A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Plastic breath: Quantification of microplastics and polymer additives in airborne particles.

The widespread extensive use of synthetic polymers has led to a substantial environmental crisis caused by plastic pollution, with microplastics detected in various environments and posing risks to both human health and ecosystems. The possibility of plastic fragments to be dispersed in the air as particles and inhaled by humans may cause damage to the respiratory and other body systems. Therefore, there is a particular need to study microplastics as air pollutants. In this study, we tested a combination of analytical pyrolysis, gas chromatography-mass spectrometry, and gas and liquid chromatography-mass spectrometry to identify and quantify both microplastics and their additives in airborne particulate matter and settled dust within a workplace environment: a WEEE treatment plant. Using this combined approach, we were able to accurately quantify ten synthetic polymers and eight classes of polymer additives. The identified additives include phthalates, adipates, citrates, sebacates, trimellitates, benzoates, organophosphates, and newly developed brominated flame retardants.

Gas chromatography-stable isotope ratio mass spectrometry prior solid phase microextraction and gas chromatography-tandem mass spectrometry: development and optimization of analytical methods to analyse garlic (Allium sativum L.) volatile fraction.

Garlic (Allium sativum L.) is not only appreciated for its flavour and taste, but it is also recognized for various health properties. The European Commission, through the attribution of the Protected Designation of Origin (PDO) certification mark, has officially recognized some specific varieties of garlic. To protect not only the commercial value but also the reputation of this appreciated product, effective tools are therefore required. For the first time, a new compound specific isotope analysis method based on carbon stable isotopic ratio measurement of the three major volatile garlic compounds allyl alcohol (AA), diallyl disulphide (DD) and diallyl trisulphide (DT) through head-space solid phase microextraction (HS-SPME) followed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was developed. A within-day standard deviation (Srwithin-day) of 0.3 ‰, 0.4 ‰ and 0.2 ‰ for δ(13C) and a between-day standard deviation (Srbetween-day) of 0.8 ‰, 1.0 ‰ and 0.6 ‰ of AA, DT and DD was estimated. For the first time, the ranges of isotopic variability for the three volatile compounds of red garlic from two neighbouring Italian regions (Abruzzo and Lazio) were defined analysing 30 samples. The same dataset was also considered in analysing the percentage composition of the previously mentioned three volatile compounds through HS-SPME followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The two analytical approaches were combined in this explorative study, aiming to provide potential parameters to discriminate garlic samples based on their geographical origin.

Comparative chemical profiling of leaf essential oils from Cinnamomum kanehirae and related species using steam distillation and solvent extraction: Implications for plant-based classification.

Cinnamomum kanehirae Hayata, belonging to Lauraceae family, is an indigenous and endangered species of considerable economic importance in Taiwan. It plays a crucial role as the host for the economically valuable saprotrophic fungus, Taiwanofungus camphorates. However, accurate species identification poses a challenge due to the similarity in morphological features and frequent natural hybridization with closely related species. Acquiring high-quality and pure leaf oils becomes imperative for precise species identification and producing superior goods. In this study, our objective was to establish methodologies for analyzing the chemical composition of leaf essential oils and subsequently apply this knowledge to differentiate among three Cinnamomum species. Gas chromatography-mass spectrometry (GC/MS) was employed to scrutinize the chemical makeup of leaf essential oils from three closely related species: C. kanehirae, C. micranthum, and C. camphora. We utilized Steam Distillation (SD) and steam distillation-solvent extraction (SDSE) methods, with the SDSE-Hexane approach chosen for optimization, enhancing extraction efficiency and ensuring essential oil purity. Through the SDSE-Hexane method, we identified seventy-four compounds distributed across three major classes: monoterpenes hydrocarbons (0.0-7.0 %), oxygenated monoterpenes (3.8-90.9 %), sesquiterpenes hydrocarbons (0.0-28.3 %), and oxygenated sesquiterpenes (1.6-88.1 %). Our findings indicated the presence of more than one chemotype in both C. kanehirae and C. camphora, whereas no specific chemotype could be discerned in C. micranthum. Furthermore, clustering based on chemotypes allowed for the differentiation of samples from the three species. Notably, we demonstrated that the chemical compositions of grafted C. kanehirae remained largely unaffected by the rootstock. Conversely, natural hybrids between C. kanehirae and C. camphora exhibited profiles more closely aligned with C. kanehirae. The optimized extraction method and the chemotype-based classification system established in this study present valuable tools for essential oil preparation, species identification, and further exploration into the genetic variation of Cinnamomum.

