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Objective: To establish a reliable and sensitive method for evaluating quality of Yiqi Jiangzhi Granules (YQJZG). Methods: Ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) was employed for simultaneous determination of eight marker components. Separation was performed on an AQUITY UPLC® HSS T3 column, the mobile phase consisted of acetonitrile as the organic phase and 0.1% (volume percentage) formic acid as the aqueous. Eight marker components, ginsenoside Rg1 (GRg1), ginsenoside Re (GRe), ginsenoside Rb1 (Gb1), typhaneoside (TEO), isorhamnetin-3-O-neohespeidoside (IN), hesperidin (HPD), aurantio-obtusin-6-O-β-D-glucoside (AG) and curcumin (CCM), were detected by multiple reaction monitoring (MRM) mode. The Chinese Pharmacopoeia (2020 edition) was regarded as the guidance document for this method validation. Results: The method showed good linearity (R
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Gumiganghwal-tang is a traditional herbal medicine widely used for its anti-inflammatory,analgesic,and antipyretic effects.However,the safety and efficacy of its active ingredients based on an in vivo pharmacokinetic (PK) study have yet been investigated.We have established a sensitive and accurate UPLC-ESI-MS/MS method and conducted a PK study on 14 constituents of Gumiganghwal-tang through human plasma analysis.Analytical conditions were optimized according to the physicochemical prop-erties of the 14 compounds to facilitate efficient separation and eliminate overlap or interference be-tween peaks.KINETEX-C18 and lnertsil-C8 columns were used as UPLC stationary phases,and acetonitrile and aqueous formic acid were used as mobile phases.All the analytes were quantified with a triple quadrupole mass spectrometer using electrospray ionization in multiple reaction monitoring mode.The chromatograms of 14 bioactive compounds showed excellent elution and sensitivity,and each peak was selectively separated and quantified without interference with each other or impurities.The established analytical method was based on international guidelines and was successfully used to perform PK studies of 14 herbal ingredients in humans after oral administration with Gumiganghwal-tang tablets.The oral absorption of most active components of Gumiganghwal-tang was relatively rapid and remained considerably long in the body to be quantified in plasma up to 48 h after administration.
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The purpose of this study was to compare pharmacokinetic (PK) parameters obtained using two newly developed assays,HPLC-UV and UPLC-ESI-MS/MS.Selection of assay and results obtained therefrom are very important in PK studies and can have a major impact on the PK-based clinical dose and usage settings.For this study,we developed two new methods that are most commonly used in biosample analysis and focused on PK parameters obtained from them.By HPLC-UV equipped with a Luna-C8 column using UV detector,cefprozil diastereomers were separated using water containing 2％ (V/V) acetic acid and acetonitrile as a mobile phase.By UPLC-ESI-MS/MS equipped with a HALO-C18column,cefprozil diastereomers were separated using 0.5％ (V/V) aqueous formic acid containing 5 mM ammonium-formate buffer and methanol as a mobile phase.Chromatograms showed high resolution,sensitivity,and selectivity without interference by plasma constituents.Both intra-and inter-day precisions (CV,％)were within 8.88％ for HPLC-UV and UPLC-ESI-MS/MS.Accuracy of both methods was 95.67％-107.50％.These two analytical methods satisfied the criteria of international guidance and could be successfully applied to PK study.Comparison of PK parameters between two assays confirmed that there is a dif-ference in the predicted minimum plasma concentrations at steady state,which may affect clinical dose and usage settings.Furthermore,we confirmed possible correlation between PK parameters and various biochemical parameters after oral administration of 1000 mg cefprozil to humans.
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The present investigation reports the chemical composition of the Rhus typhina L. stem identified via mass spectrometry and NMR as gallic acid, 1-O-galloyl-β-D-glucose, tryptophan, scopolin, methyl gallate, fustin, quercetin, rutin, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose. The antioxidant properties and the chemical composition contents of the R. typhina L. stem grown in different regions in China were de-termined. To determine the antioxidant activity, a total phenolic content analysis, 2, 2-diphenyl-1-pi-crylhydrazyl radical scavenging activity assay, ferric reducing antioxidant power assay, andβ-carotene linoleic acid model system were conducted. The results showed that the Rhus typhina L. stem possessed high antioxidant capacities due to its high phenolic content. The contents of the nine isolated compounds were determined by UPLC-ESI-MS/MS. The calibration curves of the nine isolated compounds were linear within the concentration range and the average recoveries were high. The result showed that 1-O-galloyl-β-D-glucose, gallic acid, methyl gallate, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose could be the compounds mainly responsible for the antioxidant capacity of the R. typhina L. stem. This reveals that the R. typhina L. stem is a good source of antioxidants.
