Chronic heart failure (CHF) is an ongoing clinical syndrome with cardiac dysfunction that can be traced to alterations in cardiac metabolism. The identification of metabolic biomarkers in easily accessible fluids to improve the early diagnosis of CHF has been elusive to date. In this study, we took multidimensional analytical techniques to discover potentially new diagnostic biomarkers by focusing on the dynamic changes of metabolites in serum during the progression of CHF. Using mass-spectrometry-based untargeted metabolomics, we identified 23 cardiac metabolites that were altered in a rat model of myocardial infarction induced CHF. Among these differential metabolites, branched-chain amino acids (BCAAs) in serum, especially leucine and valine, showed a high capability to differentiate between CHF and sham-operated rats, of which area under the receiver operating characteristic curve was greater than 0.75. Combining with targeted analysis of the amino acids and related proteins and genes, we confirmed that BCAA metabolic pathway was significantly inhibited in rat failing hearts. On the basis of the time series data of serum samples, we characterized the fluctuation pattern of circulating BCAAs by the disease progression model. Finally, the time-resolved diagnostic potential of serum BCAAs was evaluated by the machine-learning-based classifier, and high diagnostic accuracy of 93.75% was achieved within 3 weeks after surgery. These findings provide a promising metabolic signature that can be further exploited for CHF early diagnostic development.

Radix Bupleuri is a hepatoprotective traditional Chinese medicine (TCM) used for thousands of years in clinical, which was reported to be linked with liver damage. Previous studies have revealed that saikosaponins are the major types of components that contribute to the hepatotoxicity of Radix Bupleuri. However the underlying molecular mechanism is far from being understood. In order to clarify whether these structural analogues exert toxicity effects through the same molecular targets, a systematic comparison study was done in this paper. The effects of SSa, b2, c, and d on isolated rat liver mitochondria and human hepatocyte L02 cells were explored, respectively. The collective results indicated that although saikosaponins share the similar structures but they have quite different mechanisms. SSb2 and SSd showed most serious damage on the function of mitochondria and survival rate of cell, respectively. SSb2 could cause mitochondrial permeability transition pore (mPTP) opening and collapse of mitochondrial membrane potential (ΔΨm) by impairing the mitochondrial respiratory chain complex III. While SSd destroyed plasma membrane and led to the release of lactate dehydrogenase (LDH) mainly through activating caspase-1. Furthermore, the combine index (CI) demonstrated that the combined hepatotoxicity of SSb2 and SSd could be additive. This finding might yield more in depth understanding of hepatotoxicity of Radix Bupleuri possess many different saikosaponins.

Stroke remains a major public health problem worldwide; it causes severe disability and is associated with high mortality rates. However, early diagnosis of stroke is difficult, and no reliable biomarkers are currently established. In this study, mass-spectrometry-based metabolomics was utilized to characterize the metabolic features of the serum of patients with acute ischemic stroke (AIS) to identify novel sensitive biomarkers for diagnosis and progression. First, global metabolic profiling was performed on a training set of 80 human serum samples (40 cases and 40 controls). The metabolic profiling identified significant alterations in a series of 26 metabolites with related metabolic pathways involving amino acid, fatty acid, phospholipid, and choline metabolism. Subsequently, multiple algorithms were run on a test set consisting of 49 serum samples (26 cases and 23 controls) to develop different classifiers for verifying and evaluating potential biomarkers. Finally, a panel of five differential metabolites, including serine, isoleucine, betaine, PC(5:0/5:0), and LysoPE(18:2), exhibited potential to differentiate AIS samples from healthy control samples, with area under the receiver operating characteristic curve values of 0.988 and 0.971 in the training and test sets, respectively. These findings provided insights for the development of new diagnostic tests and therapeutic approaches for AIS.

Ten limonoids were isolated from the root bark ofDictamnus angustifolius. Their metal chelating properties, the abilities to halt copper redox cycling, cell viabilities and neuroprotection studies against oxidative stress were evaluated.

