Key Points:
- GLP-1 receptor agonists, initially developed for type 2 diabetes, are now widely used for obesity and metabolic diseases. Their rapid market growth is drawing heightened regulatory attention.
- The structural complexity of GLP-1 analogs, along with the emergence of new formulations, is prompting the integration of advanced analytical tools like LC–MS/MS and HRMS.
- With stricter global regulations such as ICH M10 and increasing demand for biosimilar comparability, laboratories are focusing on method validation, data integrity, and automation. Technologies like automated sample prep, digital data systems, and process analytical technology (PAT) are important for meeting compliance and ensuring high-throughput, robust analysis.
As glucagon-like peptide-1 (GLP-1) receptor agonists gain prominence in the treatment of obesity and metabolic disease, their rapid rise is drawing intensified scrutiny and sparking fierce competition. The FDA has begun cracking down on unapproved versions of drugs like semaglutide, produced by compounding pharmacies, citing safety and quality concerns (1). At the same time, insurers are pushing back on coverage due to high costs and limited long-term data, creating significant access challenges for patients.
Meanwhile, pharmaceutical companies are racing to capture market share, with both established firms and emerging players investing heavily in next-generation GLP-1 analogs and alternative delivery platforms. The result is a fast-moving landscape, with new clinical data, regulatory actions, and commercial partnerships emerging almost weekly. This article aims to outline current and emerging trends in the analysis of GLP-1 receptor agonists, with a focus on liquid chromatography (LC), mass spectrometry (MS), and regulatory expectations.
The Emergence of a New Class
GLP-1 receptor agonists represent a transformative class of peptide therapeutics with expanding clinical applications. Originally developed for type 2 diabetes mellitus (T2DM), these medications have gained prominence for their effects on glucose regulation, weight loss, and cardiovascular risk reduction (2). GLP-1 is an incretin hormone secreted by intestinal L-cells in response to food intake. It stimulates glucose-dependent insulin secretion, suppresses glucagon release, slows gastric emptying, and reduces appetite.
Approved GLP-1 receptor agonists include exenatide, liraglutide, dulaglutide, semaglutide, and the dual GLP-1/gastric inhibitory polypeptide (GIP) agonist tirzepatide. These peptide-based drugs are large and structurally complex, with multiple amino acid modifications to prolong half-life and enhance receptor selectivity. Their therapeutic potential now extends beyond diabetes into the management of obesity. As new formulations—including oral peptides and combination therapies—enter the market, the analytical demands on pharmaceutical laboratories continue to grow.
To address these challenges, instrument and consumable manufacturers are optimizing their chromatographic solutions. According to Sean Orlowicz, principal market development manager—pharmaceutical at Phenomenex: “Core–shell particles are very helpful in the complex analysis required to separate the main active pharmaceutical ingredient (API) from very closely related compounds such as failed amino-acid sequences and recombinant impurities.”
Daniel Esser, product manager analytical chromatography, and Julia Bartmann, product manager preparative chromatography, both at YMC, also highlighted the importance of selecting appropriate stationary phases: “Hybrid-silica-based and bioinert-coated columns can significantly reduce nonspecific adsorption—crucial when working with sensitive GLP-1 related analytes.”
The Role of Mass Spectrometry and Liquid Chromatography
As the analytical landscape for GLP-1 drugs continues to undergo a significant transformation, one major trend is the growing integration of high-resolution mass spectrometry (HRMS) in analytical workflows. GLP-1 analogs are large, complex peptides that often feature post-translational modifications and closely related isoforms. HRMS provides the resolving power needed to distinguish these subtle differences. Orbital ion trap and time-of-flight (TOF) systems enable accurate mass measurements with high mass accuracy (typically below 5 ppm), which is essential for impurity profiling, structural elucidation, and identity confirmation. In addition, advanced software for deconvolution and data processing enhances the comprehensive characterization of degradation products and peptide mapping (3).