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The root bark of Dictamnus dasycarpus is one of common traditional Chinese medicines (TCMs). Quinoline alkaloids are one of the main active substances in this TCM and possess many biological activities including anti-titumor, anti-inflammation, anti-bacteria, anti-oxidation, and anti-platelet aggregation activities. In this study, eight quinoline alkaloids 1-8 were firstly separated from the root barks of D. dasycarpus. It was difficult to isolate more quinoline alkaloids from the remaining fraction 8 in D. dasycarpus by this conventional chemical separation, so the target analysis method combined LC-MS guided-separation of quinoline alkaloids from fraction 8 was established. MS/MS fragmentation patterns of eight quinoline alkaloids reference standard compounds 1-8 were studied by ultra-performance liquid chromatography-electrospary ionization-mass spectrometry (UPLC-ESI-MS/MS). Based on the feature fragment ion 200, the parent ion scan mode was established for the target analysis of quinoline alkaloids in fraction 8. Finally, 8-methoxyflindersine (9) and N-metilatanina (10) were discovered and isolated quickly from fraction 8 guided by LC-MS, and their structures were identified by NMR and MS. Among them, compound 10 was isolated from the genus Dictamnus for the first time. These results indicated that this method is not only quick and sensitive for analyzing the quinoline alkaloids, but also to effectively guided-separate this kind of alkaloids in the root barks of D. dasycarpus.
Subject(s)
Alkaloids , Chromatography, High Pressure Liquid , Dictamnus , Chemistry , Ions , Phytochemicals , Plant Roots , Chemistry , Quinolines , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
To investigate the effect of hot air circulation drying and spray drying on the quality of Tianshu capsule from the view point of chemical compositions. UPLC-DAD was used to establish the fingerprint of Tianshu capsules for the first time, and the main chemical constituents were identified by UPLC-Q-TOF-MS/MS. A total of 62 compounds were identified in this method, 21 of which were reported in Tianshu capsules for the first time. The results showed that there were no significant differences in the identification of the chemical constituents types between these two methods, but the contents of some constituents were different. The common patterns generated by the 10 batches of hot air cycle drying samples were used as the control fingerprint, and the similarity of the spray drying samples fingerprints was 0.877, with high similarity of the fingerprints between these two methods. UPLC-DAD combined with UPLC-Q-TOF-MS/MS technology was used for the first time to evaluate the chemical constituents of Tianshu capsule rapidly, comprehensively and accurately, providing technical support for the quality evaluation of Tianshu capsule.
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The study developed a method for the determination of 14 components in Bazibushen capsule by UPLC-ESI-MS/MS. Waters ACQUITY BEH C18 column (50 mm×2.1 mm, 1.7 μm) was used and the column temperature was 40℃. A linear gradient elution of eluents A (acetonitrile) and B (0.1% acetic acid) was used for the separation. The source temperature was set at 150℃. The capillary voltage was set at 2.0 kV. The source offset voltage was kept at 50 V. The desolvation temperature was set at 500℃. The desolvation flow was 800 L·h-1. The cone flow was 150 L·h-1. The nebuliser pressure was 7.0 Bar. Multiple reaction monitoring mode (MRM) is adopted. All of the 14 components showed good linearity (r2 > 0.999 1) in the test ranges. The LOQs for the compounds ranged from 0.11-4.52 ng·mL-1, respectively. The RSDs were 0.8%-2.1%. The overall recoveries were between 97.89% and 101.9% for all compounds. The method is simple, rapid, accurate and highly reproducible, and may be used in the determination of 14 components in Bazibushen capsule.
