifugation and drying, samples were reconstituted, then processed on the AssayMAP Bravo platform with RP-S cartridges. Key steps included:

• Conditioning cartridges with 80% acetonitrile and water.
• Loading samples in water at 3 µL/min.
• Washing with water and 10% acetonitrile in 10 mM ammonium formate (pH 10).
• Elution with 5% formic acid in 80% acetonitrile.

The final eluate was analyzed by UHPLC on an AdvanceBio Peptide Mapping column (2.1×150 mm, 2.7 µm) at 80 °C coupled to an Agilent 6495D triple quadrupole mass spectrometer. MRM transitions 1204.4→396.2 and 1204.4→299.2 were used for tirzepatide quantitation.

Instrumentation Used

• AssayMAP Bravo automated sample preparation platform.
• Agilent 1290 Infinity II Bio UHPLC system with AdvanceBio Peptide Mapping column.
• Agilent 6495D Triple Quadrupole LC/TQ mass spectrometer.

Key Results and Discussion

• The optimized precipitation solvent (ACN:MeOH 1:1) maximized peptide recovery.
• Pure water was the best loading buffer; 10% ACN in ammonium formate (pH 10) provided optimal wash conditions.
• Elution with 5% formic acid in 80% ACN delivered the highest sensitivity.
• Assay sensitivity improved fivefold over precipitation alone, achieving an LLOQ of 0.05 ng/mL.
• Calibration curves were linear (R² > 0.996) from 0.05 to 1000 ng/mL, covering 4.5 orders of magnitude.
• Intra- and inter-day precision and accuracy met regulatory criteria (≤20% at LLOQ, ≤15% at other QC levels).

Benefits and Practical Applications

The automated workflow reduces manual steps and variability, enabling high-throughput, reliable bioanalytical support for clinical and preclinical tirzepatide studies.

Future Trends and Potential Applications

As GLP-1 analog therapies expand, combining automated platforms with high-sensitivity MS will facilitate personalized dosing and multiplexed peptide panels. Ongoing miniaturization and method integration will further enhance throughput and data quality.

Conclusion

This automated LC-MS/MS assay delivers robust, sensitive, and reproducible quantitation of tirzepatide in plasma, supporting comprehensive pharmacokinetic assessments in drug development.

References

1. Mounjaro (tirzepatide) injection, solution. DailyMed. May 13, 2022.
2. Zepbound (tirzepatide) injection, solution. DailyMed. Nov 9, 2023.