Simultaneous Screening of 172 Veterinary Drugs by Modified QuEChERS-LC-MS/MS in TCM Galli Gigerii Endothelium Corneum.

An analytical method was developed for the screening of 172 veterinary drugs in traditional Chinese medicine Galli Gigerii Endothelium Corneum by high-performance liquid chromatography tandem mass spectrometry. The samples were pretreated by a modified QuEChERS method. A Zorbax Eclipse plus C18 column (1.8 µm, 3.0 × 150 mm2, Agilent) was used for the separation of analytes by gradient elution. All analytes were detected by electrospray ionization mass spectrometry with multiple reaction monitoring mode. Good linearity with R ≥ 0.99 was exhibited for all analytes within the respective range. The recoveries of all monitored analytes ranged from 55.4 to 127.6% at three spiked levels (limit of quantitation-LOQ, 2-fold LOQ, 10-fold LOQ), with relative standard deviations <17.8%. The estimated LOQ levels were 0.2-20 µg/kg. The application of this method provides a reference for the safety control of traditional Chinese medicines.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics.

Pheretima, also called "earthworms", is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines (CPMs) in Chinese Pharmacopoeia (2020 edition). However, its zoological origin is unclear, both in the herbal market and CPMs. In this study, a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms (restricted search, open search, and de novo) was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima, including Pheretima aspergillum (PA), Pheretima vulgaris (PV), and Metaphire magna (MM). We identified 10,477 natural peptides in the PA, 7,451 in PV, and 5,896 in MM samples. Five specific signature peptides were screened and then validated using synthetic peptides; these demonstrated robust specificity for the authentication of PA, PV, and MM. Finally, all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills, revealing the inconsistent Pheretima species used in these CPMs. In conclusion, our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines, especially non-model species with poorly annotated protein databases.

A MS-feature-based medicinal plant database-driven strategy for ingredient identification of Chinese medicine prescriptions.

Identification of the individual herbs that constitute the Chinese medicine prescription (CMP) is a key step to control the quality and ensure the efficacy of traditional Chinese medicine (TCM), but also a challenging task for analysts from all over the world. In this study, a MS-feature-based medicinal plant database-driven strategy was proposed for quick and automatic interpretation of CMP ingredients. The single herb database consisting of stable ions of sixty-one common TCM medicinal herbs was first constructed. And then, the data of CMP was imported into a self-built searching program to achieve quick and automatic identification with four steps including level 1 candidate herb screening based on stable ions (step 1), level 2 candidate herb screening based on unique ions (step 2), difficult-to-distinguish herb differentiation (step 3) and results integration (step 4). The identification model was optimized and validated with homemade Shaoyaogancao Decoction, Mahuang Decoction, Banxiaxiexin Decoction, and their related negative prescriptions and homemade fakes. Another nine batches of homemade and commercial CMPs were applied to this new approach and most of composed herbs in the corresponding CMPs were correctly identified. This work provided a promising and universal strategy for the clarification of CMP ingredients.

Low-Temperature Cleanup Followed by Dispersive Solid-Phase Extraction for Determination of Nine Polar Plant Growth Regulators in Herbal Matrices Using Liquid Chromatography-Tandem Mass Spectrometry.

Polar plant growth regulators, used alone or doped in fertilizers, are most effective and widely utilized plant growth regulators (PGRs) in agriculture, which play important roles in mediating the yield and quality of crops and foodstuffs. The application scope has been extended to herbal medicines in the past 2 decades and relevant study is inadequate. The aim of this study is to establish a QuPPe-based extraction method containing low-temperature and d-SPE cleanup procedure followed by the detection on a selective multiresidue ultrahigh-performance liquid chromatography - triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in three herbal matrices. This simple, accurate, versatile and robust method was verified according to the validation criteria of the SANTE/12682/2019 guideline document. The analytical range was from 2.5 to 200 µg/L, and the average recoveries were in the range of 64.6-117.8% (n = 6). The optimized method was applied to 135 herbal medicines thereof. Result showed that the detection frequency of chlormequat was the highest in the investigated PGRs, with the positive rate of 15.6%. Improvement of the detection method for polar PGRs will enrich the coverage of PGRs, which is conducive to safeguard public health and ensure drug safety. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-023-04254-3.

Isolation and characterization of a new oxyphenisatin analogue, oxyphenisatin propionate, from a processed plum intended as a weight loss product.