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This paper is aimed to develop a method for the determination of five effective components in medicinal material of Tripterygium using ultra performance liquid chromalography coupled with electrospray ionization tandem mass spectrometry(UPLC-ESI-MS/MS), which then was used to study their contents in raw materials from different areas and different sources.The separation was performed on a Waters ACQUITY UPLC-CSH-C18S column(2.1 mm×100 mm,1.7 μm), usingacetonitrile-0.2% ammonium form ateaqueous solutionas mobile phase. The target components were detected in multiple-reaction monitoring(MRM) mode by mass spectrometry with electrospray ionization (ESI) source operated in positive ionization mode. The quantitative results showed that good linearity was achieved in their respective linear ranges and fine determination coefficient (r > 0.997 8),and the overall recoveries ranged from 96.72%-103.2% with the RSD ranging from 1.0%-2.4%.The method is sensitive and accurate, and suitable for the effective components quantification in medicinal material of Tripterygium; contents of five effective components from different sources vary significantly, so the quality and safety of medicinal material of Tripterygium needs to be improved. It is very important to control the quality with multi-index for clinic safety.
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OBJECTIVE:To establish the method for pharmacokinetic study of luteolin and cynaroside in rats and to determine pharmacokinetic parameters. METHODS:16 SD rats were randomly divided into luteolin group (sublingual iv,1.34 mg/kg) and cynaroside group(sublingual iv,0.64 mg/kg). 0.5 ml blood were collected before administration and 0,15,30 min and 1,2,3,4,6, 8,12,24,48 h after administration respectively to prepare plasma. UPLC-TQ-MS was adopted to determine plasma concentration, and pharmacokinetic parameters were calculated. A CORTECSTM UPLC? C18(100 mm×2.1 mm,1.6 μm)column was used with mobile phase consisted of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.4 ml/min,the column temperature was set at 40 ℃,and quercetin was used as internal standard. RESULTS:The linear range of luteolin and cynaroside were 2.5-500 ng/ml (r=0.998 2) and 10-2 500 ng/ml (r=0.993 5). The lowest quantitation limits were 1 and 2.5 ng/ml,and extraction were 70.75%-87.72% and 75.40%-91.18%(n=6);RSD of inter-day and intra-day were all lower than 10%(n=3). Pharmacokinetic parameters as t1/2 were (1.88 ± 0.32) and (1.57 ± 0.08) h;CL were (0.77 ± 0.18) and (0.06 ± 0.01) L/(h·kg);AUC0-6 h were (189.60±40.04)and(1 093.14±187.36)ng·h/ml;AUC0-∞ were(195.18±38.37)and(1 097.11±188.07)ng·h/ml. CONCLU-SIONS:The method can be used for pharmacokinetic study of luteolin and cynaroside in rats,and the pharmacokinetics of them in rats are in line with two-compartment model.
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Objective: To establish an ultra performance liquid chromatography-tandem quadrupole mass spectrometry ( UPLC-MS/MS) method to determine the concentration of carbamazepine and phenobarbital in human plasma, and apply in the clinical moni-toring. Methods:Diazepam was used as the internal standard, and the samples were precipitated by acetonitrile. An ACQUITY UPL-C? BEH C18 (50 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 40℃ with a Waters XEVO TQD. The mobile phase consisted of acetonitrile (containing 10 mmol·L-1 ammonium formate) and water (containing 10 mmol·L-1 ammonium for-mate and 0. 1% formic acid) with gradient elution pumped at a flow rate of 0. 4 ml·min-1 . ESI was applied and the samples were scanning analyzed by positive ion multi-reaction monitoring mode. The plasma was precipitated by 200 μl acetonitrile and centrifugated at 12 000 × g for 10 min and tranfer it into an Ep tube. The sample size was 20 μl. Results:The retention time of carbamazepine was 1. 23 min. Excellent linear calibration curves of carbamazepine were obtained within the concentration range of 0. 25-25μg·ml-1(r=0. 999 7). The lower limit of quantification of carbamazepine was 0. 01 μg·ml-1. The retention time of phenobarbital was 1. 11 min. Excellent linear calibration curves of carbamazepine were obtained within the concentration range of 0. 5-50 μg·ml-1(r=0. 999 6). The lower limit of quantification of carbamazepine was 0. 05 μg·ml-1. The recovery of three concentrations of carbamazepine was (82. 1 ± 6. 83)%, (82. 91 ± 4. 3)% and (84. 35 ± 3. 09)%, and the recovery of three concentrations of phenobarbital was (84. 27 ± 6. 91)%, (84. 32 ± 7. 74)% and (89. 07 ± 6. 24)%, respectively. The intra- and inter-day RSDs were all less than 10%. There were no endogenous substances existing in the incubation system, therefore, there was no interference with the determination. Conclu-sion:The simple, accurate and rapid method is suitable for the determination of carbamazepine and phenobarbital in human plasma, which can contribute greatly to the therapeutic drug monitoring service for patients.
