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ObjectiveUsing the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the plasma level of Lyso-GL3 in patients with Fabry disease and to analyze the clinical application of the method.MethodsThirty-nine patients with a genetic diagnosis of Fabry disease were included, and plasma levels of Lyso-GL3 were measured by LC-MS/MS analysis, and detailed clinical information of the patients was obtained including: α-galactosidase A activity, genetic variants, quantification of urine protein, mean arterial pressure, and estimation of glomerular filtration rate, and the differences in the levels of Lyso-GL3 in different clinical phenotypes and genotypes were statistically analyzed, as well as the association with clinical indicators.ResultsLyso-GL3 showed good linearity within 0.7856-400 ng/mL(r=0.9992).Further analysis of 39 Fabry disease patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine showed a median Lyso-GL3 concentration of 23.6 ng/mL(4.3-92.9 ng/mL); Lyso-GL3 levels were significantly higher in patients with both the frameshift and the splicing mutations, as well as in patients with the nonsense mutations, than in patients with the missense mutations (median value 119.7 ng/mL vs. 11.9 ng/mL, P=0.006, and median value 97.0 ng/mL vs. 11.9 ng/mL, P=0.015, respectively). Whereas, association analysis revealed that Lyso-GL3 was not significantly associated with urinary protein, mean arterial pressure and estimated glomerular filtration rate.ConclusionsThe using of LC-MS/MS to quantify plasma Lyso-GL showed significant differences in Lyso-GL3 concentrations between classical and atypical phenotypes, suggesting that plasma Lyso-GL3 may help with clinical phenotypes. However, Lyso-GL3 levels is found to be overlapped between genotypes. No significant linear correlation was found between Lyso-GL3 and renal clinical indicators, suggesting the urgent need in finding a more accurate tool to assess renal involvement and prognosis in patients with Fabry disease.
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ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang（LJZT）， and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， and the resulting compounds were identified by using databases， such as MassBank， PubChem， ChemSpider， Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform（TCMSP）， and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT， including liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature， database and MS information， a total of 79 compounds were identified from LJZT， including 31 flavonoids， 15 terpenoids， 14 nitrogen-containing compounds， 6 phenylpropanoids， 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges， and the precision， stability， reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5， 2.602 1， 0.082 6， 0.128 1， 1.778 6， 0.015 7， 0.006 7， 0.030 4， 0.003 2 mg·g-1， respectively. ConclusionThe established method can quickly， sensitively and accurately analyze the chemical constituents in LJZT， clarify that the material basis of LJZT is mainly flavonoids， terpenoids and nitrogen-containing compounds， and simultaneously determine the contents of the 9 components， which can lay a foundation for the research on quality control， mechanism and clinical application of LJZT.
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ObjectiveTo investigate the mechanism of Atractylodis Macrocephalae Rhizoma（AMR） in the treatment of slow-transmission constipation（STC） by observing the effects of AMR on short-chain fatty acids and intestinal barries in STC mice. MethodForty-eight male KM mice were randomly divided into blank group， model group， AMR low-， medium-， high-dose groups（2.5， 5， 10 g·kg-1） and mosapride group（2.5 mg·kg-1）. Except for the blank group， all groups were gavaged with loperamide suspension（5 mg·kg-1） twice daily for 14 d to construct the STC mouse model. At the same time， each drug administration group was given the corresponding drug by gavage for consecutive 14 d， the blank and model groups were gavaged with equal volume of distilled water. The effects of the treatment of AMR on body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice were observed， the pathological changes of mouse colon were observed by hematoxylin-eosin（HE） staining and periodic acid-Schiff（PAS） staining， the levels of gastrin（GAS） and motilin（MTL） in serum were detected by enzyme-linked immunosorbent assay（ELISA）， gas chromatography-mass spectrometry（GC-MS） was used to detect the contents of short-chain fatty acids（SCFAs） in mouse feces， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to determine the mRNA and protein expression levels of zonula occludens-1（ZO-1）， Occludin， and Claudin-1 in the colon of mice. ResultCompared with the blank group， the body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice in the model group were significantly decreased（P<0.05， P<0.01）， the arrangement of colonic tissues was disordered， and the number of goblet cells was reduced， the levels of GAS and MTL in serum were significantly decreased（P<0.01）， and the levels of SCFAs in the feces were on a decreasing trend， with the contents of acetic acid， propionic acid， butyric acid， isobutyric acid and valeric acid were significantly decreased（P<0.05， P<0.01）， the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colonic tissues were significantly decreased（P<0.01）. The above results suggested that STC mouse model was successfully constructed. Compared with the model group， the body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice in AMP administration groups all increased significantly（P<0.05， P<0.01）， the mucosal layer of the colonic tissues was structurally intact without obvious damage， and the number of goblet cells increased， serum levels of GAS and MTL were significantly increased（P<0.01）， the contents of SCFAs in the feces were all on a rising trend， with the contents of acetic， propionic， butyric and isobutyric acids rising significantly（P<0.05， P<0.01）， the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colonic tissues were significantly increased（P<0.05， P<0.01）. ConclusionAMR is able to improve the constipation symptoms in STC mice， and its mechanism may be related to increasing the contents of SCFAs in the intestine as well as promoting the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colon.
