Shaohua Zhao

Identification of metabolites of deoxyschizandrin in rats by UPLC-Q-TOF-MS/MS based on multiple mass defect filter data acquisition and multiple data processing techniques.

Deoxyschizandrin is an active lignin ingredient originating from Schisandra chinensis (Turcz.) Baill or Schisandrae Sphenantherae Fructus. In the present study, a novel and efficient strategy was developed for the in vivo screening and identification of deoxyschizandrin metabolites using ultra high performance liquid chromatography combined with triple TOF mass spectrometry (UPLC-TOF/MS/MS). This strategy was characterized by the following: a novel and unique multiple mass defect filter (MMDF) combined with an on-line data acquisition method that is dependent on dynamic background subtraction (DBS) was developed to trace all of the probable metabolites of deoxyschizandrin. The MMDF and DBS methods could trigger an IDA scan for the low-level metabolites that are masked by background noise and endogenous components. A combination of data processing methods including extracted ion chromatography (XIC), mass defect filtering (MDF), product ion filtering (PIF) and neutral loss filtering (NLF) were employed to identify the metabolites of deoxyschizandrin. Next, the structures of the metabolites were elucidated based on an accurate mass measurement, the fragmentation patterns of the parent drug and relevant drug bio-transformation knowledge. Finally, an important parameter ClogP was used to estimate the retention time of isomers. Based on the proposed strategy, 51 metabolites (including 49 phase I and 2 phase II metabolites) were identified in rats after the oral administration of deoxyschizandrin. Among these metabolites, 41 metabolites were characterized in the rat urine, and 28 metabolites were identified in the rat bile. The results indicated that oxidization was the main metabolic pathway and that the methoxy group and the biphenyl cyclooctene were the metabolic sites. Conjugation with sulfate and cysteine groups produced two phase-II metabolites. This study firstly reported the description of deoxyschizandrin metabolism in vivo. This study provided a practical strategy for rapidly screening and identifying metabolites, and this methodology can be widely applied for the structural characterization of the metabolites of other compounds.

Tentative identification of new metabolites of epimedin C by liquid chromatography–mass spectrometry

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze-dried powder.

A simple and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze-dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C18 column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion-pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra- and inter-day RSDs were all less than 10% with the relative error (RE) within ± 9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze-dried powder.

Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC-MS/MS

A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of 10 major active constituents in rat urine after oral administration of Shensong Yangxin Capsule (SSYX) using diazepam as an internal standard (IS). The urine samples were pretreated and extracted by solid-phase extraction prior to UPLC. Chromatographic separation was achieved on a Waters C18 (2.1 × 50 mm, 1.7 µm) column using a gradient elution program with 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min. Detection and quantitation were accomplished by a hybrid quadrupole mass spectrometer using electrospray ionization source and multiple reaction monitoring in the positive ionization mode. The mass transition ion-pairs (m/z) for quantitation were all optimized and the total run time was 4.50 min. The specificity, linearity, accuracy, precision, recovery, matrix effect and stabilities were all validated for the analytes in urine samples. The validation results indicated that this method was simple, rapid, specific and reliable. The proposed method was successfully applied to investigate the urinary excretion kinetics of 10 compounds in rat after oral administration of SSYX.

Identification of Multiple Constituents in Chinese Medicinal Prescription Shensong Yangxin Capsule by Ultra-Fast Liquid Chromatography Combined with Quadrupole Time-of-Flight Mass Spectrometry

A practical method using ultra-fast liquid chromatography in tandem with quadrupole time-of-flight mass spectrometry combined with dynamic background subtraction technology was developed for the rapid separation and identification of the complicated constituents in the Shensong Yangxin capsule (SSYX). The chromatographic separation was performed on a C18 column (2.1 × 100 mm, 2.6 μm) with a gradient elution program using methanol and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL min(-1). Accurate mass measurements of the molecular ions in the full scan and the characteristic fragment ions triggered by information-dependent acquisition provided reliable identification criteria. Thus, 99 compounds, including saponins, phenolic acids, tanshinones, lignans, terpenoids, alkaloids and flavonoids, were unambiguously or tentatively identified in 40 min by comparing their retention times and accurate mass measurements for each molecular ion and its subsequent fragment ions with those of authentic standards or literature data. Simultaneously, all the compounds were further assigned to the individual raw materials. In conclusion, these results will provide a basis for quality control and further study of SSYX, and the proposed technique based on high-resolution mass spectrometry would be expected to be adaptable to the analysis of complicated constituents in various complex matrices.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF 15 ACTIVE CONSTITUENTS IN JIWEILING FREEZE-DRIED POWDER BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Qualitative characterization and quantitative analysis of 15 bioactive constituents in Jiweiling freeze-dried powder (JWL) have been achieved by reversed-phase high performance liquid chromatography coupled with tandem mass spectrometry, including five flavonoids and ten ginsenosides. Chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18 HPLC column, with gradient elution of 0.1% formic acid aqueous solution and acetonitrile as mobile phase. The compounds were detected in the negative ion mode with multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer equipped with an electrospray ionization interface. By using of this method, 15 bioactive components were unequivocally identified on the basis of their characteristic fragmentation patterns. All calibration curves showed good linearity (r2 > 0.998) within the test ranges. The LOD, LOQ, specificity, precision, and accuracy for the method were validated. The proposed method was successfully applied to analyze the quality of 12 batches of JWL. The results indicated that this analytical method was simple and suitable for the identification and quality assessment of JWL.

Total synthesis of epiberberine.

Epiberberine, a natural bioactive protoberberine alkaloid, was totally synthesized in short, convenient and low-cost, four-step reactions including cyclization, condensation, reduction, and ring-closing, with an overall yield of 26.1%.