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Objective:To establish an ultra performance liquid chromatography-tandem quadrupole mass spectrometry method for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma. Methods: An ACQUITY UPLC? BEH C18 (50 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 40℃. The mobile phase consisted of acetonitrile and water (con-taining 0. 1% formic acid) with gradient elution at a flow rate of 0. 4 ml·min-1 . Warfarin-d5 was used as the internal standard. The analytes were detected on a triple-quadrupole mass spectrometer equipped with an ESI interface in a positive mode. Results:The reten-tion time of warfarin and 7-hydroxywarfarin was 1. 8 min and 1. 5 min, respectively. Excellent linear calibration curve of warfarin and 7-hydroxywarfarin was obtained within the concentration range of 25-2 000 ng · ml-1 ( r =0. 999 3 ) and 5-500 ng · ml-1 ( r =0. 999 6), respectively. The lower limit of quantification of warfarin and 7-hydroxywarfarin was 5 ng·ml-1 and 2. 5 ng·ml-1 with the average recovery of 96. 9%-105. 3% and 97. 1% -103. 3%, respectively. The intra-and inter-day standard deviations were both less than 10%. Conclusion: The method is accurate and simple, and suitable for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma.
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Objective:To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry ( UPLC-MS/MS) method to determine CYP2C9 activity in vitro. Methods:An ACQUITY UPLC? BEH C18 (100 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water ( containing 0. 1% formic acid and 0. 5%ammonia water) (40∶60, v/v). The flow rate was 0. 2 ml·min-1. Chlorpropamide was used as the internal standard. The MS condi-tions were as follows:ESI with positive ion detection mode. Self-prepared CYP2C9?1, ?2, ?3 and ?13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000g, the organic layer was then dried using nitrogen, the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS/MS. Results: The reten-tion time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl-1(r=0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0. 01 ng·μl-1 with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9?2,?3 and?13 after tol-butamide was incubated with CYP2C9?1,?2,?3 and?13 was 47. 3%, 11% and 0. 3% of wild type CYP2C9?1. Conclusion:The method is simple and stable, and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.
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OBJECTIVE: To study the pharmacokinetics amlodipine fixed-dose combination amlodipine/benazepril 5/10 mg capsule in healthy volunteers and to provide evidence for its clinical application. METHODS: Twenty-four healthy volunteers were divided to 3 groups with male and female half by half randomly, a single oral dose 1, 2, 3 capsules (5, 10 or 15 mg amlodipine respectively) was given to 8 healthy volunteers and 14 d consecutive administration (Qd) multiple oral doses 2 capsules (10 mg amlodipine) were given to the 8 healthy volunteers the mid-dose group in this open randomized design. Plasma concentration amlodipine was determined by ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLCESI-MS/MS), the main pharmacokinetic parameters were calculated by DAS2.0 program using non compartment analysis (published by Mathematical Pharmacology Professional Committee China, Shanghai, China). RESULTS: After the healthy volunteers received a single dose 1, 2, 3 capsules (5, 10, 15 mg amlodipine), the main pharmacokinetic parameters amlodipine were as follows: t1/2 were (33.5 ± 6.2), (36.1 ± 7.6), (39.8 ± 12.5) h; tmax were (6.0 ± 1.1), (7.0 ± 3.4), (5.6 ± 3.0) h; ρmax (4.37 ± 1.37), (7.23 ± 1.81), (14.71 ± 3.14) ng · mL-1; AUC0→144 were (167.7 ± 35.9), (283.8 ± 47.6), (574.9 ± 159.0) ng · h · mL-1; AUC0→∞ were (176.8 ± 40.2), (304.1 ± 54.8), (628.2 ± 197.5) ng · h · mL-1; V/F were (1 389.6 ± 231.5), (1 724.4 ± 269.2), (1 414.2 ± 342.6) L; MRT0-144 were (47.8 ± 4.0), (51.1 ± 9.9), (52.9 ± 13.9) h; CL/F were (29.5 ± 6.5), (34.0 ± 7.2), (25.9 ± 7.6) L · h-1, respectively. In the dose profile 5-15 mg, AUC0→144, AUC0-∞ and ρmax amlodipine increased with dose, but not proportionally; the statistical results showed that t1/2, V/F, CL/F, MRT0-144 and tmax had no significant difference (P > 0.05) between three groups, whereas there were increasing tendency t1/2 and MRT0-144 between three groups, the t1/2 values single high dose group increased by 6 and 3 h compared with single middle and low dose groups. Steady state concentration was achieved after 14 d consecutive administration test drug, ρmaxss was (24.03 ± 5.56) ng · mL-1, ρminss was (15. 