LC–MS/MS continues to gain prominence in the bioanalysis of GLP-1 drugs, particularly in pharmacokinetic (PK) and pharmacodynamic (PD) studies. Immunoassays often struggle with cross-reactivity and limited dynamic range, but LC–MS/MS circumvents these limitations by offering high selectivity and specificity. Triple quadrupole instruments operating in multiple reaction monitoring (MRM) mode offer good quantification capabilities, achieving lower limits of quantification (LLOQs) in the low picomolar range.
Kadar and colleagues recently developed a highly sensitive LC–MS/MS assay to support a human microdose study for PF-06882961 (danuglipron), an oral GLP-1 receptor agonist (4). By employing response surface methodology for instrument parameter optimization, they achieved a LLOQ of 0.200 pg/mL, demonstrating the feasibility of using LC–MS/MS over accelerator mass spectrometry for such studies.
According to Esser and Bartmann, reversed-phase LC remains the dominant technique for peptide purity analysis, while size-exclusion chromatography (SEC) is widely used to assess aggregation and molecular weight distribution. They also note that “customer interest is growing in specialized chromatographic approaches, depending on analytical needs such as purity profiling, identity confirmation, or aggregation monitoring.”
Nitish Sharma, assistant professor at NIPER Ahmedabad, explained: “There has been a transition from low-resolution triple quadrupole systems to high-resolution platforms such as orbital ion trap and QTOF for peptide mapping, identifying impurities or isoforms, and batch-to-batch comparability. Orthogonal analytical methods like SEC-multi-angle light scattering (MALS), dynamic light scattering (DLS), capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), and ion mobility MS are also gaining importance.”
Orlowicz notes that “with major GLP-1 APIs like liraglutide going generic, and soon semaglutide… countries like India and China are entering the market in a large way. It is important that […] chromatographic products must be scalable from standard high performance liquid chromatography (HPLC) to ultrahigh-pressure liquid chromatography (UHPLC), and vice versa… supporting method transfer across the global economy.”
The Role of Sample Prep
Another important area of development is the streamlining of sample preparation and its automation. Manual sample preparation, including protein precipitation and solid-phase extraction (SPE), introduces variability and bottlenecks in high-throughput environments. Automated systems integrated with LC platforms allow parallel processing of dozens to hundreds of samples, significantly reducing human error and increasing throughput. Techniques such as 96-well plate SPE and microextraction by packed sorbent (MEPS) are widely used to enhance scalability while minimizing sample and solvent consumption (5).
Regulatory Guidance
The regulatory landscape itself is evolving, with harmonization efforts culminating in the ICH M10 guideline, finalized in 2022 (6). This guideline outlines comprehensive validation criteria for bioanalytical methods, including parameters such as selectivity, sensitivity, accuracy, precision, matrix effect, and stability. Harmonized requirements facilitate regulatory submissions and simplify method transfer between laboratories in different regions. For biosimilars and combination therapies—such as those involving dual GLP-1/GIP agonists—regulators demand comprehensive comparability data to demonstrate analytical similarity.
With regulatory scrutiny increasing, there is a strong focus on developing stability-indicating methods. Sharma explained, “GLP-1 and its analogues are prone to physical degradation (oxidation, deamidation) and peptide aggregation. Monitoring degradation products at low levels over time requires highly sensitive and specific methods. Detection of low-level aggregates and distinguishing between covalent and non-covalent aggregates are key challenges.” Regulatory authorities require robust analytical methods capable of detecting and quantifying degradation products under a range of stress conditions, including exposure to acid, base, heat, light, and oxidative environments. These forced degradation studies are essential for demonstrating method specificity. LC methods coupled with UV or MS detection are validated based on mass balance and impurity profiling, forming a cornerstone of stability testing (7).
Sharma outlined valuable strategies: “The most effective method development strategies balance sensitivity, specificity, and robustness. Using orthogonal analytical techniques like reversed-phase (RP)-HPLC for purity, SEC for aggregation, ion exchange chromatography (IEC) for charge variants, CE-SDS for size separation, and HRMS for peptide mapping ensures comprehensive characterization.”
