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How to analyze the amino acid composition of semaglutide by HPLC?
Release time:
2025-05-28
Smeglutide is a new long-acting glucagon like peptide-1 receptor agonist (GLP-1RA), which is composed of 17 kinds of 31 amino acids. Its structure is similar to the natural human glucagon like peptide-1 (GLP-1), and has up to 94% amino acid sequence homology. It is mainly used to treat type 2 diabetes and its related complications. At the same time, it also shows broad application prospects in the fields of weight loss and cardiovascular disease prevention. At present, it has been approved by the European Union to reduce the risk of heart disease, stroke, and even cancer in overweight/obese adults with cardiovascular disease. In the field of weight loss, semaglutide also performs well, significantly reducing the risk of major adverse cardiovascular events in patients, including cardiovascular death, non fatal myocardial infarction, etc.
Figure 1. Information on Simeglutide Compounds
In the pharmaceutical research of chemically synthesized peptide drugs, amino acid composition and ratio analysis are important quality control projects. Amino acid composition analysis is an effective method for identifying peptide/protein drugs. Specific peptides/proteins are composed of specific amino acids, and determining the content of each amino acid is an effective means of controlling product quality.
Figure 2. Molecular structure diagram of semaglutide
According to the guiding principles, the sample of semaglutide is subjected to hydrochloric acid hydrolysis and sodium hydroxide hydrolysis treatment respectively. Combined with BIKAI OPA-FMOC automatic pre column automatic derivative amino acid analysis technology, the amino acid composition of semaglutide can be determined, and the determination results can be compared and analyzed with the expected amino acid composition and ratio of the product.
1. Equipment：
BIKAI 1511 HPLC System（Quaternary pump - P40、Autosampler - A10CV、UV Detector - D10、Column oven - C10V2）
2. Software：
V5.4.5.3
3. Determinand：
Amino acid mixed standard (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, alanine, aminoisobutyric acid, tyrosine, valine, methionine, n-valine, tryptophan, phenylalanine, isoleucine, leucine, lysine, proline); Simeglutide Injection
4. Derivative reagents：
OPA：Weigh 40mg of ortho benzaldehyde, add 3.5ml of borate buffer, 0.5ml of acetonitrile, and 64 μ l of 3-mercaptopropionic acid, dissolve and mix well
FMOC：Weigh 40mg of FMOC, add 8ml of acetonitrile, dissolve and mix well
5. Sample hydrolysis
Hydrochloric acid hydrolysis: Using 6mol/L HCl hydrolysis, 16 amino acids are produced, glutamine is hydrolyzed to glutamic acid, and tryptophan is destroyed.
Sodium hydroxide hydrolysis: using 5mol/L NaOH hydrolysis to produce tryptophan.
6. Chromatographic conditions：
Mobile phase A: 0.05mol/L sodium acetate solution triethylamine tetrahydrofuran (950:0.14:5, adjusted to pH 6.8 with 2% acetic acid)
Mobile phase B: 0.16mol/L sodium acetate solution (adjusted to pH 6.8 with 2% acetic acid) - methanol acetonitrile (200:400:400)
Elution gradient:
- Column：BIKAI C18（4.6*250mm，5μm）
- Flow rate：1mL/min
- Column temperature：40℃
- Injection volume：20µL
- Detection wavelength：338nm（0-27min）&262nm（27-41min）
Derivative method: Use an automatic sampler to add, mix, aspirate and other programs to achieve automatic derivation.
7. Test results
Precision:
Linear：
Sample testing：
Seventeen amino acids were detected in the Simeglutide sample, which are consistent with the theoretical amino acid composition. The relative error between the amino acid ratio and the theoretical value is within 10%, indicating accurate results.
Result spectrum：
Figure 3: Atlas of 19 Amino Acid Reference Samples
Figure 4: Atlas of Smeaglutide Acid Hydrolysis Solution
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