Establishing the chromatographic fingerprint of traditional Chinese medicine standard decoction based on quality by design approach: A case study of Licorice

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic‐acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high‐performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0‐5 min, 5–19% A; 5–10 min, 19% A; 10–50 min, 19–42% A; 50–54 min, 42–100% A; 54–60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.     

Comprehensive characterization and quantification of multiple components in Dan‐Huang‐Qu‐Yu capsule using a multivariate data processing approach based on microwave‐assisted extraction with UHPLC and Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry

Dan‐Huang‐Qu‐Yu capsule, a Chinese herbal medicine compound preparation, is widely used for chronic pelvic inflammatory disease. In this study, a rapid, selective, and sensitive microwave‐assisted extraction ultra‐high‐performance liquid chromatography‐Q Exactive quadrupole‐orbitrap high‐resolution mass spectrometry method was developed for analyzing its chemical compositions. A total of 85 compounds, including 22 flavonoids, 8 terpenoids, 5 quinones, 5 phthaleolactone, 23 organic acids, and 22 other compounds were identified from Dan‐Huang‐Qu‐Yu capsule. Among them, 35 major compounds were unambiguously detected by comparing them with reference standards and selected as quality control markers, which were simultaneously determined in Dan‐Huang‐Qu‐Yu capsule. The established method was successfully validated and applied for simultaneous determination of 35 bioactive compounds in Dan‐Huang‐Qu‐Yu capsule from ten sample batches. The quantitative data of the analytes were analyzed by principal component analysis for quality assessment of Dan‐Huang‐Qu‐Yu capsule. Six compounds (e.g., astragaloside IV, salvianolic acid B, ellagic acid, chlorogenic acid, N‐butylidenephthalide, and luteolin) were screened out and regarded as chemical markers for quality control of Dan‐Huang‐Qu‐Yu capsule. The established method has been proved to be a novel and useful tool for rapid research of Dan‐Huang‐Qu‐Yu capsule. This research will provide reference for the scientific research of traditional Chinese medicines.     

Identification and characterization of forced degradation products and stability‐indicating assay for notoginsenosidefc by using UHPLC‐Q‐TOF‐MS and UHPLC‐MS/MS: Insights into stability profile and degradation pathways

Notoginsenoside Fc, a protopanaxadiol‐type saponin, shows multi‐pharmacological activities. Chemical stability evaluation plays a crucial role in drug development. In this study, the forced degradation behavior of Notoginsenoside Fc was investigated under hydrolytic and oxidative conditions. A specific ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the separation, identification, and characterization of the degradation products of Notoginsenoside Fc. Fifty potential degradation products were formed via deglycosylation, dehydration, hydration, isomerization, side‐chain cleaving, oxidation, and superoxidation. Notoginsenoside Fc was subjected to different pH solutions, temperatures, and time periods to assess its stability. A sensitive ultra high performance liquid chromatography‐tandem mass spectrometry was developed for the quantification of Notoginsenoside Fc, notoginsenoside ST‐4, notoginsenoside Ft1, and relative quantification of notoginsenoside Ft2, 20(R)‐notoginsenoside Ft2, notoginsenoside SFt3, and notoginsenoside SFt4. The assay was linear over the concentration range (R² > 0.997) with the lowest limit of quantification of 0.02 μg/mL for Notoginsenoside Fc, Notoginsenoside ST‐4, and Notoginsenoside Ft1. The intra‐day precision, inter‐day precision, and accuracy of the three analytes were within accepted levels. The degradation kinetics of Notoginsenoside Fc in pH 1 and 3 solutions fits to first‐ and second‐order kinetics, respectively. The degradation of Notoginsenoside Fc is pH‐, temperature‐, and time‐dependent.     

Accurate discrimination of Gastrodia elata from different geographical origins using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis

Gastrodia elata from different geographical origins varies in quality and pharmacological activity. This study focused on the classification and identification of Gastrodia elata from six producing areas using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis. Before recognition analysis, a principal component analysis was applied to ascertain the discrimination possibility with high‐performance liquid chromatography fingerprints. And then, boosting partial least‐squares discriminant analysis and conventional partial least‐squares discriminant analysis were applied in this study. Experimental results indicated that the adaptive iteratively reweighted penalized least‐squares algorithm could eliminate the baseline drift of high‐performance liquid chromatography chromatograms effectively. And compared with partial least‐squares discriminant analysis, the total recognition rates using high‐performance liquid chromatography fingerprint combined with boosting partial least‐squares discriminant analysis for the calibration sets and prediction sets were improved from 94 to 100% and 86 to 97%, respectively. In conclusion, high‐performance liquid chromatography combined with boosting partial least‐squares discriminant analysis, which has such advantages as effective, specific, accurate, non‐polluting, has an edge for discrimination of traditional Chinese medicine from different geographical origins. And the proposed methodology is a useful tool to classify and identify Gastrodia elata from different geographical origins.     