Phytochemical investigation of the root bark of Dictamnus angustifolius G. Don ex Sweet (Rutaceae) resulted in the isolation and characterization of three degraded limonoids (1–3), five limonoids (4–8) and four triterpenoids (9–12). All structures were elucidated on the basis of spectral analysis. Among them, compounds 4, 6, 8, 9 and 12 were isolated from the genus Dictamnus for the first time. Compounds 10 and 11 were obtained from D. angustifolius for the first time. Moreover, the chemotaxonomic significance of these compounds was summarized.

Crude rhubarb subjected to different processing procedures will produce different therapeutic effects that are possibly due to processing-induced variation in chemical composition. In this study, a chemometric approach based on ultra fast liquid chromatography with ion trap/time-of-flight mass spectrometry was established to systematically investigate the chemical variations of rhubarb induced by different processing methods. The approach was validated based on pooled quality-control samples from two perspectives: the individual properties of variables and the bulk properties of samples. Orthogonal partial least squares discriminant analysis was introduced to compare the differences between crude and processed rhubarb products. A total of 20 significantly different markers were screened out and unambiguously/tentatively characterized. This research proved that a chemometric method based on ultra fast liquid chromatography with ion trap/time-of-flight mass spectrometry can comprehensively analyze the chemical variation of herbal medicine and provide evidence for a deeper understanding of the pharmacological activities of processed rhubarb products.

A sensitive and simple liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol-0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both <9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC-MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration.

As a specific item mentioned in traditional Chinese medicine theory, processing can fulfill different requirements of therapies. Crude and wine-processed rhubarbs are used as drastic and mild laxatives, respectively. In this study, a practical method based on ultra-fast liquid chromatography coupled with diode-array detection and ion trap time-of-flight mass spectrometry was developed to screen and analyze multiple absorbed bioactive components and metabolites in the serum of both normal and acute blood stasis rats after oral administration of crude or wine-processed rhubarbs. A total of 16 compounds, mainly including phase II metabolites, were tentatively identified. Possible explanations for the processing-induced changes in pharmacological effects of traditional Chinese medicines were first explored at serum pharmacochemistry level.

BACKGROUND:Brucine is a widely prescribed glycine antagonist, but a complete understanding of its metabolic pathway is still lacking. The present work represents the first investigation of in vivo metabolism of brucine in rats using LC-ESI-ion trap-TOF-MS.RESULTS:A total of 12 Phase I and five Phase II metabolites were tentatively identified. Brucine can be metabolized by hydrolysis, demethylation and methoxylation, in addition to diverse oxidations in a Phase I manner followed by glucuronidation in Phase II metabolism. Both the renal and biliary routes were observed for the excretion of brucine and its metabolites.CONCLUSION:Our results update the metabolism and disposition data on brucine, which provides basic information for better understanding of the pharmacological and toxicological activities of brucine-containing medicines.

Metabolomics refers to the comprehensive analysis of metabolites in biological systems, and has been employed to study patients with asthma based on their urinary metabolite profile. We hypothesize that airway allergic asthma would affect metabolism in the lungs, and could be detected in bronchoalveolar lavage (BAL) fluid (BALF) using a combined liquid chromatography- and gas chromatography-mass spectrometry (MS) platform. The objective of this study was to investigate changes of lung metabolism in allergic asthma by metabolomic analysis of BALF. BALB/c mice were sensitized and challenged with ovalbumin to develop experimental asthma. Dexamethasone was administered to study the effects of corticosteroids on lung metabolism. Metabolites in BALF were measured using liquid chromatography-MS and gas chromatography-MS, and multivariate statistical analysis was performed by orthogonal projections to latent structures discriminant analysis. Metabolomic analysis of BALF from ovalbumin-challenged mice revealed novel changes in metabolic pathways in the lungs as compared with control animals. These metabolite changes suggest alterations of energy metabolism in asthmatic lungs, with increases of lactate, malate, and creatinine and reductions in carbohydrates, such as mannose, galactose, and arabinose. Lipid and sterol metabolism were affected with significant decreases in phosphatidylcholines, diglycerides, triglycerides, cholesterol, cortol, and cholic acid. Dexamethasone treatment effectively reversed many key metabolite changes, but was ineffective in repressing lactate, malate, and creatinine, and induced additional metabolite changes. Metabolomic analysis of BALF offers a promising approach to investigating allergic asthma. Our overall findings revealed considerable pathway changes in lung metabolism in asthmatic lungs, including energy, amino acids, and lipid metabolism.

