Quality assessment of Astragali Radix based on pseudo-targeted metabolomics and chemometric approach

Astragali Radix is widely used because of its dual use in medicine and food, and its quality evaluation is of great importance. In this study, a pseudo-targeted metabolomics approach based on scheduled multiple reaction monitoring was developed, and a total of 114 compounds with good linearity, sensitivity, and reproducibility were selected for relative quantification, and the chemical differences between Astragali Radix of different growth patterns were further compared by chemometric analysis. With the help of multivariate and univariate analysis, 26 differential compounds between wild/semi-wild Astragali Radix and cultivated Astragali Radix were determined. Then five marker compounds were screened out by lasso regression, and further verified by systematic clustering, random forest, support vector machine, and logistic regression. In addition, malonyl-substituted flavonoids showed relatively higher content in wild/semi-wild Astragali Radix. Thus, the malonyl substitution was characteristic for flavonoids in wild/semi-wild Astragali Radix. In conclusion, the application of pseudo-targeted metabolomics and various statistical methods could offer multi-dimensional information for the holistic quality evaluation of Astragali Radix.     

Quantitative Analysis and Differential Evaluation of Radix Bupleuri Cultivated in Different Regions Based on HPLC-MS and GC-MS Combined with Multivariate Statistical Analysis

The quality of Radix Bupleuri is greatly affected by its growing environment. In this study, Radix Bupleuri samples that were harvested from seven different regions across northwest China were examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) to reveal significant differences in quality contributed by the cultivation region. An HPLC-MS method was firstly established and used in the multiple reaction monitoring mode for the quantitative analysis of five saikosaponins in Radix Bupleuri so as to evaluate the difference in the absolute content of saikosaponins attributable to the cultivation region. The effect on the components of Radix Bupleuri was further investigated based on the profiles of the representative saponins and volatile compounds, which were extracted from the Radix Bupleuri samples and analyzed by HPLC-MS and GC-MS. Multivariate statistical analysis was employed to differentiate the Radix Bupleuri samples cultivated in different regions and to discover the differential compositions. The developed quantitative method was validated to be accurate, stable, sensitive, and repeatable for the determination of five saikosaponins. Further statistical tests revealed that the collected Radix Bupleuri samples were distinctly different from each other in terms of both saponins and volatile compounds, based on the provinces where they were grown. In addition, twenty-eight saponins and fifty-eight volatile compounds were identified as the differentially accumulated compositions that contributed to the discrimination of the Radix Bupleuri samples. The Radix Bupleuri samples grown in Shouyang county showed the highest content of saikosaponins. All of the results indicated that the cultivation region significantly affected the accumulation and diversity of the main chemical components of Radix Bupleuri. The findings of this research provide insights into the effect of the cultivation region on the quality of Radix Bupleuri and the differentiation of Radix Bupleuri cultivated in different regions based on the use of HPLC-MS and GC-MS combined with multivariate statistical analysis.     

Fragmentation study of iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae by rapid resolution liquid chromatography with diode‐array detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid resolution liquid chromatography (RRLC) coupled with diode array detection (DAD) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) method was applied to the mass spectral study of a series of naturally occurring iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae, which provides higher speed and increased sensitivity without loss of resolution. With dynamic adjustment as the key role of the fragmentor voltage and confirmed with authentic standards, valuable structural information regarding the nature of both the glycoside skeletons was thus obtained. Most compositions were found to possess organic acid moiety such as cinnamoyl, caffeoyl and ferulyol. Besides extensive fragmentation of the carbohydrate moiety, losses of the hydroxyl and glucose residue units showed in the spectra, permitting the exploration of the skeleton and the identity of substituents in the molecule. Ten major iridoid glycosides and 10 phenylpropanoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in Radix Scrophulariae obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparative analysis of nucleosides,nucleobases, and amino acids in different parts of Angelicae Sinensis Radix by ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry

