Zhanying Hong

NMR and LC/MS-based global metabolomics to identify serum biomarkers differentiating hepatocellular carcinoma from liver cirrhosis

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. However, current biomarkers that discriminate HCC from liver cirrhosis (LC) are important but are limited. More reliable biomarkers for HCC diagnosis are therefore needed. Serum from HCC patients, LC patients and healthy volunteers were analyzed using NMR and LC/MS‐based approach in conjunction with random forest (RF) analysis to discriminate their serum metabolic profiles. Thirty‐two potential biomarkers have been identified, and the feasibility of using these biomarkers for the diagnosis of HCC was evaluated, where 100% sensitivity was achieved in detecting HCC patients even with AFP values lower than 20 ng/mL. The metabolic alterations induced by HCC showed perturbations in synthesis of ketone bodies, citrate cycle, phospholipid metabolism, sphingolipid metabolism, fatty acid oxidation, amino acid catabolism and bile acid metabolism in HCC patients. Our results suggested that these potential biomarkers identified appeared to have diagnostic and/or prognostic values for HCC, which deserve to be further investigated. In addition, it also suggested that RF is a classification algorithm well suited for selection of biologically relevant features in metabolomics.

Comparative normal/failing rat myocardium cell membrane chromatographic analysis system for screening specific components that counteract doxorubicin-induced heart failure from Acontium carmichaeli.

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K+ channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.

Simultaneous determination of rupatadine and its metabolite desloratadine in human plasma by a sensitive LC–MS/MS method: Application to the pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of rupatadine and its metabolite desloratadine in human plasma. After the addition of diphenhydramine, the internal standard (IS), plasma samples were extracted with a mixture of methyl tert-butyl ether and n-hexane (1:1, v/v). The analysis was performed on a Ultimate AQ-C18 (4.6mm x 100mm, 5microm) column using a mobile phase consisting of a 80/20 mixture of methanol/water containing 0.0005% formic acid pumped at 0.3mlmin(-1). The analytes and the IS were detected in positive ionization mode and monitoring their precursor-->product ion combinations of m/z 416-->309, 311-->259, and 256-->167, respectively, in multiple reaction monitoring mode. The linear ranges of the assay were 0.1-50 and 0.1-20ngml(-1) for rupatadine and desloratadine, respectively. The lower limits of reliable quantification for both rupatadine and desloratadine were 0.1ngml(-1), which offered high sensitivity and selectivity. The within- and between-run precision was less than 7.2%. The accuracy ranged from -9.2% to +6.4% and -7.2% to +7.2% for rupatadine and desloratadine in quality control samples at three levels, respectively. The method has been successfully applied to a pharmacokinetic study of rupatadine and its major metabolite after oral administration of 10, 20 and 40mg rupatadine tablets to healthy Chinese volunteers.

An ultra-fast LC method for the determination of iodiconazole in microdialysis samples and its application in the calibration of laboratory-made linear probes

Iodiconazole is a very potent antifungal agent used to treat serious fungal infections. After transdermal administration, several factors affect the exposure of iodiconazole, resulting in large variability and demanding further elucidation of drug distribution. For determination of iodiconazole in dermal microdialysate, a new, efficient, reliable and robust ultra-fast liquid chromatography (UFLC, Shimadzu) assay using UV detection at 230 nm has been developed and validated. Iodiconazole was separated on a Shimadzu Prominence UFLC C18 column (2.2 microm, 50 mm x 2.0 mm i.d.) using acetonitrile-0.025% triethylamine solution, adjusted to pH 3.6 with phosphoric acid (65:35, v/v), at a flow rate of 0.5 ml/min. The retention time was 1.37 min for iodiconazole and 1.78 min for the internal standard, an isomeric compound of iodiconazole. Intra- and inter-day precision ranged from 5.3% to 7.8% and 3.7% to 8.4%, respectively. The UFLC method was used to measure iodiconazole concentrations in microdialysis samples obtained during the calibration of laboratory-made linear probes. The validation and sample analysis results show that the method is precise, accurate and well suited to support the dermal microdialysis experiments.