GC/MS-based untargeted metabolomics reveals the differential metabolites for discriminating vintage of Chenxiang-type baijiu.

The "outstanding and unique aged aroma" of Chinese Chenxiang-type baijiu (CXB)-Daoguang 25 (DG25) mainly originates from a "extraordinary storage technology" of Mujiuhai (a wooden container), so it is mysterious and interesting. In this study, an untargeted GC/MS-based metabolomics was used to reveals the volatile differential metabolites for discriminating six different vintages of DG25 combing with chemometrics. A total of 100 volatile metabolites (including unknowns) were extracted and identified, including esters (41%), alcohols (10%) and acids (7%) so on. Finally, 33 differential metabolites were identified as aging-markers. Among them, 25 aging-markers showed a downtrend, including 17 esters such as ethyl acetate, ethyl hexanoate and ethyl palmitate so on. Moreover, it was interesting and to further study that furans showed a significant downtrend. Statistically speaking, ethyl benzoate played an important role in discriminating vintage of 1Y and 3Y, and the other 24 differential metabolites with downtrend discriminating the unstored (0Y-aged) DG25. Eight differential metabolites, such as ethyl octanoate, benzaldehyde, 3-methylbutanol and 1,1-diethoxyaccetal so on increased during aging of DG25, and they played a statistical role in discriminating the 5Y-, 10Y- and 20Y-aged DG25. This study provides a theoretical basis way for the formation mechanism of aging aroma for CXB.

The dynamic changes in volatile metabolites provide a new understanding for the special flavor formation in z. Mioga flower buds during the growth stages.

Although Z. mioga flower buds are popular among consumers for its unique spicy flavor, high nutritional and medicinal value, there are few reports on the formation and changes of the flavor during its growth and maturation process. The understanding of the profile of volatile compounds would help to unravel the flavor formation for Z. mioga flower buds during growth. The volatile changes in Z. mioga flower buds were analyzed by GC-MS and a total of 182 volatile compounds identified, and the terpenoids accounted for the most abundant volatile substances. Almost all the identified volatiles presented an intuitive upward trend throughout the growth period and reached the maximum at the later stage of development (DS3 or DS4). Regarding the PCA and HCA results, there were significant differences found among the four stages, and the DS3 was the critical node. The top 50 differential volatiles screened by OPLS-DA and PLS-DA were all up-regulated, and the correlation analysis indicated that terpenoids might synergize with other chemical types of volatiles to jointly affect the flavor formation of Z. mioga flower buds during growth. The association network for flavor omics revealed that the most important sensory flavor for Z. mioga flower buds were woody and sweet, and the main contribution compounds for the unique flavor contained ß-guaiene, ß-farnesene, δ-cadinene and citronellyl isobutanoate. Taken together, the results of this study provided a reference for flavor quality evaluation of flower buds and determination of the best harvest period.

Unravelling the metabolomic diversity of pigmented and non-pigmented traditional rice from Tamil Nadu, India.