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Background Volatile organic compounds (VOCs) in exhaled breath are closely associated with respiratory diseases and are linked to various metabolic reactions in the human body. A quantitative analytical method can provide technical support for studying VOCs related to various diseases. Objective To establish a thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) method for the determination of 27 VOCs in exhaled breath. Methods VOCs in exhaled breath were collected using a Bio-VOC sampler and enriched with Tenax TA thermal desorption tubes before TD-GC-MS analysis. Standards were collected using thermal desorption tubes and optimized for thermal desorption conditions as well as chromatographic and mass spectrometric conditions: The separation of the 27 VOCs was achieved by an optimized temperature program, the improvement of sensitivity by optimizing quantitative ions, and the increase of VOCs desorption efficiency by optimizing thermal desorption time and temperature. Limit of detection, limit of quantification, accuracy, precision, and stability of the proposed method were investigated by spiking with a blank gas bag, and exhaled breath samples from 20 healthy individuals were collected for an application study of the proposed method. Results The thermal desorption temperature was 280 ℃, and desorption time was 6 min. A VF-624ms chromatographic column was selected for the separation of target substances. The initial temperature of heating program was 35 ℃, maintained for 1 min, and then increased to 100 ℃ at a heating rate of 3 ℃·min−1 for 1 min, followed by increasing to 210 ℃ at a heating rate of 28 ℃·min−1 for 5 min. A quantitative analysis was conducted with a single ion monitoring (SIM) mode. Under these conditions, the 27 VOCs showed good linear relationships in their respective concentration ranges and the correlation coefficients were higher than 0.9990. The limits of detection of the method were in the range of 0.01-0.13 nmol·mol−1, the limits of quantification were in the range of 0.02-0.44 nmol·mol−1, and the spiked recoveries were in the range of 80.1%-120.5%, with intra-batch and inter-batch precision ≤ 18.8% and 17.9% respectively. All substances can be stored at room temperature (23-28 °C) for 7 d and at 4 °C for 14 d. The proposed method was applied to exhaled breath samples from 20 subjects with detection rates≥ 80% (except for trans-2-pentene and decane) and a concentration range of 0.00-465.50 nmol·mol−1. Conclusion The established TD-GC-MS method for quantification of VOCs in exhaled breath is characterized by high sensitivity and good accuracy, and is suitable for quantitative determination of VOCs in exhaled breath, which can provide technical support for the study of exhaled breath VOCs.
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ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS）， to evaluate the establishment of a mouse model of liver Yin deficiency by thyroid tablet suspension combined with 10% carbon tetrachloride（CCl4） from the perspective of non-targeted metabolomics， in order to lay the foundation for the establishment of a traditional Chinese medicine（TCM） syndrome model. MethodA total of 24 mice were randomly divided into blank group and model group. The model group was given thyroid tablet suspension（0.003 2 g·kg-1） by gavage for 14 consecutive days， and 10% CCl4（5 mL·kg-1） was intraperitoneally injected once a week to establish a liver Yin deficiency model， while the blank group was injected with an equal amount of olive oil intraperitoneally and gavaged with an equal amount of distilled water， and was fed with normal feed. After the modeling was completed， 6 mice in each group were randomly selected， the levels of alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， cyclic adenosine monophosphate（cAMP）， cyclic guanosine monophosphate（cGMP）， interleukin（IL）-6， IL-10， tumor necrosis factor-α（TNF-α）were measured in the mice serum， and malondialdehyde（MDA）， superoxide dismutase（SOD）， total protein（TP）， hydroxyproline（HYP） and other indicators were measured in the mice liver. Liver tissue sections were taken for hematoxylin-eosin（HE） staining and observing pathological changes. The remaining 6 mice in each group were subjected to UPLC-Q-TOF-MS combined with principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to screen differential metabolites in the liver Yin deficiency mouse model， Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to analyze the corresponding metabolic pathways of differential metabolites. ResultCompared with the blank group， mice in the model group showed liver Yin deficiency manifestations such as reduced body weight， fatigue and sleepiness， disheveled and lusterless hair， irritability. The levels of ALT， cAMP/cGMP， IL-6， AST， MDA， cAMP， TNF-α significantly increased（P<0.05， P<0.01）， while the levels of SOD， IL-10 and cGMP significantly decreased（P<0.05， P<0.01）， and the changes of HYP and TP were not statistically significant. Hepatic steatosis and distortion of the radial arrangement of the liver plate cells were seen in the section images of the model group， endogenous substances were clearly separated， and 252 differential metabolites were identified in the serum samples， which were mainly involved in the metabolic pathways of purine metabolism， steroid hormone biosynthesis and pyrimidine metabolism. A total of 229 differential metabolites were identified in the liver samples， mainly involving nucleotide metabolism， purine metabolism， steroid hormone biosynthesis， pyrimidine metabolism， antifolate resistance， insulin resistance， primary bile acid biosynthesis， prostate cancer， sulfur relay system， arachidonic acid metabolism and other metabolic pathways. ConclusionThe successful establishment of liver Yin deficiency model in mice by CCl4 combined with thyroid hormone is evaluated through the investigation of serum and liver metabolomics， combined with biochemical indicators， which provides a biological basis and experimental foundation for the Yin deficiency syndrome model of TCM.
