Erxin Shang

Rapid Determination of Amino Acids in Fruits of Ziziphus jujubaby Hydrophilic Interaction Ultra-High-Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry

In this study, a sensitive and rapid method for the simultaneous determination of free amino acids without derivatization using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) was developed. The method was performed on an ultra-high-performance liquid chromatography (UHPLC) separation system coupled with a triple-quadrupole mass spectrometry (TQ-MS) instrument. Sufficient separation of 23 underivatized amino acids was achieved on an Acquity BEH Amide column (2.1 mm × 100 mm, 1.7 μm) in a single run of 12 min. Then the method was applied for the analysis of the free amino acids in 46 batches of Ziziphus jujuba fruits which comprised 39 cultivars from 26 cultivation regions. Multivariate statistical analysis was also used to investigate the differences in free amino acid profiles among the samples. This study showed that HILIC-UHPLC-TQ-MS is an effective technique to analyze underivatized amino acids in the food samples.

Characterization of Triterpenic Acids in Fruits of Ziziphus Species by HPLC-ELSD-MS

The fruits of Ziziphus species have been utilized as food as well as crude drugs for their health benefits in China for thousands of years. This paper reported a reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous characterization and quantitation of 11 triterpenic acids in chloroform extracts of jujube fruits by using an evaporative light scattering detector (ELSD) and electrospray ionization-mass spectrometry (ESI-MS). The results showed that the contents of triterpenic acids in the fruits of Ziziphus jujuba var. spinosa were higher than those in the fruits of Z. jujuba, especially for the compound pomonic acid. Differences were also found among the different parts of Z. jujuba var. spinosa fruits with the sarcocarp having a higher amount of triterpenic acids than the seed and hard core.

High-performance liquid chromatography—Two wavelength detection of triterpenoid acids from the fruits of Ziziphus jujuba containing various cultivars in different regions and classification using chemometric analysis

A simple and sensitive HPLC-DAD method has been developed for the first time to simultaneously determine 10 triterpenoid acids (ceanothic acid, alphitolic acid, zizyberanal acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, oleanolic acid, ursonic acid and zizyberenalic acid) in the dried fruit of Ziziphus jujuba (called Dazao) which has been widely used as one of the traditional Chinese medicines (TCMs). This HPLC assay was performed on a reversed-phase C(18) column (250 mm x 4.6mm, 5 microm) with the column temperature at 35 degrees C. The mobile phase was composed of A (acetonitrile) and B (0.05% aqueous phosphoric acid, v/v). The flow rate was 1.0 ml/min and the detection wave length was set at 205 nm for reference compounds 1-9 and 238 nm for reference compound 10. All calibration curves showed good linear regression (r(2)>0.9999) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.43-1.72% and 0.53-2.45%, respectively, and overall recoveries of 94.98-104.09% for the 10 compounds analyzed. The validated method was successfully applied for the simultaneous determination of the 10 triterpenoid acids in 42 batches of Dazao which contained 36 cultivars from 22 cultivation regions, and were investigated and authenticated as Z. jujuba. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of the 10 triterpenoid acids. The presented HPLC-DAD method conjugated with chemometrics approach was demonstrated to be very helpful in using Dazao resources, and was possibly useful in chemotaxonomic characterization.

UPLC-QTOF/MS-based screening and identification of the constituents and their metabolites in rat plasma and urine after oral administration of Glechoma longituba extract.

Glechoma longituba is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo integrated metabolism of its multiple bioactive components remains unknown. In this paper, ultra-performance liquid chromatography (UPLC) coupled to quadrupole time-of-flight (QTOF) and the MetaboLynx™ software combined with mass defect filtering (MDF) together provide unique high throughput capabilities for drug metabolism study, with excellent MS mass accuracy and enhanced MS(E) data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of G. longituba extract after oral administration to rats. The results showed that 21 parent components of G. longituba extract were absorbed into the blood circulation of the rats and a total of 80 metabolites of 9 parent compounds were tentatively detected in vivo by their MS spectra obtained at low or high collision energy scan with the comparison of the authentic standards and literature data. The developed method was simple and reliable, revealing that it could be used to rapid screen and identify the structures of active components responsible for pharmacological effects of G. longituba and to better clarify its action mechanism. This work suggests that the integrative metabolism approach makes a useful template for drug metabolism research of TCM.