A new oxyphenisatin analogue was detected from a processed plum claiming to be a weight loss product without any side effects during the daily inspecting and monitoring of illegal adulterants in health supplements. An abundant peak caused our interest firstly owing to its identical fragments of m/z 224 and 196 in the MS/MS experiments with those of oxyphenisatin acetate. The chemical structure of the unknown compound was characterized by ultra-high performance liquid chromatography equipped with diode array detector and quadrupole time-of-flight tandem mass spectrometry (UHPLC-DAD-Q-TOF/MS), followed by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy experiments. Based on the data, it was defined that the two symmetrical acetyl groups in oxyphenisatin acetate were replaced by two propionyl groups for the unknown structure. Finally, the new oxyphenisatin analogue was identified as 3,3-bis[4'-(propionyloxy)phenyl]-1,3-dihydroindole-2-one and designated as oxyphenisatin propionate. Thereafter, the content of the new analogue was quantitatively determined to be 681 mg/kg, which would inevitably cause adverse health effect because there was not specification for daily consumption of this product. To the best of our knowledge, this is the first report for identification of oxyphenisatin propionate.

MRTCM: A comprehensive dataset for probabilistic risk assessment of metals and metalloids in traditional Chinese medicine.

Traditional Chinese medicine (TCM) is still considered a global complementary or alternative medical system, but exogenous hazardous contaminants remain in TCM even after decocting. Besides, it is time-consuming to conduct a risk assessment of trace elements in TCMs with a non-automatic approach due to the wide variety of TCMs. Here, we present MRTCM, a cloud-computing infrastructure for automating the probabilistic risk assessment of metals and metalloids in TCM. MRTCM includes a consumption database and a pollutant database involving forty million rows of consumption data and fourteen types of TCM potentially toxic elements concentrations. The algorithm of probabilistic risk assessment was also packaged in MRTCM to assess the risks of eight elements with Monte Carlo simulation. The results demonstrated that 96.64% and 99.46% had no non-carcinogenic risk (hazard indices (HI) were < 1.0) for animal and herbal medicines consumers, respectively. After twenty years of exposure, less than 1% of the total carcinogenic risk (CRt) was > 10-4 for TCM consumers, indicating that they are at potential risk for carcinogenicity. Sensitivity analysis revealed that annual consumption and concentration were the main variables affecting the assessment results. Ultimately, a priority management list of TCMs was also generated, indicating that more attention should be paid to the non-carcinogenic risks of As, Mn, and Hg and the carcinogenic risks of As and Cr in Pheretima and Cr in Arcae Conch. In general, MRTCM could significantly enhance the efficiency of risk assessment in TCM and provide reasonable guidance for policymakers to optimize risk management.

Integrated Metabolome and Lipidome Strategy to Reveal the Action Pattern of Paclobutrazol, a Plant Growth Retardant, in Varying the Chemical Constituents of Platycodon Root.

Platycodon root, a medicinal food homology species which has been used in Asian countries for hundreds of years, is now widely cultivated in China. Treatment with paclobutrazol, a typical plant growth retardant, has raised uncertainties regarding the quality of Platycodon root, which have been rarely investigated. In the present study, metabolomic and lipidomic differences were revealed by ultra-high performance liquid chromatography coupled to ion mobility-quadrupole time of flight mass spectrometry (UPLC-IM-QTOF-MS). A significant decrease of platycodigenin-type saponins was observed in the paclobutrazol-treated sample. Carrying out a comprehensive quantitative analysis, the contents of total saponins and saccharides were determined to illustrate the mode of action of paclobutrazol on Platycodon root. This study demonstrated an exemplary research model in explaining how the exogenous matter influences the chemical properties of medicinal plants, and therefore might provide insights into the reasonable application of plant growth regulators.

A Strategy for Rapid Discovery of Marker Peptides Associated with Fibrinolytic Efficacy of Pheretima aspergillum Based on Bioinformatics Combined with Parallel Reaction Monitoring.

