Quality Evaluation of Tripterygium Glycoside Tablets Based on Quantitative Band-Selective 2D 1H-13C HSQC and 1H NMR Fingerprinting.

Tripterygium glycoside tablets (TGTs) are preparations extracted and purified from Tripterygium wilfordii Hook. F and are extensively utilized in the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). However, variations in production processes among manufacturers can lead to challenges in quality control and clinical utilization of TGTs. A band-selective 2D 1H-13C HSQC quantification method was applied for the determination of 13 active ingredients in TGTs. This method was validated following the guidelines of USP-NF 2022. The results demonstrated that the quantitative method exhibited excellent signal resolution, as well as sufficient accuracy, sensitivity, and stability. In addition, the 1H NMR spectra of TGTs from three manufacturers underwent analysis using principal component analysis and orthogonal partial least-squares discriminant analysis. The results revealed significant differences among the TGTs from the three manufacturers, with manufacturer 2 and manufacturer 3 demonstrating superior product consistency compared to manufacturer 1. A quality evaluation system for TGTs was developed based on band-selective 2D 1H-13C HSQC and 1H NMR, encompassing both quality markers and fingerprinting. This system offers reliable approaches and insights for enhancing the quality control of natural products.

Distribution of active ingredients and quality control of Forsythia suspensa with AP-MALDI mass spectrometry imaging.

The fruits of Forsythia suspensa (F. suspensa) have been used as a traditional Chinese medicine for 2000 years. Currently, the quality control of F. suspensa strictly follows the instructions of Chinese Pharmacopeia, which mainly controls the content of forsythoside A, phillyrin, and volatile oil. In this study, air pressure MALDI mass spectrometry imaging (AP-MALDI MSI) was used to evaluate the quality of F. suspensa fruits and the distribution of dozens of active ingredients. The variation of active ingredients was measured for more than 30 batches of samples, regarding harvest time, cultivated environment, shelf-life, and habitat. Fifty-three active ingredients could be detected in F. suspensa fruits with AP-MALDI MSI. Seven active ingredients were upregulated, four ingredients downregulated, and 15 ingredients did not change in ripe fruits. A sharp variation of active ingredients in late September was observed for the Caochuan fruits harvested in 2019, which is closely related to the appearance of the ginger color of the pericarp under the microscope observation. The microscope observation is a reliable way to classify ripe and green fruits instead of outlook. Just considering forsythoside A and phillyrin, it is found that wild fruits are better than cultivated fruits, but cultivated fruits have high contents of other ingredients. The shelf-life of F. suspensa fruits is proposed to be 3 years, considering the 26 ingredients investigated. It was found that Luoning wild fruits are better than those from Caochuan with a new evaluation method. Mass spectrometry imaging is an easy, objective, and effective method to evaluate the quality of F. suspensa fruits.

Bio-soft matter derived from traditional Chinese medicine: Characterizations of hierarchical structure, assembly mechanism, and beyond.

Structural and functional explorations on bio-soft matter such as micelles, vesicles, nanoparticles, aggregates or polymers derived from traditional Chinese medicine (TCM) has emerged as a new topic in the field of TCM. The discovery of such cross-scaled bio-soft matter may provide a unique perspective for unraveling the new effective material basis of TCM as well as developing innovative medicine and biomaterials. Despite the rapid rise of TCM-derived bio-soft matter, their hierarchical structure and assembly mechanism must be unambiguously probed for a further in-depth understanding of their pharmacological activity. In this review, the current emerged TCM-derived bio-soft matter assembled from either small molecules or macromolecules is introduced, and particularly the unambiguous elucidation of their hierarchical structure and assembly mechanism with combined electron microscopic and spectroscopic techniques is depicted. The pros and cons of each technique are also discussed. The future challenges and perspective of TCM-derived bio-soft matter are outlined, particularly the requirement for their precise in situ structural determination is highlighted.

An Analysis of Polysaccharides from Eight Plants by a Novel Heart-Cutting Two-Dimensional Liquid Chromatography Method.

