Sensitive Quantitation of Glucagon-Like Peptide-1 (GLP-1) Analog Tirzepatide in Plasma

Importance of the topic

The rise of glucagon like peptide 1 receptor agonists such as tirzepatide for the treatment of obesity and type 2 diabetes has created a critical need for sensitive and specific bioanalytical assays. Accurate quantitation of these large peptide therapeutics in plasma supports pharmacokinetic and toxicokinetic studies that drive drug discovery and regulatory approval.

Objectives and study overview

This work presents an automated LC MS workflow combining Agilent AssayMAP Bravo sample preparation, 1290 Infinity II bio UHPLC, and 6495D triple quadrupole mass spectrometry to measure tirzepatide in human plasma. The aim was to develop a rapid method without the need for immunoaffinity reagents that achieves a low limit of quantitation and a wide linear range.

Methodology and instrumentation

Sample preparation was performed on the Agilent AssayMAP Bravo platform with the following steps

• Protein precipitation using a 1 1 mixture of acetonitrile and methanol spiked with semaglutide as internal standard
• Automated purification on AssayMAP RP S cartridges with conditioning equilibrating loading washing and elution steps
• Reconstitution of dried eluates and injection into LC MS

Used instrumentation

• Agilent AssayMAP Bravo protein sample prep platform
• Agilent 1290 Infinity II bio high speed pump multisampler and column thermostat
• Agilent 6495D triple quadrupole LC MS with Jet Stream electrospray
• Agilent AdvanceBio Peptide Mapping column for chromatographic separation

Main results and discussion

An extensive optimization was carried out to maximize recovery sensitivity and reproducibility

1. Precipitation solvent composition was compared including pure acetonitrile pure methanol and a 1 1 mixture with the latter providing the best peptide extraction
2. Loading buffer experiments showed that pure water offered optimal binding to the RP S cartridge
3. Washing conditions were optimized to an aqueous ammonium formate buffer with 10 acidified acetonitrile to remove interferences
4. Elution was most effective using 80 acetonitrile containing 5 formic acid

The optimized method achieved a lower limit of quantitation of 0.05 ng/mL using 100 uL of plasma and maintained linearity up to 1000 ng/mL with correlation coefficients above 0.999. Intra and interday precision and accuracy across low mid and high quality control levels met regulatory acceptance criteria of ±15.

Benefits and practical applications

This automated LC MS workflow provides

• High sensitivity and specificity without immunoassay reagents
• Rapid method development and high throughput capability
• Wide dynamic range to support pharmacokinetic profiling
• A universal approach applicable to other GLP 1 analogs

Future trends and possibilities

Advances in bioanalytical technologies may include integration of microflow LC MS for further sensitivity gains higher throughput robotic sample handling and broader multiplexing of peptide therapeutics. The method can be extended to new peptide modalities and adapted for clinical safety and efficacy monitoring.

Conclusion

The described automated LC MS platform delivers robust quantitation of tirzepatide in human plasma with excellent sensitivity accuracy and precision. Eliminating the need for antibody reagents accelerates assay setup and supports drug discovery and development efforts for GLP 1 based therapies.

Reference

• Mounjaro tirzepatide injection solution DailyMed 13 May 2022
• Zepbound tirzepatide injection solution DailyMed 9 Nov 2023