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ObjectiveTo establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QqQ-MS） for determination of the active ingredients in Erdongtang， and to predict the targets and pathways of anti-insulin resistance action of this formula. MethodThe analysis was performed on an ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-3 min， 90%-87%A； 3-6 min， 87%-86%A； 6-9 min， 86%-83%A； 9-11 min， 83%-75%A； 11-18 min， 75%-70%A； 18-19 min， 70%-52%A； 19-22 min， 52%A； 22-25 min， 52%-5%A； 25-27 min， 5%-90%A； 27-30 min， 90%A）. The contents of active ingredients in Erdongtang was detected by electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode under positive and negative ion modes. On this basis， network pharmacology was applied to predict the targets and pathways of Erdongtang exerting anti-insulin resistance effect. ResultThe 20 active ingredients in Erdongtang showed good linear relationships within a certain mass concentration range， and the precision， stability， repeatability and recovery rate were good. The results of determination showed that the ingredients with high content in 15 batches of samples were baicalein（1 259.39-1 635.78 mg·L-1）， baicalin（1 078.37-1 411.52 mg·L-1）， the ingredients with medium content were mangiferin（148.59-217.04 mg·L-1）， timosaponin BⅡ（245.10-604.89 mg·L-1）， quercetin-3-O-glucuronide（89.30-423.26 mg·L-1）， rutin（46.91-1 553.61 mg·L-1）， glycyrrhizic acid（55.97-391.47 mg·L-1）， neomangiferin（37.45-127.03 mg·L-1）， nuciferine（0.89-63.48 mg·L-1）， hyperoside（6.96-136.78 mg·L-1）， liquiritin（30.89-122.78 mg·L-1）， liquiritigenin（26.64-110.67 mg·L-1）， protodioscin（58.57-284.26 mg·L-1）， the ingredients with low content were wogonin（7.16-20.74 mg·L-1）， pseudoprotodioscin（5.49-22.96 mg·L-1）， ginsenoside Rb1（7.31-23.87 mg·L-1）， ginsenoside Rg1（10.78-28.33 mg·L-1）， ginsenoside Re（7.78-24.76 mg·L-1）， ophiopogonin D（2.08-4.29 mg·L-1）， methylophiopogonanone A（0.74-1.67 mg·L-1）. The results of network pharmacology indicated that the mechanism of anti-insulin resistance exerted by Erdongtang might be related to the phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt） signaling pathway. ConclusionThe established UHPLC-QqQ-MS has the advantages of simple sample processing， strong exclusivity and high sensitivity， and can simultaneously determine the contents of the main ingredients from seven herbs in Erdongtang， which can lay the foundation for the development of Erdongtang compound preparations. The results of the network pharmacology can provide a reference for the mechanism study of Erdongtang in the treatment of type 2 diabetes mellitus.
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ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata（ALRP） in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg-1 of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.
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ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.
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ObjectiveTo identify the prototypical components and metabolites absorbed into blood and cerebrospinal fluid of Schisandrae Chinensis Fructus（SCF） based on sequential metabolism combined with liquid chromatography-mass spectrometry. MethodBlood and cerebrospinal fluid samples of integrated metabolism， intestinal metabolism and hepatic metabolism were collected from male SD rats after gavage and in situ intestinal perfusion administration， and ultra-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry（UPLC Q-Exactive Orbitrap MS） was used to analyze and compare the differences in the spectra of SCF extract， blank plasma， administered plasma， blank cerebrospinal fluid and administered cerebrospinal fluid with ACQUITY UPLC BEH Shield RP18 column（2.1 mm×100 mm， 1.7 µm）， the mobile phase was acetonitrile（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-7 min， 95%B； 7-12 min， 95%-35%B； 12-17 min， 35%-15%B； 17-20 min， 15%-12%B； 20-22 min， 12%-5%B； 22-23 min， 5%B； 23-25 min， 5%-95%B； 25-28 min， 95%B）. And heated electrospray ionization（HESI） was used with positive and negative ion modes， the scanning range was m/z 100-1 500. The prototypical constituents and their metabolites absorbed into blood and cerebrospinal fluid of SCF were identified according to the retention time， characteristic fragments， molecular formulae and the information of reference substances. ResultA total of 42 chemical components were identified in the extract of SCF， including lignans， flavonoids， amino acids， tannins， and others， of which lignans were the main ones. A total of 27 prototypical components and 14 metabolites were identified in plasma samples from different sites. A total of 15 prototypical components and 9 metabolites were identified in cerebrospinal fluid. The main metabolic reactions involved in the formation of metabolites were mainly demethylation， methylation， demethoxylation and hydroxylation. ConclusionThrough the systematic identification of the prototypical components and metabolites of SCF in rats， it provides data support for further better exploring the material basis of SCF in the treatment of central nervous system diseases.
