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Species of Pithecellobium (Fabaceae) are used in traditional medicine to treat diabetes, cough, bronchitis, and inflammation. This study aims to evaluate the content and determine the antioxidant activity, phenolic compounds content, and cytotoxicity of the extract and the fractions of Pithecellobium diversifolium. This is unprecedented research with an exotic species from the Caatinga, northeastern Brazil, using High-performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS). The MeOH fractions of leaves and stem barks showed a high content of flavonoids (198.1 ± 106.50 and 542.7 ± 2.52 mg EqQ/g). The CH2Cl2 fraction of peels showed a high content of total phenolic compounds (516.7 ± 3.00 mg EqAG /g). The DPPH test showed that the CH2Cl2 fraction (leaves) held an EC50 of 0.08 ± 0.02, a higher value than that observed for the standards used in the testButylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), and ascorbic acid. The AcOEt and MeOH fractions of peels presented moderate cytotoxicity with values below 500 µg/mL. The MeOH fraction of leaves showed seven major compounds: myricetin, quercetin, quercetin-arabinofuranoside, apigenin-triglycosides, and apigenin-diglucoside, being the last three unpublished in studies involving the genus. The tests conducted in this study show the potential of P. diversifolium as a promising source of biomolecules with therapeutic applicability.
Espécies de Pithecellobium (Fabaceae) são usadas na medicina tradicional para tratar diabetes, tosse, bronquite e inflamação. Este estudo teve como objetivo avaliar o teor e determinar a atividade antioxidante, o teor de compostos fenólicos e a citotoxicidade do extrato e das frações de Pithecellobium diversifolium, uma pesquisa inédita com uma espécie exótica da Caatinga do Nordeste do Brasil, utilizando a instrumentação Clae-IES. As frações MeOH das folhas e cascas do caule apresentaram alto teor de flavonoides (198,1 ± 106,50 e 542,7 ± 2,52 mg EqQ/g). A fração CH2Cl2 das cascas apresentou um elevado teor de compostos fenólicos totais (516,7 ± 3,00 mg EqAG/g). O teste DPPH mostrou que a fração CH2Cl2 (folhas) apresentou um EC50 de 0,08 ± 0,02, valor superior ao observado para os padrões utilizados no teste Butil hidroxianisol (BHA), Butil hidroxitolueno (BHT) e ácido ascórbico. As frações AcOEt e MeOH das cascas apresentaram citotoxicidade moderada com valores inferiores a 500 µg/mL. A fração MeOH das folhas apresentou sete compostos majoritários: miricetina, quercetina, quercetina-arabinofuranosídeo, apigenina-triglicosídeos e apigenina-diglucosídeo, sendo os três últimos inéditos em estudos envolvendo o gênero. Os testes realizados demonstram o potencial de P. diversifolium, uma promissora fonte de biomoléculas com aplicabilidade terapêutica.
Las especies de Pithecellobium (Fabaceae) se utilizan en la medicina tradicional para tratar diabetes, tos, bronquitis e inflamación. Este estudio tuvo como objetivo evaluar el contenido y determinar la actividad antioxidante, el contenido de compuestos fenólicos y la citotoxicidad del extracto y de las fracciones de Pithecellobium diversifolium, un estudio inédito con una especie exótica de la Caatinga de la región Nordeste de Brasil, que utilizó la instrumentación HPLC-ESI. Las fracciones MeOH de hojas y cortezas de tallo mostraron un alto contenido de flavonoides (198,1 ± 106,50 y 542,7 ± 2,52 mg EqQ/g). La fracción CH2Cl2 de las cortezas presentó un alto contenido de compuestos fenólicos totales (516,7 ± 3,00 mg EqAG/g). El ensayo DPPH mostró que la fracción CH2Cl2 (hojas) tenía EC50 de 0,08 ± 0,02, valor superior a lo observado para los estándares utilizados en el ensayo Butilhidroxianisol (BHA), butilhidroxitolueno (BHT) y ácido ascórbico. Las fracciones AcOEt y MeOH de las cortezas presentaron una citotoxicidad moderada con valores inferiores a 500 µ g/mL. La fracción MeOH de las hojas contiene siete compuestos principales: miricetina, quercetina, quercetina-arabinofuranosido, apigenina-triglucósidos y apigenina-diglucósido, de los cuales los tres últimos son inéditos en estudios sobre el género. Las pruebas realizadas demuestran el potencial de P. diversifolium, una fuente prometedora de biomoléculas con aplicabilidad terapéutica.
