Effect of Atractylodis Macrocephalae Rhizoma on Short-chain Fatty Acids and Intestinal Barrier in Mice with Slow-transit Constipation / 中国实验方剂学杂志

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.

Determination of 27 volatile organic compounds in exhaled breath by thermal desorption-gas chromatography-mass spectrometry / 环境与职业医学

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.

Identification of Dalbergia odorifera and Its Counterfeits by HS-GC-MS / 中国实验方剂学杂志

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.

Exploring Central Regulatory Effect of Chaishao Liujuntang on Chronic Atrophic Gastritis Rats with Liver Depression and Spleen Deficiency Syndrome Based on Metabolomics / 中国实验方剂学杂志

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.

Analysis of Differences in Secondary Metabolites from Dendrobium nobile Stems Cultivated on Epiphytic Rocks in Different Growth Years Based on Metabolomics / 中国实验方剂学杂志

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.

Effects of PM2.5 sub-chronic exposure on liver metabolomics in mice / 环境与职业医学

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.

Age Discrimination Based on Volatile Components of Arisaema Cum Bile / 中国实验方剂学杂志

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.

Qualitative and Quantitative Analysis of Five Indoles or Indazole Amide Synthetic Cannabinoids in Suspected E-Cigarette Oil by GC-MS / 法医学杂志

OBJECTIVES@#To establish the GC-MS qualitative and quantitative analysis methods for the synthetic cannabinoids, its main matrix and additives in suspicious electronic cigarette (e-cigarette) oil samples.@*METHODS@#The e-cigarette oil samples were analyzed by GC-MS after diluted with methanol. Synthetic cannabinoids, its main matrix and additives in e-cigarette oil samples were qualitatively analyzed by the characteristic fragment ions and retention time. The synthetic cannabinoids were quantitatively analyzed by using the selective ion monitoring mode.@*RESULTS@#The linear range of each compound in GC-MS quantitative method was 0.025-1 mg/mL, the matrix recovery rate was 94%-103%, the intra-day precision relative standard deviations (RSD) was less than 2.5%, and inter-day precision RSD was less than 4.0%. Five indoles or indazole amide synthetic cannabinoids were detected in 25 e-cigarette samples. The main matrixes of e-cigarette samples were propylene glycol and glycerol. Additives such as N,2,3-trimethyl-2-isopropyl butanamide (WS-23), glycerol triacetate and nicotine were detected in some samples. The content range of synthetic cannabinoids in 25 e-cigarette samples was 0.05%-2.74%.@*CONCLUSIONS@#The GC-MS method for synthesizing cannabinoid, matrix and additive in e-cigarette oil samples has good selectivity, high resolution, low detection limit, and can be used for simultaneous qualitative and quantitative analysis of multiple components; The explored fragment ion fragmentation mechanism of the electron bombardment ion source of indole or indoxamide compounds helps to identify such substances or other compounds with similar structures in cases.

Proteomic Research on Characteristic Processing Materials of Porcine Cardiac Blood and Porcine Blood from Menghe Medical School and Their Effect on Anti-microglial Inflammatory Damage of Salviae Miltiorrhizae Radix et Rhizoma / 中国实验方剂学杂志

