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Objective: The main objective was to develop stability indicating UPLC technique for simultaneous estimation of Benazepril and Hydrochlorothiazide in bulk and formulation.Methods: 0.1% Triethylamine phosphate: Methanol (25:75v/v) was used as the mobile phase. Benazepril linearity was found to be 4-20 µg/ml and Hydrochlorothiazide linearity was found to be 5-25 g/ml. The detection wavelength was 236 nm, and the retention period of Benazepril was 3.4 min and Hydrochlorothiazide was 5.4 min with a flow rate of 1.0 ml/min. According to the ICH guidlines, the proposed method was validated and stress studies revealed that the drugs are prone to alkali and peroxide stress conditions.Results: The calibration curve was plotted, and the regression equations for Benazepril were y = 2,01,491.67x+60,532.30 with a correlation coefficient (r2) of 0.9997 and Hydrochlorothiazide were y = 64,635.86x-74,607.10 with a correlation coefficient (r2) of 0.9994. According to the accuracy research, the percent recovery of Benazepril is 99.09-100.69 % and that of Hydrochlorothiazide is 98.27-101.88%, both of which are within the ICH recommendations. Benazepril has a limit of detection of 0.08 g/ml-0.24 g/ml and Hydrochlorothiazide has a limit of quantitation of 0.03 g/ml-0.10 g/ml. The procedure was found to be straightforward, linear, rapid, exact, repeatable, and robust. It was determined that the % RSD was within ICH norms. Stress degradation tests showed the drug's vulnerability to oxidative, thermal, photolytic, acid, basic, and neutral hydrolysis stress conditions. Under the circumstances of alkali and peroxide stress, it was discovered that the drug degraded most quickly.Conclusion: The developed chromatographic technique under consideration was suitable for the accurate, precise, and quick simultaneous measurement of hydrochlorothiazide and benazepril in both their bulk and medicinal dose forms.
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AIM To establish a UPLC method for the simultaneous content determination of schisandrol A,gomisin J,schisandrol B,angeloylgomisin H,angeloylgomisin Q,schisanhenol,deoxyschizandrin and schisandrin C in Waigan Fenghan Granules.METHODS The analysis was performed on a 40℃thermostatic WELCH Ultimate?-C18 column(100 mm×2.1 mm,1.8 μm),with the mobile phase comprising of acetonitrile-0.1%formic acid flowing at 0.3 mL/min in a gradient elution manner,and the detection wavelength was set at 267 nm.RESULTS Eight Schisandra lignans showed good linear relationships within their own ranges(R2≥0.998 3),whose average recoveries were 96.47%-104.96%with the RSDs of 0.62%-1.60%.CONCLUSION This simple,rapid and accurate method can be used for the quality control of Hugan Tablets.
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AIM To establish an UPLC-MS/MS method for simultaneous content determination of protocatechuic acid,epicatechin,chlorogenic acid,quercitrin,gaultherin and gaultheroside A in Gaultheria leucocarpa var.yunnanensis.METHODS The analysis was performed on a 40℃thermostatic Waters BEH C18 column(100 mm×2.1 mm,1.7 μm),with the mobile phase comprising of water(containing 0.1%formic acid)-acetonitrile(containing 0.1%formic acid)flowing at 0.3 mL/min in a gradient elution manner,and electron spray inoization source was adopted in positive and negative ion scanning with multiple reaction monitoring(MRM)mode.Hierarchical cluster analysis(HCA)and principal component analysis(PCA)was used to screen important components that affect the quality of medicinal materials.RESULTS Six constituents showed good linear relationships within their own ranges(R2≥0.998 2),whose average recoveries were 98.76%-101.88%with the RSDs of 1.0%-2.5%.The constituents of G.leucocarpa in the roots and aerial parts were quite different.Gaultherin,epicatechin and protocatechuic acid may be the quality mark constituents of G.leucocarpa.CONCLUSION This accurate and efficient method can be used for the quality control of G.leucocarpa.
