Chen Dai

Rapid identification of ophiopogonins and ophiopogonones in Ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry.

This study was to develop and evaluate a practical approach of mass defect filtering (MDF), a post-acquisition data processing technique, for the rapid classification of complicated peaks into well-known chemical families based on the exact mass acquired by high resolution mass spectrometry. The full-scan LC-MS/MS data of the Ophiopogon japonicus extract was acquired using high performance liquid chromatography coupled with hybrid quadrupole-time of flight (LCMS-Q-TOF) system which features high resolution, mass accuracy, and sensitivity. To remove the interferences of the complex matrix, MDF approach was developed and employed to rapidly pick out the peaks of ophiopogonins and ophiopogonones from full-scan mass chromatograms. The accuracy of MDF was evaluated in reference to the result of structural identification. After the MDF based classification, both target and non-target components in Ophiopogon japonicus extract were characterized based on the detailed fragment ions analysis in the hybrid ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF). By this approach, more than 50 ophiopogonins and 27 ophiopogonones were structurally characterized. The present results of rapid detection and identification of ophiopogonins and ophiopogonones suggest that the proposed MDF approach based on the high-resolution mass spectrometry data would be expected adaptable to the analysis of other herbal components.

Quantitative Analysis of Neurochemical Panel in Rat Brain and Plasma by Liquid Chromatography–Tandem Mass Spectrometry

The dopamine, serotonin, and kynurenine metabolic pathways play pivotal roles on brain function, and their disturbances are closely related to various neurological diseases. Comprehensive measurements of these metabolites is thus essential for monitoring the global neurochemical responses to pathological challenges or drug intervention. However, simultaneous measurement of various neurochemcial metabolites represents a great challenge. We developed herein an original and feasible method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). A chemical derivatization approach using benzoyl chloride (benzoylation) was developed to achieve better chromatographic behavior and mass-detecting sensitivity. The developed method enables a rapid quantification of 11 metabolites spanning dopamine, serotonin, and kynurenine metabolic pathways within 10.5 min. With this method, we were able to simultaneously monitor inflammation induced alternations of all these metabolites in a rat brain and in particular their dynamics in plasma matrix. The balance between the serotonin and kynurenine branches of tryptophan metabolism was disrupted by lipopolysaccharide (LPS)-induced inflammation, characterized with the overproduction of neurotoxic metabolite 3-hydroxykynurenine and decreased levels of serotonin. The measured levels of this panel of neurotransimtters ranged from 4.3 ng to 10.6 μg per gram of brain tissue. All these results suggest that the presently developed method is sufficiently sensitive and robust to simultaneously monitor a large panel of metabolites with diverse properties and a large range of concentration differences. Therefore, this method will be expected to be highly useful for comprehensive studies of the pathophysiological roles and mechanisms of these critical neurotransmitters.

Integral pharmacokinetics of multiple lignan components in normal, CCl4-induced hepatic injury and hepatoprotective agents pretreated rats and correlations with hepatic injury biomarkers.

Although pharmacokinetic alternations by hepatic injury have been extensively studied, little is known about the potential influence of hepatoprotective agents treatment. This study was aimed to investigate the holistic pharmacokinetics of multiple lignans, CYP3A regulations, and their correlations with hepatic injury biomarkers, in hepatic injured rats pretreated with or without schisandra lignan extract (SLE) and dimethyl-diphenyl-bicarboxylate (DDB). Integral pharmacokinetics of multiple lignans based on an AUC-weighting approach was determined in normal, CCl4 induced hepatic injury rats pretreated with or without SLE and DDB. Protein expression and activities of CYP3A were determined. Pharmacokinetic parameters and CYP3A activities were correlated with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. CCl4 induced acute hepatic injury resulted in a nearly 8-fold enhancement of integral plasma exposures of multiple lignans, which was caused by the significant down-regulation of CYP3A. SLE and DDB pretreatment exhibited potent hepatoprotective effects, accompanied with the restored expression and activity of CYP3A, and the recovery of the respective and integral pharmacokinetics of lignans components. The integral AUC(0-tn) and CYP3A activities correlated well with ALT and AST. This study suggested that the pharmacokinetic regulating effects of hepatoprotective agents on themselves and co-prescribed drugs should be of concern, and hepatic injury biomarkers may serve as good predictors.