ObjectiveProteomics was used to investigate the protein differences between porcine cardiac blood（PCB） and porcine blood（PB） from Menghe medical school and to compare the effects of both on the microglial inflammation of Salviae Miltiorrhizae Radix et Rhizoma（DS）. MethodNanoliquid chromatography-quadrupole orbitrap mass spectrometry（nLC-MS/MS） and bioinformatics were utilized to compare the proteomic differences of PCB and PB in simulated gastrointestinal digestion. Furthermore， Western blot was used to verify the contents of some shared proteins and differential proteins identified in PCB and PB. In addition， BV2 neuroinflammation model constructed by corticosterone（CORT） and lipopolysaccharide（LPS） was applied to detect the intervention effects of PCB and PB on the levels of tumor necrosis factor（TNF）-α and interleukin（IL）-6 in BV2 inflammatory cells of DS. ResultA total of 69 common proteins and 68 differential proteins were identified in PCB and PB， among which the common proteins included transferrin（Tf） with brain-targeting effect， and the differential proteins in the two were 41 and 27， respectively. Western blot validation showed that the difference in the content of the same protein Tf between PCB and PB was not statistically significant， while the difference in the contents of the specific proteins of creatine kinase M and heart-type fatty acid binding protein（H-FABP） were statistically significant（P<0.05）. Moreover， in vitro experimental studies revealed that compared with the same concentration of DS group， in addition to the 100 mg·L-1 PB-DS group， PCB-DS and PB-DS groups could significantly inhibit the levels of TNF-α and IL-6 in BV2 inflammatory cells（P<0.05， P<0.01）， and PCB-DS group had more significant anti-inflammatory effect than PB-DS group with the same concentration（P<0.05， P<0.01）. ConclusionBoth of PCB and PB can enhance the inhibitory effect of DS on the release of inflammatory factors， thus playing a neuroprotective role， and PCB promotes DS inhibition more significantly， which may be due to the existence of the two involved in energy metabolism-related differential proteins， which can lay a foundation for revealing the scientific connotation of the processing of PCB-DS and PB-DS.

Effect of Wine Processing on Odour Formation of Polygonatum cyrtonema Rhizoma by GC-MS / 中国实验方剂学杂志

ObjectiveBy exploring the volatile components， polysaccharide composition and changes in the contents of five carbohydrate components of Polygonatum cyrtonema rhizoma before and after processing， and then the effect of yellow rice wine on the odour formation of P. cyrtonema rhizoma was investigated. MethodThe volatile components of P. cyrtonema rhizoma before and after processing were detected by headspace gas chromatography-mass spectrometry（HS-GC-MS）， and sample data were subjected to principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） using SIMCA 14.1， then the differences between these components of P. cyrtonema rhizoma before and after processing were screened according to the principle of variable importance in the projection（VIP） value>1. Crude carbohydrate components in raw and wine-processed P. cyrtonema rhizoma were subjected to oxime and silylation， the carbohydrate components were analyzed by gas chromatography-mass spectrometry（GC-MS/MS）， and the relative contents of various components were calculated by peak area normalization， then quantitative analysis of four carbohydrate components was also carried out. ResultA total of 23 volatile components were identified from the raw products and the wine-processed products， including 15 components in raw products and 20 components in wine-processed products. Among them， 2-methylbutyraldehyde and isovaleraldehyde had a sweet odor and their contents increased after processing， but the contents of hexanal and caproic acid decreased， new components such as 2-acetylfuran and 5-methylfuranal were produced after processing. PCA and OPLS-DA results showed that there were significant differences between raw products and the wine-processed products， a total of 13 differential compounds were screened out， of which 7 showed an upward trend in relative content and 6 showed a downward trend. A total of 7 carbohydrate components， including 5 monosaccharides and 2 disaccharides， were identified in raw products and the wine-processed products. The results of determination showed that the contents of fructose， glucose， mannose and sucrose in P. cyrtonema rhizoma increased after wine-processing， and their increases were 4.54， 1.51， 2.93， 3.66 times， respectively. ConclusionAfter processing， the increase of aromatic flavor of P. cyrtonema rhizoma may be related to the increase of the contents of aldehydes such as 2-methylbutyraldehyde and isovaleraldehyde， while the decrease of raw flavor may be related to the decrease of the contents of volatile components such as hexanal and hexanoic acid， the increase of sweet flavor may be related to the increase of the contents of monosaccharides and oligosaccharides such as fructose and sucrose.