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AIM To establish a UPLC-MS/MS method for the simultaneous content determination of liquiritin apioside,alibiflorin,swertiamarin,methyl gallate,benzoylpaeoniflorin,sweroside,6′-O-β-D-glucosylgentiopicroside,isoliquiritigenin,loganic acid,liquiritigenin,gallic acid,paeoniflorin,oxypaeoniflorin,gentiopicroside,glycyrrhizic acid,isoliquiritoside and liquiritin in Yangxue Ruanjian Capsules.METHODS The analysis was performed on a 40℃thermostatic Waters BEH C18column(2.1 mm×100 mm,1.7 μm),with the mobile phase comprising of 2 mmol/L ammonium acetate(containing 0.1%formic acid)-acetonitrile flowing at 0.3 mL/min in a gradient elution manner,and electron spray ionization source was adopted in negative ion scanning with multiple reaction monitoring mode.RESULTS Seventeen constituents showed good linear relationships within their own ranges(r>0.999 6),whose average recoveries were 91.33%-104.03%with the RSDs of 1.58%-3.50%.CONCLUSION This rapid,accurate and stable method can be used for the quality control of Yangxue Ruanjian Capsules.
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AIM To identify the chemical components of Longmu Qingxin Mixture by UPLC-Q-TOF-MS and study its material basis for the treatment of attention deficit hyperactivity disorder.METHODS The sample was detected by mass spectrometry in positive and negative ion mode on a Waters CORTECS? UPLC? T3 chromatographic column.The data were analyzed with Peakview 1.2 software and matched with the Natural Products HR-MS/MS Spectral Library 1.0 database,and the components were identified in combination with literature reports.The material basis of Longmu Qingxin Mixture for the treatment of attention deficit hyperactivity disorder was analysed according to the identified components.RESULTS Forty chemical components were identified,including 11 flavonoids,6 monoterpene glycosides,4 triterpene saponins,3 phenolic acids,6 alkaloids etc.,which mainly derived from Radix Astragali,Radix Paeoniae Alba,Radix Scutellariae,licorice root,Ramulus Uncariae cum,etc.,baicalein,formononetin,astragaloside Ⅳ and rhynchophylline may be the material basis for the therapeutic effect of Longmu Qingxin Mixture.CONCLUSION UPLC-Q-TOF-MS can quickly identify the chemical components of Longmu Qingxin Mixture.Flavonoids,triterpene saponins and alkaloids may be the material basis for Longmu Qingxin Mixture for the treatment of attention deficit hyperactivity disorder,which can provide the basis for its material basis research,quality standard establishment and pharmacological study of the dismantled formula.
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AIM To investigate the variation rules of main secondary metabolites in Hedysari Radix before and after rubbing strip.METHODS UPLC-MS/MS was adopted in the content determination of formononetin,ononin,calycosin,calycosin-7-glucoside,medicarpin,genistein,luteolin,liquiritigenin,isoliquiritigenin,vanillic acid,ferulic acid,γ-aminobutyric acid,adenosine and betaine,after which cluster analysis,principal component analysis and orthogonal partial least squares discriminant analysis were used for chemical pattern recognition to explore differential components.RESULTS After rubbing strip,formononetin,calycosin,liquiritigenin and γ-aminobutynic acid demonstrated increased contents,along with decreased contents of ononin,calycosin-7-glucoside and vanillic acid.The samples with and without rubbing strip were clustered into two types,calycosin-7-glucoside,formononetin,γ-aminobutynic acid,vanillic acid,calycosin-7-glucoside and formononetin were differential components.CONCLUSION This experiment clarifies the differences of chemical constituents in Hedysari Radix before and after rubbing strip,which can provide a reference for the research on rubbing strip mechanism of other medicinal materials.
