This blog explores the development and validation of a highly precise, accurate, and robust LC-MS/MS method for quantifying semaglutide in human plasma, addressing challenges, extraction techniques, validation results, and its application in clinical study.
Semaglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist used to improve glycemic control in type 2 diabetes mellitus, manage obesity, and reduce the risk of major adverse cardiovascular events in select adults. Due to its significant health benefits, particularly in enhancing insulin sensitivity and promoting weight loss, the market for Semaglutide has expanded rapidly in recent years. The global market size increased from $20.54 billion in 2023 to $23.07 billion in 2024, reflecting a compound annual growth rate (CAGR) of 12.3%.
Given the increasing clinical and commercial relevance of Semaglutide, we have developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method that ensures high precision, accuracy, and reproducibility for Semaglutide quantification.
Challenges and Solutions
The bioanalysis of Semaglutide presents unique challenges due to its high molecular weight, peptide structure, and tendency for carryover effects.
| Challenges | Solutions |
|---|---|
| Semaglutide is a long-acting GLP-1 analogue consisting of 31 amino acids with a high molecular weight of 4113 Da, making ionization challenging in a triple quadrupole mass spectrometer. | We optimized the MS parameters to selectively detect the quadruply-charged parent ion, ensuring higher ionization efficiency and improved detection. |
| Large peptides have low ionization efficiency in LC-MS/MS due to mass range limitations. | The MS settings were fine-tuned to ensure optimal signal intensity and improve peak response, leading to higher sensitivity. |
| Peptides are prone to adherence in analytical columns and LC systems, leading to residual contamination and inaccurate quantification. | A smaller-particle C18 column with an extended LC gradient was employed to eliminate carryover effects. Additionally, system wash procedures were optimized to prevent residual accumulation. |
Extraction and Analytical Method
The method for quantifying Semaglutide involves protein precipitation followed by solid-phase extraction using an isotope-labeled internal standard (ISTD). Chromatography was achieved using a 50 mm C18 column with a mobile phase consisting of formic acid and an organic solvent, with a total run time of 13.0 minutes.
This method was validated for linearity, stability, carryover effects, reinjection reproducibility, precision, accuracy, and selectivity. The linearity ranged from 2 to 100 ng/mL. The method demonstrated excellent precision and accuracy at all concentration levels.
Global statistics of Three precision and accuracy run
The table below presents the global statistical summary from three independent precision and accuracy runs:
| DQC(1/5) | HQC | MQC | LMQC | LQC | LOQQC | |
|---|---|---|---|---|---|---|
| Mean (pg/mL) | 314.3757 | 83.9325 | 42.4444 | 12.6953 | 6.0477 | 1.9850 |
| SD± | 12.84830 | 5.51510 | 1.67288 | 0.47119 | 0.24385 | 0.12994 |
| Precision (%CV) | 4.1 | 6.6 | 3.9 | 3.7 | 4.0 | 6.5 |
| Nominal value (pg/mL) | 299.634 | 79.684 | 42.498 | 12.750 | 5.950 | 2.023 |
| Accuracy % | 104.9 | 105.3 | 99.9 | 99.6 | 101.6 | 98.1 |
| n | 18 | 18 | 18 | 18 | 18 | 18 |
Method Validation Parameters
To ensure compliance with regulatory guidelines, the method was validated across multiple parameters, including:
- Selectivity and Sensitivity: The method effectively differentiated Semaglutide from endogenous plasma components, ensuring high specificity and no matrix interference.
- Linearity: The method demonstrated an excellent linear range of 2–100 ng/mL, with correlation coefficients (r²) consistently >0.99.
- Precision and Accuracy: All QC samples exhibited %CV values below 10%, confirming high precision and accuracy.
- Stability: Semaglutide exhibited benchtop stability for 16 hours, freeze-thaw stability for five cycles, and interim long-term stability for 33 days.
Application in Clinical study
The validated method, with a linearity range of 2–100 ng/mL, has been successfully applied for the analysis of study samples in pivotal study. Additionally, an alternative method using the same extraction procedure was developed with a linearity range of 0.5–75 ng/mL, which has been successfully implemented in pilot study. This method demonstrated more than 95% acceptance based on incurred sample analysis criteria.
Conclusion
A selective, precise, and accurate LC-MS/MS method is now available for the quantification of Semaglutide in human plasma.
About Lambda
Lambda & Novum delivers full-service CRO services to the innovator, biotech, and generic pharmaceutical industries worldwide. With a strong global presence in India, the USA, Canada, the UK, Spain and Poland, we bring specialized expertise to every project. Our focus on secure IT infrastructure and automation ensures timely project delivery and strict adherence to international regulatory standards. Lambda’s exemplary regulatory track record includes over 60 successful inspections and audits by esteemed authorities, including the USFDA, EMEA, MHRA, EU member states, and other global regulatory bodies, in the past five years.
Lambda’s commitment to excellence has garnered significant recognition, including the ‘Best Indian CRO’ award from Frost & Sullivan (USA) and the ‘Great Indian Workplace’ title from UBS Transformance. Recent accolades include the ‘Regulatory Excellence’ Award at the CPhI Pharma Awards 2023 and the ‘Industry Partner of the Year’ Award at the Global Generics & Biosimilar Awards 2023.
Lambda offers a wide range of bioanalytical capabilities specializing in cell-based assays, biomarkers, immunogenicity, and pharmacokinetics (PK). With state-of-the-art facilities and a team of experienced scientists, Lambda provides comprehensive solutions for the analysis of small molecules, biomarkers, biologics, and therapeutic trace elements.
