Effect of Rhei Radix et Rhizoma and Eupolyphaga Steleophaga on liver protection mechanism based on pharmacokinetics and metabonomics / 中草药·英文版

OBJECTIVE@#Based on metabonomics technology of high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and hydrogen nuclear magnetic resonance spectroscopy (1H NMR), the pharmacokinetic characteristics and therapeutic mechanism of Rhei Radix et Rhizoma (RhRR, Dahuang in Chinese), Eupolyphaga Steleophaga (EuS, Tubiechong in Chinese) combined with RhRR acting on acute liver injury were explored.@*METHODS@#Models of acute liver injury were established, and the pharmacokinetic methods of five components of RhRR-EuS in rats were found by HPLC-MS/MS. The liver tissues of different groups of mice were analyzed by 1H NMR spectroscopy combined with multivariate statistical analysis to investigate the metabolomics of RhRR-EuS and RhRR.@*RESULTS@#Pharmacokinetic results showed there were different levels of bimodal phenomenon in different groups, and the absorption of free anthraquinone in RhRR increased after compatibility with EuS. In addition, the pathological state of acute liver injury in rats can selectively promote the absorption of emodin, chrysophanol, physcion and aloe emodin. Through 15 differential metabolites in the liver tissue of acute liver injury mice, it was revealed that RhRR-EuS and RhRR could protect the liver injury by regulating the metabolism of glutamine and glutamic acid, alanine, aspartic acid and glutamic acid, and phosphoinositide. However, the regulation of RhRR was weaker than that of RhRR-EuS.@*CONCLUSION@#For the first time, we studied the pharmacokinetics and metabolomics differences of RhRR-EuS and RhRR in rats and mice with acute liver injury, in order to provide theoretical reference for clinical treatment of liver disease by DHZCP.

Method establishment and clinical practice for concentration determination of caffeine and its metabolites in urine / 中国药房

OBJECTIVE To establish a method for concentration determination of caffeine and its three metabolites， theophylline， paraxanthine and theobromine in urine， and apply it in clinical practice. METHODS Using caffeine-13C3-d3 as internal standard （IS）， and the urine samples were protein precipitated with acetonitrile； HPLC-MS/MS method was adopted to determine the concentrations of caffeine and its three metabolites. The determination was performed on Waters ACQUITY UPLC® BEH HILIC column with mobile phase consisting of 60 mmol/L ammonium acetate （A）-acetonitrile （B） （gradient elution） at the flow rate of 0.5 mL/min. The column temperature was set at 38 ℃ ， and the sample size was 2 μL. The electrospray ionization detection was operated in a positive mode by multiple reaction monitoring. The detection ions for quantitative analysis were m/z 195.1→110.0 for caffeine， m/z 181.1→124.0 for theophylline， m/z 181.1→124.0 for paraxanthine， m/z 181.1→138.0 for theobromine， and m/z 198.1→ 140.1 for IS. The above method was used to determine the concentrations of caffeine and its three metabolites in the urine of 19 infants with apnea of prematurity （AOP）. RESULTS The linear ranges of mass concentration of caffeine， theophylline， paraxanthin and theobromine were 0.200-200， 0.050-50.0，0.050 0-50.0， and 0.100-100 μg/mL， respectively. The lower limits of quantification were 0.200， 0.050， 0.050 and 0.100 μg/mL （r＞0.990）， respectively. RSDs of intra-day and intra- day precision were not above 10.37%， and matrix factors were 85.68%-109.90%； extraction recoveries were 93.53%-109.40% （RSD≤15%）， and RSDs of stability tests were all lower than 15%. The concentrations of caffeine and its three metabolites in the urine of 19 cases were （27.346±7.951）， （0.351±0.223）， （0.428±0.395） and （0.472±0.374） μg/mL， respectively. CONCLUSIONS The established HPLC-MS/MS method is simple， sensitive and can be used for the determination of caffeine and its three metabolites in urine samples of AOP.