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@#Objective To develop a high performance liquid chromatography(HPLC)method for determination of aluminium adjuvant content in vaccine,and verify and preliminarily apply the method.Methods The 8-hydroxyquinoline derivatization method was used for determination. The chromatographic column was phenyl-hexyl column[Luna 5u PhenylHexyl(250 mm × 4. 6 mm)],and the mobile phase was composed of ammonium acetate solution-acetonitrile(with 8-hydroxyquinoline)(60 ∶ 40)containing 20 mg/L ascorbic acid,while eluted at a flow rate of 1. 0 mL/min with the isocratic eluent. The excitation wavelength and the emission wavelength of the fluorescence detector were 380 nm and 520 nm respectively. The column temperature was 40 ℃,and the sample injection was 50 μL. The developed method was verified for the specificity,linear range,accuracy,repeatability,stability and durability,and used to determine the aluminum content in 12 batches of vaccines. The results were compared with those determined by titration in general principle 3106of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results No interference peaks appeared in the sample chromatogram,and the non-aluminum adjuvant vaccine components and phosphate buffer had no interference with the determination. The linearity of aluminum standard was good in the concentration range of 6. 25 ～ 100 μg/mL,r = 0. 999 6. The average results of spike recoveries of aluminum content in inactivated hepatitis A vaccine,recombinant hepatitis B vaccine,adsorbed acellular DTP vaccine and inactivated enterovirus 71 vaccine were 98. 32%,100. 85%,101. 09% and 99. 31%,respectively in the verification for accuracy. The relative standard deviations(RSDs) of the determination results of aluminum content in the solution of six samples of the four vaccines in the same batch were 1. 09%,1. 42%,0. 97% and1. 30%,respectively. The RSDs of aluminum content of four vaccine samples stored at room tempe-rature for 0,2,4,6 and8 h were 0. 82%,0. 73%,0. 40% and 0. 48%,respectively. When the ratio of ammonium acetate solution to 8-hydroxyquinoline acetonitrile solution in mobile phase changed within 5%,the fluctuation range of aluminum content of four vaccines was less than 2%. There was no significant difference between the developed HPLC method and the titration method of Chinese Pharmacopoeia(VolumeⅢ,2020 edition)for determination of aluminum content in the 12 batches of vaccine samples.Conclusion A HPLC method for determination of aluminum adjuvant content in vaccines has been successfully established with good specificity,linearity,accuracy,repeatability,stability and durability,simple operation,high degree of automation and less interference of manual factors. It can realize the determination of aluminium content in single dose,which provides an effective means for the rapid and large-scale determination of aluminum content in vaccine products and monitoring the dispensing of semi-final products in the production process.
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@#Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.