64 ± 3.93) ng · mL-1, AUC0~24ss was (463.7 ± 121.1) ng · h · mL-1 and DF was (0.4 ± 0.1), respectively. Statistical analys is indicated that these ρmax, AUC0-24ss values multiple dose group were obviously higher than the ρmax, AUC0-∞ the single 10 mg dose group with p values less than 0.05 which confirmed that obvious accumulation amlodipine in human plasma occurred after multiple doses administration. CONCLUSION: The established UPLC-ESI-MS/MS method is simple, rapid, sensitive and accuracy, and can be used for the study clinical pharmacokinetics of amlodipine. The dynamics amlodipine in vivo are nonlinear in the dosage 5-15 mg, no gender difference is found in the main pharmacokinetic parameters amlodipine, and there is obvious accumulation amlodipine besylate in human plasma found after repeated administration. Amlodipine besylate is well tolerated and no serous adverse reaction is observed during the trail.
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This study was aimed to establish a method for the rapid content determination of Ixerin Z and 11,13α-dihydroixerin Z in Ku-Die-Zi (KDZ) injection by UPLC-ESI-MS/MS. The separation was performed on a Waters ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7μm) by using a gradient elution with the mobile phase of acetonitrile-water at the flow rate of 0.4 mL·min-1. The column temperature was set at 40℃. Multi-reaction moni-toring (MRM) scanning was employed for quantification in ESI negative mode. The results showed that two sesquit-erpene lactones in KDZ injection were totally separated within 2 min. The linear range of Ixerin Z was 5.70-182.50 ng·mL-1, and the linear range of 11,13α-dihydroixerin Z was 4.60-131.25 ng·mL-1. The correlation coefficient r was more than 0.999 0. The recovery rates (n = 6) were 98.18% and 97.52%, with RSDs < 1.5%. The established method was successfully applied for simultaneous content determination of Ixerin Z and 11,13α-dihydroixerin Z in 6 batches of KDZ injection from 2 factories, which had some variations on the content determination results. It was concluded that the method was rapid, accurate and sensitive, which can be used for the content determination of two sesquiterpene lactones in KDZ injection.
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A simple and selective ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) assay was developed for the determination of the human plasma protein binding of four bioactive flavonoids (such as orientin and vitexin) in Polygonum orientale. Protein precipitation was used for sample preparation. Equilibrium dialysis technique was applied to determine the plasma protein binding under physiological conditions. The separation was achieved through a Waters C18 column with a mobile phase composed of 0.1%formic acid in acetonitrile and 0.1%aqueous formic acid using step gradient elution at a flow rate of 0.35 mL/min. A Waters ACQUITY?TQD system was operated under the multiple reaction monitoring (MRM) mode of positive electrospray ionization. All of the recovery, precision, accuracy and stability of the method met the requirements. Good correlations (r40.99) of the four compounds were found, which suggested that these compounds can be simultaneously determined with acceptable accuracy. Results showed that the plasma protein bindings of the four bioactive flavonoids were in the range of 74-89% over the six concentrations studied. The binding parameters containing protein binding affinity, protein binding dissociation constant, and protein binding site were studied. The maximum ability to bind with protein was also determined in the assay in order to understand the drug-protein binding of each compound better.