Characteristic Analysis of "Imprinting Template" for Pungent Herbs Based on Molecular Connectivity Index and Matching Frequency Total Statistical Moment / 中国实验方剂学杂志

ObjectiveTo analyze the fingerprint of six pungent herbs based on the molecular connectivity index（MCI）and the matching frequency total statistical moment method， and to study the division and integration of the "imprinting template" of their volatile components， so as to find the common "imprinting template" characteristics of the pungent herbs. MethodThe volatile components of six pungent herbs were extracted by steam distillation， and their fingerprints were established by gas chromatography-mass spectrometry（GC-MS） with a programmed temperature increase（80 ℃ for 5 min， 5 ℃·min-1 to 200 ℃ for 5 min， 2 ℃·min-1 to 230 ℃ for 10 min）， a splitting ratio of 20∶1， an electron bombardment ion source（EI） and the detection range of m/z 35-650， and the average MCI and total statistical moment parameters of the fingerprints were calculated. Then the matching frequency method was used to classify， integrate and confirm the chromatographic peaks of the fingerprints of six pungent herbs. ResultThe average zero order， first-order and second-order MCI values of the volatile components of Pogostemonis Herba， Artemisiae Argyi Folium， Atractylodis Rhizoma， Asari Radix et Rhizoma， Magnoliae Flos and Schizonepetae Herba were 9.02， 5.28 and 5.05， respectively. The average values of peak number， total zero-order moment， total first-order moment and total second-order moment were 60， 169×107， 22.49 min and 36.82 min2， respectively. The 20 integrated imprinting templates were obtained by the matching frequency method for the six pungent herbs， among which three were common imprinting templates with the retention times of （25.97±0.21），（26.90±0.20），（31.64±1.24） min， respectively， and the representative components were valencene，β-elemene， caryophyllin， etc. ConclusionMCI combined the matching frequency total statistical moment can divide and integrate the characteristics of imprinting templates of six pungent herbs， and find their common chromatographic imprinting characteristics， which can provide a reference for the determination of effective substances of pungent herbs.

Analysis of Effect of Characteristic Processing with Rice-washed Water on Volatile Components in Raw and Bran-fried Products of Atractylodis Macrocephalae Rhizoma by HS-GC-MS / 中国实验方剂学杂志

ObjectiveBy comparing the differences in composition and content of volatile components between Atractylodis Macrocephalae Rhizoma（AMR）and bleaching AMR， bran-fried AMR and bran-fried bleaching AMR， the effect of processing with rice-washed water on the volatile components in AMR and bran-fried AMR were investigated. MethodHeadspace gas chromatography-mass spectrometry（HS-GC-MS）was used to determine the volatile components in raw products， bran-fried products and their processed products with rice-washed water. GC conditions were programmed temperature（starting temperature of 50 ℃， rising to 140 ℃ at 10 ℃·min-1， maintained for 5 min， then rising to 210 ℃ at 4 ℃·min-1）， splitting ratio of 10∶1， high purity helium as the carrier gas and a solvent delay time of 3 min. MS conditions were an electron bombardment ion source（EI） with an electron collision energy of 70 eV， ion source temperature of 230 ℃， and the detection range of m/z 20-650. The relative contents of the components were determined by the peak area normalization method， the obtained sample data were subjected to principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） by SIMCA 14.1 software， and the differential components of AMR and bleaching AMR， and bran-fried AMR and bran-fried bleaching AMR were screened according to variable importance in the projection（VIP） value>1 and P<0.05. ResultA total of 71 volatile components were identified， including 53 in AMR， 50 in bleaching AMR， 51 in bran-fried AMR， and 44 in bran-fried bleaching AMR. OPLS-DA results showed that there were significant differences between AMR and bleaching AMR， bran-fried AMR and bran-fried bleaching AMR， but not between AMR samples from different origins. The compound composition of AMR and bleaching AMR， bran-fried AMR and bran-fried bleaching AMR did not change， but the contents of monoterpenes and sesquiterpenes changed significantly. ConclusionSignificant changes in the contents of volatile components were observed in AMR and bleaching AMR， bran-fried AMR and bran-fried bleaching AMR， among them， 1，2-dimethyl-4-methylidenecyclopentene， 9，10-dehydro-isolongifolene， γ-elemene， zingiberene， atractylone， silphinene， modhephene and （1S，4S，4aS）-1-isopropyl-4，7-dimethyl-1，2，3，4，4a，5-hexahydronaphthalene can be used as candidate differential markers of volatile components of AMR before and after processing with rice-washed water.