Research of pharmacokinetics of thalidomide in SD rats with inflammatory bowel disease / 中国药理学通报

Aim To investigate the absorption of thalidomide in inflammatory bowel disease(IBD)SD rats compared with healthy rats to provide a basis for the safety and efficacy of thalidomide in clinical practice. Method The IBD rat model was induced by the 2, 4, 6-trinitrobenzene sulfonic acid, by which the rats were continuously intervened for six days. The general state and disease activity index of the rats were recorded every day. The rats in IBD group after modeling and control group were administered with thalidomide at a dose of 10 mg·kg-1. The blood sample of the rats was collected at different time points. After a comprehensive evaluation of morphological and histopathological results, the samples of rats with IBD were determined by proven high performance liquid chromatography-mass spectrometry,and the pharmacokinetic parameters were calculated and compared with the healthy rats. Results The body weight of rats in IBD group was obviously lower than that in control group and the disease activity index, score of colonic macroscopic morphous and histopathology were obviously higher than those in control group. The success rate of modeling was 62.5%. The pharmacokinetic results showed that the Cmax(P<0.05)and AUC(P<0.01)of the IBD group both increased by 2 to 3 times, but there was no significant difference in t1/2,Tmax,MRT and other parameters. Conclusions The rate and extent of oral thalidomide absorption of rats in the model of inflammatory bowel disease significantly increase compared to those of healthy rats, which may provide new considerations for clinical practice of thalidomide in the treatment of IBD.

Tissue distribution of Qingfei Paidu Decoction based on HPLC-MS/MS / 中国中药杂志

The tissue distribution of Qingfei Paidu Decoction was studied by HPLC-MS/MS in vivo. Hypersil GOLD C_(18) column(2.1 mm×50 mm, 1.9 μm) was used for gradient elution with acetonitrile as the mobile phase A and 0.1% formic acid solution as the mobile phase B. High-resolution liquid chromatography-mass spectrometry in both positive and negative ion scanning mode and multiple response monitoring(MRM) mode was employed to analyze the behaviors of the active components of Qingfei Paidu Decoction in diffe-rent tissues. The results showed that 19, 9, 17, 14, 22, 19, 24, and 2 compounds were detected in plasma, heart, liver, spleen, lung, kidney, large intestine, and brain, respectively. The compounds belonged to 8 groups, covering 14 herbs in the prescription. After administration with Qingfei Paidu Decoction, the compounds were rapidly distributed in various tissues, especially in the lung, liver, large intestine, and kidney. The majority of the compounds displayed secondary distribution. This study comprehensively analyzed the distribution rules of the main active components in Qingfei Paidu Decoction and provided a basis for the clinical application.

Screening of quantitative preparation quality markers of Zhibao Sanbian Wan / 药学学报

On the basis of the qualitative preparation quality markers of Zhibao Sanbian Wan (ZBSBW), we screened out the quantitative markers and evaluated the content consistency of ZBSBW. A method capable of simultaneously determining 34 compounds in ZBSBW was established based on HPLC-MS/MS, and 16 batches of ZBSBW were simultaneously analyzed by this method. Furthermore, we explored a general strategy for analyzing the component migration in preparation, plasma, brain tissue and cerebrospinal fluid. The methodological investigation was confirmed by linear range, recovery (85.10%-105.07%), precision (RSD: 1.37%-4.58%), stability, and repeatability (3.00%-12.45%), the established method was suitable for the detection and quantification of the compounds in ZBSBW. The contents of compounds in ZBSBW were all lower than 1 mg·g-1, and the contents and daily dose of nystose were the highest, followed by echinacoside, paeoniflorin, osthole and paeonol. The results of systematic clustering showed that the contents were consistent for ordinary preparations of ZBSBW. The principal component analysis showed that the components of berberine, ginsenoside Re, ginsenoside Rg1, pinoresinol diglucoside and tenuifolin had large variation, which contributed significantly to the grouping. The contents of echinacoside, verbascoside, polygalaxanthone Ⅲ, β-ecdysterone, osthole, alisol B 23-acetate, liquiritin and glycyrrhizic acid were stable from batch to batch. The animal experiment results showed that osthole, paeonol and liquiritin in ZBSBW could be absorbed into the blood and enter the brain tissue by passing through the blood-brain barrier. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (No. 2020B071). The above compounds contributed the quantitative preparation quality markers of ZBSBW. In conclusion, the HPLC-MS/MS method established in this study was sensitive, accurate and rapid, and could be used for simultaneous quantification of 34 compounds and content consistency evaluation of multiple batches of preparations in ZBSBW. The result provided a methodological basis for the screening of quantitative preparation quality markers and material basis research of ZBSBW.