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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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ObjectiveTo establish a rapid and stable liquid chromatography-mass spectrometry（LC-MS） for simultaneous analysis of 17 chemical components in Gnaphalium affine aboveground parts with flowers， so as to provide experimental basis for improving the quality standard of this herb. MethodUltra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry（UPLC-Q-Exactive Orbitrap MS） was used for the quantitative analysis of 17 constituents in 15 batches of G. affine from different origins， the separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of methanol（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-1.0 min， 8%A； 1.0-4.0 min， 8%-26%A； 4.0-9.0 min， 26%A； 9.0-14.0 min， 26%-34%A； 14.0-14.5 min， 34%-45%A； 14.5-15.0 min， 45%-60%A； 15.0-18.0 min， 60%-90%A； 18.0-19.0 min， 90%A； 19.0-19.01 min， 90%-8%A； 19.01-20.0 min， 8%A）， the flow rate was 0.3 mL·min-1， the column temperature was 40 ℃ and the injection volume was 2 μL. And the electrospray ionization was used with full scanning in both positive and negative ion modes， and the scanning range was m/z 100-1 000. ResultThe established method has been verified by the methodology and could be used for the simultaneous quantification of 17 components in G. affine. The content ranges of the 17 components（quinic acid， gallic acid， protocatechuic acid， neochlorogenic acid， chlorogenic acid， cryptochlorogenic acid， caffeic acid， 1，3-O-dicaffeoylquinic acid， isochlorogenic acid A， isoquercitrin， 1，5-O-dicaffeoylquinic acid， apigenin-7-O-glucoside， astragalin， isochlorogenic acid C， luteolin， apigenin and hispidulin） in 15 batches of G. affine samples was 39.60-179.12， 0.17-0.84， 2.41-8.38， 4.33-31.50， 13.63-180.38， 2.43-14.75， 1.16-19.68， 0.49-5.63， 55.77-445.16， 0.23-10.26， 62.04-530.10， 1.11-18.01， 11.36-90.61， 12.22-65.98， 7.22-69.84， 3.37-45.65， 0.30-2.59 μg·g-1， respectively. The content of organic acids was higher than that of flavonoids in G. affine， and the contents of 1，5-O-dicaffeoylquinic acid， isochlorogenic acid A， quinic acid and chlorogenic acid were higher. Meanwhile， the content of flavonoids in the samples from Guizhou was higher than that from Jiangsu， while the content of organic acids in the samples from Jiangsu was higher than that from Guizhou. ConclusionThe established method can be used for the rapid and accurate determination of 17 components in G. affine， which clarifies the content range of the main components in this herb， and can provide a reference for the selection of quality control markers of G. affine.
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AIM:To explore the anti-ulcerative colitis mechanism of Buzhongyiqi pills based on the network pharmacology and experimental verification. METHODS: UPLC-QE-MS was used for qualitative analysis of Buzhongyiqi pills. Targets of the chemistry constituents and the disease were retrieved from GeneCards. Then the protein-protein interaction (PPI) network was constructed and core targets were screened for GO term enrichment and KEGG pathway enrichment. Ulcerative colitis mouse model was established to verify the key targets. RESULTS: A total of 41 constituents migrating of Buzhongyiqi pills were identified. A total of 123 common targets of the constituents and the disease and 24 core targets were screened out.KEGG enrichment and PPI network analysis showed that Buzhongyiqi pills may play a role in the treatment of ulcerative colitis through Akt, PI3K and other pathways. Furthermore, the results of animal experiments showed that Buzhongyiqi pills could significantly improve the depression behaviors of ulcerative colitis, reduce the levels of IL-6 and TNF-α in serum, inhibition Akt/PI3K signaling, and reduce the protein expression of PI3K. CONCLUSION: Buzhongyiqi pills may play a role in the treatment of ulcerative colitis by inhibition Akt / PI3K signaling pathway, and inhibiting PI3K and reduce the levels of IL-6 and TNF-α in the mice.
