Slit2 signaling contributes to cholestatic fibrosis in mice by activation of hepatic stellate cells.

Liver Cholestasis is a widespread disease of broad etiologies and ultimately results in fibrosis, which is still lacking effective therapeutic strategies. Activation of hepatic stellate cells (HSCs) is the key event of liver fibrosis. Here, we aimed to investigate the effect and mechanism of the Slit2 signaling in cholestasis-induced liver fibrosis. Our findings revealed that the serum levels and hepatic expression of Slit2 were significantly increased in patients with primary biliary cirrhosis (PBC). Additionally, Slit2-Tg mice were much more vulnerable to BDL-induced liver injury and fibrosis compared to WT control. Slit2 up-regulation by Slit2 recombinant protein induced proliferation, and inhibited apoptosis of human HSCs cell line LX-2 via p38 and ERK signaling pathway, resulting in the activation of HSCs. In contrast, Slit2 down-regulation by siRNA silencing inhibit the activation of HSCs. In conclusion, Slit2 is involved in the activation of HSCs and liver fibrogenesis, highlighting Slit2 as a potential therapeutic target for liver fibrosis.

[Identification of metabolites of Chenxiang Huaqi pill in rats based on UPLC-Q-TOF-MS].

To analyze the metabolites of Chenxiang Huaqi pill in rats by using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS). The separation was performed on Phenomenex Kinetex C18 column, with the acetonitrile -0.1% formic acid as the mobile phase for gradient elution at a flow rate of 0.8 mL·min⁻¹. The data were collected by the positive ion mode of ESI source. The plasma and urine total ion chromatograms of the rats in blank group and treatment group were used to analyze the targeted ion chromatograms. The results showed that 24 compounds were detected in the plasma and urine, including 5 prototype components and 19 metabolites. The major metabolic pathways included hydration, glucuronidation, demethylation, hydrolysis, hydroxylation and sulfation. The method was rapid, simple and sensitive, and can be used to rapidly identify the metabolites of Chenxiang Huaqi pill that can be absorbed in rats, providing a reference for the study of the absorption and metabolism mechanism of Chenxiang Huaqi pill in vitro.

Analysis of lignans in Magnoliae Flos by turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry.

In this study, a method coupling turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry was developed for analyzing the lignans in Magnoliae Flos. By the online pretreatment of turbulent flow chromatography solid-phase extraction, the impurities removal and analytes concentration were automatically processed, and the lignans were separated rapidly and well. Seven lignans of Magnoliae Flos including epieudesmin, magnolin, 1-irioresinol-B-dimethyl ether, epi-magnolin, fargesin aschantin, and demethoxyaschantin were identified by comparing their retention behavior, UV spectra, and mass spectra with those of reference substances or literature data. The developed method was validated, and the good results showed that the method was not only automatic and rapid, but also accurate and reliable. The turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry method holds a high potential to become an effective method for the quality control of lignans in Magnoliae Flos and a useful tool for the analysis of other complex mixtures.

Chemical fingerprint of Ganmaoling granule by double-wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

A method incorporating double-wavelength ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C18 column (2.1 × 50 mm, 1.8 µm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4-O-caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.

Palladium catalyzed dual C-H functionalization of indoles with cyclic diaryliodoniums, an approach to ring fused carbazole derivatives.

Palladium(II)-catalyzed dual C-H functionalization of indoles with cyclic diaryliodoniums was successfully achieved, providing a concise method to synthesize dibenzocarbazoles. In a single operation, two C-C bonds and one ring were formed. The reaction was ligand free and tolerated air and moisture conditions.

Development and validation of a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry assay for the determination of neostigmine in small-volume beagle dog plasma and its application to a pharmacokinetic study.

A simple, rapid and high sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of neostigmine in small-volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96-well filtration plate, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol-water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 µL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor-to-product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1-100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra-run and inter-run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs.

Chiral separation of bupivacaine hydrochloride by capillary electrophoresis with high frequency conductivity detection and its application to rabbit serum and pharmaceutical injection.

