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ObjectiveTo investigate the effect of total alkaloids of Corydalis saxicola on a rat model of lipopolysaccharide（LPS）-induced depression， as well as the pharmacokinetic characteristics of 8 of its major components. MethodTwenty-four male SD rats were randomly divided into normal group， model group， fluoxetine group（10 mg·kg-1） and total alkaloids of C. saxicola group（210 mg·kg-1）， with 6 rats in each group. In addition to the normal group， the rats were injected intraperitoneally with LPS to establish the inflammation model of depression， and the drug administration was started 1 week after modeling， and the administration groups were gavaged according to the corresponding dose， and the normal and model groups were intragastric administration with equal volume of distilled water， and the administration was performed along with the modeling. After two weeks of continuous administration， the effect of total alkaloids of C. saxicola on the behavior of depressed rats were tested by sucrose preference， forced swimming and open field experiments， the levels of tumor necrosis factor-α（TNF-α）， interleukin（IL）-1β and IL-6 in serum of rats were determined by enzyme-linked immunosorbent assay（ELISA）， the histopathological changes of rat hippocampus were observed by hematoxylin-eosin（HE） staining. After the last administration， blood was collected from orbit according to the set time， and ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry（UPLC-QqQ-MS） was established to simultaneously detect the concentrations of dehydrocavidine， tetrahydropalmatine， coptisine， palmatine， jatrorrhizine， berberine， berberrubine and epiberberine in plasma， and drug-time curves were drawn. The pharmacokinetic parameters were analyzed by DAS 2.0 software. ResultCompared with the normal group， the model group exhibited a decrease in sucrose preference rate， total distance traveled in the open field， as well as an increase in swimming immobility time and serum inflammatory factor expression（P<0.01）. In contrast， compared with the model group， rats in each administration group showed an increase in sucrose preference rate and total distance traveled in the open field， a decrease in swimming immobility time， and a reduction in serum inflammatory factor expression（P<0.05， P<0.01）. Additionally， HE staining results revealed that neurons in the hippocampus of rats from the model group were characterized by loss， disorganization and residual vacuoles， whereas those from the total alkaloids of C.saxicola group displayed an increase in number with orderly arrangement and clear cytoplasm. Pharmacokinetic results showed that the time to peak（tmax） and half-life（t1/2） of the 8 active ingredients were 0.19-2.06 h and 3.71-8.70 h after continuous administration of total alkaloids of C. saxicola. Among them， the area under the curve（AUC0-∞） of tetrahydropalmatine was the highest and the t1/2 was the shortest， and the AUC0-∞ of coptisine， palmatine， jatrorrhizine， berberine， berberrubine and epiberberine were low. The curves of dehydrocavidine， coptisine， palmatine， berberine and epiberberine showed obvious double peak phenomenon. ConclusionTotal alkaloids of C. saxicola can improve the depression-like behavior of rats， inhibit the expression of inflammatory factors in serum， improve the pathological injury of hippocampus， and has the antidepressant effect. Meanwhile， the effective site is absorbed quickly and eliminated slowly in the depressed model rats， and the efficacy is maintained for a long time.
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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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Objective@#To establish a non-derivatization-PriME-ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneou determination of 9 kinds of biogenic amines in yellow rice wine.@*Methods@#Yellow rice wine samples were purified by PriME HLB solid phase extraction column purification, separated using Waters XSelect HSS T3 column (150 mm×2.1 mm, 3 μm), and qualified using multiple reaction monitoring mode, electrospray ion source positive ion and external standard method.@*Results@#There was a good linear relationship for the 9 kinds of biogenic amines at 2.0 to 500.0 μg/L (r≥0.996). The limit of detection was 0.1 to 0.2 mg/L, and the limit of quantitation was 0.3 to 0.6 mg/L. The spike recovery rate of 9 kinds of biogenic amines ranged from 83.5% to 108.6% at 0.1 and 1.0 mg/L, with relative standard deviations of 2.8% to 8.7%.@*Conclusion@#Non-derivation-prime ultra-high performance liquid chromatography-tandem mass spectrometry can be used for the rapid quantitative detection of biogenic amines in yellow rice wine.