Digitalization and data integrity have also become central to the analytical process. Compliance with data integrity principles, such as ALCOA+ (Attributable, Legible, Contemporaneous, Original, Accurate, plus Complete, Consistent, Enduring, and Available), is mandated by regulatory bodies. Integration of chromatography data systems (CDS) with laboratory information management systems (LIMS) supports audit trails, automated reporting, and electronic signatures, thereby improving traceability and compliance. Cloud-based platforms further enhance real-time data monitoring and accessibility (8).
Finally, process analytical technology (PAT) and real-time release testing (RTRT) are becoming increasingly relevant. PAT frameworks are applied to peptide synthesis and formulation using spectroscopic tools to monitor critical process parameters (CPPs) and critical quality attributes (CQAs). Techniques such as near-infrared (NIR) and Raman spectroscopy enable real-time or near-real-time analysis and complement traditional LC testing. These approaches align with quality by design (QbD) principles and reduce reliance on end-product testing.
Looking to the Future
Emerging areas of growth in analytical chemistry related to GLP-1 drug analysis include hydrogen-deuterium exchange mass spectrometry (HDX-MS), which Sharma said, “provides insights into higher-order structure and conformation of degradants—critical for understanding long-acting or modified GLP-1 analogs.” He also highlights deuterated HCl hydrolysis followed by gas chromatography (GC)–MS as “a powerful method for analyzing diastereomeric amino acids in peptide manufacturing.”
As more complex matrices come to market, Orlowicz added, “Oral dosage formulations are the next great frontier for GLP-1s. These formulations also bring about new chromatographic challenges. Solubility concerns will require new excipient considerations, which will need to be chromatographically resolved.”
GLP-1 receptor agonist analysis is evolving rapidly to meet the challenges of increasing demand, structural complexity, and regulatory scrutiny. The integration of HRMS, LC–MS/MS, and automated sample preparation technologies is enhancing analytical performance. At the same time, regulatory frameworks are pushing for greater method robustness, traceability, and data integrity. Continued innovation in GLP-1 analytics will empower drug developers and manufacturers to deliver high-quality therapeutics with greater speed and confidence.
References
(1) FDA, “FDA’s Concerns with Unapproved GLP-1 Drugs Used for Weight Loss,” https://www.fda.gov/drugs/postmarket-drug-safety-information-patients-and-providers/fdas-concerns-unapproved-glp-1-drugs-used-weight-loss (accessed 2025-05-28).
(2) Davis, N. Weight-loss jabs linked to reduced risk of 42 conditions including dementia. The Guardian, January 20, 2025. https://www.theguardian.com/society/2025/jan/20/weight-loss-jabs-linked-to-reduced-risk-of-42-conditions-including-dementia
(3) Domon, B.; Aebersold, R. Mass Spectrometry and Protein Analysis. Science 2006, 312, 212–7. DOI: 10.1126/science.1124619
(4) Kadar, E. P.; Eng, H.; Kalgutkar, A. S.; Holliman, C. L.; Steeno, G. S. Development of a Sensitive LC-MS/MS Assay to Support Human Microdose Study for an Oral Agonist of the GLP-1 Receptor. Bioanalysis 2024, 16, 545–555. DOI: 10.1080/17576180.2024.2349421
(5) Wells, D. High Throughput Bioanalytical Sample Preparation: Methods and Automation Strategies (ISSN Book 5) 1st Edition; Elsevier Science, 2003.
(6) ICH, M10 Bioanalytical Method Validation and Study Sample Analysis, Final version (2022).
(7) Blessy, M.; Patel, R. D.; Prajapati, P. N.; Agrawal, Y. K. Development of forced degradation and stability indicating studies of drugs-A review. J. Pharm. Anal. 2014, 4, 159–165. DOI: 10.1016/j.jpha.2013.09.003
(8) FDA, Data Integrity and Compliance with Drug CGMP, Questions and Answers Guidance for Industry (CDER, December 2018).