Conductivity detection was employed to detect the enantiomers of bupivacaine hydrochloride (Bup), which were separated by high performance capillary electrophoresis. A computer-aided technique was used to calculate the binding energies, and the interaction between Bup enantiomers and cyclodextrins (CDs) is preliminarily discussed. Factors affecting the separation efficiency such as the types and concentration of chiral selectors, running buffer, pH value, separation voltage and capillary inside diameter and length were studied. Under optimized conditions, a baseline separation of Bup enantiomers was achieved in less than 15 min in 4mM NH4Ac-NaAc-HAc (pH 4.00) -0.48mM sulfobutyl ether-beta-cyclodextrin running buffer at a separation voltage of 12 kV. The lowest detectable concentration was 0.052 microg/mL. The proposed method was applied to chiral separation of Bup enantiomers in rabbit serum and pharmaceutical injections.

Metabolism of 4F-MDMB-BICA in zebrafish by liquid chromatography-high resolution mass spectrometry.

In this study, in vivo metabolic studies of the synthetic cannabinoid 4F-MDMB-BICA were investigated using zebrafish models. The metabolites were identified and structurally illustrated by liquid chromatography-high resolution mass spectrometry. Fourteen phase-I metabolites and four phase-II metabolites were generated from zebrafish. The main metabolic pathways of the phase-I metabolism included N-dealkylation, N-dealkylation combined with hydroxylation, amide hydrolysis, oxidative defluorination, oxidative defluorination to butyric acid, acetic acid formation at the indole side chain, hydroxylation, ester hydrolysis followed by hydroxylation, dehydrogenation, dehydrogenation, and N-dealkylation, and oxidative defluorination subsequently combined with dehydrogenation. The main biotransformations of the phase-II metabolism were glucuronidation and sulfation. Two phase-I metabolites (A1 and A11) and four phase-II metabolites (A2, A3, A4, and A12) were reported for the first time. A14, which was confirmed in human biological samples, was detected only in zebrafish samples but not found in human liver microsome incubation study. The current study indicates that the zebrafish model is a promising tool for elucidating the metabolism of NPS in the future.

A single-step preparation of carbohydrate functionalized monoliths for separation and trapping of polar compounds.

A single-step copolymerization strategy was developed for the preparation of carbohydrate (glucose and maltose) functionalized monoliths using click reaction. Firstly, novel carbohydrate-functionalized methacrylate monomers were synthesized through Cu(I)-catalyzed 1,3-dipolar cycloaddition (alkyne-azide reaction) of terminal alkyne with azide of carbohydrate derivatives. The corresponding carbohydrate functionalized monolithic columns were then prepared through a single-step in-situ copolymerization. The physicochemical properties and performance of the fabricated monolithic columns were evaluated using scanning electron microscopy, Fourier-transform infrared spectroscopy, and nano-liquid chromatography. For the optimized monolithic column, satisfactory column permeability and good separation performance were demonstrated for polar compounds including nucleoside, phenolic compounds and benzoic acid derivatives. The monolithic column is also highly useful for selective and efficient enrichment of glycopeptides from human IgG tryptic digests. This study not only provided a novel hydrophilic column for separation and selective trapping of polar compounds, but also proposed a facile and efficient approach for preparing carbohydrate functionalized monoliths.

Determination of 37 fentanyl analogues and novel synthetic opioids in hair by UHPLC-MS/MS and its application to authentic cases.

The recent emergence of new fentanyl analogues and synthetic opioids on the drug market poses a global public health threat. However, these compounds cannot typically be identified using existing analytical methods. In this study, we aimed to develop and validate a rapid and sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 37 fentanyl analogues and novel synthetic opioids in hair samples. Hair samples (20 mg) were extracted by cryogenic grinding in an extraction medium of methanol, acetonitrile, and 2 mmol/L ammonium acetate (pH 5.3). Following centrifugation of the samples, the analytes were separated using a WATERS Acquity UPLC HSS T3 column. The limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.5 to 2.5 pg/mg and from 2 to 5 pg/mg, respectively. The intraday and interday precisions were within 13.32% at LOQ, low, medium, and high levels. The accuracies were within the range of 85.63-116.1%. The extraction recoveries were in the range of 89.42-119.68%, and the matrix effects were within the range of 44.81-119.77%. Furthermore, the method was successfully applied to the detection and quantification of fentanyl and sufentanil in hair samples from two authentic cases. Thus, this method has great potential for detecting fentanyl analogues and novel synthetic opioids in forensic work.