Rice metabolomics is widely used for biomarker research in the fields of pharmacology. As a consequence, characterization of the variations of the pigmented and non-pigmented traditional rice varieties of Tamil Nadu is crucial. These varieties possess fatty acids, sugars, terpenoids, plant sterols, phenols, carotenoids and other compounds that plays a major role in achieving sustainable development goal 2 (SDG 2). Gas-chromatography coupled with mass spectrometry was used to profile complete untargeted metabolomics of Kullkar (red colour) and Milagu Samba (white colour) for the first time and a total of 168 metabolites were identified. The metabolite profiles were subjected to data mining processes, including principal component analysis (PCA), Orthogonal Partial Least Square Discrimination Analysis (OPLS-DA) and Heat map analysis. OPLS-DA identified 144 differential metabolites between the 2 rice groups, variable importance in projection (VIP) ≥ 1 and fold change (FC) ≥ 2 or FC ≤ 0.5. Volcano plot (64 down regulated, 80 up regulated) was used to illustrate the differential metabolites. OPLS-DA predictive model showed good fit (R2X = 0.687) and predictability (Q2 = 0.977). The pathway enrichment analysis revealed the presence of three distinct pathways that were enriched. These findings serve as a foundation for further investigation into the function and nutritional significance of both pigmented and non-pigmented rice grains thereby can achieve the SDG 2.

Integrated identification-quantification (ID-Quant) workflow utilizing UPLC-QTOF-MS for the therapeutic drug monitoring of multi-component antibiotics without pure standards: Validation using teicoplanin.

The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MSE full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.

Chemical profiling of principle active and toxic constituents in herbs containing aristolochic acids.

Objective: To clear the amounts of the principal active/toxic components in herbs containing aristolochic acids (HCAAs), which are still used as medicine and/or seasoning in many ethnic minority areas of China. Methods: In this study, six major active and toxic components in HCAAs were extracted with ultrasonic extraction. With 6-O-methyl guanosine as internal standard, the target compounds were analyzed qualitatively and quantitatively by using ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) with multiple reaction monitoring-information dependent acquisition-enhanced production ion scanning mode (MRM-IDA-EPI) combined with dynamic background subtraction (DBS) function. Results: The method showed good linearity in the linear range of the six analytes. The limit range of detection was from 0.01 ng/mL to 0.27 ng/mL. All of the detection repeatability, extraction repeatability and accuracy of the method were good. After extraction, the samples remained stable at 15 °C within 24 h. Six analytes were all found in samples except aristolactam (AL) in sample 2, and the contents varied greatly. The contents of these compounds decreased in fruits, leaves and stems of Aristolochia delavayi successively. Conclusion: This method has the advantages of less sample dosage, simple operation, short analysis cycle, high sensitivity, specificity and accuracy. It laid a good foundation for guiding the safety of HCAAs, the in-depth study of pharmacological and toxicological effects and the scientific and standardized processing and compatibility of HCAAs.

Chitin quantitation (as glucosamine) in food raw materials by HPLC-C18-DAD with off-line derivatization.

This HPLC method is suitable for chitin quantitation (reported as glucosamine) in food raw materials like insects (mealworm larvae, crickets), shrimps, mushrooms and fungi in a research (non-routine) laboratory using a C18 column with HPLC system <600 bar with UV detection capability (at 265 nm). To remove interferences, the sample is defatted (Soxhlet) and deproteinized (by alkali) prior to acid hydrolysis in 6 M HCl. A five-point linear calibration (5-100 µg/mL) is used. The use of fluorescence detection (λex = 260 nm, λem = 350 nm) is also possible with this method [1].•18 min HPLC run time•LOD = 0.05 µg/mL and LOQ = 5 µg/mL.

A new anti-inflammatory lupane in Ziziphus jujuba (L.) Gaertn. var. hysudrica Edgew.