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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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OBJECTIVE To study the effects of methimazole on the urinary metabolomics of hyperthyroidism rats， and to preliminarily investigate its possible mechanism. METHODS Thirty SD rats were randomly divided into control group， model group and methimazole group， with 10 rats in each group. Except for the control group， the rats in the other two groups were given Levothyroxine sodium tablets 160 mg/kg by intragastric administration for 15 days； at the same time， methimazole group was additionally given methimazole 3.6 mg/kg daily by intragastric administration every day. The basic condition of the rats was observed， and the body weight and anal temperature were measured. After the last medication， the serum levels of triiodothyronine （T3）， tetraiodothyronine （T4）， free triiodothyronine （FT3）， free tetraiodothyronine （FT4）， and thyroid stimulating hormone （TSH） were determined； 24-hour urine was collected on the 15th day after administration. UPLC-TOF-MS was used to analyze the urine metabolomics of rats. Principal component analysis and orthogonal partial least squares-discriminant analysis were used to screen out related differential metabolites， and potential metabolic pathways were analyzed by using HMDB and KEGG. RESULTS Compared with the control group， the rectal temperature， serum levels of T3， T4， FT3 and FT4， the expressions of differential metabolites sebacic acid， cholic acid 3-O-glucuronic acid and N6， N6， N6-trimethyl-L-lysine in urine were significantly up-regulated， while body weight， serum level of TSH， the expressions of deoxycytidine and 2-oxo-4-methylthiobutanoic acid in urine were significantly down-regulated （P＜0.01）. Compared with model group， above indexes of rats were reversed significantly in methimazole group （P＜0.01 or P＜0.05）. Above five differential metabolites were mainly involved in four signaling pathways: pentose and glucuronate interaction， lysine degradation， cysteine and methionine metabolism， and pyrimidine metabolism. CONCLUSIONS Methimazole might improve hyperthyroidism by modulating the four pathways of pentose and glucuronate interaction， lysine degradation， cysteine and methionine metabolism， and pyrimidine metabolism.
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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.
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ObjectiveTo investigate the mechanism of anti-pulmonary fibrosis of cannabidiol by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）. MethodSD rats were randomly divided into blank group， model group， prednisone group（3.15 mg·kg-1） and cannabidiol low， medium and high dose groups（12， 36， 108 mg·kg-1）， with 8 rats in each group. The rat model of pulmonary fibrosis was established by intratracheal injection of bleomycin（5 mg·kg-1）， which was administered continuously for 28 days after successful modeling. The pathological changes of rat lung tissue were observed， and enzyme-linked immunosorbent assay（ELISA） was used to detect the expression levels of matrix metalloproteinase-7（MMP-7）， type Ⅱ alveolar cell surface antigen（KL-6）， pulmonary surfactant-associated protein A（SP-A） and SP-D in serum. The expression levels of type Ⅰ collagen（Col-Ⅰ） and fibronectin（FN） in lung tissues were detected by immunohistochemistry， and the expression of mucin 5 subtype AC（MUC5AC） was detected by immunofluorescence. UPLC-Q-TOF-MS was used to search for potential biomarkers and related metabolic pathways of cannabidiol in treating pulmonary fibrosis. ResultCompared with the blank group， there were a large number of inflammatory cell infiltration and continuous fibrosis lesions in the lung tissue of rats in the model group. Compared with the model group， the inflammatory infiltration and blue collagen deposition in the lung tissue of rats in the prednisone and cannabidiol groups were reduced. Compared with the blank group， the expressions of MMP-7， KL-6， SP-A and SP-D in serum of the model group were significantly increased（P<0.01）， while the expressions of MMP-7， KL-6， SP-A and SP-D in the prednisone and cannabidiol high dose groups were significantly decreased by comparing with the model group（P<0.05， P<0.01）. Compared with the blank group， the expression levels of Col-Ⅰ and FN in the lung tissues of the model group were significantly increased， and the fluorescence intensity of MUC5AC was significantly increased（P<0.01）. Compared with the model group， the expression levels of Col-Ⅰ and FN in the lung tissues of the prednisone and cannabidiol high dose groups were significantly decreased（P<0.05， P<0.01）， and the expression of MUC5AC was significantly decreased（P<0.01）. Compared with the blank group， a total of 18 differential compounds were screened out in the model group， which could be used as potential biomarkers， and cannabidiol could call back 16 of them， mainly involving 4 metabolic pathways（linoleic acid metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， arachidonic acid metabolism， and niacin and niacinamide metabolism）. Compared with the blank group， the relative contents of potential biomarkers arachidonic acid and linoleic acid were significantly increased in the model group（P<0.05， P<0.01）， while the relative contents of 5，6-EET， L-tyrosine and niacinamide were significantly decreased（P<0.01）. Compared with the model group， cannabidiol could significantly reduce the relative contents of arachidonic acid and linoleic acid， and significantly increase the relative contents of 5，6-EET， L-tyrosine and niacinamide（P<0.01）. ConclusionCannabidiol has an intervention and remission effect on pulmonary fibrosis， and its mechanism may be related to linoleic acid metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， arachidonic acid metabolism， niacin and niacinamide metabolism.