Content determination of four kinds of alkaloids in Uncaria by HPLC-MS-AIS / 四川精神卫生

ObjectiveTo determine the contents of four kinds of indole alkaloids （rhynchophylline， isorhynchophylline， corynoxeine， isocorynoxeine） in Uncaria by high-performance liquid chromatography-mass spectrometry-automatic internal standard （HPLC-MS-AIS）. MethodsChromatographic separation was performed using C18 column （3.0 mm×50 mm， 3.3 μm）， and the mobile phase， comprising 0.1% formic acid aqueous solution-acetonitrile （82∶18， V/V）， was eluted at a flow rate of 0.5 mL/min and column temperature of 30℃. Mass spectrometric detection was performed using an electrospray ionization source and positive multiple-reaction monitoring mode at a voltage capillary of 4 000 V. The mass-to-charge ratio （m/z） transition was 385.25/160.10 for rhynchophylline， 385.30/160.10 for isorhynchophylline， 383.25/160.15 for corynoxeine and 383.25/160.15 for isocorynoxeine， respectively. The injection volume was kept constant at 2 μL. ResultsThe linear concentration ranges of rhynchophylline， isorhynchophylline， corynoxeine and isocorynoxeine were 2.30~600.00 ng/mL （r=0.999 3）， 2.30~600.00 ng/mL （r=0.999 2）， 2.47~650.00 ng/mL （r=0.999 4） and 2.47~650.00 ng/mL （r=0.999 2）， respectively. The relative standard deviation （RSD） of precision and stability were all lower than 5.00%， the accuracy ranged from 92.40% to 104.10%， and the average recovery was 95.90%~104.60%. ConclusionHPLC-MS-AIS method is simple and accurate for the determination of four kinds of alkaloids in Uncaria， and can be used as a new method for quality control of Uncaria.

Determination of sodium valproate and vancomycin in human serum by HPLC-MS/MS / 药学实践杂志

Objective To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of sodium valproate and vancomycin in human serum. Methods Valproic acid-d6 and kanamycin B were used as the internal standard of sodium valproate and vancomycin, the serum samples were treated by acetonitrile precipitation protein method. The mobile phase was 0.1% formic acid aqueous solution-acetonitrile for gradient elution. The flow rate was 0.5 ml/min, with column temperature at 25 ℃. The sample volume was 4 μl and total analysis time was 12 min. The positive and negative ion mode was monitored by electrospray ion source and the multiple reaction monitoring mode was used for quantitative analysis. The specificity, standard curve, lower limit of quantification, precision, recovery, matrix effect, and stability of the method were examined. Results Sodium valproate and vancomycin had good linear relationships in the range of 1 - 200 μg/ml and 0.5 - 100 μg/ml, respectively. The quantitative lower limits were 1 μg/ml and 0.5 μg/ml, respectively. The extraction recoveries were above 70%. The inter- and intra-batch precision RSD values were less than 10%. The stability was good and there was no obvious matrix effect. Conclusion This method is simple, quick, sensitive, specific and accurate, which could be used to simultaneously determine the concentration of sodium valproate and vancomycin in human serum.

Bioequivalence study of buthlphthalide injection in Chinese healthy volunteers / 中国临床药理学与治疗学

AIM: To establish a method to investigate pharmacokinetics and bioequivalence of buthlphthalide injection. METHODS: An open, randomized, and two-cycle crossover study was conducted in 24 healthy volunteers. Plasma concentrations of buthlphthalide were determined by LC-MS/MS after administering a single dose of reference drug or test drug. Main pharmacokinetic parameters were calculated by Phoenix WinNonlin 6.4 software. RESULTS: For the test drug and the reference drug, the main pharmacokinetic parameters of flurbiprofen were as follows: AUC