Metabonomic Analysis of Wenfei Huaxian Granules Against Idiopathic Pulmonary Fibrosis in Mice / 中国实验方剂学杂志

ObjectiveTo investigate the changes of differential metabolites in the serum of mice at different stages of bleomycin sulfate（BLM）-induced pulmonary fibrosis modeling and administration， and the mechanism of Wenfei Huaxian granules（WHG）against idiopathic pulmonary fibrosis. MethodMice were randomly divided into control group， control group of 14 days， model group， model group of 14 days， low-dose WHG group and high-dose WHG group. BLM（0.04 U per mouse）was injected into the trachea of mice in the model group， model group of 14 days， low-dose WHG group and high-dose WHG group， and sterile normal saline was injected into the trachea of mice in the control group and control group of 14 days. Mice of low-dose WHG group and high-dose WHG group were given different doses of WHG by gavage every day after injection of BLM， and mice of control group， control group of 14 days， model group and model group of 14 days were given sterile water by gavage every day. The peripheral blood of mice in the control group of 14 days and model group of 14 days were taken to prepare serum after injection of BLM for 14 days， and the peripheral blood and other materials of mice in the other groups were taken after continuous administration for 28 days. The bronchoalveolar lavage fluid（BALF）was collected for leucocyte differential count， the pathological examination and hydroxyproline（HYP）content determination of lung tissues of mice were performed， and the small molecule metabolites in serum samples of mice in each group were determined by ultra-high performance liquid chromatography-mass spectrometry（UHPLC-MS）. Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA）were conducted to screen differential metabolites and their biological functions were analyzed. ResultCompared with the control group， a large number of continuous fibrotic foci appeared in the lung tissue of mice in the model group， the alveolitis score， fibrosis degree score and HYP content increased significantly（P<0.01）， and the total number of leukocytes， macrophages and lymphocytes in BALF increased significantly（P<0.05）. A total of 33 differential metabolites were screened between the control group of 14 days and model group of 14 days， mainly lipid metabolites， which were mainly involved in oxidative damage and inflammatory process. A total of 34 differential metabolites， mainly amino acid metabolites， were screened between the control group and model group， mainly involving nucleic acid damage and inflammatory process. Compared with the model group， the HYP content， fibrosis score and alveolitis score in the lung tissue of mice from high-dose WHG group decreased significantly（P<0.05， P<0.01）， and the total number of lymphocytes in BALF decreased significantly（P<0.05）. Compared with the model group， 27， 40 differential metabolites were identified in the serum of mice from the low-dose WHG group and high-dose WHG group separately. There were totally 9 common differential metabolites between the model group and low-dose WHG group/high-dose WHG group， which mainly involved in the metabolic pathways of inflammation related lipids metabolism， arginine and tryptophan metabolism， and the change trends in low-dose WHG group and high-dose WHG group were significantly back-regulated compared with the model group. ConclusionWHG can alleviate BLM-induced pulmonary fibrosis， collagen deposition and inflammatory reaction in mice， and its anti-fibrotic effect may be related to the adjusting of inflammatory factors， nitric oxide and oxidative stress related metabolic pathways.