The pharmacokinetics and relative bioavailability/bioequivalence of two formulations of cetirizine hydrochloride (CAS 83881-51-0) were assessed in this paper. Using a two-treatment, two-period, two-sequence, randomized crossover design, test and reference formulations were administered as individual single doses to 18 Chinese healthy male volunteers under non-fed conditions, with a 7-day washout period between dosing. Fourteen blood samples were drawn from each subject over a 34-h period. Cetirizine concentrations in plasma were determined by a validated high performance liquid chromatographic-ultraviolet (HPLC/UV) assay, and pharmacokinetic parameters, C-max, AUC(0-t), AUC(0-infinity) and t(1/2) were calculated from the plasma concentration-time data of each individual and during each period by applying noncompartmental analysis. Pharmacokinetic data for test and reference formulations were analyzed statistically to test for bioequivalence of the two formulations. After oral administration the values of C-max, t(max), t(1/2), MRT, AUC(0-0) AUC(0-infinity) for test and reference formulations were 0.80 +/- 0.14 and 0.80 +/- 0.23 mu g/ml, 0.8 +/- 0.4 and 1.1 +/- 0.7 h, 7.59 +/- 0.68 and 7.63 +/- 0.93 h, 10.59 +/- 0.94 and 10.92 +/- 1.26 h, 6.00 +/- 1.04 and 5.98 +/- 1.39 mu g . h/ml, respectively. ANOVA and Cl test showed no significant (p > 0.05) variation in these pharmacokinetic parameters of test and reference formulations. When the AUC(0-t) values for both formulations for natural log-transformed data were compared, the test formulation showed a bioavailability of 100.9 +/- 7.7%, as compared to the reference formulation. These values are within the acceptance limit of 80 - 125%. No adverse events were observed in any of the subjects during the two runs of the study. Both clinical and laboratory parameters of all subjects showed no clinically significant changes. This study shows that both formulations were well tolerated and the test formulation can be considered a pharmaceutically and therapeutically equivalent alternative to the reference formulation.

The integration of liquid chromatography-mass spectrometry (LC-MS) with derivatization is a relatively new and unique strategy that could add value and could enhance the capabilities of LC-MS-based technologies. The derivatization process could be carried out in various analytical steps, for example, sampling, storage, sample preparation, HPLC separation, and MS detection. This review presents an overview of derivatization-based LC-MS strategy over the past 10 years and covers both the general principles and applications in the fields of pharmaceutical and biomedical analysis, biomarker and metabolomic research, environmental analysis, and food-safety evaluation. The underlying mechanisms and theories for derivative reagent selection are summarized and highlighted to guide future studies.

Liquid chromatography–electrospray ionization tandem mass spectrometry has been used for rapid, selective, and sensitive quantitative analysis of gallic acid in rat plasma. Sample pretreatment involved a one-step extraction using ethyl acetate with protocatechic acid as internal standard. Separation was on a C18 column using an isocratic mobile phase, consisting of methanol-0.1% aqueous formic acid (40:60, v/v) at 0.2 mL min−1. The stability of gallic acid was evaluated in acidified and non-acidified plasma. The method was validated then successfully applied to a pharmacokinetic study in rats after oral administration of rhubarb extract.

Differences in the contents of sixteen bioactive components (three tannins, five anthraquinones, six flavonoids and two neolignans) between Da-Cheng-Qi decoction (DCQD) and its three constitutional herbal medicines (Radix et Rhizoma Rhei, Cortex Magnoliae officinalis, and Fructus Aurantii Immaturus) were compared using validated HPLC/DAD methods. The results indicated that there existed some kinds of interactions between these constitutional natural medicines during the DCQD preparation procedure, which could either enhance or depress the extractive rates of bioactive components.