Effect of particle size on the oral absorption of isoliquiritigenin nanocrystals

Abstract As one of the most promising formulations for poorly water-soluble drugs, nanocrystals have been attracting increasing attention in recent years. Isoliquiritigenin (ISL) is a flavonoid with a chalcone structure, and possesses many biological activities. However, its clinical application is significantly limited mainly due to its low oral bioavailability caused by poor hydrophilicity. To address this, ISL nanocrystals were developed in this study to improve its oral bioavailability. Three types of nanocrystals with differing particle size; R1, R2, and R3, were prepared by anti- solvent precipitation or anti-solvent precipitation combined with sonication, which was optimized by single-factor experiments. These nanocrystals were characterized based on their physical properties, in vitro release, and in vivo absorption performance. The mean particle size of R1, R2, and R3 was 555.7, 271.0, and 46.2, respectively. The dissolution ratio of ISL in the nanocrystals was significantly improved, with the quickest rate recorded in R2. Peak concentration and area under the concentration-time curve of R2 after oral administration in rats was 5.83- and 2.72-fold higher than that of the ISL solution, respectively. These findings indicate that the dissolution and absorption of ISL can be significantly enhanced by nanocrystals, and the dissolution behavior and pharmacokinetic properties of nanocrystals is significantly influenced by particle size.

Detection of Synthetic Cannabinoid CUMYL-PEGACLONE in E-Cigarette Oil and Hair / 法医学杂志

OBJECTIVES@#To establish a combined high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) method to detect the synthetic cannabinoid CUMYL-PEGACLONE in e-cigarette oil and hair.@*METHODS@#HPLC-MS/MS and GC-MS were used to establish the detection method of CUMYL-PEGACLONE, and the hair of drug-involved persons and the seized e-cigarette oil were detected.@*RESULTS@#The main mass spectrometry characteristic ions m/z of CUMYL-PEGACLONE measured by GC-MS were 91, 179, 197, 254 and 372. CUMYL-PEGACLONE had a good linear relationship in the mass concentration range of 2-50 ng/mL, and the linear correlation coefficient (r) was greater than 0.99. The limit of detection (LOD) of CUMYL-PEGACLONE in hair was 0.01 ng/mg, and the limit of quantitation (LOQ) was 0.02 ng/mg. The LOD of CUMYL-PEGACLONE in e-cigarette oil was 1 ng/mg, and the LOQ was 2 ng/mg. The average recoveries of CUMYL-PEGACLONE under the attempt at high, intermediate and low levels in blank human hair and e-cigarette oil matrix were 98.2%-132.4% and 93.5%-110.6%, and the intraday and intraday precision were 1.2%-12.9% and 0.7%-2.9%. CUMYL-PEGACLONE was detected in the hair of 15 drug-involved persons. Except for 1 person who was lower than LOQ, the concentration of CUMYL-PEGACLONE in the hair of other 14 persons was 0.035-0.563 ng/mg. The mass fraction of CUMYL-PEGACLONE in 2 e-cigarette oil were 0.17% and 0.21%, respectively.@*CONCLUSIONS@#The established HPLC-MS/MS and GC-MS methods are applied to the detection of HPLC-MS/MS in drug-related cases, which provides strong evidence support for the handling authority to quickly investigate these cases, and also provides a reference for the identification of such substances in future.

Determination of bioactive components in the fruits of Cercis chinensis Bunge by HPLC-MS/MS and quality evaluation by principal components and hierarchical cluster analyses / 药物分析学报

The fruits of leguminous plants Cercis Chinensis Bunge are still overlooked although they have been reported to be antioxidative because of the limited information on the phytochemicals of C.chinensis fruits.A simple,rapid and sensitive HPLC-MS/MS method was developed for the identification and quantitation of the major bioactive components in C.chinensis fruits.Eighteen polyphenols were iden-tified,which are first reported in C.chinensis fruits.Moreover,ten components were simultaneously quantified.The validated quantitative method was proved to be sensitive,reproducible and accurate.Then,it was applied to analyze batches of C.chinensis fruits from different phytomorph and areas.The principal components analysis (PCA) realized visualization and reduction of data set dimension while the hierarchical cluster analysis (HCA) indicated that the content of phenolic acids or all ten components might be used to differentiate C.chinensis fruits of different phytomorph.