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Objective:To establish UPLC fingerprint method and 2 contents determination methods of Buddleja officinalis; To provide a reference for improving the quality control standard and evaluation of Buddleja officinalis from different habitats.Methods:UPLC method was used to establish the fingerprints of 17 batches of Buddleja officinalis. The similarity evaluation, clustering analysis, principal component analysis and orthogonal partial least squares discriminant analysis were used to compare the quality differences of Buddleja officinalis from different habitats. The contents of acteoside and linarin in Buddleja officinalis were determined.Results:There were 12 common peaks in UPLC fingerprints of Buddleja officinalis, six of which were identified as echinacoside, acteoside, cynaroside, isoacteoside, linarin, and apigenin. The fingerprint similarity of 17 batches of Buddleja officinalis was more than 0.9; Buddleja officinalis from different habitats were classified into 2 groups. Five differential markers were determined by OPLS-DA analysis. The order of significance was acteoside > peak 3 > echinacoside > isoacteoside > linarin. Edgeworthia chrysantha was identified by the method of fingerprint as counterfeit. The results of content determination showed that the content of Buddleja officinalis in Hubei and Sichuan was the high and stable.Conclusion:The method can effectively analyze the differences of Buddleja officinalis from different habitats, and provide reference for the quality control of Buddleja officinalis.
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Objective:To explore the effective components and potential mechanisms of Xiefei Lishui Prescription in the treatment of heart failure.Methods:Ultra high-performance liquid chromatography tandem four stage rod time of flight mass spectrometry (UPLC-Q-TOF-MS) technology was used to analyze and identify the active components of Xiefei Lishui Prescription. Drug targets were predicted through the Swiss Target Prediction database, and disease targets were collected from Gene Cards, Dis GENET, and TTD databases. The intersection of drug targets and disease targets was screened using a STRING database for protein interaction to identify core targets. The core targets were included in the DAVID database for GO enrichment and KEGG analysis. Finally, molecular docking validation was performed between the drug components and the corresponding core targets.Results:The results identified 10 active components of Xiefei Lishui Prescription, and 8 potential active components were screened using network pharmacology for the treatment of heart failure with Xiefei Lishui Prescription, corresponding to 160 related action targets. A total of 1 305 disease-related targets were collected, and a total of 51 targets ad 17 core targets were included in the string database for protein interaction analysis. GO functional enrichment and KEGG analysis indicated that the mechanism of Xiefei Lishui Prescription in treating heart failure may be related to pathways such as protein binding, ATP binding, and negative regulation of the VEGF signaling pathway and T cell receptor pathway during apoptosis. The molecular docking results showed that baicalin exhibited good binding activity with ESR1, sorghum isoflavones with ESR1, and quercetin with AKT1, EGFR, IL2, and ABCB1.Conclusion:Xiefei Lishui Prescription may exert therapeutic effects on heart failure through multiple pathways by targeting ESR1, AKT1, EGFR, and other targets.
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Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances.Here,we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC-MS/MS).This enabled simultaneous quantification of 1136 acyl-carnitines(C0-C26)within 10-min with good sensitivity(limit of detection＜0.7 fmol),linearity(cor-relation coefficient＞0.992),accuracy(relative error＜20％),precision(coefficient of variation(CV),CV＜15％),stability(CV＜15％),and inter-technician consistency(CV＜20％,n=6).We also established a quantitative structure-retention relationship(goodness of fit＞0.998)for predicting retention time(tR)of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters(tR,ion-pairs,and collision energy).Furthermore,we quantified 514 acylcarnitines in human plasma and urine,mouse kidney,liver,heart,lung,and muscle.This provides a rapid method for quantifying acyl-carnitines in multiple biological matrices.