Objectives: To investigate extracts of the stem bark of Ziziphus jujuba (L.) Gaertn. var. hysudrica Edgew. (Rhamnaceae) for anti-inflammatory activity and isolate the active principle(s). Methods: The dry powder was macerated separately in three types of solvents to prepare methanol extract (ME), ethyl acetate extract (EE), and chloroform extract (CE). Following in vitro anti-inflammatory screening, the most active extract was selected to isolate the active compound. Both, the active extract and isolated compound were further tested on rats using the carrageenan-induced inflammation model. The blood and paw tissue were subjected to qPCR, and histopathology, respectively. Key findings: CE showed comparatively higher anti-inflammatory activity (85.0-95.0 %) in all in vitro assays, except the heat-induced membrane stabilization model (p < 0.05), and upon column chromatography, it yielded a pure crystalline compound. The compound was a pentacyclic triterpenoid (Lupane), named as hydroxymethyl (3ß)-3-methyl-lup-20(29)-en-28-oate (Hussainate). CE (500 mg/kg) and Hussainate (1.0 mg/kg) reduced edema in 5 h after carrageenan administration. The activity of Hussainate was found to be comparable to that of dexamethasone (standard). The possible activity mechanism was the downregulation of tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-II), NF-κB, and IL-1ß. Conclusions: This study reveals that chloroform extract of the stem's bark of Z. jujuba may be used to prepare standardized anti-inflammatory herbal products using Hussainate as an active analytical marker. Hussainate may be used as a lead to develop anti-inflammatory drugs.

Systematic review on fingerprinting development to determine adulteration of Chinese herbal medicines.

BACKGROUND: It has been a current research hospots using fingerprinting technology for quality control of Chinese herbal medicines (CHMs), which provides a scientific basis for establishment of overall quality control in accordance with the characteristics of CHMs. The fingerprinting technology for CHMs is diverse, and the research field covers many disciplines, such as analytical chemistry, pharmacology, pharmaceutics, biochemistry, and molecular biology. PURPOSE: To effectively understand the key areas and future directions of research regarding the fingerprint and adulteration of CHMs. METHODS/RESULTS: this paper analyzed 879 articles in this field in the Web of Science Core Collection from 2000 to 2023 with CiteSpace and VOSviewer, and systematically assessed the research process, hotspots, topic distribution among disciplines, etc. The most prominent contributors of fingerprint and adulteration of CHMs research are mainly from China, India, the United States, England, and Brazil. The knowledge domains of fingerprint and adulteration of CHMs research focus mainly on the topics of molecular authentication, DNA barcoding, HPLC, near-infrared spectroscopy, manage data, chemometrics, and electrochemical fingerprinting. Most countries have recognized the pharmaceutical potential of natural products, and have paid more attention to the fingerprint and adulteration of CHMs in the past decade. Future the research tends to focus more on molecular identification and authentication, and electrochemical and chromatographic fingerprinting in controlling the adulteration of CHMs. CONCLUSION: This research provides a valuable reference for scholars in related fields to analyze existing research results, understand the development trend, and explore new research directions.

Magnetic-stirring-enhanced mechanical amorphous dispersion extraction for the hydrophobic phytochemical constituents using an aqueous solution from a medicinal plant.

A method involving chitosan-assisted magnetic-stirring-enhanced mechanical amorphous dispersion extraction was developed and utilized to extract hydrophobic anthraquinones from Rhei Radix et Rhizoma prior to ultrahigh performance liquid chromatography analysis. Incorporating natural chitosan as a dispersant facilitated the extraction of hydrophobic anthraquinones using purified water, considerably enhancing the eco-friendliness of the extraction methodology. To optimize extraction efficiency, an extensive evaluation of the crucial parameters influencing rhubarb yield was conducted. Furthermore, a response surface methodology was applied to optimize the extraction conditions. Under these optimized conditions, the method exhibited linearity ranges of 0.1-100 µg/mL, with correlation coefficients between 0.9990 and 0.9998. The method's intraday (n = 6) and interday (n = 6) precision levels were maintained at ≤3.58%, which was considered to be within acceptable limits. The computed detection and quantification limits were 16.54-24.60 and 54.91-82.04 ng/mL, respectively. Consequently, this optimized method was effectively employed to extract five specific compounds (aloe-emodin, emodin, rhein, chrysophanol, and physcion) from Rhei Radix et Rhizoma, achieving recoveries ranging from 86.43% to 102.75%.