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ObjectiveExosomes are microvesicles which could be secreted by all cell types with diameters between 30 and 150 nm. It was widely distributed in body fluids including blood, urine, and breast milk. Exosomes are considered as potential biomarkers and drug carriers by reason of containing nucleic acids, lipids, proteins and other bioactive molecules. Milk-derived exosomes have been widely used as drug delivery carriers to treat targeted diseases with a lower cost, higher biocompatibility and lower immunogenicity. Until now, there is no research about the milk-derived exosomes phosphorylation to reveal the difference of protein phosphorylation in different species of milk. To investigate the pathways and proteins with specific functions, phosphorylated proteomic analysis of milk-derived exosomes from different species is performed, and provide new ideas for exploring diversified treatments of disease. MethodsWhey and exosomes derived from bovine, porcine and caprine milk were performed for proteomics and phosphoproteomics analysis. The relationship between milk exosome proteins from different species and signaling pathways were analyzed using bioinformatics tools. ResultsA total of 4 191 global proteins, 1 640 phosphoproteins and 4 064 phosphosites were identified from 3 species of milk-derived exosomes, and the exosome proteins and phosphoproteins from different species were significantly higher than those of whey. Meanwhile, some special pathways were enriched like Fcγ-mediated phagocytosis from bovine exosomes, pathways related with neural and immune system from caprine exosomes, positive and negative regulation of multiple activities from porcine exosomes. ConclusionIn this study, the proteomic and phosphoproteomic analyses of exosomes and whey from bovine, porcine and caprine milk were carried out to reveal the difference of composition and related signaling pathways of milk exosome from different species. These results provided powerful support for the application of exosomes from different milk sources in the field of disease treatment.
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@#Objective To characterize the capsid proteins of recombinant adeno-associated virus 6(rAAV6)vectors by reversed phase liquid chromatography-mass spectrometry(RPLC-MS),including primary structure and post-translational modification(PTM).Methods The mobile phase A consisted of 0. 1% aqueous solution of difluoroacetic acid(DFA),while the mobile phase B was 0. 1% DFA acetonitrile solution. The column temperature was maintained at 80 ℃,and the gradient elution lasted for 10 min(0→10 min,mobile phase B 15%→45%). The ESI-Q-TOF mass spectrometry detection operated in positive ion mode with the scanning range of 400-4 000 m/z,the scanning frequency of 2 Hz,the cone voltage at 80 V,the capillary voltage at 3. 0 kV,and the ion source temperature at 120 ℃.Results The measured relative molecular mass of the AAV capsid proteins VP1,VP2,and VP3 was 81 255. 9,66 062. 9,and 59 488. 6,respectively. The deviations from the theoretical values were 8. 1 ppm for VP1,3. 8 ppm for VP2,and 36 ppm for VP3. Mass peptide profile analysis of the enzymatically digested rAAV6 sample indicated a sequence coverage of about 89% with detected PTMs mainly including deamidation,N-terminal acetylation,ubiquitination,and phosphorylation;no glycosylation modification sites were found. Tandem mass spectrometry confirmed the N-terminal and C-terminal sequences of the rAAV6 capsid protein as well as the N-terminal PTM.Conclusion The complete relative molecular mass of rAAV6 capsid protein was analyzed by RPLCMS technique,and the PTM of rAAV6 capsid protein was analyzed by tandem mass spectrometry at the peptide level,which has a certain significance for the quality control of AAV gene therapy products and the improvement of production process.