Development of a Systematic Approach to Identify Metabolites for Herbal Homologs Based on Liquid Chromatography Hybrid Ion Trap Time-of-Flight Mass Spectrometry: Gender-Related Difference in Metabolism of Schisandra Lignans in Rats

Metabolic research for herbal medicine (HM) is a formidable task, which is still in its infancy due to complicated components in HM, complex metabolic pathways, and lack of authentic standards. The present work contributes to the development of a powerful technical platform to rapidly identify and classify metabolites of herbal components based on a liquid chromatography hybrid ion trap time-of-flight mass spectrometry. Taking Schisandra lignans extract as an example, the metabolic studies were completed both in vitro and in vivo. In the in vitro study, metabolites for five representative Schisandra lignans were identified and structurally characterized. The major metabolic pathways were summed as demethylation, hydroxylation, and demethylation and hydroxylation. In the in vivo study, 44 metabolites were detected in rat urine. These metabolites were identified and classified rapidly according to the metabolic rules obtained in the in vitro studies, and hydroxylation was confirmed as the primacy metabolic pathway for lignans in rat urine. In addition, “relative cumulative excretion” (RCE) for the metabolites in female and male rats were calculated according to their relative intensities in the urine samples collected at 0 to 12, 12 to 24, and 24 to 36 h. As a result, great gender-related difference on RCE was observed. For most metabolites, RCE in female rats was significantly lower than that in male rats. In conclusion, the presently developed methodology and approach on metabolic research for Schisandra lignans will find its wide use in metabolic studies for herbal medicines.

PERIPHERAL IMMUNOMODULATION WITH GINSENOSIDE RG1 AMELIORATES NEUROINFLAMMATION-INDUCED BEHAVIORAL DEFICITS IN RATS

Neuroinflammatory disturbances have been closely associated with depression and many other neuropsychiatric diseases. Although targeting neuroinflammatory mediators with centrally acting drugs has shown certain promise, its translation is faced with several challenges especially drug delivery and safety concerns. Here, we report that neuroinflammation-induced behavioral abnormality could be effectively attenuated with immunomodulatory agents that need not to gain brain penetration. In a rat model with intracerebral lipopolysaccharide (LPS) challenge, we validated that ginsenoside Rg1 (Rg1), a well-established anti-inflammatory agent, was unable to produce a direct action in the brain. Interestingly, peripherally restricted Rg1 could effectively attenuate the weight loss, anorexic- and depressive-like behavior as well as neurochemical disturbances associated with central LPS challenge. Biochemical assay of neuroimmune mediators in the periphery revealed that Rg1 could mitigate the deregulation of the hypothalamic-pituitary-adrenal axis and selectively blunt the increase in circulating interleukin-6 levels. Furthermore, these peripheral regulatory effects were accompanied by dampened microglial activation, mitigated expression of pro-inflammatory mediators and neurotoxic species in the central compartment. Taken together, our work suggested that targeting the peripheral immune system may serve as a novel therapeutic approach to neuroinflammation-induced neuropsychiatric disorders. Moreover, our findings provided the rationale for employing peripherally active agents like Rg1 to combat mental disturbances.

Effect of mobile phase additives on qualitative and quantitative analysis of ginsenosides by liquid chromatography hybrid quadrupole-time of flight mass spectrometry.

This study was to systematically investigate the effect of mobile phase additives, including ammonia water, formic acid, acetic acid, ammonium chloride and water (as a control), on qualitative and quantitative analysis of fifteen representative ginsenosides based on liquid chromatography hybrid quadrupole-time of flight mass spectrometry (LC-Q-TOF/MS). To evaluate the influence of mobile phase additives on qualitative performance, the quality of the negative mode MS/MS spectra of ginsenosides produced by online LC-Q-TOF/MS analyses, particularly the numbers and intensities of fragment ions, were compared under different adduct ion states, and found to be strongly affected by the mobile phase additives. When 0.02% acetic acid was added in the mobile phase, the deprotonated ginsenosides ions produced the most abundant product ions, while almost no product ion was observed for the chlorinated ginsenoside ions when 0.1mM ammonium chloride was used as the mobile phase additive. On the other hand, sensitivity, linear range and precision were adopted to investigate the quantitative performance affected by different mobile phase additives. Validation results of the LC-Q-TOF/MS-based quantitative performance for ginsenosides showed that ammonium chloride not only provided the highest sensitivity for all the target analytes, but also dramatically improved the linear ranges, the intra-day and inter-day precisions comparing to the results obtained using other mobile phase additives. Importantly, the validated method, using 0.1mM ammonium chloride as the mobile phase additive, was successfully applied to the quantitative analysis of ginsenosides in rat plasma after intragastric administration of Ginsenoside Extract at 200mg/kg. In conclusion, 0.02% acetic acid was deemed to be the most suitable mobile phase additive for qualitative analysis of ginsenosides, and 0.1mM ammonium chloride in mobile phase could lead to the best quantitative performance. Our results reveal that choosing the appropriate mobile phase additive is an important step in optimizing the analytical conditions, and the best quantitative method may not be suitable for the qualitative analysis.