Identification and quantification of the bioactive components in Osmanthus fragrans roots by HPLC-MS/MS / 药物分析学报

The roots of O.fragrans are also a valuable resource in addition to its flowers and fruits.In this study,the HPLC-MS/MS method used for analyzing the chemical constituents in O.fragrans roots extract was developed,which showed high sensitivity for both qualitative and quantitative analyses.Thirty-two compounds were first discovered in O.fragrans roots,one compound of which was reported for the first time.The simultaneous determination method for acteoside,isoacteoside,oleuropein and phillyrin was validated to be sensitive and accurate.Then it was applied to determine the content of bioactive components in O.fragrans roots from different cultivars.The content of oleuropein and phillyrin in the twelve batches was relatively stable,while the content of acteoside and isoacteoside varied greatly.Moreover,the therapeutic material basis and mechanism of O.fragrans roots exerting its traditional pharmacodynamics were analyzed by network pharmacology.The results showed that O.fragrans roots might be effective for the treatment of inflammation,cardiovascular diseases,cancer,and rheumatoid arthritis,which is consistent with the traditional pharmacodynamics of O.fragrans roots.This work can provide an analytical method for the comprehensive development of O.fragrans roots.

Screening of quantitative preparation quality markers of Yulian Decoction / 中国中药杂志

On the basis of the qualitative preparation quality markers of Yulian Decoction, we screened out the quantitative markers and explored a general strategy for analyzing the component migration in Chinese herbal pieces, preparations, and plasma. A method capable of simultaneously determining 28 chemical components in Yulian Decoction was established based on HPLC-MS/MS. This method was used to determine the migrated components in herbal pieces-lyophilized powder preparations-rat plasma after administration of Yulian Decoction. Liquid chromatography was performed under the following conditions: C_(18)-reversed phase chromatographic column(2.1 mm × 100 mm, 1.8 μm); acetonitrile-water(containing 0.1% formic acid) as the mobile phase for gradient elution; the flow rate of 0.2 mL·min~(-1). Electrospray ionization source was adopted for mass spectrometry detection, in which positive and negative ion modes and multiple reaction monitoring were applied. Confirmed by the methodological investigation in linear range, recovery(95.48%-103.4%), precision(RSD, 0.45%-3.8%), stability, and repeatability(RSD, 5.6%-14%), the established method was suitable for the detection and quantification of the components in Yulian Decoction. The results showed that in the lyophilized powder of Yulian Decoction, berberine was greater than 5% in mass fraction, magnoflorine, epiberberine, coptisine, palmatine, and limonin in the range of 1%-5%, and dehydroevodiamine, evodiamine, rutaecarpine, costunolide, and dehydrocostus lactone in the range of 0.002%-1%. Of the 28 components detected in pieces, 27 were found to migrate to the lyophilized powder, and 11 were detected in rat plasma. Fifteen components were preliminarily determined as quantitative preparation quality markers for Yulian Decoction, including berberine, epiberberine, coptisine, palmatine, evodiamine, rutaecarpine, limonin, costunolide, dehydrocostus lactone, magnoflorine, jatrorrhizine, columbamine, groenlandicine, chlorogenic acid, and neochlorogenic acid. In conclusion, the HPLC-MS/MS general strategy was established for analyzing the migration of multiple components in Chinese herbal pieces, preparations, and plasma, which can provide the basis for the screening of quantitative preparation quality markers and multi-index quality control of Yulian Decoction.

Systematic chemical characterization of Xiexin decoctions using high performance liquid chromatography coupled with electrospray ionization mass spectrometry / 中国天然药物

Xiexin decoctions (XXDs) display beneficial anti-inflammatory and anti-diabetic effects, which raises interests on this group of formulae for broad clinical applications. However, there was no report about systematic analysis of XXDs to elucidate the constitution of chemical components, which hampers further investigations on the therapeutic values of XXDs. In this work, crude herbs were extracted and prepared to obtain the XXDs for systemic analysis on their chemical compositions, according to the information described in the ancient Zhang Zhongjing's herbal formulae. LC-MS analysis of five XXDs was carried out to facilitate recognition of the source herbs for compounds in the mixture. A total number of 93 compounds were identified through our methods and their chemical classes encompassed five major groups, including protoberberine alkaloids, flavonoids, stilbenes, anthraquinones and saponins. Our current work provided important information about material basis for pharmacological studies on XXDs and would help shed light on relationships between chemical compositions and therapeutic effects.