Application of a validated UHPLC-MS/MS method for 28 fentanyl-analogue and novel synthetic opioids in whole blood in authentic forensic cases.

The abuse of fentanyl and its analogues, which are potent, short-acting, synthetic narcotic analgesics, has become a major issue. Many cases containing fentanyl-analogue and novel synthetic opioids have also been reported. Hence, we report the determination of fentanyl, fentanyl-analogue and novel synthetic opioids in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method is characterized by the use of a simple, fast and inexpensive liquid-liquid extraction (LLE) for sample preparation, a rapid run time (8 min) and a low required volume of whole blood (100 µl). The limits of detection (LODs) ranged from 0.005 to 0.03 ng/ml, and the lower limits of quantitation (LLOQs) ranged from 0.05 to 0.2 ng/ml. The method was shown to be liner over a concentration range of 0.2-40 ng/ml for fentanyl, norfentanyl and norcarfentanil and 0.05-40 ng/ml for all other target analytes. Recoveries were within the range of 72.09%-103.22%, and the matrix effects were in the 67.95%-113.32% range. Moreover, the method was applied to the detection and quantification of fentanyl and its analogues in whole blood from real forensic cases. This methodology has great potential for use in the determination of fentanyl and its analogues in forensic cases.

Toxicological effects of Nux Vomica in rats urine and serum by means of clinical chemistry, histopathology and 1H NMR-based metabonomics approach.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried ripe seeds of Nux Vomica (Strychnos nux-vomica L.), a traditional Chinese medicine, have been used to treat multifarious symptoms. However, the clinical applications of Nux Vomica are limited by its severe toxicity. In this study, Nux Vomica was subjected to nuclear magnetic resonance (NMR) metabonomics and pathological examination to determine relevant biomarkers in target organs and to explain the underlying toxicity mechanism. MATERIALS AND METHOD: Thirty-six male Sprague-Dawley rats were randomly divided into three groups of twelve rats. The control group was oral gavaged with distilled water, and two experiment groups were treated with Nux Vomica at a dose of 0.315 and 0.630g/kg body weight. On days 14 and 21, serum, urine, liver and kidney tissues were collected for histopathological examination, biochemical analysis and 1H-NMR analysis. RESULTS: The metabolites changes of rats treated with Nux Vomica are obviously differ from that of controls. In serum, low-dose group compared with control shows the significantly changes included elevated concentration of glucose, TMAO, and creatine, with decreased lipids, 3-HB, lactate, and unsaturated fatty acid. Change in taurine was only observed in the separation comparison of high-dose group and control. In urine, the variation metabolites included elevations in glucose, creatine, and TMAO as well as decreased lactate, succinate, α-ketoglutaric acid, citrate and hippurate in low-dose group compared with control. Only alanine and creatine were decreased significantly in high-dose group compared with control. CONCLUSION: Nux Vomica induced disruptions in glycolysis, lipid and amino acid metabolism, and toxic effects were aggravated in liver and kidney tissues as dosing time was prolonged. 1H NMR-based metabonomics combined with biochemical and histopathological methods can be applied to elucidate the toxicity mechanism of Nux Vomica decoction that caused liver and kidney injuries in rats.

Rapid determination of 3', 4'-dimethoxy flavonol-3-ß-d-glucopyranoside in rat plasma by LC-MS/MS method followed by protein precipitation.

A reliable liquid chromatography/tandem mass (LC-MS/MS) method was developed for quantitation of 3', 4'-dimethoxy flavonol-3-ß-d-glucopyranoside (DF3G) in rat plasma using pantoprazole as the internal standard. An Agilent C18 column (100 mm × 3 mm, 3.5 µm) was performed for chromatographic separation with the mobile phase composed of methanol-water (containing formic acid) at a flow rate of 0.4 mL/min. A triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source in positive ion mode was applied to quantitative analysis by multiple reacting monitoring (MRM). The MRM precursor-product ion transition of DF3G and Pantoprazole (IS) were m/z 461.1 → 299.2 and 383.9 → 200.2, respectively. Calibration curves were recovered in a concentration range of 1-500 ng/mL for plasma with a limit of lower quantification (LLOQ) of 1 ng/mL. The intra-day and inter-day precision were not >10%. The accuracy of DF3G ranged from -10% to 0.53% in quality control (QC) samples at three concentrations. DF3G was not degraded during the analysis and the storage period. All the data were validated in accordance with the FDA bioanalytical method validation guideline. The LC-MS/MS method was successfully employed in the pharmacokinetic study of the DF3G after oral administration and intravenous injection in rats.