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ObjectiveTo screen the differential markers by analyzing volatile components in Dalbergia odorifera and its counterfeits， in order to provide reference for authentication of D. odorifera. MethodThe volatile components in D. odorifera and its counterfeits were detected by headspace gas chromatography-mass spectrometry（HS-GC-MS）， and the GC conditions were heated by procedure（the initial temperature of the column was 50 ℃， the retention time was 1 min， and then the temperature was raised to 300 ℃ at 10 ℃ for 10 min）， the carrier gas was helium， and the flow rate was 1.0 mL·min-1， the split ratio was 10∶1， and the injection volume was 1 mL. The MS conditions used electron bombardment ionization（EI） with the scanning range of m/z 35-550. The compound species were identified by database matching， the relative content of each component was calculated by the peak area normalization method， and principal component analysis（PCA）， orthogonal partial least squares-discrimination analysis（OPLS-DA） and cluster analysis were performed on the detection results by SIMCA 14.1 software， and the differential components of D. odorifera and its counterfeits were screened out according to the variable importance in the projection（VIP） value>2 and P<0.05. ResultA total of 26， 17， 8， 22， 24 and 7 volatile components were identified from D. odorifera， D. bariensis， D. latifolia， D. benthamii， D. pinnata and D. cochinchinensis， respectively. Among them， there were 11 unique volatile components of D. odorifera， 6 unique volatile components of D. bariensis， 3 unique volatile components of D. latifolia， 6 unique volatile components of D. benthamii， 8 unique volatile components of D. pinnata， 4 unique volatile components of D. cochinchinensis. The PCA results showed that， except for D. latifolia and D. cochinchinensis， which could not be clearly distinguished， D. odorifera and other counterfeits could be distributed in a certain area， respectively. The OPLS-DA results showed that D. odorifera and its five counterfeits were clustered into one group each， indicating significant differences in volatile components between D. odorifera and its counterfeits. Finally， a total of 31 differential markers of volatile components between D. odoriferae and its counterfeits were screened. ConclusionHS-GC-MS combined with SIMCA 14.1 software can systematically elucidate the volatile differential components between D. odorifera and its counterfeits， which is suitable for rapid identification of them.
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ObjectiveBased on ultra performance liquid chromatography-mass spectrometry（UPLC-MS） and non-targeted metabolomics technology to discuss the central regulatory effect of Chaishao Liujuntang on chronic atrophic gastritis（CAG） rats with liver-depression and spleen-deficiency， and to look for the correlation between cerebral cortex， hypothalamus and metabolic status of gastric tissues. MethodA CAG rat model with liver-depression and spleen-deficiency was established by chemical induction， hunger and satiety disorders， chronic restraint and tail clamping stimulation， lasting for 16 weeks. Twenty-eight Wistar rats were randomly divided into a blank group of 8 rats and a model group of 20 rats. After the completion of modeling， 4 rats in the model group were taken to observe the pathological changes of gastric mucosa. The remaining model rats were randomly divided into a model group of 8 rats and a Chaishao Liujuntang group of 8 rats. Chaishao Liujuntang group rats were given 5.1 g·kg-1 by gavage， and the remaining rats were given equal volume sterilized water by gavage for 4 weeks. Macroscopic characteristics， behavioral indicators and histopathological changes of the gastric mucosa of rats in each group were observed and compared. UPLC-MS non-targeted metabolomics was used to explore the metabolic regulation effect of Chaishao Liujuntang on the cerebral cortex， hypothalamus and stomach tissues of CAG rats with liver-depression and spleen-deficiency. Pearson correlation coefficient method was used to analyze the correlation between different tissue metabolites. ResultCompared with the model group， the macroscopic characteristics of rats in Chaishao Liujuntang group were improved， such as hair color， mental state and stool properties， and the number of times of crossing and standing in the open field experiment was significantly increased， and the static time of forced swimming was significantly reduced（P<0.01）， and the gastric mucosa atrophy was reduced. The metabolic data from the cerebral cortex of rats in each group identified a total of 3 common potential biomarkers， but not enriched in pathways， 26 common potential biomarkers were identified in the hypothalamus， and the key metabolic pathways involved were mainly enriched in purine metabolism， glycerol phospholipid metabolism， D-glutamine and D-glutamic acid metabolism. Seventeen common potential biomarkers were identified in the stomach， and the key metabolic pathways involved were mainly enriched in thiamine metabolism， valine， leucine and isoleucine biosynthesis， and taurine and taurine metabolism. Correlation analysis of metabolites in different tissues revealed that multiple amino acids and their derivatives mediated metabolic connections between the cerebral cortex， hypothalamus and stomach of rats. ConclusionThe metabolic disorders in the cerebral cortex， hypothalamus and stomach of CAG rats with liver-depression and spleen-deficiency have their own characteristics， mainly manifested by changes in the content of glycerol phospholipids， fatty acids and bile acid metabolites. Moreover， Chaishao Liujuntang may play a central regulatory role in CAG rats with liver-depression and spleen-deficiency by correcting the metabolic disorders of amino acids.