An LC-MS/MS method for simultaneous analysis of the cystic fibrosis therapeutic drugs colistin,ivacaftor and ciprofloxacin / 药物分析学报

Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung in-fections in cystic fibrosis patients.In this study,we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin,colistin and ivacaftor in rat plasma,human epithelial cell lysate,cell culture medium,and drug transport media.An aliquot of 200 μL drug-containing rat plasma or cell culture medium was treated with 600 μL of extraction solution(acetonitrile containing 0.1％ formic acid and 0.2％ trifluoroacetic acid (TFA)).The addition of 0.2％ TFA helped to break the drug-protein bonds.Moreover,the addition of 0.1％ formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility.After vortexing and centrifuging,the sample components were analyzed by HPLC-MS/MS.The multiple re-action monitoring mode was used to detect the following transitions:585.5-101.1 (colistin A),578.5-101.1 (colistin B),393.2-337.2 (ivacaftor),332.2-314.2 (ciprofloxacin),602.3-101.1 (polymyxin 81 as internal standard (IS)) and 595.4-101.1 (polymyxin B2 as IS).The running time of a single sample was only 6 min,making this a time-efficient method.Linear correlations were found for colistin A at 0.029-5.82 μg/mL.colistin B at 0.016-3.14 μg/mL.ivacaftor at 0.05-10.0 μg/mL,and ciprofloxacin at 0.043-8.58 μg/mL.Accuracy,precision,and stability of the method were within the acceptable range.This method would be highly useful for research on cytotoxicity,animal pharmacokinetics,and in vitro drug delivery.

Population pharmacokinetics of capecitabine in Chinese breast cancer patients / 中国临床药理学与治疗学

AIM: To investigate the population pharmacokinetic characteristics of capecitabine and its possible influencing factors in Chinese patients of breast cancer. METHODS: 78 cases of Chinese patients with breast cancer were chosen as the objects in this study. Following treatment with capecitabine (0.6 g, 0.15 g/piece, 4 pieces, orally), blood samples were collected and concentrations of capecitabine in plasma were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method. The nonlinear mixed-effects software (NONMEM) was used to analyze the data and the population pharmacokinetic model was constructed accordingly. RESULTS: The final established model of absorption and elimination is one-compartment model. The clearance (CL/F) in pharmacokinetic formula of the model is as follows: CL/F=291×e

Bioequivalence of moxifloxacin hydrochloride tablets in healthy Chinese subjects / 中国临床药理学与治疗学

AIM: To study the pharmacokinetic characteristics of single-dose oral moxifloxacin hydrochloride tablets under fasting and fed conditions, and use moxifloxacin hydrochloride tablets produced by Bayer Pharma AG as a reference to compare the pharmacokinetic parameters of the two preparations, and evaluate the human bioequivalence of the two preparations. METHODS: A single-center, randomized, open, two-period, and self-crossover design was adopted to conduct a fasting and fed bioequivalence study of 23 healthy subjects each. The 0.4 g dose preparations were taken orally per cycle on fasting or fed administration. The plasma concentrations of moxifloxacin at different times after administration were determined by HPLC-MS/MS. The main pharmacokinetic parameters were calculated, and the bioavailability of the test preparation relative to the reference preparation was evaluated. RESULTS: After subjects in the fasting group took the test preparation T and the reference preparation R, the main pharmacokinetic parameters of moxifloxacin hydrochloride were: C

