Caisheng Wu

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC-HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).

Identification and characterization of active compounds and their metabolites by high-performance liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry after oral administration of a herbal extract of Epimedium koreanum Nakai to rats

Epimedium is an important traditional Chinese medicine that is widely used throughout China as a tonic, aphrodisiac, and antirheumatic medicine. Flavonoids are considered to be the active compounds in Epimedium. In the study reported here, high-performance liquid chromatography coupled with Fourier transform-ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was developed to identify active compounds and their metabolites after oral administration of a herbal extract of Epimedium koreanum Nakai to rats, using parent mass list triggered data-dependent multiple-stage accurate mass analysis at a resolving power of 100 000 in the external calibration mode. Nine flavonoids were identified in rats. The chemical formulae with unsaturation numbers calculated from accurate m/z values of precursor and product ions were used to assign the structures of metabolites and the chemical sites of metabolism. The mass accuracies obtained for all full-scan MS and MS(n) spectra were within 3 ppm (<1 ppm in most cases). The majority of the metabolites identified have been previously reported, but three compounds were noted for the first time in rats. By contrasting the analytical results obtained from the herbal extract with those obtained from biological specimens, the profile of flavonoid biotransformation in Epimedium was obtained and the metabolic pathways of these components, in rats, are described. The results should be of use in targeting potential active ingredients in Epimedium.

Use of on-line stop-flow heart-cutting two-dimensional high performance liquid chromatography for simultaneous determination of 12 major constituents in tartary buckwheat (Fagopyrum tataricum Gaertn).

The use of two-dimensional liquid chromatography (2D-LC) for quantification studies presents challenges with respect to repeatability, precision, and robustness. The present study used an on-line stop-flow heart-cutting 2D-LC system to determine 12 chemical constituents in tartary buckwheat. A combination of various stationary phases was developed and bridged using two switch valves as the interface. Hydrophilic interaction chromatography was chosen for separation in the first dimension ((1)D), and mixed mode stationary phases (an amide polar-embedded phase and alkyl-phenyl phase) were used in parallel for separation in the second dimension ((2)D). The mobile phase comprised acetonitrile and water containing 0.03% aqueous phosphoric acid. The sample was separated into two fractions on the (1)D column (HILIC-10 column) using 5% acetonitrile. One fraction, mainly comprising flavonoids, was directly eluted onto the head of (2)D column (Polar Advantage II column) and further separated using a linear gradient of 11-23% acetonitrile. The second fraction, containing phenylpropanoid glycosides, was trapped on the (1)D column. This retained fraction was back-flushed onto the (2)D column (Phenyl-1 column) and separated using a linear gradient of 35-43% acetonitrile. An on-line stop-flow heart-cutting 2D-LC system was successfully developed with column switching and back-flush. This 2D-LC system was validated and was able to simultaneously determine 12 major components in tartary buckwheat: seven flavonoids, four phenylpropanoid glycosides, and N-trans-feruloyltyramine. The system showed good performance with respect to linearity (r>0.996), repeatability (RSD, relative standard deviation<3.4%), intra-day and inter-day precision (RSD<4.6%), recovery (91.2-108%), limit of detection (LOD) (0.05-0.21μg/mL), and limit of quantification (LOQ) (0.10-0.41μg/mL). The on-line stop-flow heart-cutting 2D-LC system offers a potential approach to analyze compounds, which have similar structures but different polarities, in herbal medicines.

Sphingolipids as New Biomarkers for Assessment of Delayed-Type Hypersensitivity and Response to Triptolide

Background Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism. Methods High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation. Results As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation. Conclusions These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.

Lipidomic profiling of plasma in patients with chronic hepatitis C infection.

AbstractChronic hepatitis C virus (HCV) infection is a global health issue. Although its progression is reported to be closely associated with lipids, the way in which the plasma lipidome changes during the development of chronic HCV infection in humans is currently unknown. Using an improved quantitative high-throughput lipidomic platform, we profiled 284 lipids in human plasma samples obtained from healthy controls (n = 11) and patients with chronic HCV infection (n = 113). The intrahepatic inflammation grade (IG) of liver tissue was determined by biopsy. Two types of mass spectrometers were integrated into a single lipidomic platform with a wide dynamic range. Compared with previous methods, the performance of this method was significantly improved in terms of both the number of target sphingolipids identified and the specificity of the high-resolution mass spectrometer. As a result, 44 sphingolipids, one diacylglycerol, 43 triglycerides, 24 glycerophosphocholines, and 5 glycerophospho-ethanolamines were successfully identified and quantified. The lipid profiles of individuals with chronic HCV infection were significantly different from those of healthy individuals. Several lipids showed significant differences between mild and severe intrahepatic inflammation grades, indicating that they could be utilized as novel noninvasive indicators of intrahepatic IG. Using multivariate analysis, healthy controls could be discriminated from HCV patients based on their plasma lipidome; however, patients with different IGs were not well discriminated. Based on these results, we speculate that variations in lipid composition arise as a result of HCV infection, and are caused by HCV-related digestive system disorders rather than progression of the disease. FigureFlowchart of the lipidomic platform