Characterization of nucleosides and nucleobases in fruits of Ziziphus jujuba by UPLC-DAD-MS.

The fruit of Ziziphus jujuba , named dazao in Chinese, has been utilized as food as well as crude drugs in China for thousands of years. To explore the profiles of the nucleosides and nucleobases in this fruit, an ultraperformance liquid chromatograph coupled with a photodiode array detector and electrospray ionization-mass spectrometer method (UPLC-DAD-MS) has been established and validated in this paper. The validated method was successfully applied for the simultaneous characterization and quantitation of 9 nucleosides and nucleobases in 49 dazao samples, which comprised 43 cultivars from 26 cultivation regions. Furthermore, principal component analysis (PCA) was performed to classify the samples on the basis of the contents of the nine analyzed compounds. The results showed that almost all of these dazao samples were rich in nucleosides and nucleobases, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of jujube products.

Ultra-performance liquid chromatography-tandem mass spectrometry analysis of the bioactive components and their metabolites of Shaofu Zhuyu decoction active extract in rat plasma.

A rapid, sensitive and selective ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometric (UPLC-ESI-MS/MS) method was developed for analysis and identification of the bioactive components and their metabolites in rat plasma following oral administration Shaofu Zhuyu decoction active extract. The analysis was carried out on an AcQuity UPLC chromatographic instrument and a QTOF mass spectrometer using positive and negative electrospray ionization (ESI), respectively. The results showed that sixteen peaks were detected and twelve peaks, including flavones, organic acids and terpene glycosides, were identified by comparing with reference compounds. Furthermore, nine metabolites, including quercetin glucuronide sulfates, quercetin diglucuronides, isorhamnetin sulfates, isorhamnetin glucosides, and isorhamnetin glucuronides were detected and identified in rat plasma based on the mass fragmentation behaviors and literature reports. These results provided a meaningful basis for evaluating the bioactive components and their action mechanisms of complex traditional Chinese medicines (TCMs).

Hydrophilic interaction ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry for determination of nucleotides, nucleosides and nucleobases in Ziziphus plants

In this study, a rapid and sensitive analytical method was developed for the determination of 20 nucleobases, nucleosides and nucleotides in Ziziphus plants at trace levels by using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC-UHPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under the optimized chromatographic conditions, good separation for 20 target compounds were obtained on a UHPLC Amide column with sub-2μm particles within 10min. The overall LODs and LOQs were between 0.11-3.12ngmL(-1) and 0.29-12.48ngmL(-1) for the 20 analytes, respectively. It is the first report about simultaneous analysis of nucleobases, nucleosides and nucleotides in medicinal plants using HILIC-UHPLC-TQ-MS/MS method, which affords good linearity, precision, repeatability and accuracy. The developed method was successfully applied to Ziziphus plant (Z. jujuba, Z. jujuba var. spinosa and Z. mauritiana) samples. The analysis showed that the fruits and leaves of Ziziphus plants are rich in nucleosides and nucleobases as well as nucleotides, and could be selected as the healthy food resources. Our results in present study suggest that HILIC-UHPLC-TQ-MS/MS method could be employed as a useful tool for quality assessment of the samples from the Ziziphus plants as well as other medicinal plants or food samples using nucleotides, nucleosides and nucleobases as markers.

Simultaneous qualitative and quantitative analysis of triterpenic acids, saponins and flavonoids in the leaves of two Ziziphus species by HPLC-PDA-MS/ELSD.