Absolute bioavailability of salidroside in Beagle dog / 药学实践杂志

Objective To develop a HPLC-MS/MS method for the absolute bioavailability study of salidroside in Beagle dogs. Methods Gastrodin was used as internal standard. Plasma samples were treated by protein precipitation and separated by Symmetry RP18 column (100 mm×4.6 mm, 3.5 μm). 0.1% formic acid in water(A) and 0.1% formic acid in acetonitrile: methanol (20 : 80, V/V) (B) were used as the mobile phase for isocratic elution with 35% mobile phase B. The flow rate was 0.4 ml/min. Column temperature was 40 ℃. Injection volume was 2 μl. By electrospray ionization source (ESI) and multi-reaction monitoring (MRM) mode, the MRM ion pairs of salidroside and gastrodin were identified as m/z 299.1→118.9 and m/z 285.1→122.9, separately. Blood samples were collected at different time points after oral or intravenous administration of salidroside. The harvested plasma samples were analyzed by HPLC-MS/MS method to assess the pharmacokinetics and absolute bioavailability of salidroside. Results Excellent linearity(r>0.998 6) was found in the concentration range of 10−10 000 ng/ml for salidroside and the lowest quantitative concentration was 10 ng/ml. The recovery was 89.5%−91.8%. The intra-day precision (RSD) was less than 9.7%, and the inter-day precision (RSD) was less than 7.3%. After a single oral dose of 15 mg/kg or an intravenous injection of 1.5 mg/kg of salidroside, cmax was (9 680±3725) and (9 310±1 645) ng/ml; tmax was (1.25±0.67) and (0.011±0.017) h, AUC0−t was (20 535.4±5 200.0) and (4 646.7±720.5) ng·h/ml, AUC0−∞ was (20 607.9±5 266.2) and (4 691.6±715.2) ng·h/ml; t1/2 was (1.31±0.63) and (0.98±0.13) h, respectively. Conclusion The LC-MS/MS method established in this study was simple, rapid, sensitive and reliable. It meets the regulatory requirements of biological analysis for pharmacokinetic properties of salidroside in Beagle dogs. The absolute bioavailability of salidroside in Beagle dogs is (43.9±11.2)%.

Identification of chemical constituents in ethyl acetate soluble extract of Sinopodophylli Fructus based on HPLC-MS~n / 中国中药杂志

A high performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry(HPLC-DAD-ESI-IT-TOF-MS~n, HPLC-MS~n) method was established for qualitative analysis of the chemical components of ethyl acetate extract from Sinopodophylli Fructus. The analysis was performed on a Kromasil 100-5 C_(18)(4.6 mm×250 mm, 5 μm) column, with a mobile phase consisted of 0.1% formic acid(A) and acetonitrile(B) for gradient at a flow rate of 1.0 mL·min~(-1). Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. With use of reference substance, characteristic fragmentation and their HR-MS data, 102 components were identified, including 67 flavonoids and 35 lignans. Among them, 45 compounds were reported in Sinopodophylli Fructus for the first time and 19 compounds were identified as new compounds. PharmMapper was used to predict the bioactivity of compounds that were first reported in Sinopodophylli Fructus, and 20 compounds of them were identified to have potential anticancer activity. The results showed that there were many isomers in the ethyl acetate extract of Folium Nelumbinis, and a total of 19 groups of isomers were found. Among them, C_(21)H_(20)O_8 had the highest number of isomers(18 compounds), all of which were α-peltatin or its isomers; C_(21)H_(20)O_7 ranked second, with 10 compounds, all of which were 8-prenylquercetin-3-methyl ether or its isomers. In conclusion, an HPLC-MS~n method was established for qualitative analysis of the ethyl acetate extract(with anti-breast cancer activity) from Sinopodophylli Fructus in this study, which will provide the evidence for clarifying pharmacological active ingredients of the ethyl acetate extract from Sinopodophylli Fructus against breast cancer.

Identification of PD-1 small molecule inhibitors and validation in Panax ginseng / 药学学报

Tumor immune therapy has been remarkably successful in recent years and several kinds of PD-1/PD-L1 (programmed death-1/programmed death-ligand 1) antibody drugs have been approved by the FDA for treatment of advanced malignant neoplasms. However, as biomacromolecules these antibody drugs have certain drawbacks such as high cost, injection-only administration and immunogenicity; thus, we turned to small molecules that have lower immune risks and better modifiability. Considering the structural diversity of natural products, we chose to investigate the active components in Panax ginseng, a famous and highly valued traditional Chinese medicine. Nine compounds were separated and identified in this research using a HPLC-coupled MS system, and 3 PD-1 binding compounds were identified using the SPR method. The PD-1/PD-L1 inhibitory ability of ginsenoside Rg1, as a representative ginsenoside, was verified by cytopharmacological methods. This research provides a new method for the identification of immune blockade inhibitors in natural products.