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ObjectiveTo identify the chemical components of Houpo Wenzhongtang in vivo and in vitro and to analyze the pharmacokinetic properties of the index components in rats with deficiency-cold of spleen and stomach. MethodThe chemical components of Houpo Wenzhongtang was analyzed and identified by ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS/MS）. Six rats were randomly selected from 18 SD rats as the blank group， and the remaining rats were given lard and cold vinegar for a long time to construct a rat model with deficiency-cold of spleen and stomach. After successful modeling， the rats were randomly divided into the model group and Houpu Wenzhongtang group（13.5 g·kg-1， calculated as crude drug）. The administration group was given the corresponding dose of Houpu Wenzhongtang by gavage， and the blank group and the model group were given the same amount of distilled water by gavage. Enzyme-linked immunosorbent assay（ELISA） were used to measure gastrin（GAS） and motilin（MTL） levels in each group. At the same time， plasma samples were collected at different time points after administration， and blood-entry prototype components and metabolites of Houpo Wenzhongtang were analyzed by UPLC-Q-TOF-MS/MS. On this basis， plasma concentrations of magnolol， honokiol， alpinetin and hesperidin in Houpo Wenzhongtang were determined by ultra performance liquid chromatography coupled with triple quadrupole/linear ion trap mass spectrometry（UPLC-QTRAP-MS/MS）， and the pharmacokinetic parameters were calculated using DAS 2.0 software. ResultA total of 79 chemical components， including 44 flavonoids and 11 lignans， were identified in Houpo Wenzhongtang. Meanwhile， 18 blood-entry prototype components and 27 metabolites were identified， the main metabolic pathways of metabolites were glucuronidation， sulfation， oxidation and hydrolysis， and phase Ⅰ and phase Ⅱ were the two primary forms of metabolism. Pharmacokinetic results showed that among the four index components， the time to peak（tmax） values of magnolol and honokiol were consistent and exhibited similar drug metabolism characteristics， the tmax of alpinetin was the shortest， and the absorption rate was the fastest， which had the earliest peak plasma concentration levels， and hesperidin had the shortest mean residence time（MRT0-t） and the highest metabolic rate in rats. ConclusionThis study clarifies the blood-entry prototype components and their metabolites of Houpo Wenzhongtang in the rat model of deficiency-cold of spleen and stomach， and reveals the pharmacokinetic characteristics of the main active ingredients， which can provide a scientific basis for the study of pharmacodynamic material basis of this formula and its clinical application in treating the syndrome of deficiency-cold of spleen and stomach.
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ObjectiveTo determine the pharmacodynamic substance basis of Epimedii Folium（EF） and Epimedii Wushanensis Folium（EWF） in promoting osteogenic differentiation， and to establish a method to analyze the material basis of Chinese materia medica based on the correlation between chemical fingerprint and cellular metabolomics. MethodThe chemical fingerprints of 15 batches of EF with 4 species and 3 batches of EWF were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap-MS）， and partial least squares-discriminant analysis（PLS-DA） was used to analyze the peak areas of chemical fingerprints of samples. The effects of different samples on proliferative activity of MC3T3-E1 osteoblast precursors， as well as the activity of alkaline phosphatase（ALP） in osteoblasts were detected by cell counting kit-8（CCK-8） and enzyme-linked immunosorbent assay（ELISA）. At the same time， UPLC-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was used to analyze the effects of different samples on the metabolomics of MC3T3-E1 cells， then metabolic peak table of osteogenic differentiation cells was constructed， and pharmacodynamic index mean Y0 was introduced into the peak table. PLS was used to calculate mean Y0 of each group， and the mean Y0 was added to the peak table of chemical fingerprint to construct the correlation between chemical fingerprint and cell metabolome， the pharmacodynamic components of EF and EWF that promote bone differentiation were screened according to variable importance in the projection（VIP） value>1. The pharmacodynamic effects of EF and EWF were evaluated according to the mean Y0 of each group. ResultThe chemical fingerprints of EF with different origins and EWF were completely separated. Compared with the blank group， the activity of MC3T3-E1 cells in EF and EWF groups was significantly increased， the activity of ALP in the Epimedium brevicornu（Gansu province）， E. koreanum and E. pubescens groups was significantly increased（P<0.05）. The results of cell metabolomics showed that the blank group and the model group had an obvious trend of separation. EF with different origins and EWF had different distance from the model group， indicating that EF with different origins and EWF had different effect on promoting osteogenic differentiation. Chemical fingerprint-cell metabolomics integration analysis screened 9 components closely related to the efficacy of EF and EWF， including diphylloside B， epimedin C， icariin， baohuoside Ⅰ， yinyanghuo B， β-anhydroicaritin， magnoflorine， cryptochlorogenic acid and quercetin. E. koreanum had the strongest effect on promoting osteogenic differentiation. ConclusionThis study determined that the material basis of EF and EWF promoting osteogenic differentiation were mostly flavonoids， alkaloids and organic acids， which provided ideas and methods for the screening of pharmacodynamic components and the prediction of therapeutic effect of Chinese materia medica.