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Objective To establish a highly sensitive,stable,and universally applicable ultra-high-performance liquid mass spectrometry tandem method(UPLC-MS/MS)for simultaneous determination of nirmatrelvir and ritonavir blood concentrations in human plasma.Methods The separation was performed on an ACQUITY UPLC BEH C18 column(2.1 mm× 50 mm,1.7 μm)with gradient elution,and the mobile phase consisted of 0.1％formic acid-water and 100％acetonitrile at the flow rate of 0.3 mL·min-1.The column temperature was 45℃,and the injection volume was 2 μL.Electrospray ionization as ion source(ESI+)was used as the ion source and multiple reactions monitoring mode(nirmatrelvir m/z 500.20→319.10,nirmatrelvir-D9 m/z 508.59→328.10,ritonavir m/z 721.30→426.10,13C,2H3-ritonavir m/z 725.30→426.10)was adopted.Thirty patients with coronavirus disease 2019(COVID-19)treated with nirmatrelvir and ritonavir at the People's Hospital of Changxing County in Jan.2023 were selected to measure their steady-state trough concentrations of nirmatrelvir and ritonavir after 3 days of treatment.Results The linear range of nirmatrelvir was 0.100-10.0 μg·mL-1(R2=0.997 2),and the linear range of nirmatrelvir was 0.050-5.00 μg·mL-1(R2=0.995 2).The recovery rates of nirmatrelvir and lopinavir were both＞90％and the intra-batch and inter-batch precision relative standard deviations(RSDs)were both＜10％.Additionally,the recovery ranges for nirmatrelvir and lopinavir were 91.5％-97.0％,and the matrix effects ranged from 92.4％to 97.7％.The results of clinical samples showed that the plasma concentrations of nirmatrelvir and ritonavir in patients with COVID-19 varied greatly among individuals.Conclusion The method for simultaneous determination of nirmatrelvir and ritonavir concentrations in human plasma established in this study is convenient,highly specific,highly accurate,with high precision,which is suitable for monitoring the concentrations of nirmatrelvir and ritonavir in patients.
ABSTRACT
Objective To observe the effects of Levo-tetrahydropalmatine(l-THP)on the expression,regression and relapse of conditioned place preference(CPP)in ketamine induced rats,and to detect the content of dopamine(DA)in the striatum(caudate putamen,CPu)of the rat brain at different time points.Methods Ketamine addiction rat model was established by CPP.The effects of l-THP on the expression,regression and relapse of ketamine induced rat CPP were investigated using CPP score as the index.The content of DA in CPu of rats was determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS)after ketamine administration and l-THP intervention at 30 min,60 min,90 min,120 min and 150 min.Results It indicated that 1-THP could decrease the expression of CPP in ketamine induced rats,promote the process of CPP resolution and inhibit the process of relapse.In addition,l-THP combined with ketamine administration significantly inhibited the ketamine-induced increase in DA content in the CPu of the rats.Conclusion The mechanism of l-THP inhibiting the reward effect of ketamine may be related to blocking DA receptors and reducing the release of DA neurotransmitters.l-THP has potential implications for the treatment of ketamine addiction.
ABSTRACT
Objective To establish the method for simultaneous determination of six index components in the water extract of Weile Prescription;To optimize the water extraction process.Methods UPLC-MS/MS was used with Waters CORTECS C18 column(2.1 mm×100 mm,1.6 μm)as the chromatographic conditions;the mobile phase was 0.1%formic acid water-acetonitrile with gradient elution;the flow rate was 0.25 mL/min;the column temperature was 40℃;the sample volume was 2 μL.Electrospray negative ion source,positive and negative ion switching multi-reaction monitoring(MRM)mode were detected.Taking the content of six index components(gallic acid,vitexin,paeoniflorin,naringin,hesperidin and glycyrrhizic acid)and extraction rate as evaluation indexes,the weight coefficient of each index was determined by G1-entropy weight method,and the optimum parameters of extraction process were determined by orthogonal experiment design with the amount of water,extraction time and extraction times as investigation factors.Results There was a good linear relationship of the six components in the water extract of Weile Prescription in the concentration range(r>0.999),and the average recovery rate was 96.83%-102.56%,RSD<4.0%.The best technological parameters were as follows:Chinese decoction pieces were soaked in 12 times of water for 2 h,and extracted twice,each time for 1.5 h.Conclusion The UPLC-MS/MS method established in the study for simultaneous determination of six components in Weile Prescription is rapid,simple and sensitive,and the optimized extraction process is stable and feasible,which provides experimental basis for the development and research of the preparation.