Simultaneous screening and determination of 18 illegal adulterants in herbal medicines and health foods for male sexual potency by ultra‐fast liquid chromatography‐electrospray ionization tandem mass spectrometry

An ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated to simultaneously screen, confirm, and determine 18 illegal adulterants in herbal medicines and health foods for male sexual potency. The separation was achieved on a Shim-Pack XR-ODS II column (2.0 × 100 mm, 2.2 μm) with acetonitrile and aqueous solution (12 mmol/L ammonium formate, 0.01% acetic acid) as mobile phase at a flow rate of 0.4 mL/min with a gradient elution. The column temperature was maintained at 40°C and the run time was within 18 min. The 18 illegal adulterants were detected in electrospray ionization positive mode by multiple-reaction monitoring. All the calibration curves showed good linearity with correlation coefficient (r) higher than 0.996 within the tested concentration ranges. The extraction recoveries and relative recoveries were in the range of 79.5-114% and 82.0-120%, respectively. The RSD of repeatability and intermediate precision was all less than 18% and the accuracy was in the range of 81.7-118%. The intra-day and inter-day stability was in the range of 86.8-110%. The validated method was successfully applied to screen, confirm, and determine 16 samples. Nine products were confirmed to contain illegal adulterants and the contents of adulterants were related to the therapeutic dosages.

Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction.

In this study, a sensitive and specific ultra-performance liquid chromatography-tandem mass method has been developed and validated for the identification and determination of 7 flavonoids in dog plasma for the first time: epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2″-O-rhamnosyl icariside II, and baohuoside I. Chromatographic separation was accomplished on an Agilent Zorbax-SB C(18) column (50 mm × 2.1mm, 1.8 μm) with a gradient elution system composed of 0.3% acetic acid and 0.3% acetic acid in acetonitrile at a flow rate of 0.4 mL/min. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring mode with an electrospray ionization source. All of the calibration curves showed good linearity (r>0.99) within the tested concentration ranges. The lower limits of quantification of the seven analytes were all lower than 0.0654 ng/mL. The relative standard deviations for intra- and inter-batches of the seven analytes were less than 13.7% and 14.9%, respectively, at four concentration levels of quality control samples, and the recoveries were between 92.8% and 114.5%, respectively. In addition, the seven flavonoids were found to be stable in dog plasma samples under short- and long-term storage and processing conditions. The validated method was successfully applied to a bioequivalence study in dog plasma after the oral administration of extracts of Herba Epimedii and Er-Xian Decoction.

Identification of active compounds and their metabolites by high‐performance liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry from Xiao‐xu‐ming decoction (XXMD)

Xiao-xu-ming decoction (XXMD) prescription is a traditional Chinese prescription that has been widely used to treat theoplegia and the sequela of theoplegia. Modern pharmacological research has also indicated that the active fraction from XXMD is able to treat cardiovascular diseases and Alzheimers disease. In the study reported here, high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was developed to identify active compounds and their metabolites after oral administration of active fraction from Xiao-xu-ming decoction to rats, using parent mass list triggered data-dependent multiple-stage mass analysis at a resolving power of 100,000 in the external calibration mode. The mass accuracies obtained for full-scan MS were within 2 ppm in most cases. Fifteen constituents were identified in the active fraction from XXMD and the biological samples of rats. The fragmentation behaviors of these constituents were summarized which would be helpful for structural characterization. The profiles of the constituents in the active fraction and biological samples of rats were obtained which provided us with much information for a better understanding of the chemical basis of the pharmacologic actions of XXMD.

Identification and characterization of flavonoids from semen zizyphi spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry.

Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS n (HPLC-PDA/LTQ-MS n ) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C18 column by gradient elution using CH3CN/H2O–CH3COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.

Identification of the metabolites after oral administration of extract of ziziphi spinosae semen to rats or dogs by high‐performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry

Ziziphi spinosae semen (ZSS) is a traditional Chinese medicine that has been widely used to treat insomnia and anxiety. Modern pharmacological studies have demonstrated that flavonoids are the main active compounds in ZSS. However, the metabolites and the metabolic pathways of flavonoids in ZSS have not been investigated thoroughly. In this study, a method based on high-performance liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS) was established to identify the metabolites of flavonoids after oral administration of extract of ZSS to rats or dogs, using parent mass list-triggered data-dependent multiple-stage mass analysis at a resolving power of 50,000 in the external calibration mode. The mass accuracies obtained for all full-scan analyses were less than 4 ppm (<2 ppm in most cases). A total of 15 compounds were detected in biological samples of rats and dogs, and nine compounds were identified. The metabolic pathways of flavonoids of ZSS in rats and dogs were proposed. The results may help better understand the material basis and pharmacological mechanism of ZSS.