Influence of segmental and selected ion monitoring on quantitation of multi-component using high-pressure liquid chromatography-quadrupole mass spectrometry: Simultaneous detection of 16 saponins in rat plasma as a case.

Quadrupole (Q) mass spectrometers are the most popular analytical tools due to their reliability, effectiveness, and low cost. However, they are not suitable for quantitative analysis of multi-component since the sensitivity will get worse rapidly with the increasing number of m/z detected. The present work, for the first time, attempted to analyze of 16 saponins simultaneously using an approach of segmental and selected ion monitoring (SSIM) based on LC-Q/MS, and systematically investigated the influence of different SSIM modes on signal level/noise level (S/N), lower limits of quantification (LLOQ), upper limits of quantification (ULOQs), etc. Our results showed that a proper SSIM mode could not only provide much higher sensitivity for all the targeting analytes, but also dramatically broadened their dynamic ranges. The developed methodology could effectively break the application bottleneck on the quantitative analysis of multi-component with LC-Q/MS, and would be applied widely in related fields for multi-component analysis, such as environmental monitoring, metabonomics, Chinese herbal medicine research.

Structural identification of the metabolites for strictosamide in rats bile by an ion trap-TOF mass spectrometer and mass defect filter technique

We report herein, a facile metabolite identification workflow on the antimicrobial strictosamide, which is derived from accurate mass measurement by a hybrid ion trap-TOF mass spectrometer. In step 1, the parent drug and metabolites in rat bile were separated on an HPLC column followed by ion trap-TOF mass spectrometer analysis after a single oral dose of 50mg/kg strictosamide. In step 2, mass defect filter technique, which enables high-resolution mass spectrometers to be utilized for detecting drug metabolites based on well-defined mass defect ranges, was used to find metabolites in the mass spectrum. In step 3, the differences of accurate masses and their mass fragmentation pattern among the parent drug and metabolites used to assign structures for the metabolites successfully. As a result, five metabolites of strictosamide were found in rat bile, and all the metabolites were reported for the first time.

Study on the plasma protein binding rate of Schisandra lignans based on the LC-IT-TOF/MS technique with relative quantitative analysis

The main objective of the current study was to develop a universal method for a protein binding assay of complicated herbal components, and to investigate the possible relationship between compound polarity and protein binding using Schisadra lignans as an example. Firstly, the rat, dog and human plasma were spiked with three different concentrations of Schisandra chinensis extract (SLE), and ultramicrofiltration was used to obtain the unbound ingredients. Secondly, thirty-one Schisandra lignans in total plasma and ultrafiltered fluid were measured by LC-IT-TOFMS. Lastly, a relative exposure approach, which entailed calculating the relative concentrations of each Schisandra lignan from the corresponding calibration equation created from the calibration samples spiked with the stock solution of SLE, was applied in order to overcome the absence of authentic standards. The results showed that Schisandra lignans exhibited a high capability to bind with plasma protein, furthermore, the protein binding ratio of the lignan components increased proportionally with their individual chromatographic retention time, which indicated that the ratio of protein binding of lignans might increase accordingly with decreasing polarity. This study suggested that the compound polarity might be an important factor affecting the plasma protein binding of herbal components.

Post acquisition data processing techniques for lipid analysis by quadrupole time-of-flight mass spectrometry

This study describes the effectiveness of post-acquisition data processing techniques in detecting the lipid species rapidly from the massive data generated by high resolution mass spectrometry. The filtering approaches by product ions or neutral losses enabled glycerophospholipids and sterol conjugates to be identified based on the investigation of their fragmentation patterns, and the filtration by mass defect facilitated the detection of fatty acyl residues and bile acids by limiting the range of mass defect values. After application of these filtering techniques to mass spectra, the background noise was significantly filtered out and characteristic peaks of lipid species were efficiently sorted out. Totally 145 individual lipids were identified and structurally elucidated. Validation results of the LCMS-Q-TOF-based quantitative performance for all the peaks showed that the accuracy, expressed as relative errors (RE%), was lower than ±15%, and values (RSD%) of the inter-batch and intra-batch precision were lower than 15% in the assay. The developed method was integrated to the evaluation of plasma lipid profile from high fat diet versus energy restricted diet fed rats. A unique discrimination of the groups was successfully achieved through a principal component analysis (PCA).