Study on the stereoselective excretion of tetrahydropalmatine enantiomers in rats and identification of in vivo metabolites by liquid chromatography-tandem mass spectrometry

The objective of this work was to study the stereoselectivity in excretion of tetrahydropalmatine (THP) enantiomers by rats and identify the metabolites of racemic THP (rac-THP) in rat urine. Urine and bile samples were collected at various time intervals after a single oral dose of rac-THP. The concentrations of THP enantiomers in rat urine and bile were determined using a modification of an achiral-chiral high-performance liquid chromatographic (HPLC) method that had been previously published. The cumulative urinary excretion over 96 h of (-)-THP and (+)-THP was found to be 55.49 +/- 36.9 microg and 18.33 +/- 9.7 microg, respectively. The cumulative biliary excretion over 24 h of (-)-THP and (+)-THP was 19.19 +/- 14.6 microg and 12.53 +/- 10.4 microg, respectively. The enantiomeric (-/+) concentration ratios of THP changed from 2.80 to 5.15 in urine, and from 1.36 to 1.80 in bile. The mean cumulative amount of (-)-THP was significantly higher than that of (+)-THP both in urine and bile samples. However, the enantiomeric (-/+) concentration ratios in rat urine and bile were significantly lower than those ratios in rat plasma. These findings suggested the excretion of THP enantiomers was stereoselective rather than a reflection of chiral pharmacokinetic aspects in plasma and (-)-THP was preferentially excreted in rat urine and bile. Three O-demethylation metabolites and the parent drug rac-THP were detected by liquid chromatography-tandem mass spectrometry in rat urine. One metabolite was obtained by preparative HPLC and identified as 10-O-demethyl-THP.

A practical strategy for characterization of the metabolic profile of chiral drugs using combinatory liquid chromatography–mass spectrometric techniques: Application to tetrahydropalmatine enantiomers and their metabolites in rat urine

The characterization and quantification of the metabolites of chiral drugs still remain a great challenge due to the complexity of the metabolites and most of them are not commercially available. In this study, a practical approach based on the combinatory liquid chromatography-mass spectrometric techniques has been proposed for the evaluation of metabolism profiles and urinary excretion kinetics of chiral drugs and their metabolites. Racemic tetrahydropalmatine (rac-THP), a biologically active ingredient isolated from a traditional Chinese herb Rhizoma Corydalis, was chosen as the model chiral drug. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to characterize the metabolites of THP enantiomers in rat urine after administration of (+)-THP or (-)-THP. Accurate mass measurement was used to determine the elemental composition of metabolites and thus to confirm the proposed structures of these metabolites. More than 30 potential metabolites were found in rat urine, most of which were identified for the first time, and the metabolic pathways in vivo were involved in demethylation, oxidation, glucuronide conjugation and sulfation, etc. And the tridesmethlyzed metabolite and didesmethlyzed coupled with oxidation metabolite were found only in (+)-THP treated rats. Afterwards, a liquid chromatography tandem mass spectrometry (LC-QqQ/MS) assay was developed and validated for the determination of the urine level of THP enantiomers and their metabolites. Semi-quantification of three phase I metabolites and two phase II metabolites were performed. Enantiomeric (-/+) cumulative urinary excretion ratios of THP and its five metabolites were obtained, which indicated the stereoselective aspects of metabolites of THP enantiomers in vivo. The study demonstrated the enormous potential of this strategy for the qualitative characterization, quantitative assay and the stereoselectivity of chiral drugs and their metabolites.

Innovative microwave-assisted oximation and silylation procedures for metabolomic analysis of plasma samples using gas chromatography–mass spectrometry

Analysis of plasma metabolomic samples by gas chromatography-mass spectrometry always requires comprehensive pretreatment including oximation and silylation. Although heating block (HB) is a commonly used method, it is time consuming. This study describes an extremely time-effective microwave-assisted (MA) oximation and silylation approach for metabolomic study of plasma samples. The Box-Behnken design was employed to optimize the MA conditions by means of oximation at 65 W for 100 s and then silylation through 180 s incubation with 230 W microwave irradiation. The results showed that microwave irradiation decreased the sample preparation time from approximately 180 min to 5 min without loss of information for the metabolites in plasma samples. Both the HB method and the developed MA method were applied in plasma metabolomic study of sulfur mustard intoxication. Partial least-squares discriminant analysis (PLS-DA) was used to globally understand the metabolic changes, and multi-criteria assessment was used to select the most significant and reliable variables as potential biomarkers. The data obtained by the MA method were in good correlation with the HB method. Compared with HB method, the newly developed MA oximation and silylation of plasma metabolome samples was more efficient and time-effective and may prove to be an attractive alternative for high-throughput sample preparation in plasma metabolomics.