Innovative orthogonal two-dimensional reversed-phase liquid chromatography × supercritical fluid chromatography with a phenyl/tetrazole stationary phase for the preparative isolation of diarylheptanoids.

In this investigation, we successfully isolated and purified natural diarylheptanoids using an orthogonal offline two-dimensional RPLC × SFC approach, employing only the phenyl/tetrazole stationary phase. First, a styrene-divinylbenzene matrix medium pretreatment liquid chromatography system effectively processed chlorophyll-containing plant extract solution with a recovery rate of 33.8 %, obviating the need for concentration steps. Subsequently, an offline two-dimensional RPLC × SFC employing only the phenyl/tetrazole stationary phase achieved a remarkable 96.38 % orthogonality and was established and utilized in the preparative separation and purification of natural products. Finally, the constructed single stationary phase highly orthogonal RPLC × SFC system was successfully applied in the preparative separation and purification of natural diarylheptanoids from the Saxifraga tangutica target fraction and yielded four diarylheptanoids with purities exceeding 95 %.

Concept of a six-fold multiplex planar bioassay to distinguish endocrine agonist, antagonist, cytotoxic and false-positive responses.

To analyze a complex sample for endocrine activity, different tests must be performed to clarify androgen/estrogen agonism, antagonism, cytotoxicity, anti-cytotoxicity, and corresponding false-positive reactions. This means a large amount of work. Therefore, a six-fold planar multiplex bioassay concept was developed to evaluate up to the mentioned six endpoints or mechanisms simultaneously in the same sample analysis. Separation of active constituents from interfering matrix via high-performance thin-layer chromatography and effect differentiation via four vertical stripes (of agonists and end-products of the respective enzyme-substrate reaction) applied along each separated sample track were key to success. First, duplex endocrine bioassay versions were established. For the androgen/anti-androgen bioassay applied via piezoelectric spraying, the mean limit of biological detection of bisphenol A was 14 ng/band and its mean half maximal inhibitory concentration IC50 was 116 ng/band. Applied to trace analysis of six migrate samples from food packaging materials, 19 compound zones with agonistic or antagonistic estrogen/androgen activities were detected, with up to seven active compound zones within one migrate. For the first time, the S9 metabolism of endocrine effective compounds was studied on the same surface and revealed partial deactivation. Coupled to high-resolution mass spectrometry, molecular formulas were tentatively assigned to compounds, known to be present in packaging materials or endocrine active or previously unknown. Finally, the detection of cytotoxicity/anti-cytotoxicity and false-positives was integrated into the duplex androgen/anti-androgen bioassay. The resulting six-fold multiplex planar bioassay was evaluated with positive control standards and successfully applied to one migrate sample. The streamlined stripe concept for multiplex planar bioassays made it possible to assign different mechanisms to individual active compounds in a complex sample. The concept is generic and can be transferred to other assays.

Antimicrobial, antibiofilm, and antiviral investigations using egyptian phoenix dactylifera L. pits extract.