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ObjectiveTo establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QqQ-MS） for determination of the active ingredients in Erdongtang， and to predict the targets and pathways of anti-insulin resistance action of this formula. MethodThe analysis was performed on an ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-3 min， 90%-87%A； 3-6 min， 87%-86%A； 6-9 min， 86%-83%A； 9-11 min， 83%-75%A； 11-18 min， 75%-70%A； 18-19 min， 70%-52%A； 19-22 min， 52%A； 22-25 min， 52%-5%A； 25-27 min， 5%-90%A； 27-30 min， 90%A）. The contents of active ingredients in Erdongtang was detected by electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode under positive and negative ion modes. On this basis， network pharmacology was applied to predict the targets and pathways of Erdongtang exerting anti-insulin resistance effect. ResultThe 20 active ingredients in Erdongtang showed good linear relationships within a certain mass concentration range， and the precision， stability， repeatability and recovery rate were good. The results of determination showed that the ingredients with high content in 15 batches of samples were baicalein（1 259.39-1 635.78 mg·L-1）， baicalin（1 078.37-1 411.52 mg·L-1）， the ingredients with medium content were mangiferin（148.59-217.04 mg·L-1）， timosaponin BⅡ（245.10-604.89 mg·L-1）， quercetin-3-O-glucuronide（89.30-423.26 mg·L-1）， rutin（46.91-1 553.61 mg·L-1）， glycyrrhizic acid（55.97-391.47 mg·L-1）， neomangiferin（37.45-127.03 mg·L-1）， nuciferine（0.89-63.48 mg·L-1）， hyperoside（6.96-136.78 mg·L-1）， liquiritin（30.89-122.78 mg·L-1）， liquiritigenin（26.64-110.67 mg·L-1）， protodioscin（58.57-284.26 mg·L-1）， the ingredients with low content were wogonin（7.16-20.74 mg·L-1）， pseudoprotodioscin（5.49-22.96 mg·L-1）， ginsenoside Rb1（7.31-23.87 mg·L-1）， ginsenoside Rg1（10.78-28.33 mg·L-1）， ginsenoside Re（7.78-24.76 mg·L-1）， ophiopogonin D（2.08-4.29 mg·L-1）， methylophiopogonanone A（0.74-1.67 mg·L-1）. The results of network pharmacology indicated that the mechanism of anti-insulin resistance exerted by Erdongtang might be related to the phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt） signaling pathway. ConclusionThe established UHPLC-QqQ-MS has the advantages of simple sample processing， strong exclusivity and high sensitivity， and can simultaneously determine the contents of the main ingredients from seven herbs in Erdongtang， which can lay the foundation for the development of Erdongtang compound preparations. The results of the network pharmacology can provide a reference for the mechanism study of Erdongtang in the treatment of type 2 diabetes mellitus.
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ObjectiveTaking Achyranthis Bidentatae Radix（ABR） from different origins as samples， to quantitatively analyze the chemical composition and chromaticity of ABR with different processing degrees， and clarify the correlation and change law between color and composition in the processing process of ABR， so as to provide reference for the quality evaluation of processed products of ABR. MethodThe colorimeter is used to measure the chromaticity values of three kinds of processing degrees of ABR in different origins to show the color value change trend during the processing process， and the color parameters of wine-processed and salt-processed products of ABR with different processing degrees were analyzed by principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA） and other analysis methods. The contents of eight representative components of ABR were measured by high performance liquid chromatography（HPLC）， the correlation between chromaticity and each representative component was analyzed by Pearson correlation analysis， and the applicability of the selected eight representative components was further verified by Fisher linear discriminant analysis， and the wine-processed and salt-processed products of ABR with different processing degrees were grouped according to the degree of processing， and 48 samples of wine-processed and salt-processed products with different processing degrees were used as training samples. Taking the contents of 5-hydroxymethylfurfural， polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone， ginsenoside Ro， chikusetsusaponin Ⅳa and polysaccharides as variables， the discriminant function was established respectively， and 12 samples of wine-processed and salt-processed products of ABR with different processing degrees were back-tested to verify the discriminant function and test the reliability of the function. ResultPCA and OPLS-DA results showed that ABR samples with different processing degrees were classified into clusters， and the results could significantly distinguish different processed products. During the process of wine and salt processing， the contents of 5-hydroxymethylfurfural， ginsenoside Ro， and chikusetsusaponin Ⅳa gradually increased with the deepening of the processing degree， while the contents of polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone and polysaccharides showed a gradual decreasing trend， indicating these 8 components increased and decreased to different degrees in the process of wine and salt processing. The results of Pearson correlation analysis showed that the 5-hydroxymethylfurfural content of the samples with different processing degrees of wine-processed and salt-processed products were negatively correlated with the brightness value（L*） and the total color difference value（E*ab）（P<0.01）， and positively correlated with the red-green value（a*） and the yellow-blue value（b*）（P<0.01）， and that the content of polypodine B and polysaccharides were positively correlated with L* and E*ab（P<0.01）. The discriminant functions of wine-processed and salt-processed products of ABR were established by Fisher linear discriminant analysis， and their accuracy rates in the training samples were 93.75% and 95.83%， respectively. Twelve test samples of wine-processed and salt-processed products with different processing degree were back substitution， and the correct rate was 100%. ConclusionThe trend of composition and color changes of ABR with different processing degrees in different production areas is relatively consistent， and the color value can better distinguish ABR with different processing degrees， and the color of ABR is related to some representative components in the processing process， indicating that the color can provide reference for the identification of the processing degree of ABR and the prediction of component content.