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Two methods including gas chromatography tandem mass spectrometry (GC-MS/MS) and high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) were established to detect common alkyl sulfonates and aryl sulfonates genotoxic impurities. Four alkyl sulfonates and methyl benzenesulfonate were determined by GC-MS/MS using butyl methanesulfonate as the internal standard, the chromatographic column was HP-5MS UI (30 mm × 0.25 mm, 0.25 µm), the carrier gas was helium, the flow rate was 1.0 mL·min-1 in a constant flow mode, the sample inlet temperature was set to 250 ℃, the split ratio was 10∶1, and the initial temperature of the heating program was 80 ℃, maintained for 1 minute, and then increased to 240 ℃ at a heating rate of 30 ℃·min-1 for 2 minutes. The mass spectrometry detector was an electron bombardment ion source (EI source), the data collection condition was multi reaction monitoring mode (MRM), and method validation using the raw material of clinical drug citalopram hydrobromide as a sample. The results showed that the linear range of four alkyl sulfonates and methyl benzenesulfonate were good at 3-50 ng·mL-1 and 9-150 ng·mL-1, with a correlation coefficient of r > 0.999, The spiked recovery was 80%-120%. The detection limits were 1 and 3 ng·mL-1; Ten aryl sulfonates determined by LC-MS/MS, the chromatographic column was CSH Fluoro phenyl (100 mm × 2.1 mm, 1.7 µm), the mobile phase was methanol (B)-5 mmol·L-1 ammonium formate (D), with a flow rate of 0.2 mL·min-1, and gradient elution was performed. The gradient program (T/% B) was set as 0/20, 25/90, 35/90, 42/20. The mass spectrometer detector was electro spray ionization with positive ionization mode (ESI+), the data collection was in dynamic multi reaction monitoring mode (dMRM), and the method was validated using the raw material of the clinical drug citalopram hydrobromide as a sample. The results showed that the linear range of aryl sulfonates were good at 9-2 000 ng·mL-1, 3-100 ng·mL-1 and 0.9-30 ng·mL-1, respectively. The correlation coefficient r > 0.999, the spiked recovery was 80%-120%. The detection limits were 30, 1 and 0.3 ng·mL-1. Two detection methods did not detect potential sulfonate genotoxicity impurities in the above APIs. The established analytical methods are reliable and effective, which can provide reference for drug quality control and detection.
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italic>Schisandra chinensis is a traditional Chinese medicine with the functions of reinforcing deficiency, strengthening, and inducing astringency, appliable to treat the chronic cough and deficiency in breath, palpitation, and insomnia, etc. A hybrid mass spectrometry scanning strategy (high-definition data-independent/data-dependent acquisition, HDDIDDA), enabling the ion mobility separation and alternating data-independent acquisition/data-dependent acquisition, was established, which, in combination with in-house library-driven automatic peak annotation workflows facilitated by the UNIFI software, was utilized to systematically characterize the multi-classes of chemical components from S. chinensis. The use of an HSS T3 column (100 mm × 2.1 mm, 1.8 μm), 0.1% formic acid in H2O-acetonitrile as the mobile phase running at the flow rate of 0.3 mL·min-1, and column temperature at 35 ℃, could enable good separation of the S. chinensis components within 42 min. HDDIDDA scan in both the positive and negative ion modes was employed for data acquisition. Based on the automatic peak annotation, reference standards comparison, MS2 data interpretation, and literature analysis, we were able to identify or tentatively characterize 105 compounds in the S. chinensis decoction, involving 56 terpenoids, 42 lignans, five glycosides, one organic acid, and one flavonoid. HDDIDDA scanning can improve the coverage of data acquisition and improve the accuracy of identification, while CCS prediction analysis provides the possibility to distinguish isomers by the ion mobility technology. The results provide reference for the intelligent material basis research of TCM.
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ObjectiveThrough the correlation analysis between intestinal absorption profile and inhibition of macrophage foaming， the pharmacodynamic components of Zhuriheng dripping pills（ZRH） were explored to provide a basis for establishing its quality standard. MethodIntestinal absorption fluids with 0， 5， 10， 15， 20 times clinical equivalent doses were prepared by a rat everted gut sac（EGS）， and the oxidized low density lipoprotein（ox-LDL）-induced RAW264.7 macrophage foaming model was used to investigate the effect of intestinal absorption fluid with different doses on the accumulation of lipids in RAW264.7 cells by oil red O staining and cholesterol content determination， and to screen for the optimal dose. Ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used to analyze and identify intestinal absorption fractions of ZRH intestinal absorption fluids， and partial least squares-discriminant analysis（PLS-DA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were performed on different doses of ZRH intestinal absorption fluids using SIMCA 13.0 with peak area as the independent variable and the pharmacodynamic indicators as the dependent variables to screen the compounds with variable importance in the projection（VIP） value>1.0 as contributing components， and Pearson correlation analysis was used to determine the spectral effect relationship， determined the compounds and positive correlation with pharmacodynamic were as active ingredients. Molecular docking was used to verify the binding energy of peroxisome proliferator-activated receptor α（PPARα）， PPARγ， PPARβ， human retinoid X receptor α（RXRA） and nuclear transcription factor-κB（NF-κB） with the active ingredients in ZRH intestinal absorption fluids. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was performed to detect the mRNA levels of PPARγ， scavenger receptor A1（SRA1） and adenosine triphosphate-binding cassette transporter A1（ABCA1） in RAW264.7 cells， Westen blot was used to detect the expression level of PPARγ protein in RAW264.7 cells， and enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of interleukin（IL）-1β and NF-κB in RAW264.7 cells. ResultAccording to the results of oil red O staining and cholesterol content determination， the ZRH intestinal absorption fluids could significantly reduce macrophage foaming， and intestinal absorption fluids with 15， 20 times clinical equivalent doses had the best effect， the 15-fold ZRH intestinal absorption fluid was finally determined as the study subject. Spectral effect relationship showed that 52 corresponding peaks in the ZRH-containing intestinal fluid were positively correlated with the efficacy， including organic acids， phenylpropanoids， iridoids， flavonoids， bile acids， coumarins and chromones. Target validation results showed that 86.9%-96.2% of the total components processed good binding activities with the key targets of PPARα， PPARγ， PPARβ， RXRA and NF-κB， and the docking energy values were all less than -6.0 kcal·mol-1（1 cal≈4.19 J）. The results of validation showed that， compared with the normal group， the model group showed a significant increase in the levels of SRA1 and PPARγ mRNA expression， a significant decrease in ABCA1 mRNA expression， a significant increase in the level of PPARγ protein expression， and a significant increase in the levels of IL-1β and NF-κB（P<0.01）， compared with the model group， the 15-fold intestinal absorption fluid group showed a significant decrease in the levels of SRA1 and PPARγ mRNA expression（P<0.05， P<0.01）， ABCA1 mRNA expression level was significantly up-regulated， the levels of IL-1β and NF-κB were significantly reduced（P<0.01）， and PPARγ protein expression level was significantly reduced（P<0.05）. ConclusionThis study identifies 52 components and their metabolites in ZRH intestinal absorption fluid that are positively correlated with the inhibition of macrophage foaming， which may be related to the regulation of the PPARs pathway in cells and the reduction of the levels of inflammatory factors， and can provide a reference for the quality control and clinical application of ZRH.