Natural polysaccharides are important active biomolecules. However, the analysis and structural characterization of polysaccharides are challenging tasks that often require multiple techniques and maps to reflect their structural features. This study aimed to propose a new heart-cutting two-dimensional liquid chromatography (2D-LC) method for separating and analyzing polysaccharides to explore the multidimensional information of polysaccharide structure in a single map. That is, the first-dimension liquid chromatography (1D-LC) presents molecular-weight information, and the second-dimension liquid chromatography (2D-LC) shows the fingerprints of polysaccharides. In this 2D-LC system, the size-exclusion chromatography-hydrophilic interaction chromatography (SEC-HILIC) model was established. Coupling with a charged aerosol detector (CAD) eliminated the need for the derivatization of the polysaccharide sample, allowing the whole process to be completed within 80 min. The methods were all validated in terms of precision, linearity, stability, and repeatability. The capability of the new 2D-LC method was demonstrated in determining various species of natural polysaccharides. Our experimental data demonstrated the feasibility of the whole systematic approach, opening the door for further applications in the field of natural polysaccharide analysis.

[Study on changes of Styrax varieties].

Styrax is a commonly used imported traditional Chinese medicinal material in China. It was introduced to China in the Han Dynasty and was first described as a traditional Chinese medicine in Miscellaneous Records of Famous Physicians(Ming Yi Bie Lu). In this paper, by combing ancient and modern Chinese and foreign herbal medicine books and modern literature, combined with the results of field investigations on the origin of Styrax, the changes of Styrax involving the name, quality evaluation, origin, place of origin, and harvesting and processing were systematically verified. The results show that since ancient times, the origin and place of origin of Styrax have been unclear. The medical scientists of all dynasties in China have evaluated the quality of Styrax from four aspects: texture, viscosity, odor concentration, and color. The varieties of Styrax changed twice. The first change may have occurred during the Sui and Tang Dynasties, and the base changed from Styrax officinalis to Liquidambar orientalis. The second change was in modern times, and the base changed from L. orientalis to L. styraciflua. At the same time, the place of origin changed for the first time, from Turkey, Syria, and other countries in southern Asia Minor to Honduras, Guatemala, and other countries in Central America and southern North America. This paper studied the historical evolution of Styrax in terms of quality evaluation, origin, place of origin, character, and harvesting and processing. At the same time, it summarized the application of Styrax in the western countries, which can provide a historical basis for the further development and utilization of Styrax.

Purification and Structural Characterization of Polysaccharides from Polygonum multiflorum Thunb. and Their Immunostimulatory Activity in RAW264.7 Cells.

Polygonum multiflorum Thunb. (PM) and derived products are broadly utilized in Chinese traditional medicine. According to our previous research, PM mostly contains polysaccharides, which display a wide range of biological activities. Two water-soluble polysaccharides (PMPs-1 and PMPs-2) were obtained from PM by DEAE-Cellulose and Sephadex G-100 column chromatography. Colorimetry, HPGPC-MALLS-RID, HPLC-PDA, methylation, FT-IR, NMR, and SEM were used to characterize these polysaccharides. PMPs-1 and PMPs-2 had average molecular weights of 255.5 and 55.7 kDa, respectively. PMPs-1 consisted of Man, Glc, Gal, and Ara at 0.9:78.6:1.0:1.6 and was a glucan with → 4)-Glcp-(1 → as a backbone. Meanwhile, PMPs-2, an acidic polysaccharide, comprised Rha, GalA, Glc, Gal, and Ara at 3.2:20.3:2.7:1.0:8.3. PMPs-1 and PMPs-2 significantly improved the proliferation of RAW 264.7 cells and induced NO, TNF-α, and IL-6 release. This study reveals that these two polysaccharides can be explored as novel immunomodulators and provide a basis for further development of PM in food and pharmaceutical industries.

Enhanced identification and localization of metabolites in Scutellariae Radix using ion mobility enabled MALDI-Q-TOF/MS imaging.

Image 1.

Toxicity Study on Crude Alkaloid Extracts of Houttuyniae herba Based on Zebrafish and Mice.