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ObjectiveBased on response surface methodology combined with principal component analysis（PCA）， the optimal decocting process of Moringa oleifera leaf standard decoction was optimized， and its multi-index quality evaluation system was established， in order to provide scientific basis for the quality control of this standard decoction. MethodResponse surface methodology and PCA were used to optimize the decoction process by taking the relative peak areas of 8 characteristic peaks and dry extract yield as indexes. Based on this， the quality of 15 batches of the standard decoction was evaluated by high performance liquid chromatography（HPLC） characteristic chromatogram， determination of major components（neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin， astragalin）， determination of active parts（total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine）， dry extract yield， specific gravity and pH. ResultThe optimal decocting process was to soak M. oleifera leaves（100.00 g） for 30 min and decoct twice with the first decoction of 12 times the amount of water for 30 min and the second decoction of 10 times the amount of water for 20 min. Standard decoction containing 0.2 g·mL-1 of crude drug was defined by x¯±30%， the specific gravity was 0.722-1.340， pH was 3.86-7.16， dry extract yield was 23.1%-42.9%， and the alcohol-soluble extract content was 8.26%-15.34%. Calculated according to the dried products of the standard decoction， the contents of neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin and astragalin were 1.99-3.69， 1.20-2.22， 1.44-2.67， 0.53-0.99， 2.45-4.55， 1.22-2.26 mg·g-1， the relative transfer rates relative to the herbs were 34.37%-63.83%， 62.43%-115.94%， 64.65%-120.06%， 56.98%-105.82%， 37.46%-69.57%， 41.81%-77.64%， respectively. The contents of total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine were 10.19-18.92， 11.82-21.96， 94.07-174.71， 42.69-79.27， 9.55-17.73 mg·g-1， the relative transfer rates for herbs were 25.72%-47.77%， 41.78%-77.59%， 64.90%-120.54%， 42.30%-78.57%， 34.99%-64.99%， respectively. ConclusionThe optimized decocting technology of M. oleifera leaf standard decoction is stable and feasible， and the established multi-indicator quality evaluation system can lay the foundation for the quality control of this standard decoction.
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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 a rapid screening method for 34 emerging contaminants in surface water by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS).Methods The pretreatment conditions of solid phase extraction(SPE)were op-timized by orthogonal experimental design and the surface water samples were concentrated and ex-tracted by Oasis? HLB and Oasis? MCX SPE columns in series.The extracts were separated by Kine-tex? EVO C18 column,with gradient elution of 0.1%formic acid aqueous solution and 0.1%formic acid methanol solution.Q-TOF-MS'fullscan'and'targeted MS/MS'modes were used to detect 34 emerging contaminants and to establish a database with 34 emerging contaminants precursor ion,prod-uct ion and retention times.Results The 34 emerging contaminants exhibited good linearity in the con-centration range respectively and the correlation coefficients(r)were higher than 0.97.The limit of de-tection was 0.2-10 ng/L and the recoveries were 81.2%-119.2%.The intra-day precision was 0.78%-18.70%.The method was applied to analyze multiple surface water samples and 6 emerging contaminants were detected,with a concentration range of 1.93-157.71 ng/L.Conclusion The method is simple and rapid for screening various emerging contaminants at the trace level in surface water.