Quality control of animal-derived traditional Chinese medicines has improved dramatically as proteomics research advanced in the past few decades. However, it remains challenging to identify quality attributes with routine proteomics approaches since protein with fibrinolytic activity is rarely reported in pheretima, a typical animal-derived traditional medicine. A novel strategy based on bioinformatics combined with parallel reaction monitoring (PRM) was developed here to rapidly discover the marker peptides associated with a fibrinolytic effect. Potential marker peptides were found by lumbrokinase sequences' alignment and in silico digestion. The fibrinogen zymography was used to visually identify fibrinolytic proteins in pheretima. As a result, it was found that the fibrinolytic activity varied among different portions of pheretima. Fibrinolytic proteins were distributed regionally in the anterior and anterior-mid portion and there was no significant fibrinogenolytic activity observed in the mid-posterior and posterior portion. Finally, PRM experiments were deployed to validate and quantify selected marker peptides and a total of 11 peptides were identified as marker peptides, which could be potentially used in quality control of pheretima. This strategy provides a robust workflow to benefit the quality control of other animal-derived traditional medicines.

Comprehensive profiling of Platycodonis radix in different growing regions using liquid chromatography coupled with mass spectrometry: from metabolome and lipidome aspects.

Platycodon grandiflorus (Jacq.) A. DC. is widely cultivated across the south and north of China. Its root, Platycodonis radix, is commonly used as a vegetable, functional food, and traditional herbal medicine with various biological benefits. It is critical to fully clarify the chemical composition of Platycodonis radix for the sake of the food industry and traditional herb markets. In this study, a strategy of metabolome and lipidome profiling based on ultra-high performance liquid chromatography coupled to ion mobility-quadrupole time of flight mass spectrometry (UPLC-IM-QTOF-MS) was developed to reveal the overall chemical composition of Platycodonis radix. IN particular, comprehensive lipidome profiling was first performed for Platycodonis radix, in which 170 lipid molecular species including 55.9% glycerophospholipids, 31.2% glycerolipids, and 12.9% sphingolipids were identified. Platycodonis radix from two major production regions in China, Inner Mongolia and Anhui province, were collected and analyzed by the MS based approach combined with multivariate statistical analysis from both the metabolome and lipidome aspects. This study threw focus on the profiling investigations of Platycodonis radix from different growing regions and provided new potential in the lipidome analysis of medicinal food.

Exposure assessment and risk-based limit levels evaluation of ochratoxin A in Astragali Radix in China.

Ochratoxin A (OTA) is a mycotoxin found in a variety of foods and herbal medicines, and several governmental bodies around the world have set maximum allowable levels of OTA in different foods and herbal medicines. This study aims to evaluate the health risk of OTA in Astragali Radix (AR) in China, and to evaluate the effects of different limit levels on the risk control of OTA in AR. The concentrations of OTA in 187 samples of AR were investigated, and 61 (32.6%) samples were positive. The mean, 50th and 95th percentile values of OTA in positive samples were 56.2, 5.1 and 304.5 µg/kg, respectively. A margin of exposure (MOE) approach was applied to assess the risk. Considering other food sources, long-term consumers have a relatively high risk of OTA exposure due to the ingestion of AR. Theoretical limit levels of OTA in AR were evaluated from two dimensions by weighing the costs and the benefits. The results indicated that the limit levels that might be applied to the management of OTA contamination in AR in China could be screened out through risk-based evaluation of limit levels.

A rapid and simple signature peptides-based method for species authentication of three main commercial Pheretima.

Pheretima with various activities is a commonly used animal-derived traditional medicine in Asia countries. However, almost half of them are non-pharmacopoeia species in the market due to the similar morphological characteristics between medicinal and non-medicinal species. This study aims to establish an effective method based on signature peptides for species authentication of three main commercial Pheretima, including two major Pheretima species (Amynthas aspergillum, Metaphire vulgaris) and one main adulteration (Metaphire magna). Firstly, the species of 52 batches of commercial Pheretima were authenticated based on DNA barcodes. Secondly, proteomic analysis was performed for protein characterization of three main commercial Pheretima. Furthermore, their signature peptides were screened and validated using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, a simplified sample processing method was developed. Finally, large quantities of commercial Pheretima samples were analyzed for further verifying the feasibility of the signature peptides-based method. The result showed that the established method had a great application potential for authenticity identification of commercial Pheretima. SIGNIFICANCE: The authenticity assessment of medicinal materials is a main issue in the quality control process as deceptive practices could imply severe health risks. In this study, a rapid and simple method based on signature peptides was established for species authentication of three main commercial Pheretima, which can be an effective alternative to complex DNA barcoding and difficult morphological identification, and provided a reference for improvement of Pheretima quality standards.

Comprehensive characterization of the chemical constituents in Platycodon grandiflorum by an integrated liquid chromatography-mass spectrometry strategy.