Quality improvements of cell membrane chromatographic column

Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using a silica stationary phase covered with specific cell membrane. However, its short life span and poor quality control was highlighted in a lot of research articles. In this study, special attention has been paid to the disruption, cell load and packing procedure in order to improve the quality of the CMC columns. Hereto, two newly established CMC models, HSC-T6/CMC and SMMC-7721/CMC have been developed and used in this research project. The optimization of the abovementioned parameters resulted in a better reproducibility of the retention time of the compound GFT (RSD<10%) and improved significantly the quality of the CMC columns. 3.5×10(7)cells were the optimal cell load for the preparation of the CMC columns, the disruption condition was optimized to 5 cycles (400W and 20s interval per cycle) by an ultrasonic processor reducing the total time of cell disruption to 1.5min and the packing flow rate was optimized by applying a linear gradient program. Additionally, 4% paraformaldehyde (PFA) was employed to improve the column quality and prolong the column life span. The results showed that the retention time was longer with PFA treated columns than the ones obtained with the control groups.

Simultaneous determination of tetrahydropalmatine and tetrahydroberberine in rat urine using dispersive liquid–liquid microextraction coupled with high‐performance liquid chromatography

Dispersive liquid-liquid microextraction (DLLME) coupled with high-performance liquid chromatography (HPLC)-UV detection was applied in rat urine for the extraction and determination of tetrahydropalmatine (THP) and tetrahydroberberine (THB), both active components in Rhizoma corydalis. Various parameters affecting the extraction efficiency, such as the type and volume of extraction and dispersive solvent, pH, etc. were evaluated. Under the optimal conditions (extraction solvent: 37 μL of chloroform, dispersive solvent: 100 μL of methanol, alkaline with 100 μL of 1 mol/L NaOH, and without salt addition), the enrichment factors of THP and THB were more than 30. The extraction recoveries were 69.8-75.8% and 72.7-77.6% for THP and THB in rat urine, respectively. Both THP and THB showed good linearity in the range of 0.025-2.5 μg/mL, and the limit of quantification was 0.025 μg/mL (S/N=10, n=6). The intra-day and inter-day precision of THP and THB were <12.6%. The relative recoveries ranged from 95.5 to 107.4% and 96.8 to 100.9% for THP and THB in rat urine, respectively. The method has been successfully applied to rat urine samples. The results demonstrated that DLLME is a very simple, rapid and efficient method for the extraction and preconcentration of THP and THB from urine samples.

Investigation of the therapeutic effectiveness of active components in Sini decoction by a comprehensive GC/LC-MS based metabolomics and network pharmacology approaches

As a classical formula, Sini decoction (SND) has been fully proved to be clinically effective in treating doxorubicin (DOX)-induced cardiomyopathy. Current chemomics and pharmacology proved that the total alkaloids (TA), total gingerols (TG), total flavones and total saponins (TFS) are the major active ingredients of Aconitum carmichaelii, Zingiber officinale and Glycyrrhiza uralensis in SND respectively. Our animal experiments in this study demonstrated that the above active ingredients (TAGFS) were more effective than formulas formed by any one or two of the three individual components and nearly the same as SND. However, very little is known about the action mechanisms of TAGFS. Thus, this study aimed to use for the first time the combination of GC/LC-MS based metabolomics and network pharmacology for solving this problem. By metabolomics, it was found that TAGFS worked by regulating six primary pathways. Then, network pharmacology was applied to search for specific targets. 17 potential cardiovascular related targets were found through molecular docking, 11 of which were identified by references, which demonstrated the therapeutic effectiveness of TAGFS using network pharmacology. Among these targets, four targets, including phosphoinositide 3-kinase gamma, insulin receptor, ornithine aminotransferase and glucokinase, were involved in the TAGFS regulated pathways. Moreover, phosphoinositide 3-kinase gamma, insulin receptor and glucokinase were proved to be targets of active components in SND. In addition, our data indicated TA as the principal ingredient in the SND formula, whereas TG and TFS served as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae with the combination use of metabolomics and network pharmacology may be a good strategy.