Qualitative analysis of volatile organic components in chemicals using headspace gas chromatography-mass spectrometry / 中国职业医学

Objective To establish a rapid qualitative analysis method for volatile organic components in chemicals. Methods Headspace gas chromatography-mass spectrometry was used to qualitatively determine 19 volatile organic components, including benzene, 1,2-dichloroethane, and n-hexane, in chemicals. Different sample amounts, heating temperatures, heating times, and sample volumes were analyzed to assess their effects on detection results and optimize sampling conditions. Results Based on the set chromatography, the optimal sampling process of this method was as follows： 5.0 g sample in a 20.0 mL headspace bottle, incubated at 40 ℃ for 30 minutes in a constant-temperature drying incubator, and a 1.00 mL headspace gas injection. The within-run and between-run relative standard deviations of all components ranged from 0.00% to 21.05% and 0.00% to 33.33%, respectively. The samples stored in sealed glass containers were stable at room temperature for at least 60 days. Conclusion This method offers simplicity, good reproducibility, and stability, making it suitable for rapid qualitative analysis of volatile organic components in chemicals.

Impurity spectra of lansoprazole enteric-coated preparations by high performance liquid chromatography-high resolution orbital trap mass spectrometry / 中国药科大学学报

@#Using high performance liquid chromatography and high resolution orbital trap mass spectrometry, a two-dimensional online desalting detection method was established to determine the structure of the impurities detected under the official testing conditions of lansoprazole enteric solution preparation, and a chromatographic method compatible with mass spectrometry was established to determine and presume the structure of the impurities that could not be separated by the the official testing method.The identification of impurity was to presume its structure according to the presence of impurity reference product, so as to investigate the difference of impurity spectrum of products from different manufacturers.The one-dimensional chromatographic conditions for the 2D online desalting method were the same as those in China Pharmacopoeia (2020) under relevant substances.Two-dimensional chromatography was performed on a Waters C18 T3 column (2.1 mm × 100 mm, 1.7 μm) with 0.1% formic acid in water-acetonitrile mobile phase and gradient elution.The chromatographic conditions for the compatible mass spectra were based on an Agilent Extend C18 (4.6 mm × 150 mm, 5 μm) column with mobile phase A: 25 mmol/L ammonium acetate and B: 25 mmol/L ammonium acetate-acetonitrile (1∶4) [pH adjusted to 6.5 with glacial acetic acid], with gradient elution. Nine impurities were detected by two-dimensional online desalting method, with 5 known impurities (A-E) and 4 unknown ones.14 impurities were detected by the compatible mass spectrometry method, with 9 unknown impurities (4 consistent with the results of two-dimensional online desalting method, and 5 newly detected).The structures and sources of the unknown impurities were deduced.The two detection methods of lansoprazole preparation by high-performance liquid chromatography-high resolution orbital trap mass spectrometry have guiding significance for quality control and process evaluation.

Determination of deuterium incorporation of new drug donafenib tosilate by two methods of LC-MS with natural isotope abundance correction and 1H NMR relative quantitative method / 药学学报

An LC-MS method with natural isotope abundance correction and a 1H NMR relative quantitative method were established to determine the deuterium incorporation of donafenib tosilate, a new deuterated drug molecule. First, the peak areas of isotopic impurities (non-deuterated and incompletely deuterated impurities) and deuterated drug were recorded through the single ion monitoring (SIM) mode of the established LC-MS method and then corrected in terms of the natural isotope abundance offered by ChemDraw soft, removing the nature isotope interference from 13C, 37Cl, etc. The corrected areas were subsequently used to calculate mol% of isotopologues (D0, D1, D2, D3) and Atom% D, namely, deuterium incorporation. In addition, a 1H qNMR experiment was conducted with the aromatic proton at δ 8.63 and the residual proton of isotopic impurities at δ 2.79 as quantitative peaks. The mixture of DMSO-d6 and D2O (10∶1) was employed as the solvent to change the spin-coupling between the residual proton and active hydrogen so that the residual proton could be measured as the single peak, and the sensitivity was greatly improved. The acquisition parameters were also optimized, and Atom% 1H and the deuterium incorporation were then calculated. The two methods were applied to samples of three commercial batches, and the testing results were almost consistent. Both methods proved accurate, sensitive, fast and independent of standard substances and accurate weighing, which could be applied to the determination of the deuterium incorporation of donafenib tosilate and provide a reference for other deuterated drugs.

