Welch Amino Acid Method Kit Featured in Semaglutide Research

At Welch Materials, we take pride in providing chromatographic solutions that enable innovation in the life sciences. We are pleased to share that our amino acid method kit has been successfully employed in a recent peer-reviewed study published by Precise Biopharma Private Limited, in the World Journal of Biology Pharmacy and Health Sciences.

The article, titled “Development and Validation of HPLC Method for Determination of Amino Acid Mole Ratio in Semaglutide”, highlights the robust analytical capability of Welch’s amino acid analysis solution in complex peptide characterization workflows.

This collaboration marks a significant milestone not only for Welch, but also for the broader field of peptide therapeutics, where precise amino acid profiling remains critical for drug development, quality control, and regulatory compliance.

The research conducted by Precise Biopharma focused on developing a precise, accurate, and robust HPLC method for quantifying the molar ratio of amino acids in Semaglutide, a GLP-1 receptor agonist used in the treatment of type 2 diabetes and obesity. As a long-acting peptide containing 34 amino acid residues, Semaglutide presents analytical challenges due to its structural complexity, chemical modifications, and high molecular weight.

The objective of the study was to establish and validate an analytical method capable of determining the amino acid content in Semaglutide. The goal was to ensure that the molar ratios of amino acids meet the requirements for its intended analytical application.

To achieve this, the researchers selected Welch’s amino acid method kit, which integrates high-performance column technology with optimized pre-column derivatization protocols and mobile phase systems, enabling high-resolution separation and quantification of amino acids.

Welch’s amino acid method kit is designed to simplify and standardize amino acid analysis using pre-column derivatization with PITC reagents. The kit includes:

• A high-efficiency amino acid analysis column.
• A set of derivatization reagents (A and B, 10 mL/vial each), along with the diluent (20 mL/vial).
• An amino acid standard (1 mL).

Key Advantages:

• Rapid derivatization reaction – completed in just 30 minutes at room temperature.
• High stability of derivatives, ensuring consistent and reliable detection.
• Single, well-defined reaction product for each amino acid.
• Short overall analysis time, improving laboratory efficiency and throughput.

In the published article, semaglutide was subjected to HCl hydrolysis at 110°C for 16 hours, followed by (after cooling down to room temperature) evaporation in a water bath at 75°C and vacuum was applied in the headspace vial. The hydrolyzed sample solution was then mixed with diluted derivatization reagents A and B from the method kit.

The separation was carried out on a Welch Ultisil Amino Acid (4.6×250 mm, 5 μm) column under a gradient program using sodium acetate trihydrate solution: acetonitrile (93:7) as mobile phase A and acetonitrile: water (80:20) as mobile phase B. Detection was performed using PDA/UV detector.

The method successfully resolved and quantified 14 amino acids in semaglutide, with a high linearity (correlation coefficient not less than 0.99 for all amino acids) and low relative standard deviation (%RSD of each amino acid peak less than 5.0%).

The HPLC method developed using Welch’s amino acid method kit was rigorously validated for the determination of amino acid mole ratios in Semaglutide. Validation parameters included system suitability, specificity, linearity and range, accuracy, precision, and intermediate precision (ruggedness).

• System suitability was established by evaluating the repeatability of amino acid peak areas across three injections of the standard solution, with %RSD < 5.0%.
• Specificity was determined by observing all amino acid peaks obtained from standard solution well resolved from each other, and passing peak purity test.
• Linearity was evaluated over a concentration range from 12.5% to 200% of the specification level, using five concentration levels for each amino acid. Each level was injected in triplicate. Regression analysis yielded correlation coefficients (R²) not less than 0.99 for all amino acids, and the slope, intercept, and %Y-intercept within acceptance values.
• Accuracy was confirmed by recovery studies conducted at 80%, 100%, and 120% of the standard concentration. Each level was injected in triplicate.
• Method Precision was assessed through six independent preparations of Semaglutide test samples, with the %RSD across the six preparations below 5.0%.
• Intermediate Precision (Ruggedness) was achieved by repeating the same test by a second analyst using different columns, instruments, and days. All twelve determinations (from method precision and intermediate precision combined) showed cumulative %RSD within 5.0%.

Welch Materials is committed to advancing chromatographic science through close collaboration with industry partners and research institutions. The successful use of our amino acid method kit by Precise Biopharma underscores the impact of high-quality consumables and technical support on achieving analytical excellence.

In a landscape where biopharmaceutical innovation is rapidly expanding, particularly in the field of peptide and protein therapeutics, the ability to develop reliable analytical methods remains essential. From formulation to final release, amino acid analysis plays a pivotal role in ensuring product safety, efficacy, and regulatory compliance.