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ObjectiveTo establish a qualitative analysis method for the chemical constituents of the reference sample of Xiao Xumingtang， and to establish the fingerprint of 15 batches of Xiao Xumingtang， so as to evaluate the quality consistency among batches. MethodAccording to the key information of Xiao Xumingtang in the Key Information Table of Ancient Famous Classical Formulas（25 Formulas）， the reference sample of this formula was prepared， and it was detected by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）. The chemical components were identified by self-constructed database， consulting relevant literature， and comparing with the reference substances， and the components were assigned by comparing with single drug samples and negative samples lacking single drug. The fingerprint of the reference sample of Xiao Xumingtang was established using high performance liquid chromatography（HPLC）， and the common peaks were assigned and identified through single drug samples and negative samples lacking single drug. ResultBased on the information of MS fragments， relevant literature， and database retrieval， a total of 64 compounds were identified and inferred from the reference sample of Xiao Xumingtang， including 31 flavonoids， 8 terpenoids， 12 triterpenoid saponins， 2 phthalides， 3 phenylpropanoids， 2 gingerols， 5 alkaloids， and 1 cyanoside. Among them， 21 were derived from Scutellariae Radix， 10 from stir-fried Glycyrrhizae Radix et Rhizoma， 9 from Ginseng Radix et Rhizoma， 8 from Paeoniae Radix Alba， 4 from Saposhnikoviae Radix， 3 from Stephaniae Tetrandrae Radix， 3 from Chuanxiong Rhizoma， 2 from Aconiti Lateralis Radix Praeparata， 2 from Zingiberis Rhizoma Recens， 1 from Ephedrae Herba， and 1 from Armeniacae Semen Amarum. The established HPLC fingerprint of the reference sample of Xiao Xumingtang had 23 common peaks， among which， peaks 1 and 2 were derived from Paeoniae Radix Alba， peaks 3 and 7 from Saposhnikoviae Radix， peaks 4， 8 and 9 from Stephaniae Tetrandrae Radix， peaks 10， 17， 18， 20 and 21 from stir-fried Glycyrrhizae Radix et Rhizoma， peaks 11-16， 19 and 22 from Scutellariae Radix， peak 5 from Chuanxiong Rhizoma， peak 23 from Zingiberis Rhizoma Recens， peak 6 was the common component of stir-fried Glycyrrhizae Radix et Rhizoma and Scutellariae Radix. A total of 10 compounds including albiflorin（peak 1）， paeoniflorin（peak 2）， cimicifugoside（peak 3）， 5-O-methylvisammioside（peak 7）， baicalin（peak 11）， sec-O-glucosylhamaudol（peak 13）， oroxylin A-7-O-β-D-glucuronide（peak 15）， wogonoside（peak 16）， glycyrrhizic acid（peak 21） and 6-gingerol（peak 23） were identified. The similarities of 15 batches of reference samples were>0.999， indicating that the reference samples had good consistency. ConclusionThrough the identification of the chemical constituents in the reference sample of Xiao Xumingtang， it is clear that the composition of the samples is mainly composed of flavonoids and triterpenoid saponins. The established fingerprint can basically reflect the overall chemical characteristics of the reference sample of Xiao Xumingtang， which can provide a basis for the quality research of its compound preparations.
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Objective To establish a quality control method for monitoring the blood concentrations of cyclosporin A and tacrolimus by HPLC-MS/MS,and to evaluate the quality control samples using the Westgard multi-rule theory.Methods HPLC-MS/MS was used to determine the concentration of cyclosporin A and tacrolimus in human whole blood.The quality control samples of low,medium and high concentration levels in the therapeutic drug monitoring process were statistically analyzed,Levery-Jennings and Z-score quality control charts were drawn,and the Westgard multi-rule theory was applied for in-house quality control evaluation.Results The established method was fully validated with linear ranges of 10.40-1 040.00 ng·mL-1 and 0.50-49.50 ng·mL-1,the quantification limits were 10.40 and 0.50 ng·mL-1,respectively.The extraction recoveries were 108.61％-113.24％and 101.99％-109.37％,respectively.The matrix factors normalized by internal standard were 106.68％-111.27％and 95.70％-97.81％for cyclosporin A and tacrolimus,respectively.The intra-day and inter-day accuracy and precision were less than 15.0％.Other parameters were also validated and met the acceptance criteria.Levery-Jennings and Z-score quality control charts showed that there were 4 warnings(violation of the 12s rule)in the results of the 26 groups of quality control samples in the third quarter of 2022,and no phenomenon was observed to be out of control.Conclusion The established in-house quality control system for therapeutic drug monitoring of cyclosporin A and tacrolimus can effectively ensure the accuracy of blood drug concentration detection.