Quality by Design approach for the investigation of critical characteristics of Phyllanthus emblica from different vicinities / 数字中医药（英文）

@#[Objective] To explore the application of Quality by Design (QbD) tools in assessing geographical variations of Phyllanthus emblica (P. emblica) from five distinct Indian states. [Methods] In the current experiment, the Box-Behnken design with a reduced quartic model and 105 runs was employed with the use of the Design Expert software for randomized response surface mapping. Three different extraction methods (Soxhlet, maceration, and sonication) along with three solventst [distilled water, methanol, and water-methanol mixture (50 : 50 v/v)] were considered in the present study. The anti-oxidant activities, total flavonoid content (TFC), and total phenolic content (TPC) in the P. emblica were determined and analysed by gas chromatography-mass spectrometry (GC-MS) to identify the major components. [Results] The QbD overlay plot showed that the extractive value of the P. emblica was no less than 30% w/w, 2,2-diphenyl-1-picrylhydrazyl (DPPH) no less than 60% mcg/mL (micrograms per millilitre), TFC no less than 75 mg QE/g (milligrams of quercetin equivalents per gram), and TPC no less than 80 mg GAE/g (milligrams of gallic acid equivalents per gram). Moreover, the GC-MS data confirmed the presence of variation in the bioactives of P. emblica extracts. [Conclusion] The model was significant in describing the variation in extractive value, DPPH, TFC, and TPC. The QbD approach may tend to prioritize thoroughness in the extraction process, ultimately resulting in improved quality in the extracted products.

Evaluation of uncertainty in determination of cetrimonium bromide residue in polysaccharide vaccines by ultra performance liquid chromatography-mass spectrometry / 中国生物制品学杂志

@#Objective To determine the cetrimonium bromide(CTAB)residue in polysaccharide vaccines using ultra performance liquid chromatography-mass spectrometry(UPLC/MS-MS),and analyze and evaluate the uncertainty of the determination results.Methods By establishing a mathematical model,the sources and values of uncertainty introduced in the measurement process were analyzed,the uncertainty components of each influencing factor were calculated,and the standard uncertainty and expanded uncertainty were synthesized to form an uncertainty report.Results At 95% confidence interval,the expanded uncertainty was 0. 002 8 mg/kg. The determination result of CTAB residue in polysaccharide vaccine was reported as(1. 000 6 ± 0. 002 8)mg/kg(k = 2,confidence interval p = 95%).Conclusion The main factors affecting the accuracy of determination results are the preparation of standard solution and the introduction of recovery rate,which should be focused on and controlled in the experiment process to make the detection results more reliable.

Untargeted and targeted mass spectrometry reveal the effects of theanine on the central and peripheral metabolomics of chronic unpredictable mild stress-induced depression in juvenile rats / 药物分析学报

L-theanine has been shown to have a therapeutic effect on depression.However,whether L-theanine has an excellent preventive effect on depression in children and adolescents and what its mechanism is have not been well explained.Given the complexity of the pathogenesis of depression,this study investigated the preventive effect and mechanism of L-theanine on depression in juvenile rats by combining serum and hippocampal metabolomic strategies.Behavioral tests,hippocampal tissue sections,and serum and hippocampal biochemical indexes were studied,and the results confirmed the preventive effect of L-theanine.Untargeted reversed-phase liquid chromatography-quadrupole-time-of-flight mass spec-trometry and targeted hydrophilic interaction liquid chromatography-triple quadrupole mass spec-trometry were developed to analyze the metabolism changes in the serum and hippocampus to screen for potential biomarkers related to L-theanine treatment.The results suggested that 28 abnormal me-tabolites in the serum and hippocampus that were considered as potential biomarkers returned to near-normal levels after L-theanine administration.These biomarkers were involved in various metabolic pathways,mainly including amino acid metabolism and lipid metabolism.The levels of amino acids and neurotransmitters in the phenylalanine,tryptophan,and glutamic acid pathways were significantly reduced after L-theanine administration compared with chronic unpredictable mild stress-induced rats.In summary,L-theanine had a significant preventive effect on depression and achieved its preventive results on depression by regulating various aspects of the body,such as amino acids,lipids,and inflammation.This research systematically analyzed the mechanism of L-theanine in preventing depression and laid the foundation for applying L-theanine to prevent depression in children and adolescents.