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Objective:To compare and evaluate the quality of wild and different cultivation methods of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai through analysis on UPLC characteristic atlas and multi-component content determination results. Methods:UPLC was used to establish the characteristic chromatogram and multi-component content determination method of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai, and clustering analysis, orthogonal partial least squares - discriminant analysis method were used for chemical pattern recognition analysis. Results:The results showed that there were 10 common peaks in 18 batches of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai. Five components were identified, erythrothioneine(peak 1), protocatechuic acid (peak 2), protocatechualdehyde (peak 3), caffeic acid (peak 4) and Hispidin (peak 5). HCA and OPLS-DA could distinguish Sanghuang porus vaninii (Ljub.) with different cultivation methods. Conclusion:Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai in wood is closer to wild Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai than in substitute cultivation. The UPLC characteristic atlas and multi-component content determination method established in this study can provide reference for the quality evaluation of Sanghuang porus vaninii (Ljub.) L.W. Zhou & Y.C. Dai.
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Objective:To analyze the effects of processed Epimedii Folium on endogenous metabolites of mouse melanoma cells (B16 cells) before and after processing based on cell metabolomics; To investigate the changes of processed Epimedii Folium before and after processing.Methods:Ultra performance liquid chromatography tandem four-stage orbital trap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) technology was used, and the endogenous small molecules of B16 cells treated with Epimedii Folium and processed Epimedii Folium were analyzed by metabolomics. The differential metabolites between groups were obtained, and relevant metabolic pathways were analyzed based on the MetaboAnalyst 5.0 database.Results:Significant changes were observed in 13 kinds of endogenous metabolites, including alanine, carnitine C3∶0, glutamic acid-1, lactic acid, isoleucine, choline, phosphatidylcholine (34∶2, 36∶2), free fatty acids, citric acid, carnitine C4∶0, lysophosphatidylcholine 16∶0 and malic acid after the intervention of Epimedii Folium and processed Epimedii Folium. And the impact of processed products on differential metabolites was stronger than that of raw products. The main pathways involved were Warburg effect, pyruvate metabolism, malate-aspartic acid shuttle, pyruvaldehyde degradation and so on.Conclusions:Epimedii Folium and processed Epimedii Folium would have certain effects on cellular metabolic pathways. The results may be related to the pharmacological effects and changes in cold and hot properties of Epimedii Folium before and after processing.
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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.
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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 develop an ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS)method for the simultaneous quantification of dolutegravir,raltegravir,efavirenz,lamivudine and tenofovir in human plasma and to apply it to the therapeutic monitoring.Methods Dolutegravir-D5,raltegravir-D4,efavirenz-D5,lamivudine-13 C-15 N2 and tenofovir-D7 were used as internal standard,respectively.All samples were extracted using the protein precipitation method with acetonitrile and then diluted for analysis.Chromatographic separation was performed on Shim-pack XR-ODS Ⅲ(2.0 mmx50 mm,1.6 μm)column.Mobile phases A and B consisted of 0.1％formic acid in water and acetonitrile respectively.A programmed mobile phase gradient was used at a flow rate of 0.3 mL·min-1 and column temperature of 40 ℃.The tandem mass spectrometer was equipped with an electrospray ionization(ESI)source operating in multiple reaction monitoring(MRM)modes.After methodological validation,it can be used for therapeutic drug monitoring in HIV patients.Results There was good linearity in the validated concentration ranges of 62.5-3 000 ng·mL-1 for dolutegravir,10-500 ng·mL-1 for raltegravir,125-6 000 ng·mL-1for efavirenz,10-500 ng·mL-1 for lamivudine and 10-500 ng·mL-1 for tenofovir with the linear correlation coeffificients of determination(R2)of all higher than 0.998.The accuracy of both intra-day and inter-day studies ranged from 94.0％-109.3％,and the relative standard deviations were less than 7％.The IS-normalized matrix factor and extraction recoveries of all analytes were 95.7％-106.0％and 98.7％-104.5％at all concentrations.All analytes were stable in plasma at a certain storage environment.The trough blood concentrations of dolutegravir,efavirenz,lamivudine and tenofovir were 107.7-2 366.0,740.0-3 410.0,38.5-1 229.3,31.6-224.4ng·mL-1 in HIV patients,respectively.Conclusion The method is highly aceurate,easy to perform,low-cost,and suitable for therapeutic drug monitoring of dolutegravir,raltegravir,efavirenz,lamivudine and tenofovir in HIV patients.