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Objective To establish a method of LC-MS/MS for determining cordycepin(Cor)and 3′-deoxyinosine(3′-Deo)concentration in rat plasma,and to study their pharmacokinetics in rats.Methods Protein was precipitated with methanol using 2-chloadenosine(2-Chl)as an internal standard.The chromatography was performed on Kinetex C18(3 mm×100 mm,2.6 μm,Phenomenex,USA)with gradient elution in aqueous(5 mmol·L-1 ammonium acetate)-methanol solution as mobile phase.ESI ion source was used for mass spectrometry,and positive ion multiple reaction monitoring(MRM)was used for scanning detection.The pharmacokinetics of Cor and 3′-Deo after oral administration of Cor(10 mg·kg-1)were studied in rats.Results Cor at 0.5-100 ng·mL-1 and 3′-Deo at 1-200 ng·mL-1 had good linearity,and the lower limits of quantification were 0.5 and 1 ng·mL-1,respectively.After oral administration of Cor in rats,the plasma concentration of Cor was low,which was mainly converted into the metabolite 3′-Deo.The Cmax of Cor and 3′-Deo were(5.4±3.4)and(142.0±50.0)ng·mL-1,and AUC0-360min min were(658.4±459.3)and(18 034.9±4 981.1)ng·min·mL-1,respectively.Conclusion The method is simple,sensi-tive,and accurate,which is suitable for determining Cor and 3′-Deo concentration in plasma and the pharmacokinetic study.
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Objective To analyze the residual status,transfer behavior,and risk of paclobutrazole in the national mar-ket of Shenmai granules.Methods GC-MS/MS determined the residual amount of paclobutrazol in Shenmai granules,and ANOVA analyzed the distribution characteristics of sample residuals.The transfer rule of paclobutrazol from Ophiopogonis Radix to Shenmai granules was investigated by simulating the production process,and chronic exposure assessment was performed using the point evaluation model.Results The established method can accurately determine the residual amount of paclobutrazol in Shenmai granules.The residual amount of paclobutrazol in 85 batches of Shenmai granules ranged from 0.001 3 to 0.015 8 mg·kg-1,and there was a statistical difference in the residual amount among different enterprise samples.The transfer rate of paclobutrazol from decoction pieces to preparations was 29.8%.The chronic risk quotient(HQc)of paclobutrazol residues in Shenmai granules was 0.000 7%,far lower than 1.Conclusion There is a general presence of paclobutrazol residues in Shenmai granules.The risk assessment results show that the normal dosage of Shenmai granules does not pose an unacceptable risk to the general population.The residual distribution characteristics and process transfer rules can provide a reference for safety risk control in production enterprises.
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Objective To analyze the influence of different processing methods,including frying,ginger frying,and salt frying,on the volatile components of A.fructus.Methods The volatile components in different processed products of A.fructus were detected and analyzed by gas chromatography-mass spectrometry(GC-MS)based on multivariate statistical analysis.After OPLS-DA analysis,the different components were screened under the conditions of VIP＞1.5 and P＜0.05 and were qualitatively searched using the NIST 11 spectral library.Results A total of 49 different components were identified,with 14 components only changing in the seed mass and 22 components changing in the peel.The content of camphor could be significantly reduced in the seed mass after A.fructus was processed and the content of bornyl acetate significantly increased in the peel of frying A.fructus.Salt frying had a great influence on the alkanes in A.fructus,and ginger processing did not only increase the volatile components in ginger,which reflected the complexity of the processing mechanism.Conclusion At present,the specific processing mechanism is not clear,but the experimental results provide theoretical data for the "detoxification and efficiency enhancement" effect of A.fructus processing,reflecting the scientific nature of the processing,enriching the processing theory of A.fructus,and providing a reference for further in-depth research on the activity of different processed products of A.fructus.