Analysis of Chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ligustrazine was the active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma. However, the content of ligustrazine is very low. We proposed a hypothesis that ligustrazine was produced by the mutual effects between endophytic Bacillus subtilis and the Ligusticum chuanxiong Hort. This study aimed to explore whether the endophytic B. subtilis LB5 could make use of Chuanxiong Rhizoma fermentation matrix to produce ligustrazine and clarify the mechanisms of action preliminarily. Ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis showed the content of ligustrazine in Chuanxiong Rhizoma was below the detection limit (0.1 ng/mL), while B. subtilis LB5 produced ligustrazine at the yield of 1.0268 mg/mL in the Chuanxiong Rhizoma‐ammonium sulfate fermentation medium. In the fermented matrix, the reducing sugar had a significant reduction from 12.034 to 2.424 mg/mL, and rough protein content increased from 2.239 to 4.361 mg/mL. Acetoin, the biosynthetic precursor of ligustrazine, was generated in the Chuanxiong Rhizoma‐Ammonium sulfate (151.2 mg/mL) fermentation medium. This result showed that the endophytic bacteria B. subtilis LB5 metabolized Chuanxiong Rhizoma via secreted protein to consume the sugar in Chuanxiong Rhizoma to produce a considerable amount of ligustrazine. Collectively, our preliminary research suggested that ligustrazine was the interaction product of endophyte, but not the secondary metabolite of Chuanxiong Rhizoma itself.     

Rapid analysis and characterization of multiple constituents of corn silk aqueous extract using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Corn silk is a well‐known traditional Chinese medicine that has been widely used for its antidiabetic, antioxidant, antihyperlipidemic, and other effects in China for thousands of years. Numerous studies have revealed that corn silk contains multiple bioactive constituents that are beneficial for human health. However, the constituents of corn silk in vivo remain ambiguous. In this study, high‐throughput ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry technology using multivariate statistical analysis was established to systematically investigate the constituents migrating into blood from corn silk aqueous extract. As a result, 76 compounds were identified, including caffeic acid and ten of its derivatives, (E)‐p‐coumaric acid and two of its derivatives, ferulic acid and four of its derivatives, and five flavones. Among the identified constituents, 21 constituents, including nine prototype components and 12 metabolites derived from eight components, were characterized in sequence. Based on the significance of the results, the applied approach was powerful for the accurate determination and rapid screening of bioactive components from corn silk aqueous extract. The obtained results are valuable for the in‐depth understanding and further pharmacological study of corn silk aqueous extract.     

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid‐phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32–1.59 ng/mL), good recoveries (86.63–98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid‐phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time‐of‐flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.     

Probing the degradation mechanism of forsythiaside A and simultaneous determination of three forsythiasides in Forsythia preparations by a single marker

Forsythiaside A is the major component of Forsythia suspensa. This study investigated the degradation mechanism of forsythiaside A. Eight degraded components including forsythiaside I, forsythiaside H, forsythiaside E, caffeic acid, suspensaside A, β‐hydroxy forsythiaside I, β‐hydroxy forsythiaside H, and β‐hydroxy forsythiaside A were identified by using ultra‐high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry. Then, the quantitative analysis of multi‐components by a single‐marker was performed with ultra‐high performance liquid chromatography to simultaneously determine forsythiaside A, forsythiaside H, and forsythiaside I in Forsythia suspensa preparations. The result showed good linear relationships within 2.871–287.1, 0.231–23.1, and 0.983–98.3 μg/mL (r > 0.9998), with average recoveries of 97.7, 95.7, and 95.8% and relative standard deviations of 1.4, 2.4, and 1.8%, respectively. Using forsythiaside A as an internal reference, the relative retention values of forsythiaside H and forsythiaside I to forsythiaside A were calculated to be 0.89 and 0.61, respectively, and the relative correction factors were 0.816 and 0.799, respectively. The method for quantitative analysis of multi‐components by a single‐marker was applied to evaluate the overall quality of forsythia preparations. There was no significant difference in the measurement results of the method developed and the method of external standard.     

Chinese Oak Tasar Silkworm Antheraea pernyi Silk Proteins: Current Strategies and Future Perspectives for Biomedical Applications

Chinese nonmulberry temperate oak tasar/tussah, Antheraea pernyi (Ap) silk is a natural biopolymer that has attracted considerable attention as a biomaterial. The proteinaceous components of Ap silk proteins, namely fibroin and sericin may represent an alternative over mulberry Bombyx mori silk proteins. In fact, the silk fibroin (SF) of Ap is rich in Arginyl‐Glycyl‐Aspartic acid (RGD) peptides, which facilitate the adhesion and proliferation of various cell types. The possibility of processing Ap silk proteins into different distinct 2D‐ and 3D‐based matrices is described in earlier studies, such as membranes, nanofibers, scaffolds, and micro/nanoparticles, contributing to a different rate of degradation, mechanical properties, and biological performance useful for various biomedical applications. This review summarizes the current advances and developments on nonmulberry Chinese oak tasar silk protein (fibroin and sericin)‐based biomaterials and their potential uses in tissue engineering, regenerative medicine, and therapeutic delivery strategies.     

Analysis of dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and processed seeds by high‐performance liquid chromatography coupled with diode array detection and mass spectrometry

The ripened seeds of Strychnos nux‐vomica L. have been extensively used as herbal medicines in Asian countries. Dihydroindole‐type alkaloids are not only the active constituents but also the toxicants in Strychnos. However, the simultaneous determination of these alkaloids in both crude and processed Semen Strychni is still lacking. The present study represents the first quantitation and relative quantitation assay of 12 dihydroindole‐type alkaloids in Strychnos nux‐vomica unprocessed and sand‐processed seeds using high‐performance liquid chromatography coupled with diode array detection and mass spectrometry. The relative concentration of ten alkaloids was calculated by semi‐quantification using the internal standard and their amounts in unprocessed and detoxified Semen Strychni were compared. We report here for the first time the significant increase of the two alkaloids, 19‐N‐methyl‐strychnine, and 2,3‐dimethoxy‐19‐N‐methyl‐strychnine, during the processing of Semen Strychni. Our study provides new insight into the true complexity of seed processing procedure and valuable information for assessing the efficacy and safety for clinical applications of Semen Strychni‐containing drugs.