Da-Cheng-Qi decoction (DCQD) is a purgative prescription used in China and East Asia. To profile the constituents of this complex traditional Chinese medicine (TCM), a high-performance liquid chromatographic, electrospray ionization, tandem mass spectrometric (HPLC-ESI/MS/MS) analytical method was developed. After separation on a reversed-phase C18 analytical column using gradient elution, samples were analyzed by ESI-MS/MS in negative mode. As a result, a total of 37 compounds were detected, of which two tannins, three anthraquinones, two sennosides, five flavonoids and two lignans were unambiguously identified by comparison with standard compounds, and sixteen compounds were either tentatively identified or deduced according to their MS/MS data. The fragmentation pathways of many of the observed compounds, such as the tannins and lignans are reported for the first time. In addition, the identity of each peak in DCQD was explored by comparison with those of its three constituent herbs. The results indicated that tannins, anthraquinones and sennosides in DCQD originated from Radix et Rhizoma Rhei, flavonoids from Fructus Aurantii Immaturus, and lignans from Cortex Magnoliae officinalis. The present study provides an example of chemical constitution profiling in complex TCM systems using LC/MS/MS.

The aim is to develop a simple and specific method for the extraction and chemical fingerprinting of Andrographis paniculata (Burm. f.) Nees and to apply the method to this drug from different regions. High-performance liquid chromatographic (HPLC) with gradient elution is used for developing the fingerprints, and liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) technique is employed to identify the component of the fingerprints. Nine peaks are selected as common peaks, and six compounds are elucidated by MS data. Twenty-three samples of A. paniculata from different regions of China are collected and detected by HPLC fingerprinting. Comparisons of the chromatograms show that there are obvious differences in the content of each component contained between the habitat samples in China. The results of hierarchical cluster analysis show that these samples can be clustered reasonably into three groups, and the growth of A. paniculata and their internal quality are related to their habitat. The HPLC fingerprint developed allows simple identification of A. paniculata from many natural drugs.

A rapid, sensitive and specific liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) method has been developed and validated for simultaneous determination of five active constituents (including magnolol, honokiol, rhein, emodin and aloe-emodin) from Da-Cheng-Qi decoction (DCQD) in rat plasma. After the addition of gliquidone as the internal standard (IS), plasma samples were prepared by one-step protein precipitation using methanol and separated by HPLC on a short reversed phase C18 column packed with smaller particles (100mm×3.0mm, 3.5μm) using a mobile phase of methanol–0.1% formic acid aqueous solution (70:30, v/v). Analytes were determined in a triple-quadrupole mass spectrometer in the selected reaction-monitoring (SRM) mode using electrospray source with negative mode. The method was proved to be rapid, sensitive, specific, accurate and reproducible and has been successfully applied to the determination of the five compounds in rat plasma after oral administration of low dose DCQD for pharmacokinetic study.

To quantify naringenin and hesperetin in rat plasma after oral administration of Da-Cheng-Qi decoction, a famous purgative traditional Chinese medicine, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated. The HPLC separation was carried out on a Zorbax SB-C(18) column using 0.1% formic acid-methanol as mobile phase and estazolam as internal standard after the sample of rat plasma had been cleaned up with one-step protein precipitation using methanol. Atmospheric pressure chemical ionization in the positive ion mode and selected reaction monitoring method was developed to determine the active components. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-batch variation). The recoveries of naringenin and hesperetin were 72.8-76.6 and 75.7-77.2%, respectively. Linearity in rat plasma was observed over the range of 0.5-250 ng/mL (r2 > 0.99) for both naringenin and hesperetin. The accuracy and precision were well within the acceptable range and the relative standard deviation of the measured rat plasma samples was less than 15% (n = 5). The validated method was successfully applied for the evaluation of the pharmacokinetics of naringenin and hesperetin administered to six rats.