Study of Separation and Identification of the Active Ingredients in Gardenia jasminoides Ellis Based on a Two-Dimensional Liquid Chromatography by Coupling Reversed Phase Liquid Chromatography and Hydrophilic Interaction Liquid Chromatography.

In this paper, by coupling reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), a two-dimensional liquid chromatography system was developed for separation and identification of the active ingredients in Gardenia jasminoides Ellis (GJE). By applying the semi-preparative C18 column as the first dimension and the core-shell column as the second dimension, a total of 896 peaks of GJE were separated. Among the 896 peaks, 16 active ingredients including geniposide, gardenoside, gardoside, etc. were identified by mass spectrometry analysis. The results indicated that the proposed two-dimensional RPLC/HILIC system was an effective method for the analysis of GJE and might hold a high potential to become a useful tool for analysis of other complex mixtures.

Triple cascade reactions: An ultrasensitive and specific single tube strategy enabling isothermal analysis of microRNA at sub-attomole level.

Sensitive and specific analysis of microRNAs (miRNAs) in a single tube without the need of thermal cycler instrument would greatly facilitate the investigation of miRNA-associated regulatory circuits and diseases. Homogeneous isothermal amplification assays are attractive in conducting single tube assays that can minimize contamination-prone steps and simplifies assay procedures. However, the relative low amplification efficiency and high detection background remain as bottlenecks restricting their more versatile applications. In this work, we have developed a novel isothermal exponential enzymatic amplification (IEEA) strategy for miRNAs analysis. By rational triple cascade amplification cycles of target recycling, nicking-replication reaction, and DNAzyme catalysis, the strategy exhibited high signal amplification efficiency (10(4)-10(9) folds of amplification in 1h) with very low detection background and excellent specificity. As a result, the miR-27a target model was rapidly determined with a limit of detection down to 0.79 aM (S/N=3), corresponding to 94 copies of the miRNA molecule in a 200 µL sample solution. The levels of miR-27a in atherosclerotic sprague-dawley rats were accurately quantified. The strategy is anticipated to have an important impact on the development of simple and rapid molecular diagnostic applications for any short oligonucleotides.

[Simultaneous determination of 11 constituents in Citrus reticulate 'Chachi' by high performance liquid chromatography].

An HPLC method was developed for the simultaneous determination of 11 constituents, 5-hydroxymethyl furfural (5-HMF), vicenin-2, hesperidin, hesperetin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein (TEOS), nobiletin, 3, 5, 6, 7, 8, 3', 4'-heptamethoxy- flavone (HEPTA), tangeretin, 5-demethylnobiletin in Citrus reticulate 'Chachi'. The separation was conducted on a Hanbon Benatach C18 column (250 mm x 4.6 mm, 5 µm) with acetonitrile and 0.2% formic acid as mobile phases with gradient elution. The flow rate was 1.0 mL/min. The detection wavelength was 280 nm. The column temperature was 25 °C. The results showed that the correlation coefficients (r) between concentration and chromatographic peak area of the 11 constituents were over 0.998 in the selected linear ranges. The limits of detection (LODs, S/N = 3) and limits of quantification (LOQs, S/N = 10) of the 11 constituents were in the range of 0.0125-1.25 mg/L and 0.0502-4.99 mg/L, respectively. The average recoveries (n = 3) of the 11 constituents were in the range of 96.4%-102.4% and the RSDs were 0.25%-4.01%. The developed method has been successfully applied for the analysis of eight samples from different cultivation regions in Guangdong Province. This method is simple, accurate and effective for the simultaneous determination of the 11 components, and suitable for the quality control of Citrus reticulate 'Chachi'.