Houttuyniae herba has a long history of medicinal and edible homology in China. It has the functions of clearing heat and detoxifying, reducing swelling and purulent discharge, diuresis, and relieving gonorrhea. It is mainly distributed in the central, southeastern, and southwestern provinces of China. Houttuyniae herba has been designated by the National Ministry of Health of China as a dual-use plant for both food and medicine. Comprising volatile oils, flavonoids, and alkaloids as its primary constituents, Houttuyniae herba harbors aristolactams, a prominent subclass of alkaloids. Notably, the structural affinity of aristolactams to aristolochic acids is discernible, the latter known for its explicit toxicological effects. Additionally, the safety study on Houttuyniae herba mainly focused on the ethanol, methanol, or aqueous extract. In this study, both zebrafish and mice were used to evaluate the acute toxicity of the total alkaloids extracts from Houttuyniae herba (HHTAE). The zebrafish experiment showed that a high concentration (0.1 mg/mL) of HHTAE had a lethal effect on zebrafish embryos. Furthermore, the mice experiment results showed that, even at a higher dose of 2000 mg/kg, HHTAE was not toxic. In conclusion, HHTAE was of low safety risk.

Discovery and identification of interspecies peptide biomarkers in the seahorse species using liquid chromatography tandem mass spectrometry and chemometrics.

Seahorses have important edible and medicinal values including strengthening the body, tonifying the liver and kidneys, and reducing swelling. And there are abundant seahorse species on Earth. Many seahorses have large price differences due to the scarcity of resources, and some seahorses with similar appearances appear to be confused for use. While in market trading, Hippocampus is susceptible to loss of specialized morphology characteristics, making it difficult to distinguish between specific species. Here we report an effective method based on peptide biomarkers for the identification of seahorse species. Peptide biomarkers for each species were predicted using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with chemometrics software. One unique biomarker peptide for each species was synthesized and verified, and finally developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring method. The results indicate that the method has great potential for species-specific identification of seahorses and their preparations, among others.

Anti-aging activities of neutral and acidic polysaccharides from Polygonum multiflorum Thunb in Caenorhabditis elegans.

Polygonum multiflorum Thunb (PM) is used to slow the aging process. Although polysaccharides are a major constituent of PM, their anti-aging properties have not been thoroughly investigated. Therefore, this study aimed to examine the anti-aging effects of polysaccharides extracted from PM using the Caenorhabditis elegans (C. elegans) model. Two types of water-soluble heteropolysaccharides, namely a neutral polysaccharide (RPMP-N) and an acidic polysaccharide (RPMP-A), were obtained from PM. Their structures were elucidated by various methods. The effects of these polysaccharides on the lifespan, levels of antioxidants, and activities of antioxidant-related enzymes in C. elegans were also evaluated. The results showed that RPMP-A had higher GalA content compared with RPMP-N. The average molecular weights of RPMP-N and RPMP-A were 245.30 and 28.45 kDa, respectively. RPMP-N is a α-1,4-linked dextran as the main chain, and contains a small amount of branched dextran with O-6 as the branched linkage site；RPMP-A may be a complex of α-1,4-linked dextran, HG and RG-I. Treatment with RPMP-N and RPMP-A increased the mean lifespan of C. elegans, and significantly regulated oxidative stress. RPMP-A exhibited stronger anti-aging effects compared with RPMP-N. These findings suggest that RPMP-A may be a potent antioxidant and anti-aging component that can be used for developing functional food products and effective dietary supplements.

Synergistic effect of Euphorbia kansui stir-fried with vinegar and bile acids on malignant ascites effusion through modulation of gut microbiota.

Background: Toxic Euphorbia kansui (EK) is employed to treat malignant ascites effusion (MAE). EK stir-fried with vinegar (VEK) has been demonstrated to reduce toxicity due to its preserved water-expelling effect. This was demonstrated to be correlated with gut microbiota. Therein, bile acids (BAs) have a bidirectional relationship with the gut microbiota. Therefore, the aim of this study is to explore whether BA-mediated gut microbiota influences the water-expelling effect of VEK against MAE. Methods: The MAE rat model was established by intraperitoneal injection of Walker-256 tumor cells. A reliable simultaneous method for the determination of 15 bile acids in rat feces using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established and applied to analyze the fecal BAs in rats treated with VEK. The screened BA was then administered to VEK-treated MAE rats. The water-expelling effect was evaluated using histopathological analysis, biochemical examination, inflammatory factors in ascites, urine volume, ascites amount, and intestinal aquaporin expression. The microbial composition was determined using 16S rRNA sequencing, and the contents of bile acids were finally measured. Results: VEK decreased the content of fecal deoxycholic acid (DCA), lithocholic acid (LCA), and taurocholic acid (TCA) while increasing the content of ursodeoxycholic acid (UDCA). VEK alleviated liver, stomach, and intestinal injuries; oxidative damage; and inflammation, which were further ameliorated with UDCA intervention. VEK alleviated MAE by increasing the fecal water content, urine volume, and AQP3 protein expression and decreasing the urine levels of Na+, K+, and Cl-. This was retained with the intervention of UDCA. UDCA and VEK regulated the BA metabolism disorder to a certain extent. Analysis of gut microbiota showed that VEK increased the abundance of Lactobacillus and decreased that of Prevotella_9 in MAE rats. The combined administration of UDCA and VEK showed a better modulation of the microbiota structure than that of VEK alone, and the effect of this administration reached closer to the reference state. Conclusion: The water-expelling effect of VEK did not directly depend on the BA-mediated gut microbiota. However, VEK and BAs had a synergistic effect on malignant ascites effusion through the regulation of the gut microbiota. These results provided a scientific basis for the reasonable usage of VEK and the novel combination treatment strategy of VEK and UDCA.