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Objective To develop a method for rapid determination of the dissolution of cefixime capsules,to explore the consistency of the dissolution curves of the generic and reference preparations in different media,and to assess the reliability of the in vitro dissolution evaluation method according to the results of bioequivalence studies.Methods The dissolution test was performed by the paddle method at 50 r·min-1,using pH1.2 hydrochloric acid solution,pH6.8 phosphate buffer solution,pH7.5 phosphate buffer solution,and water as the dissolution media.And a high-performance liquid chromatography(HPLC)method with a core-shell column was established to determine the dissolution curves of the generic and reference preparations respectively.The bioequivalence of the generic and reference preparations was evaluated through the bioequivalence(BE)test.Results The similarity factors(f2)of the three batches of the generic and reference preparations in the four media were greater than 60.The fasting and postprandial pharmacokinetic parameters(Cmax,AUC0-t,AUC0-∞)of the generic and reference preparations in hu-mans were all in line with the bioequivalence standard.Conclusion The in vitro dissolution behavior of the generic and refer-ence preparations was consistent,and the two preparations were bioequivalent.The method is simple and quick,and it can be em-ployed to measure the dissolution of cefixime capsules,which can provide references for the consistency evaluation of cefixime capsules.
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Objective To establish an HPLC method for the simultaneous determination of 10 components in the active parts of Uygur medicine Dracocephalum Moldavica L.Methods The determination was performed on a Shim-pack ODS(4.6 mm×250 mm,5 um)column with the mobile phase consisting of acetonitrile(A)-0.5%formic acid(B)in aqueous solution in a gradient elution mode(0-30 min,17%A;30-60 min,17%→ 28%A;60-78 min,28%A)at a flow rate of 1.0 mL·min-1.The temperature of the chromatographic column was 35℃and the detection was monitored by a UV detector at 330 nm.Results Cof-feic acid,p-coumalic acid,cynaroside,luteolin-7-O-β-D-glucuronide,apigenin 7-O-glucuronide,rosmarinic acid,diosmetin7-O-β-D-glucuronide,salvianolic acid A,tilianin,apigenin were well separated under this chromatographic condition,and the linear relation-ship were good in the concentration range examined(r＞0.999 2).The overall recoveries ranged from 91.83%to 106.43%with the RSD ranging from 0.38%to 2.22%.Conclusion The established content determination method is highly accurate and reproduci-ble,and suitable for the analysis and quality control of the active parts of Dracocephalum Moldavica L.
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Objective To establish an HPLC method for the determination of alkaloids(epiberberine,coptisine,palma-tine,berberine)and catalpol in different ratios(1∶1,1∶10)of ancient and modern Qianjin Huanglian Pills,and to compare the differences in their contents.The content differences were compared to preliminarily evaluate the differences in the efficacy of Qianjin Huanglian Pills in the treatment of diabetes under different preparation processes and different ratios.Methods The alkaloid solvent was methanol∶ hydrochloric acid(100∶1).The detection conditions were as follows:C18 column,acetonitrile-0.05 mol·L-1 potassium dihydrogen phosphate solution(50∶50),detection wavelength 345 nm,column temperature 30℃,flow rate 1 mL·min-1,injection volume 10 μL.The catalpol solution was methanol∶ water(20∶80).The detection conditions were as follows:chromatographic column C18 column,methanol-0.1%phosphoric acid solution(1∶ 99),detection wavelength 210 nm,column temperature 30℃,flow rate 1 mL·min-1,injection volume 10 μL.Results The established method was spe-cific,and the separation effect of the five components was good.It exhibited a good linear relationship(R2＞0.999)in their respec-tive linear ranges.The repeatability,precision,stability,and sample recovery rate all met the requirements.The content of four alka-loids in the ancient method 1∶1 was the highest,and the content of catalpol was the lowest.The content of four alkaloids in the ancient method 1∶10 was the lowest;the content of 1∶1 in the present method was higher than that in the ancient method 1∶10,and the content of berberine in the present method 1∶10 was slightly lower than that in the present method 1∶1,and the rest were higher than that in the present method 1∶1.The PCA results showed that the chemical composition contents of the four kinds of Qianjin Huanglian pills were very different.Conclusion The method is simple,accurate,and reproducible,making it suitable for the quality control of Qianjin Huanglian Pills.It provides a theoretical basis for exploring the difference in efficacy of Qianjin Huanglian Pills.