Analysis of Intervention of Erxiantang in Ovariectomized Rats by LC-MS Serum Metabolomics / 中国实验方剂学杂志

ObjectiveTo investigate the changes of endogenous metabolites in serum of ovariectomized rats and the effect of Erxiantang on them based on liquid chromatography-mass spectrometry（LC-MS）. MethodTwenty-four healthy female SD rats were randomly divided into sham-operated group， model group and Erxiantang group（7.5 g·kg-1）， with 8 rats in each group. Bilateral ovarian tissues were excised in the model and Erxiantang groups， and small pieces of adipose tissues were excised in the abdominal cavity of the sham-operated group bilaterally， and gastric administration was started 2 weeks after surgery， and equal volumes of distilled water were gavaged in the sham-operated and model groups. After 12 weeks of administration， blood was collected from abdominal aorta， and non-targeted metabonomics was performed on rat serum by LC-MS， and orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to screen differential metabolites. Metabolic pathway analysis was performed based on Kyoto Encyclopedia of Genes and Genomes（KEGG）， and the levels of key enzymes of metabolic pathways were verified by enzyme-linked immunosorbent assay（ELISA）. ResultThe results of metabonomics showed that 82 differential metabolites between the model group and the sham-operated group were glycerophospholipids， fatty acyls， steroids and steroid derivatives， of which the most significant difference was glycerophospholipids. At the same time， Erxiantang could call back 65 out of 82 differential metabolites， of which 11 were statistically significant， mainly phosphatidylcholine（PC） and lysophosphatidylcholine（LysoPC） in glycerophospholipids， followed by corticosterone and 11-deoxycortisol in steroids and steroid derivatives. Metabolic pathway analysis showed that the pathways of glycerophospholipid metabolism and steroid hormone biosynthesis in model group were changed， and were recovered after the administration of Erxiantang. ELISA results showed that compared with the sham-operated group， serum levels of cholinephosphate cytidylytransferase（CCT）， secretory phospholipase A2（sPLA2） and lysophosphatidylcholine acyltransferase（LPCAT）， which were the key metabolic enzymes of glycerophospholipid metabolite PC and LysoPC， were significantly decreased in the model group（P<0.05， P<0.01）， and choline phosphotransferase 1（CPT1） levels decreased but the difference was not statistically significant， compared with the model group， the levels of CCT， sPLA2 and CPT1 were significantly increased in Erxiantang group（P<0.01）. In addition， compared with the sham-operated group， the levels of cholesterol（TC）， triglyceride（TG） and low density lipoprotein cholesterol（LDL-C） were significantly increased in the model group（P<0.01）， the high density lipoprotein cholesterol（HDL-C） level was decreased（P<0.05）， compared with the model group， the levels of TC， TG and LDL-C were significantly decreased and the level of HDL-C was significantly increased in Erxiantang group（P<0.01）. ConclusionEndogenous metabolites and related metabolic pathways in ovariectomized rats were altered， and Erxiantang can reverse some of the different metabolites and related pathways， such as regulating glycerophospholipid metabolism by regulating metabolic enzymes CCT， sPLA2 and CPT1 to increase the levels of PC and LysoPC， and then improve the pathological changes such as lipid metabolism disorder in ovariectomized rats.