Plasma-Excited Nebulizer Gas-Assisted Electrospray Ionization: Enhancing the Sensitivity of Pesticide in Mass Spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) is widely used in the detection of pesticide residues. However, the detection sensitivity of low-polarity pesticides by commonly used electrospray ionization may be severely suppressed, which greatly affects the limit of detection and repeatability. Herein, a plasma-excited nebulizer gas-assisted electrospray ionization (PENG-ESI) device has been developed. By introducing the discharge plasma formed by Tesla coil into the electrospray nebulizer gas channel, the sensitivity of low-polarity pesticides was significantly increased while maintaining sensitivity to polar pesticides. Under the optimized conditions, the limit of detection for S-bioallethrin was achieved at the level of 100 pg/g with good linearity (R2 > 0.99) and precision (RSD ≤ 4.61%). The matrix effect of a series of spiked matrix samples is less than 13.1%. Finally, different pyrethroid pesticide residues were successfully analyzed without separation, highlighting that the technology has potential application prospects in food quality control, environmental monitoring, and other fields.

[UPLC-QDA and machine learning for distinguishing different commodity specifications of Fritillariae Cirrhosae Bulbus and application of data augmentation technology].

This study aimed to establish a method based on machine learning technology for accurately predicting the commodity specifications of Fritillariae Cirrhosae Bulbus and explore the application of data augmentation technology in the field of drug analysis. The correlation optimized warping(COW) algorithm was used to perform peak calibration on the UPLC-QDA multi-channel superimposed data of 30 batches of samples, and the data were normalized. Through unsupervised learning methods such as clustering analysis, principal component analysis(PCA), and correlation analysis, the general characteristics of the data were understood. Then, the logistic regression algorithm was used for supervised learning on the data, and the condition tabular generative adversarial networks(CTGAN) was used to generate a large amount of data. Logistic regression classification models were trained separately using the real data and the data generated by CTGAN, and these models were evaluated. The logistic regression model trained with real data achieved cross-validation and test set accuracies of 0.95 and 1.00, respectively, while the logistic regression model trained with both real and CTGAN-generated data achieved cross-validation and test set accuracies of 0.99 and 1.00, respectively. The results indicate that machine learning can accurately predict the classification of Songbei, Qingbei, and Lubeibased on UPLC-QDA detection data. CTGAN-generated data can partially compensate for the lack of data in drug analysis, improving the accuracy and predictive ability of machine learning models.

Residual Change of Four Pesticides in the Processing of Pogostemon cablin and Associated Factors.

Before use as medicines, most traditional Chinese medicine (TCM) plants are processed and decocted. During processing, there may be some changes in pesticide residues in TCM. In recent years, reports have studied the changes of pesticides during the processes of boiling, drying and peeling of TCM materials but have rarely involved special processing methods for TCM, such as ethanol extraction and volatile oil extraction. The changes of carbendazim, carbofuran, pyridaben and tebuconazole residues in common processing methods for P. cablin products were systemically assessed in this study. After each processing step, the pesticides were quantitated by UPLC-MS/MS. The results showed amount decreases in various pesticides to different extents after each processing procedure. Processing factor (PF) values for the four pesticides after decoction, 75% ethanol extraction and volatile oil extraction were 0.02~0.75, 0.40~0.98 and 0~0.02, respectively, which indicated that residual pesticide concentrations may depend on the processing technique. A risk assessment according to the hazard quotient with PF values showed that residual pesticide amounts in P. cablin were substantially lower than levels potentially posing a health risk. Overall, these findings provide insights into the safety assessment of P. cablin.