Phoenix dactylifera L. and its wastes are known to be high in nutrients that are beneficial to human health. The study aimed to evaluate the antimicrobial, antibiofilm, and antiviral properties of Phoenix dactylifera L. pits extract (PDPE) in vitro. Gas chromatography-mass spectrometry (GC-MS) analysis indicated phenol, 2,5-bis(1,1-dimethyl ethyl), tetradecanoic acid, octaethylene glycol monododecyl ether, á-D-glucopyranosiduronic acid, and heptaethylene glycol monododecyl ether existence. The PDPE influenced pathogenic microorganisms, with inhibition zone diameters (IZDs) ranging from 10.0 to 35.0 mm. Staphylococcus aureus ATCC 5638 had the highest IZD, while Salmonella typhi DSM 17058 and Shigella sonnei DSM 5570 had the lowest. The antifungal effect observed only in spore failure or conidia formation. PDPE showed a 100% antibacterial spectrum against bacteria, with MIC values between 250 and 1000 µg/ml. MIC was only indicated with S. aureus of 500 µg/ml. MBC values ranged from 500 to 1000 g/ml, with MBC values of 500 g/ml for B. cereus, E. faecalis, S. typhi, and S. sonnei. The activity was 66.7% at 500 µg/ml, further concentrations of 125-250 g/ml had no antibacterial effect. PDPE biofilm inhibition % had the highest percentage of inhibition (98.59%) with S. aureus, B. cereus (94.12%), and E. coli (74.46%). With 50% (CC50) viral activity, the highest non-toxic PDPE dose was found to be at 123.0 µg/ml.

Development and validation of a high-performance thin-layer chromatography method for detection of sibutramine in dietary supplements.

INTRODUCTION: In the period between 1997 and 2010, sibutramine-containing drugs were widely prescribed for obesity and over-weight management. Due to safety concerns, in 2010 all medicines containing sibutramine were urgently withdrawn from the USA and European pharmaceutical market. Although sibutramine is no longer available in pharmaceutical products, there have been numerous reports of mislabeled weight-loss dietary supplements containing sibutramine.

Qualitative transformations of street-seized ecstasy over a decade: A case study in Rio de Janeiro (Brazil).

The illegal drug market is constantly evolving, with new drugs being created and existing ones being modified. Adulterants are often added to the mix, and the primary substance may be secretly replaced by a new one. Once-known tablets can now be vastly different from what they are sold as, all due to the pursuit of profit and evasion of current drug regulations. These alterations in drug composition pose a threat to society, as their effects are still not well understood. Therefore, it is crucial for police intelligence and public health development to obtain the chemical profiles of illicit drugs. This study presents the chemical fingerprinting of ecstasy tablets seized in the state of Rio de Janeiro (Brazil) between 2012 and 2021. The tablet samples were weighed, extracted, diluted with methanol, and acidified before analysis using gas chromatography high-resolution mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopy. The major constituents found were MDMA and clobenzorex, with fewer occurrences of MDA, MDEA, and 2C-B. The results also indicate that the occurrence of mega-events in the study location impacted the chemical fingerprints of ecstasy. A total of 27 combinations of cutting agents, including caffeine, ephedrine, and anesthetics, were identified. Samples composed of clobenzorex were observed throughout the evaluated period in areas near highways, suggesting that this product is mainly used by truck drivers. These findings can help police intelligence units anticipate the behavior of the illicit market during major events, identify traffic routes, and support public health initiatives.

Chemical Relationship among Genetically Authenticated Medicinal Species of Genus Angelica.

The genus Angelica comprises various species utilized for diverse medicinal purposes, with differences attributed to the varying levels or types of inherent chemical components in each species. This study employed DNA barcode analysis and HPLC analysis to genetically authenticate and chemically classify eight medicinal Angelica species (n = 106) as well as two non-medicinal species (n = 14) that have been misused. Nucleotide sequence analysis of the nuclear internal transcribed spacer (ITS) region revealed differences ranging from 11 to 117 bp, while psbA-trnH showed variances of 3 to 95 bp, respectively. Phylogenetic analysis grouped all samples except Angelica sinensis into the same cluster, with some counterfeits forming separate clusters. Verification using the NCBI database confirmed the feasibility of species identification. For chemical identification, a robust quantitative HPLC analysis method was developed for 46 marker compounds. Subsequently, two A. reflexa-specific and seven A. biserrata-specific marker compounds were identified, alongside non-specific markers. Moreover, chemometric clustering analysis reflecting differences in chemical content between species revealed that most samples formed distinct clusters according to the plant species. However, some samples formed mixed clusters containing different species. These findings offer crucial insights for the standardization and quality control of medicinal Angelica species.