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Objective To analyze the metabolic changes of myocardial tissue in rats under acute exposure to macleaya cordata by gas chromatography mass spectrometry(GC-MS),explore forensic identifications of its characteristic metabolites,and verify its toxicological mechanism in poisoning cases.Methods The rats in the exposure group were given 382 mg/kg macleaya extract solution by gavage,and the rats in the control group were given the same dose of solvent.The myocardial samples were analyzed by GC-MS,and pattern recognition was conducted through partial least squares discriminant analysis(PLSDA).The differential metabolites with characteristic changes were identified by variable importance projection(VIP value＞1)and Student's t test(P＜0.01).Results Compared with the control group,21 potential characteristic metabolites were identified.Through KEGG pathway enrichment analysis,it was found that these metabolites were mainly involved in the pathways of glycine,serine and threonine metabolism;pyruvate metabolism and glycerolipid metabolism.Conclusion Through the study of myocardial metabolism in rats exposed to macleaya cordata,we found the information on metabolites closely related to poisoning,which provides new insight and reference for studies on the mechanisms of macleaya cordata poisoning in the field of forensic medicine.
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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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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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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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Background Per- and polyfluoroalkyl substances (PFAS) are a class of persistent organic pollutants that possess potential toxicity to the human body. The production and utilization of diverse emerging PFAS have resulted in widespread human exposure. Therefore, it is imperative to establish a quantitative methodology encompassing a wide range of PFAS for a comprehensive assessment of human exposure to these compounds. Objective To establish a high-throughput quantitative method for the simultaneous determination of 53 PFAS in human serum based on ultra-high-performance liquid chromatography-Q Exactive high resolution mass spectrometry (UPLC-Q Exactive HRMS). Methods The extraction recoveries of hydrophilic-lipophilic balance (HLB) column, weak anionexchange (WAX) column, and 96-well WAX μElution plate were compared to select the SPE column with the highest recovery. The retention time and peak shape of the target compounds were compared between ACQUITY UPLC BEH C18 column and Accucore aQ column, and the more cost-effective column was chosen. The effects of adding different levels of ammonium formate (0, 2, 5 and 10 mmol·L−1) in mobile phase on peak shape and target response were compared to determine the optimal buffer salt concentration. The optimal spray voltage was obtained by comparing −2 kV and −4 kV. The proposed method was validated from the aspects of selectivity, standard curve, limits of detection, precision, accuracy, and matrix effect. The method was applied to 142 umbilical serum samples. Results The best recovery rate (64%-118%) was achieved by using 96-well WAX μElution plate. The optimal separation and peak shape were obtained by utilizing Accucore aQ column with H2O-methanol (containing 5 mmol·L−1 ammonium formate) as the mobile phase. Less in-source collision and better target response were observed when the spray voltage was set to −2 kV. All target analytes had a good linearity, with R2 > 0.99. The limits of detection ranged from 0.01 to 0.50 μg·L−1, and the recovery ranged from 69% to 127% with the precision less than 26%. A total of 31 PFAS were detected in the 142 actual samples, among which 14 PFAS had a detection frequency over 50%. Perfluorooctanoic acid showed the highest median concentration of 4.16 μg·L−1, followed by 6:2 chlorinated polyfluorinated ether sulfonate and perfluorooctane sulfonates (3.50 μg·L−1 and 1.59 μg·L−1, respectively). Conclusion In this study, we establish a UPLC-Q Excative HRMS method for simutanious determination of 53 PFAS concentrations in serum. This method has the advantages of wide coverage of PFAS, good selectivity, and easy operation, and is suitable for biological detection with a large sample size.
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ObjectiveUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to identify the metabolites of harmine in rats， in order to explore the differences in distribution of metabolites in rats after single dose（40 mg·kg-1） intragastric administration of harmine， as well to speculate the metabolic pathways. MethodSD rats were given a single dose of harmine by intragastric administration. Plasma， bile， urine and feces samples were collected after administration， and the samples were processed for determination by UPLC-Q-TOF-MS. The separation was performed on an ACQUITY UPLC™ HSS T3 columu（2.1 mm×100 mm， 1.8 μm） with acetonitrile（A）-0.1% formic acid aqueous solution（B） as mobile phase for gradient elution（0-2 min， 5%A； 2-9 min， 5%-35%A； 9-9.5 min， 35%-100%A； 9.5-12 min， 100%A； 12-12.5 min， 100%-5%A； 12.5-14 min， 5%A）， the mass spectra were obtained in positive ion mode with electrospray ionization（ESI）， the scanning range was m/z 50-1 200. The metabolites of harmine were identified based on the information of the obtained compounds and the literature data， and the metabolic pathways were hypothesized. ResultA total of 42 compounds（harmine and its metabolites） were identified in rats， including 27 in plasma， 17 in bile， 26 in urine and 13 in feces. The metabolic pathways involved in these 42 metabolites included monohydroxylation， dihydroxylation， demethylation， glucuronidation and sulfation. ConclusionHarmine can undergo phase Ⅰ and phase Ⅱ metabolic reactions in rats， and the prototype drug is metabolized rapidly in vivo， and the metabolites are mainly excreted by the kidneys， which can provide a reference basis for the pharmacodynamics and material basis of harmine.