Platycodon grandiflorum (PG), as a well-known medicine food homology species, possess various pharmacological effects and health benefits. Aiming to facilitate in-depth and global characterization of the chemical compositions of PG, a profiling method based on ultra-high performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry (UPLC/IM-QTOF-MS) was conducted. Consequently, as many as 187 compounds were plausibly or unambiguously identified. Most importantly, phospholipids (PLs) were first observed and identified in PG. Due to their widely confirmed bioactivities, an analysis scheme was developed by hydrophilic interaction liquid chromatography and electrospray ionization tandem mass spectrometry combined with the online Paternò-Büchi reaction (HILIC-PB-MS/MS). The fatty acyl chains and C=C locations of 180 PLs molecular species, which fell into four classes, were unprecedently characterized. This exposure strategy of multi-type constituents greatly enriches the chemical profiling of PG, and helps promoting the further development of therapeutic agents and nutraceutical products from PG.

Proteomics analysis in the kidney of mice following oral feeding Realgar.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar, a famous traditional Chinese mineral medicine, has been toxic to the renal system. However, the underlying mechanism of Realgar nephrotoxicity is still unclear. AIM OF THE STUDY: This study aimed to investigate the potential mechanism of Realgar-induced nephrotoxicity by using a label-free quantitative proteomic method. MATERIALS AND METHODS: 36 mice were randomly divided into four groups: Control group, 0.5-, 1.0, and 2.0 g/kg Realgar group. After one week, serum biochemical parameters and renal histopathological examination were performed. Label-free quantitative proteomics was used to identify differentially expressed proteins which were subsequently analyzed with bioinformatics methods. Western blot was utilized to verify the six representative protein expressions. RESULTS: The results showed that 2.0 g/kg Realgar significantly increased blood urea nitrogen and induced the formation of tube cast of renal tubules, while the lower-dose of 0.5 g/kg and 1.0 g/kg Realgar showed no changes. Label-free proteomic analysis identified 3138 proteins, and 272 of those proteins were screened for significant changes in a dose-dependent manner. Functional enrichment analysis suggested that these proteins could affect the apoptotic process and oxidative stress. Representative proteins in the 2.0 g/kg Realgar group, including Cat, Bad, Cycs, Nqo1, Podxl, and Hmox1, were verified by western blot. CONCLUSIONS: The results in this study suggest that apoptosis and oxidative stress might be related to the Realgar-induced nephrotoxicity in mice. Moreover, the strategy of proteomics could contribute to the understanding of the mechanisms of nephrotoxicity in mice exposed to Realgar.

Marker peptide screening and species-specific authentication of Pheretima using proteomics.

Pheretima is a common and valuable animal-derived medication used in traditional Chinese medicine. There are four species of Pheretima specified in the Chinese Pharmacopoeia (2015 edition), i.e. Pheretima aspergillum, P. vulgaris, P. guillelmi, and P. pectinifera. A recent report revealed ~ 55% of Pheretima in the commercial marketplace may be adulterated by other species, contrary to the Pharmacopoeia standard. The safety, efficacy, and authenticity of Pheretima is an important issue. Currently, the availability of specific quality-markers for the various species and effective identification methods are still limited. In this study, label-free quantification proteomics of species from Pheretima and Amynthas was carried out using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS), and marker peptides were identified based on their ion intensities using multivariate data analysis (principal component analysis and supervised partial least-squares discriminant analysis). A total of 48,476 peptides with high confidence corresponding to 13,397 proteins were identified from all samples. The marker peptides were validated by comparison with synthetic peptide reference standards using LC-MS/MS operating in a multiple-reaction monitoring mode. A multiple-peptide identification strategy was proposed for the authentication of Pheretima and subsequently applied to samples obtained from retail outlets in various regions of China. The results showed that eight out of the 15 samples tested were deemed authentic Pheretima.

Plant metabolomics for studying the effect of two insecticides on comprehensive constituents of Lonicerae Japonicae Flos.

