Due to the structure and physicochemical properties of peptide compounds, many conventional methods for qualitative and quantitative analysis of common organic compounds are not suitable for the analysis of peptides. For example, when the molecular weight of the peptide is more than 800 or 1000Da, it is difficult to have a clear melting point; Similarly, except for small peptides containing less than 4 to 5 residues, the structure of most peptides contains a large number of H2N-, -CONh - and saturated C and H atoms in very similar chemical environments, so the data of elemental analysis is not practical. Similarly, 1HNMR and 13CNMR data in infrared spectrum and nuclear magnetic resonance spectrum are also difficult to identify individually. Therefore, polypeptide compounds as drugs are generally not based on the above analytical data as the basis for purity and structure confirmation.

It should be noted that in the stereochemical analysis of polypeptide molecules, optical rotation dispersion (ORD), circular binary dispersion (CD) and two-dimensional nuclear magnetic resonance spectroscopy are increasingly used for analysis. They can elucidate the secondary and tertiary structure of peptide bonds. These studies are significant for the analysis of the stereochemistry of peptides, especially the relationship between the overall conformation and biological activity, but they are not considered as standards in the evaluation of drug quality. The detailed relationship between peptide stereochemistry and IR, UV, ORD and CD analysis has been described in Professor Peng Shiqi's book "Pharmaceutical Chemistry of Peptides" and will not be repeated here. The following is only a brief introduction to several essential analytical situations suitable for quality control of peptide compounds.

Mass spectrometry (MS) analysis

Because infrared, ultraviolet and nuclear magnetic resonance analysis have no obvious effect on the quality control of large molecular weight peptide compounds, molecular weight detection is particularly important for the quality control of peptides. Because the peptide chain is easily broken under general mass spectrometry bombardment conditions, it is difficult to obtain a complete molecular peak, so mild conditions are often used FAB-MS, ESI-MS... The peptide compounds were analyzed by mass spectrometry. Not only the molecular weight of the product can be proved from a qualified MS analysis, but also the purity of the main product can be evaluated from the presence or absence of other miscellaneous peaks.The following are the detection and analysis steps for custom peptide synthesis.

HPLC analysis

The AAA and MS values in peptide analysis are mainly used for structural proof (i.e. qualitative). Only by HPLC analysis can the specific purity of the peptide product be known. It should be noted that the retention time (R.T.) of the main peak should not be too short (such as < 5min). In this case, some impurity peaks (if present) may be combined with the main peak. Therefore, the separation condition with moderate RT value of the main peak is a reasonable condition for quality inspection. Another function of HPLC analysis is that it can also be qualitative. When the synthetic peptide is a known substance (such as a generic drug), it can be compared with the standard product by individual injection and co-injection (co-injection), similar to TLC analysis of small organic molecules.

Sequence analysis

In addition to the content composition of the residues, the order of bonding between the residues must be proved. For example, the following two peptides :H-Arg-Lys-Asp-Val-Tyr-OH... (A),H-Tyr-Arg-Val-Asp-Lys-OH... (B) They have the same structural composition, so AAA values and MW are the same. (Of course, their R.T. in HPLC analysis differed.) Among them, peptide A is a thymus pentapeptide (TP5) with immunomodulatory activity, which has been widely used clinically. B-peptides, on the other hand, have no biological activity. Therefore, the sequence connection of peptides is different, and their physicochemical properties and biological activities are very different. It is worth pointing out that the peptides isolated from animals, plants and humans must be sequenced as one of the important bases for structural determination. However, chemically synthesized peptides do not require sequence analysis. Because the reaction route in the synthesis process has made the connection order of each residue clear, it is impossible for the above-mentioned A peptide and B peptide to coexist.

Specific rotation [α]

With the exception of Gly residues, each monomer in a classical peptide contains a chiral C atom, so each peptide compound has a specific [α] value. In oligopeptide molecules with relative molecular weight below 500, the primary structure is the main body, so its [α] value is one of the necessary quality basis. However, when the length of the peptide chain reaches more than eight residues (some literature considers six residues), more than secondary structures begin to appear. At this point, the contribution of the α-C (i.e., chiral C) atom in each residue to the optical rotation of the entire molecule is not the only factor. Intramolecular secondary or tertiary structure can bring new asymmetrical environment to peptide molecules, and these asymmetries are often related to concentration, intermolecular association degree, solvent and temperature. For example, the [α] value of Somatostatin14 varied by as much as 20 degrees between batch numbers. Therefore, the [α] value is not regarded as a necessary content in the pharmacopoeia and quality control standards of new drugs in various countries.

Analysis of amino acid composition (AAA)

It can accurately express the composition of the number of peptide chain residues. Because if one or several steps in the synthesis of the condensation is not complete, it may lead to a partial peptide (delation peptides) exist. Especially in solid-phase synthesis, these peptides cannot be removed in time and are often mixed with the target peptide after final cleavage. Moreover, it is difficult to separate completely from the target peptide due to its close physical and chemical properties. At this time, the content ratio between various amino acids provided by the AAA value can determine the presence of the defective peptide and which residues are missing. The general standard is that the integer ratio error between the amino acids is allowed within 5%.