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AIM To analyze the component composition of Xeriga-4 Powder,and to determine the contents of phellodendrine,chlorogenic acid,gardenoside,berberine,rutin and curcumin.METHODS The high performance liquid chromatography-Q-exactive orbitrap mass spectrometry(HPLC-Q-Exactive-MS)qualitative analysis was performed on a 35℃thermostatic Agilent ZORBAX SB-Aq column(4.6 mm×150 mm,5 μm),with the mobile phase comprising of methanol-0.1%formic acid flowing at 0.35 mL/min in a gradient elution manner,and electron spray ionization source was adopted in positive and negative ion scanning.High performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)quantitative analysis was performed on a 35℃thermostatic Shim-pack GIST-HP C18 column(2.1 mm×100 mm,3 μm),with the mobile phase comprising of methanol-0.1%formic acid flowing at 0.25 mL/min in a gradient elution manner,and electron spray ionization source was adopted in positive and negative ion scanning with multiple reaction monitoring mode.RESULTS Total 65 constituents were identified,containing 19 alkaloids,13 organic acids,13 flavonoids,7 curcumins,6 iridoids,4 fatty acids,2 aldehydes,and 1 amino acid.Six constituents showed good linear relationships within their own ranges(r≥0.999 1),whose average recoveries were 96.44%-102.37%with the RSDs of 2.05%-3.74%.CONCLUSION This study can provide a reference for the quality control for Xieriga-4 Powder.
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AIM To study the amino acids and proteins in 16 batches of commercial fish swim-bladders with different origins.METHODS A high performance liquid chromatography method based on pre-column derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC)was developed for the determination of contents and components of 17 amino acids in fish swim-bladders.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was performed to analyze the molecular weight distribution of proteins from different fish swim-bladders,and proteins in fish swim-bladders were identified by proteomics method.RESULTS The result showed that the determination of 17 amino acids had a good linear relationship(R2≥0.998 0).The average recovery rate was 85.62%-109.60%and the relative standard deviations of precision,stability and repeatability were less than 3.5%.The total content of the 17 amino acids in 16 batches of fish swim-bladders ranged from 468.31 mg/g to 620.05 mg/g.A total of 688 proteins including 11 collagens were identified from 16 batches of fish swim-bladder samples and a plenty of low-abundance proteins at 52-95 kDa were also detected in fish swim-bladders by SDS-PAGE.CONCLUSION This study provides a good reference for the quality evaluation and further utilization of fish swim-bladders.
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Objective To establish a method for simultaneous determination of HPLC fingerprint and multi-target ingredients in Atractylodis Macrocephalae Rhizoma(AMR),in order to provide reference for its quality control.Methods HPLC-DAD multi-wavelength switching method was used to establish fingerprint of AMR,similarity evaluation combined with hierarchical clustering analysis(HCA),principal components analysis(PCA)and discriminant analysis of partial least squares(PLS-DA)were used to carry out chemometric study.The contents of differential component such as atractylenolide Ⅰ,Ⅱ,Ⅲ and atractylon were determined simultaneously.Results The HPLC fingerprint of 37 batches of AMR was established.Nine common peaks were marked,and 4 of them were identified as atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ.The similarity degrees were between 0.539 and 0.996,the quality of AMR from different origin and different batches varies greatly.Atractylon,atractylenolide Ⅰ,Ⅱ,Ⅲ and one unknown component(peak 9)are the important factors affecting the quality of AMR.Conclusion The combination methods of HPLC fingerprint and simultaneous determinations of multiple components are simple,stable,accurate and reliable,which can provide reference for the quality evaluation of AMR and the improvement of quality standard,as well as lay a foundation for the basic research of its pharmacodynamic substances and related compound.