Pesticides' overuse and misuse have been reported to induce ingredient variations in herbal medicine, which is now gaining attention in the medicinal field as a form of alternative medicine. To date, available studies on pesticide-induced ingredient variations of herbal medicine are limited only on a few compounds and remain most others unexamined. In this study, a plant metabolomics-based strategy was performed to systematically explore the effects of two frequently used insecticides on the comprehensive constituents of Lonicerae Japonicae Flos (LJF), the flower buds of Lonicera japonica Thunb. Field trials were designed on a cultivating plot of L. japonica with controls and treatments of imidacloprid (IMI) and compound flonicamid and acetamiprid (CFA). Unbiased metabolite profiling was conducted by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometer. After data pretreatment by automatic extraction and screening, a data matrix of metabolite features was submitted for statistical analyses. Consequently, 29 metabolic markers, including chlorogenic acids, iridoids and organic acid-glucosides were obtained and characterized. The relative quantitative assay was subsequently performed to monitor their variations across flowering developments. This is the first study that systematically explored the insecticide-induced metabolite variations of LJF while taking into account the inherent variability of flowering development. The results were beneficial for holistic quality assessment of LJF and significant for guiding scientific use of pesticides in the large-scale cultivation.

Heavy Metals and Probabilistic Risk Assessment via Pheretima (a Traditional Chinese Medicine) Consumption in China.

Earthworms are known to accumulate inorganic contaminants from the soil; they are also used as a traditional Chinese medicine (TCM) called Pheretima, which might cause safety problems with long-term exposure. Here, this study was conducted to determine and analyze the level of heavy metal contamination such as arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), manganese (Mn), nickel (Ni), and lead (Pb) in Pheretima and then explore the probabilistic health risks caused by 8 heavy metals in 98 batches of Pheretima using Monte Carlo simulation. A risk assessment strategy was conducted to assess heavy metal-associated health risk of Pheretima based on consumption data. For random consumption sampling, the results found that the non-carcinogenic risk of As is higher than the acceptable level, and the carcinogenic risk levels of As and Cr exceeded the acceptable risk recommended by the USEPA. Cr and As were regarded as the priority metals for risk control in the present study. Finally, it was recommended that the dosing frequency should be less than 24 d/y. In general, this study conducted a probabilistic risk assessment of heavy metals in Pheretima, which would be of significance for policy makers to take effective strategies to improve the quality and safety of Pheretima.

TaqMan real-time quantitative PCR for identification of antlers in tradition Chinese medicine.

In this study, a method was established for discriminating the true Cervus antlers from its counterfeits using TaqMan real-time quantitative PCR. The method combines the use of true Cervus antlers-specific primers, that amplify a 226 bp fragment from true Cervus antlers DNA, and mammalian-specific primers amplifying a 146 bp fragment from mammalian species DNA, which are used as endogenous control. A TaqMan probe that hybridizes in the 'Cervus antler' and also in the 'mammalian' DNA fragments is used to monitor the amplification of the target gene. The Cervus antler mitochondrial DNA was used as target gene to design the primers and TaqMan probes. The data revealed that the TaqMan real-time PCR-based assay can be used for identification of the true Cervus antlers from counterfeits in a single step. The limit of detection (LOD) was lower than 1 pg of DNA per reaction.

High performance liquid chromatography-tandem mass spectrometry method for residue determination of 39 plant growth regulators in root and rhizome Chinese herbs.

A simple, rapid, and effective method based on salting-out extraction and LC-MS/MS techniques was developed for the determination of 39 plant growth regulator (PGR) residues in two of the most common root and rhizome Chinese herbs, Codonopsis Radix (Dangshen) and Notoginseng Radix et Rhizoma (Sanqi). The extraction process was performed with acetonitrile-water (5:1) and citrate buffer extraction salt. The performance of the method was validated in accordance with the analytical quality control criteria of SANTE/11813/2017 guidelines. Analyte recoveries of 79.49-109.41% (Dangshen) and 80.17-102.81% (Sanqi) were achieved. The limit of quantifications (LOQs) were determined with the consideration of accuracy and precision. LOQs were lower than the lowest residue limits in EU pesticide regulation (10 µg/kg) for most PGRs. Moreover, the method was successfully applied in the analysis of 35 batches of Dangshen, and 60 batches of Sanqi products. The concentration of eleven PGRs were determined in analyzed samples.

Isolation and identification of a new sildenafil analogue, hydroxycarbodenafil, found as an adulterant in a health supplement.

During routine screening of illegal adulterants in health supplements, a novel sildenafil analogue was discovered, and subsequently isolated by recrystallization. Its structure was elucidated by extensive analyses of high resolution mass spectrometry (HRMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data. The analogue was finally determined as hydroxycarbodenafil, featuring a hydroxyethyl group instead of an ethyl group on piperazine ring in comparison with carbodenafil.