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ObjectiveTo study the changing characteristics of secondary metabolic compounds accumulated in Dendrobium nobile stems at different growth years， a simulated wild stone plant， in order to provide a theoretical basis for rational planning of the harvesting period of D. nobile. MethodUltra-high performance liquid chromatography-mass spectrometry（UPLC-MS/MS） was used to detect and analyze the secondary metabolites in the stems of 1-year-old， 2-year-old， and 3-year-old D. nobile. The mass spectrometry data were processed using Analyst 1.6.3 software， and all samples were subjected to principal component analysis（PCA）， cluster heat map analysis， partial least squares-discriminant analysis（PLS-DA）， and differential secondary metabolites were screened based on variable importance in projection（VIP） values>1， fold change（FC）≥2 and FC≤0.5. Then differential secondary metabolites were identified based on relative molecular weight， fragmentation ions and mass spectrometry database， and enriched pathways were identified based on the Kyoto Encyclopedia of Genes and Genomes（KEGG） database. ResultA total of 1 317 secondary metabolites were identified in the stems of D. nobile at three growth stages， with flavonoids， phenolic acids， alkaloids and terpenoids accounting for 76.55% of the total. Compared with the 1-year-old stems of D. nobile， 289 differential secondary metabolites were identified in the 2-year-old stems， of which 255 were up-regulated and 34 were down-regulated， 682 differential secondary metabolites were identified in the 3-year-old stems， of which 502 were up-regulated and 180 were down-regulated. Compared to the 2-year-old stems， the 3-year-old stems had 602 differential secondary metabolites， with 405 up-regulated and 197 down-regulated. As the growth stage of D. nobile increased， the top 10 up-regulated differential metabolites mainly included flavonoids， phenolic acids， phenylpropanoids and terpenoids， such as kaempferol derivatives， asperulosidic acid， apigenin derivatives， chrysoeriol derivatives， isorhamnetin derivatives， taxifolin derivatives， quercetin derivatives. KEGG enrichment analysis showed significant enrichment of secondary metabolites in the flavonoid biosynthesis， flavone， and flavonol biosynthesis， secondary metabolite biosynthesis， and phenylpropanoid biosynthesis pathways with the increase of growth years. ConclusionWith the increase of the growth years， the levels of secondary metabolites such as flavonoids， phenolic acids， phenylpropanoids and terpenoids in the wild-grown D. nobile have been significantly enhanced. In practical production， grading based on different growth years can be carried out to improve the medicinal and economic values of D. nobile.