The leaves of Ziziphus jujuba and Z. jujuba var. spinosa have been utilized as crude drugs for their health benefits in China for thousands of years. To control their quality, a reliable method based on high-performance liquid chromatography coupled with photo diode array and electrospray ionization tandem mass spectrometry detection (HPLC-PDA-ESI-MS/MS) was developed for exploration of the chemical profiles of these jujube leaves. As the results, fourteen constituents including three flavonoids, two saponins and nine triterpenic acids were identified or tentatively characterized. Then, twelve of them such as quercetin-3-O-rutinoside, zizyphus saponins I and II, ceanothic acid, alphitolic acid, maslinic acid, 2α-hydroxyursolic acid, zizyberanalic acid, epiceanothic acid, ceanothenic acid, betulinic acid, and oleanolic acid were selected as the chemical markers and were determined using an HPLC coupled with evaporative light scattering detection (ELSD) method. The separation was carried out on a Waters Sunfire C₁₈ column with 0.2 % acetic acid and acetonitrile as the mobile phase under gradient elution. The operating conditions of ELSD were set as 80°C for drift tube temperature and 2.7 l/min for nitrogen flow rate. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of eight batches of Z. jujuba and Z. jujuba var. spinosa leaves from different collections.

Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach

The compatibility of Angelicae Sinensis Radix (Danggui, DG) and Chuanxiong Rhizoma (Chuanxiong, CX), a famous herb pair Gui-Xiong (GX), can produce synergistic and complementary hematopoiesis. In present study, global metabolic profiling with ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) combined with pattern recognition method was performed to discover the underlying hematopoietic regulation mechanisms of DG, CX and GX on hemolytic and aplastic anemia rats (HAA) induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CP). Thirteen endogenous metabolites contributing to the separation of model group and control group were tentatively identified. The levels of LPCs including lysoPC (18:0), lysoPC (20:4), lysoPC (16:0) and lysoPC (18:2), sphinganine, nicotinic acid, thiamine pyrophosphate, phytosphingosine, and glycerophosphocholine increased significantly (p<0.05) in HAA, while the levels of oleic acid, 8,11,14-eicosatrienoic acid, ceramides (d18:1/14:0), and 17a-hydroxypregnenolone decreased significantly (p<0.05) in comparison with control rats. Those endogenous metabolites were chiefly involved in thiamine metabolism and sphingolipid metabolism. The metabolic deviations could be regulated closer to normal level after DG, CX and GX intervention. In term of hematopoietic function, GX was the most effective as shown by the relative distance in PLS-DA score plots and relative intensity of metabolomic strategy, reflecting the synergic action between DG and CX. The relative distance calculation was firstly used in metabolomics for semi-quantization.

Identification of rutin deglycosylated metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

In this paper, rutin was metabolized by human intestinal bacteria and five isolated strains including Bacillus sp. 52, Bacteroides sp. 45, 42, 22 and Veillonella sp. 32, the metabolites were identified using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS). As a result, Bacillus sp. 52 and Bacteroides sp. 45 could metabolize rutin to quercetin 3-O-glucoside and leucocyanidin. Bacteroides sp. 42 and Veillonella sp. 32 could convert rutin to leucocyanidin. Bacteroides sp. 22 could hydrolyze rutin to quercetin-3-O-glucoside. In order to further explain the metabolism pathway of rutin, the β-D-glucosidase and α-L-rhamnosidase activities of five strains were determined. Bacteroides sp. 22 could produce α-L-rhamnosidase but did not produce β-D-glucosidase or β-D-glucosidase activity was too low to be detected. The other four strains all demonstrated α-L-rhamnosidase and β-D-glucosidase activities. Furthermore, α-L-rhamnosidase and β-D-glucosidase activities of Veillonella sp. 32 and Bacteroides sp. 42 were higher than those of Bacteroides sp. 45 and Bacillus sp. 52. Based on these results, we can propose the deglycosylated rout of rutin: rutin was metabolized to be quercetin-3-O-glucoside by α-L-rhamnosidase produced from these bacteria, thereafter, quercetin-3-O-glucoside was further metabolized by β-D-glucosidase to form leucocyanidin. Because of the higher enzyme activity in Veillonella sp. 32 and Bacteroides sp. 42, quercetin-3-O-glucoside was completely metabolized to leucocyanidin by these two bacteria. Due to the lack of β-D-glucosidase activity, Bacteroides sp. 22 could not further metabolize quercetin-3-O-glucoside to leucocyanidin. This study will be helpful for understanding the deglycosylated rout of rutin and the role of different intestinal bacteria on the metabolism of natural compounds.