Systems biochemical responses of rats to Kansui and vinegar-processed Kansui exposure by integrated metabonomics.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Kansui (Euphorbia kansui L.) is an effective and commonly used traditional Chinese medicine. Even so, Kansui cannot be satisfactorily applied clinically because of toxic side effects. In China, the most common Kansui-processing method uses vinegar to reduce its toxicity. The present study was designed to investigate the toxic effects caused by Kansui and evaluate detoxification of Kansui by vinegar processing of Kansui. MATERIALS AND METHOD: Thirty male Sprague Dawley (SD) rats were randomly assigned to five groups of six rats. Two experimental groups were oral gavaged with 7.875 and 15.75 g Kansui/kg body weight, two treated with 7.875 and 15.75 g VP-Kansui/kg body weight for 14 d, and the control group concurrently subjected to oral gavage with only distilled water. On day 14, plasma, liver and kidney tissues were collected from all rats for biochemistry assessments, histopathological examination, and NMR analyses. RESULTS: The metabonome of rats treated with Kansui and vinegar-processed (VP-) Kansui was found to differ from that of controls. In liver extracts, the variational metabolites included elevated concentrations of isoleucine, leucine, valine, glutamate, and phenylalanine, with decreased taurine, glucose, and glycogen. However, changes in lysine, methionine, choline, phosphorylcholine, and tyrosine were only observed in Kansui-treated rats. In kidney extracts, prominent changes included elevations in isoleucine, leucine, valine, methionine, creatine/creatinine, and phenylalanine as well as decreased glutamine. Only Kansui treatment induced variations in alanine, lysine, acetate, choline, and phosphorylcholine. CONCLUSION: Perturbations in endogenous metabolites induced by Kansui correlated with disturbances in glycolysis and amino acid and lipid metabolism, while biochemical pathway disorders caused by VP-Kansui only involved glycolysis and amino acid metabolism. All results were confirmed by histopathological examination of liver and kidney tissues and clinical biochemistry analyses.

Chemical fingerprint and metabolic profile analysis of Citrus reticulate 'Chachi' decoction by HPLC-PDA-IT-MS(n) and HPLC-Quadrupole-Orbitrap-MS method.

A method incorporating HPLC-PDA-IT-MS(n) with HPLC-Quadrupole-Orbitrap-MS was developed for the investigation of chemical fingerprint of Citrus reticulate 'Chachi' decoction (CRCD) and metabolic profile of SD rat plasma sample after oral administration of CRCD (1.5 g herb/kg). A total of 27 chemical constituents of CRCD were identified from their MW, UV spectra, MS(n) data and retention behavior by comparing the results with those of the reference standards or literature. And 43 compounds were detected in dosed SD rat plasma samples, including 9 prototypes which were identified as hesperetin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein, nobiletin, tetramethyl-O-scutellarein, HMF (3,5,6,7,8,3',4'-heptamethoxyflavone), tangeretin and 5-demethylnobiletin and 34 metabolites underwent metabolic process of demethylation, glucuronide conjugation, sulfate conjugation or mixed modes. This is the first research for the metabolic profile of CRCD in SD rats, which could lay a foundation for the further studies of CRC or its formulation.

1H NMR-based metabonomics study of the urinary biochemical changes in Kansui treated rat.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Kansui (Euphorbia kansui L.) is a commonly used and effective traditional Chinese medicine (TCM). AIM OF THE STUDY: We combined the urinary metabolites alteration and traditional assays of Kansui-induced rats to discuss the mechanism of toxicity of Kansui. MATERIALS AND METHODS: The Sprague-Dawley rats were dosed with 7.875g Kansui/kg weight and 15.75g Kansui/kg weight. Urine samples were collected at day -1 (before treatment), and days 7, 14 and 21 for NMR analysis. Plasma and liver and kidney tissues were collected at day 14 for biochemical assays and histopathological examination, respectively. RESULTS: The metabonome of rats treated with Kansui differed markedly from that of the controls. This was confirmed by the histopathology of liver and kidney tissue and clinical biochemistry analysis. The toxicity of Kansui accumulated with dosing time, and persisted even when treatment was stopped. The corresponding biochemical pathways alterations included inhibited TCA cycle, increased anaerobic glycolysis, and perturbed amino acids metabolism. CONCLUSION: The biochemical pathways disorder conjunction with histopathology changes provides new clues to evaluate the toxicity of Kansui from a systematic and holistic view.