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ObjectiveTo establish a rapid and stable liquid chromatography-mass spectrometry（LC-MS） for simultaneous analysis of 17 chemical components in Gnaphalium affine aboveground parts with flowers， so as to provide experimental basis for improving the quality standard of this herb. MethodUltra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used for the quantitative analysis of 17 constituents in 15 batches of G. affine from different origins， the separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of methanol（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-1.0 min， 8%A； 1.0-4.0 min， 8%-26%A； 4.0-9.0 min， 26%A； 9.0-14.0 min， 26%-34%A； 14.0-14.5 min， 34%-45%A； 14.5-15.0 min， 45%-60%A； 15.0-18.0 min， 60%-90%A； 18.0-19.0 min， 90%A； 19.0-19.01 min， 90%-8%A； 19.01-20.0 min， 8%A）， the flow rate was 0.3 mL·min-1， the column temperature was 40 ℃ and the injection volume was 2 μL. And the electrospray ionization was used with full scanning in both positive and negative ion modes， and the scanning range was m/z 100-1 000. ResultThe established method has been verified by the methodology and could be used for the simultaneous quantification of 17 components in G. affine. The content ranges of the 17 components（quinic acid， gallic acid， protocatechuic acid， neochlorogenic acid， chlorogenic acid， cryptochlorogenic acid， caffeic acid， 1，3-O-dicaffeoylquinic acid， isochlorogenic acid A， isoquercitrin， 1，5-O-dicaffeoylquinic acid， apigenin-7-O-glucoside， astragalin， isochlorogenic acid C， luteolin， apigenin and hispidulin） in 15 batches of G. affine samples was 39.60-179.12， 0.17-0.84， 2.41-8.38， 4.33-31.50， 13.63-180.38， 2.43-14.75， 1.16-19.68， 0.49-5.63， 55.77-445.16， 0.23-10.26， 62.04-530.10， 1.11-18.01， 11.36-90.61， 12.22-65.98， 7.22-69.84， 3.37-45.65， 0.30-2.59 μg·g-1， respectively. The content of organic acids was higher than that of flavonoids in G. affine， and the contents of 1，5-O-dicaffeoylquinic acid， isochlorogenic acid A， quinic acid and chlorogenic acid were higher. Meanwhile， the content of flavonoids in the samples from Guizhou was higher than that from Jiangsu， while the content of organic acids in the samples from Jiangsu was higher than that from Guizhou. ConclusionThe established method can be used for the rapid and accurate determination of 17 components in G. affine， which clarifies the content range of the main components in this herb， and can provide a reference for the selection of quality control markers of G. affine.
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ObjectiveTo investigate the effects of Aconiti Coreani Radix and Typhonii Rhizoma on the urinary metabolites of gerbils with stroke by non-targeted metabolomics technique， and then to clarify the mechanism of the two， as well as their similarities and differences. MethodTwenty-four gerbils were randomly divided into control group（CG）， model group（MG）， Aconiti Coreani Radix group（RA） and Typhonii Rhizoma group（RT）. Except for the CG， ischemic stroke model was constructed using right unilateral ligation of gerbil carotid artery in the remaining groups. Except for the CG and MG， rats in the other groups received whole powder suspension（0.586 mg·g-1） was administered for 14 days. The neurological deficit in each group was scored by Longa scoring on days 0， 3， 7 and 14. After the end of administration， the serum， brain tissue and urine of gerbils in each group were collected， and the rate of cerebral infarction was detected by 2，3，5-triphenyltetrazolium chloride（TTC）， and the levels of interleukin（IL）-6， tumor necrosis factor（TNF）-α， malondialdehyde（MDA）， superoxide dismutase（SOD）， glutathione（GSH）， and nitric oxide（NO） in serum and brain tissue were determined by enzyme-linked immunosorbent assay（ELISA）. The urine metabolomics of gerbils in each group was studied by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Orbitrap-MS）， and the data were processed by multivariate statistical analysis， and differential metabolites were screened based on value of variable importance in the projection（VIP） of the first principal component>1 and t-test P<0.05. Metabolic pathway analysis of the screened differential metabolites was performed using Kyoto Encyclopedia of Genes and Genomes（KEGG） database and Metaboanalyst 5.0. ResultCompared with the CG， the neurological deficit score was significantly increased in the MG（P<0.05）， compared with the MG， the neurological deficit scores in the RA and RT were significantly reduced after 7 d and 14 d（P<0.05）. Compared with the CG， the rate of cerebral infarction was significantly increased in the MG（P<0.05）， compared with the MG， the rates of cerebral infarction in the RA and RT were significantly reduced（P<0.05）. Compared with the CG， the levels of IL-6， TNF-α， and MDA in the serum and brain tissue of gerbils from the MG were significantly increased（P<0.05）， and the levels of SOD， GSH and NO were significantly reduced（P<0.05）. Compared with the MG， Aconiti Coreani Radix and Typhonii Rhizoma could down-regulate the levels of IL-6， TNF-α and MDA， and up-regulated the levels of SOD， GSH and NO. A total of 112 endogenous differential metabolites were screened by urine metabolomics， of which 16 and 26 metabolites were called back by Aconiti Coreani Radix and Typhonii Rhizoma， and could be used as potential biomarkers for both treatments in stroke gerbils， respectively. The results of the pathway analysis showed that both Aconiti Coreani Radix and Typhonii Rhizoma had regulatory effects on arginine and proline metabolism， pyrimidine metabolism， and aminoacyl-tRNA biosynthesis. In addition， Aconiti Coreani Radix could also regulate riboflavin metabolism， Typhonii Rhizoma could also regulate purine metabolism， glycine， serine and threonine metabolism， arachidonic acid metabolism， biosynthesis of pantothenate and coenzyme A， and β-alanine metabolism. ConclusionBoth Aconiti Coreani Radix and Typhonii Rhizoma have better therapeutic effects on stroke， with Aconiti Coreani Radix having stronger effects. From the metabolomics results， the main metabolic pathways regulated by Aconiti Coreani Radix involve amino acid metabolism， oxidative stress and so on， while Typhonii Rhizoma mainly involve amino acid metabolism， lipid metabolism， energy metabolism， etc.