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@#Abstract: The differential expression and subcellular localization of single-cell proteins are closely related to the physiological state and pathological mechanisms of the body. The development of single-cell protein in situ imaging methods provides powerful tools for spatial single-cell proteomics research and single-cell protein profiling. This article summarizes the single-cell protein imaging methods developed in recent years, including the circulating immunofluorescence imaging methods based on ordered multi-round antibody incubation, mass spectrometry imaging based on metal element labeled antibodies, fluorescence imaging based on DNA-barcoded antibody, gene encoded fluorescence protein imaging and spectral imaging based on Raman spectroscopy or X-ray spectroscopy, with brief explanation of the imaging principles of these methods. It focuses on the multiple performance, imaging resolution and signal amplification performance of these methods, and analyzes their application characteristics in practical scientific research and clinical work, in the hope of providing some reference for the development of more revolutionary single-cell imaging methods, and promoting the development of biomedical and precision medicine.
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ObjectiveTo investigate the brain absorption components of Tianyuan Zhitong prescription and their distribution based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， desorption electrospray ionization mass spectrometry imaging（DESI-MSI） and hyperspectral imaging techniques. MethodTen BALB/c mice were randomly divided into blank group（n=3） and administration group（n=7）， the administration group was gavaged with 0.3 mL of Tianyuan Zhitong prescription liquid at a concentration of about 5 g·mL-1 of the raw material， and the blank group was gavaged with an equal volume of normal saline， and the whole brain of the mice were taken for the preparation of tissue homogenates and frozen sections， respectively. The tissue homogenates were qualitatively analyzed by UPLC-Q-TOF-MS for the brain absorption components in positive and negative ion modes， frozen sections were used for imaging to observe the distribution of these components in the brain. Cytoviva dark-field enhancement microscope was used to perform hyperspectral imaging scanning on the brain sections of mice from each group， and the scattered light data of at least 1 000 pixels in the visible-near-infrared（400-1 000 nm） band in the microscopic field of view were collected and average spectrum were created， which were used to compare the components in the brain tissues of mice from the blank and administration groups. ResultA total of 27 brain absorption components of Tianyuan Zhitong prescription were identified by UPLC-Q-TOF-MS， including 10 organic acids， 5 glycosides， 4 alkaloids， 1 phenol， 4 flavonoids， 2 phthalides and 1 other compound， which were mainly derived from Gastrodiae Rhizoma， Chuanxiong Rhizoma， vinegar-processed Corydalis Rhizoma， Ziziphi Spinosae Semen and processed Morindae Officinalis Radix. A total of 14 components were identified by mass spectrometry imaging， of which ferulic acid， tetrahydropalmatine and N-methyl dehydroberberine were mainly distributed in the cerebral cortex， vitamin B5， vemonoic acid and ricinoleic acid were mainly distributed in the hypothalamus， elemicin， octadecenic acid and octadecanoic acid were mainly distributed in the cortex and hypothalamus， while senkyunolide B， ligustilide， linoleic acid， 9，12-octadecadienoyl ethyl ester and spinosin were distributed in most regions of the brain tissues. Hyperspectral imaging showed that in the visible-near-infrared band range， the average spectrum of the brain tissues of mice in the administration group was significantly red-shifted， indicating that there were differences in the physical properties or contents of the chemical components in the brain between mice in the blank group and those in the administration group， and further verified the results of mass spectrometry imaging. ConclusionThrough the combination of UPLC-Q-TOF-MS and imaging techniques， the pharmacodynamic components of Tianyuan Zhitong prescription in the treatment of headache and the regional characteristics in brain tissue are clarified， which can provide reference for the selection of the index components of the research on the quality standard of this prescription and the research on the mechanism of the pharmacological effect.