A quality-comprehensive-evaluation-index-based model for evaluating traditional Chinese medicine quality.

BACKGROUND: Evaluating traditional Chinese medicine (TCM) quality is a powerful method to ensure TCM safety. TCM quality evaluation methods primarily include characterization evaluations and separate physical, chemical, and biological evaluations; however, these approaches have limitations. Nevertheless, researchers have recently integrated evaluation methods, advancing the emergence of frontier research tools, such as TCM quality markers (Q-markers). These studies are largely based on biological activity, with weak correlations between the quality indices and quality. However, these TCM quality indices focus on the individual efficacies of single bioactive components and, therefore, do not accurately represent the TCM quality. Conventionally, provenance, place of origin, preparation, and processing are the key attributes influencing TCM quality. In this study, we identified TCM-attribute-based quality indices and developed a comprehensive multiweighted multi-index-based TCM quality composite evaluation index (QCEI) for grading TCM quality. METHODS: The area of origin, number of growth years, and harvest season are considered key TCM quality attributes. In this study, licorice was the model TCM to investigate the quality indicators associated with key factors that are considered to influence TCM quality using multivariate statistical analysis, identify biological-evaluation-based pharmacological activity indicators by network pharmacology, establish real quality indicators, and develop a QCEI-based model for grading TCM quality using a machine learning model. Finally, to determine whether different licorice quality grades differently reduced the inflammatory response, TNF-α and IL-1ß levels were measured in RAW 264.7 cells using ELISA analysis. RESULTS: The 21 quality indices are suitable candidates for establishing a method for grading licorice quality. A computer model was established using SVM analysis to predict the TCM quality composite evaluation index (TCM QCEI). The tenfold cross validation accuracy was 90.26%. Licorice diameter; total flavonoid content; similarities of HPLC chromatogram fingerprints recorded at 250 and 330 nm; contents of liquiritin apioside, liquiritin, glycyrrhizic acid, and liquiritigenin; and pharmacological activity quality index were identified as the key indices for constructing the model for evaluating licorice quality and determining which model contribution rates were proportionally weighted in the model. The ELISA analysis results preliminarily suggest that the inflammatory responses were likely better reduced by premium-grade than by first-class licorice. CONCLUSIONS: In the present study, traditional sensory characterization and modern standardized processes based on production process and pharmacological efficacy evaluation were integrated for use in the assessment of TCM quality. Multidimensional quality evaluation indices were integrated with a machine learning model to identify key quality indices and their corresponding weight coefficients, to establish a multiweighted multi-index and comprehensive quality index, and to construct a QCEI-based model for grading TCM quality. Our results could facilitate and guide the development of TCM quality control research.

Quantification of sesquiterpene pyridine alkaloids from genus Tripterygium by band-selective HSQC NMR.

Sesquiterpene pyridine alkaloids (SPAs) are bioactive analogues derived from the genus Tripterygium and have anti-inflammatory and anti-rheumatic properties. Attributed to the similar sesquiterpene structures, the total SPAs showed severe peak overlap in 1D NMR and HPLC, leading to difficulties in identification and quantification. Interestingly, the application of band-selective HSQC NMR that specifically excited the region corresponding to the H-3 of SPAs prompted a signal separation of the total SPAs. Based on the high resolution, 23 SPAs were identified from the band-selective HSQC spectrum. The coupling constants (JCH, JHH) and relaxation times (T1, T2) of SPAs were measured, and it was found that they caused less than 1% attenuation of the HSQC signals, so the HSQC signals of SPAs had almost uniform responses. The concentrations of 23 SPAs were determined by standard curve method, using wilforgine as the calibration. In addition, we extended the pulse length-based concentration determination (PULCON) as a more efficient external standard method to the band-selective HSQC spectrum, and the results showed that the concentrations of alkaloids determined by PULCON were consistent with those measured by standard curve method. The developed quantification approach was validated according to the <761> of United States Pharmacopoeia (USP), demonstrating that the established band-selective HSQC approach is reliable for the rapid quantification of analogues in botanical extracts.

Progress in quality control, detection techniques, speciation and risk assessment of heavy metals in marine traditional Chinese medicine.