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ObjectiveTo qualitatively analyze the chemical constituents and their tissue distribution in Lujiao formula based on ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Orbitrap-MS）. MethodThe separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-methanol（B） in a gradient elution（0-2 min， 4%B； 2-6 min 4%-12%B； 6-38 min， 12%-70%B； 38-38.5 min， 70%B； 38.5-39 min， 70%-95%B； 39-43 min， 95%B； 43-43.1 min， 95%-4%B； 43.1-45 min， 4%B）， the flow rate was 0.3 mL·min-1 with the column temperature of 40 ℃ and the injection volume of 5 µL. The data were acquired by an electrospray ionization（ESI） in the full scanning mode of positive and negative ions， the scanning rang was set at m/z 100-1 500， the collision energies were 10， 20， 40 eV. Retention time， precise relative molecular mass and secondary mass spectrometry fragment ions were used to identify the compounds in Lujiao formula and analyze their tissue distribution， combing with self-established database and comparing with standard substances and published literature data. ResultA total of 260 compounds， including 156 flavonoids， 43 terpenoids， 18 coumarins， 13 organic acids， 7 phenylethanoids， 7 alkaloids and 16 others， were identified or hypothesized from Lujiao formula， 68 of which were identified by comparison with standard substances. And the results of tissue distribution showed that 100， 143， 129 and 126 prototype components were detected in blood， heart， liver and kidney， respectively. ConclusionThe chemical composition of Lujiao formula and their tissue distribution were systematic analyzed， which can provide reference for the quality control， clinical application， pharmacokinetics and pharmacodynamic material basis of Lujiao formula and its medicinal materials.
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Background Atmospheric fine particulate matter (PM2.5) can disrupt the metabolic homeostasis of the liver and accelerate the progression of liver diseases, but there are few studies on the effects of sub-chronic PM2.5 exposure on the liver metabolome. Objectives To investigate the effects of sub-chronic exposure to concentrated PM2.5 on hepatic metabolomics in mice by liquid chromatography-mass spectrometry (LC-MS), and to identify potentially affected metabolites and metabolic pathways. Methods Twelve male C57BL/6J (6 weeks old) mice were randomly divided into two groups: a concentrated PM2.5 exposure group and a clean air exposure group. The mice were exposed to concentrated PM2.5 using the "Shanghai Meteorological and Environmental Animal Exposure System" at Fudan University. The exposure duration was 8 h per day, 6 d per week, for a total of 8 weeks. The mice's liver tissues were collected 24 h after the completion of exposure. LC-MS was performed to assess changes in the hepatic metabolome. Orthogonal partial least squares discriminant analysis and t-test were employed to identify differentially regulated metabolites between the two groups under the conditions of variable important in projection (VIP)≥1.0 and P<0.05. Metabolic pathway enrichment analysis was performed using MetaboAnalyst 5.0 software and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Results A total of 297 differentially regulated metabolites were identified between the concentrated PM2.5 exposure group and the clean air group. Among these metabolites, 142 were upregulated and 155 were downregulated. A total of 38 metabolic pathways were altered, with 7 pathways showing significant perturbation (P<0.05). These pathways involved amino acid metabolism, glucose metabolism, nucleotide metabolism, as well as cofactor and vitamin metabolism. The 7 significant metabolic pathways were pantothenic acid and coenzyme A biosynthesis; purine metabolism; amino sugar and nucleotide sugar metabolism; arginine biosynthesis; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis; and fructose and mannose metabolism. Conclusion The results from metabolomics analysis suggest that sub-chronic exposure to PM2.5 may disrupt hepatic energy metabolism and induce oxidative stress damage. Aspartic acid, succinic acid, ornithine, fumaric acid, as well as purine and xanthine derivatives, were identified as potential early biomarkers of hepatic response to sub-chronic PM2.5 exposure.
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ObjectiveTo discriminate the age of Arisaema Cum Bile， the combination of headspace solid-phase microextraction （HS-SPME） with gas chromatography-mass spectrometry （GC-MS） was applied to explore the differences of volatile components of unfermented， 1-year fermented， 2-year fermented， and 3-year fermented Arisaema Cum Bile. MethodSamples with different fermentation durations were collected and HS-SPME-GC-MS technology was employed to detect the volatile components of each sample. The relative contents of detected volatile components were processed and analyzed by chemometrics methods such as principal component analysis （PCA）， hierarchical cluster analysis （HCA）， and partial least squares discriminant analysis （PLS-DA）. ResultThe results showed that 145 volatile components were identified. Among these volatile components， the relative contents of heterocyclic， alcohols， aldehydes and aromatics were high. PCA， HCA， and PLS-DA can effectively separate Arisaema Cum Bile with four different ages. Based on variable importance in projection （VIP） value > 1， 73 markers of differential volatile components were identified. The content of 2，6，11-trimethyldodecane and m-xylene in unfermented samples was the highest， and the content difference between them and those in fermented samples was significant （P<0.05）. 2，3-butanediol was detected only in 1-year samples， octane was detected only in 2-year samples， and ethyl heptanoate was detected only in 3-year samples. These components can be used as odor markers for Arisaema Cum Bile with different fermentation years. ConclusionThe identification method of volatile components of Arisaema Cum Bile was established by HS-SPME-GC-MS technology， which can realize the rapid identification of unfermented， 1-year fermented， 2-year fermented， and 3-year fermented samples， and provide a scientific basis for the standardization of processing technology and quality standards of Arisaema Cum Bile.