Marine traditional Chinese medicines (MTCMs) hold a significant place in the rich cultural heritage in China. It plays an irreplaceable role in addressing human diseases and serves as a crucial pillar for the development of China's marine economy. However, the rapid pace of industrialization has raised concerns about the safety of MTCM, particularly in relation to heavy metal pollution. Heavy metal pollution poses a significant threat to the development of MTCM and human health, necessitating the need for detection analysis and risk assessment of heavy metals in MTCM. In this paper, the current research status, pollution situation, detection and analysis technology, removal technology and risk assessment of heavy metals in MTCM are discussed, and the establishment of a pollution detection database and a comprehensive quality and safety supervision system for MTCM is proposed. These measures aim to enhance understanding of heavy metals and harmful elements in MTCM. It is expected to provide a valuable reference for the control of heavy metals and harmful elements in MTCM, as well as the sustainable development and application of MTCM.

Discovery of species-specific peptide markers and development of quality-evaluation strategies for deer horn gelatin using liquid chromatography-tandem mass spectrometry and a label-free methodology.

Deer horn gelatin (DHG) is a valuable nutritional dietary supplement. Due to the significant variation in the price of DHG from different sources, it is important to assess its quality and to clarify the species of its raw material. However, due to the similarity in appearance and physicochemical properties, as well as the destruction of genetic material during the manufacturing process, it is difficult to distinguish DHG from gelatin derived from other sources. Furthermore, current methods are unable to evaluate the overall quality of DHG. Using Nano LC-Orbitrap MS and data analysis software, DHG samples from five deer species were analyzed to identify peptide markers specific to alpha-2-HS-glycoprotein (AHSG) and collagen. The peptide markers were validated using HPLC-Triple Quadrupole MS, and strategies for assessing the quality of DHG were developed. Eighteen peptide markers were discovered, comprising peptides with differing specificities. Three strategies for the identification, characteristic mapping, and content determination of DHG were developed. These strategies can be used to assess the quality of deer gelatin.

Technical guidelines for risk assessment of heavy metals in traditional Chinese medicines.

BACKGROUND: Heavy metals are considered a global concern because they can deteriorate human health. This guideline aims to scientifically evaluate health risk of heavy metals in TCM and to propose a reference for decision making in developing TCM-related health policies. METHODS: Using a multidisciplinary approach, a steering committee oversaw the development of the guideline. To obtain a reasonable and accurate risk assessment, key exposure assessment parameters for TCM, e.g., exposure frequency (EF), exposure duration (ED), and daily ingestion rate (IR) were obtained from surveys. In addition, transfer rates for heavy metals from Chinese medicinal materials (CMM) to decoctions or preparations were examined. RESULTS: Based on the scientific theory of risk control, the guideline was designed systematically, and principles and procedures for the risk assessment of heavy metals in TCM were identified. The guideline can be utilized to assess the risk of heavy metals in CMM and Chinese patent medicines (CPM). CONCLUSION: This guideline may help standardize the risk assessment of heavy metals in TCM, advance regulatory standards for heavy metals in TCM, and ultimately improve human health through scientific TCM usage in clinic.

A novel method for the component identification of human blood products: Mass spectrometric analysis of human fibrinogen digested after SDS-PAGE in-gel digestion.

Human fibrinogen, as a blood product of special origin, is relatively simple to prepare and purify. Therefore, completely isolating and removing the relevant impurity proteins is difficult. Further, which impurity protein components are present is not clear. In this study, human fibrinogen products from seven enterprises were collected from the market, and the presence of impurity proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the major 12 impurity proteins were identified and screened by in-gel enzymolysis mass spectrometry, and 7 major impurity proteins with different peptide coverage were identified by enzyme-linked immunosorbent assay, in agreement with the mass spectrometry results. The seven major impurity proteins included fibronectin, plasminogen, F-XIII, F-VIII, complement factor H, cystatin-A, and α-2-macroglobulin. The final test results were in the range of undetectable to 50.94 µg/mL, with correspondingly low levels of impurity proteins between different companies and a manageable risk. Moreover, we found that these impurity proteins existed in the form of polymers, which might also be an important cause of adverse reactions. This study established a protein identification technique applicable to fibrinogen products, which provided new ideas for studying the protein composition of blood products. In addition, it provided a new means of testing for companies to monitor the flow of proteomic fractions and improve the purification yield and product quality. It laid the foundation for reducing the risk of clinical adverse reactions.