A rapid and sensitive ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method was established and employed to determine 21 nucleosides, nucleobases, and amino acids in 60 samples from different parts of Angelicae Sinensis Radix. The established methods were validated by good linearity (r² > 0.9937), limits of detection (0.12–77.75 ng/mL), limits of quantitation (0.31–272.13 ng/mL), intra‐ and interday precisions (RSD ≤ 4.84%, RSD ≤ 6.26%), stability (RSD ≤ 5.92%), repeatability (RSD ≤ 7.14%), recovery (91.4–103.4%), and matrix effects (0.92–1.03). Chemical comparative analysis revealed that the content of total analytes in four parts of Angelicae Sinensis Radix were different, and exhibited the order: Head (14.89 mg/g) > Body (10.15 mg/g) > All (8.22 mg/g) > Tail (6.23 mg/g). Principal component analysis showed that the samples could be classified into four groups in accord with four different parts of Angelicae Sinensis Radix. The results could provide a scientific basis and reference for the quality control of Angelicae Sinensis Radix, and may be conducive to further research on the pharmacological activities of Angelicae Sinensis Radix.     

Application of liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry for screening and quantitative analysis of C21 steroids in the roots and rhizomes of Cynanchum paniculatum

A liquid chromatography-mass spectrometry method is presented for the quantification of C21 steroids in the roots and rhizomes of Cynanchum paniculatum. Eight C21 steroids, including five steroidal aglycones and three steroidal glycosides, were simultaneously analyzed by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The extracted ion current chromatograms were extracted from the total ion current chromatogram using characteristic ions produced by target compounds for peak determination. Chromatographic separation was achieved on a C18 reversed-phase column within 60 min, using an acetonitrile/water gradient. For comparision, six C. paniculatum samples from different locations were investigated by the established method, and the results indicated that the different geographical origin significantly influenced the C21 steroid composition. The method was observed to have the necessary sensitivity, selectivity, precision, and accuracy, and to be suitable for quality control of herbal medicines and their preparations.     

超高效液相色谱-串联质谱法快速检测洛党参中乙基多杀菌素残留

建立了超高效液相色谱-串联质谱(UHPLC-MS/MS)快速检测洛党参中乙基多杀菌素-J(XDE-175-J)和乙基多杀菌素-L(XDE-175-L)残留的方法。样品经乙腈提取,乙二胺-N-丙基硅烷和石墨化炭黑净化后,外标法定量。结果表明,XDE-175-J和XDE-175-L的峰面积与其质量浓度分别在0.075~75μg/L和0.025~25μg/L范围内,呈良好线性关系(r20.990);在0.375、3.75、75μg/kg加标水平下,XDE-175-J的平均回收率为88.4%~113.5%,相对标准偏差(RSD,n=5)为2.0%~4.2%;在0.125、1.25、25μg/kg加标水平下,XDE-175-L的平均回收率为84.4%~99.5%,RSD(n=5)为2.5%~4.9%。乙基多杀菌素的定量下限(LOQ)为0.375μg/kg(XDE-175-J)和0.125μg/kg(XDE-175-L)。该方法简便、快捷,灵敏度高,回收率和重复性良好,能满足农药残留检测技术要求,可用于大量洛党参样品中乙基多杀菌素的残留分析。     

Qualitative and quantitative determination of polyacetylenes in different Bupleurum species by high performance liquid chromatography with diode array detector and mass spectrometry

Polyacetylenes are main toxic ingredients in Bupleurum longiradiatum, a poisonous plant that has ever been misused as substitutes for Chaihu (Bupleuri Radix). For the first time, a high-performance liquid chromatography method coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for qualitative and quantitative analysis of nine polyacetylenes in Bupleurum species. All references, including two new polyacetylenes, were isolated from B. longiradiatum and purified using a semi-preparation HPLC chromatography. The analysis was performed on a TSKgel ODS-100V C18 column (3 μm, 150 mm x 4.6 mm i.d.) using a gradient system of acetonitrile and water, with diode array detection (254 nm). The method was validated for linearity, precision, accuracy, limit of detection and quantification. A total of 27 Bupleurum samples were examined with this method, which showed a great variety in the distribution and contents of the polyacetylenes. It was found that polyacetylenes (1-8) were the main ingredients in B. longiradiatum, while a few kinds of polyacetylenes (5-8) were also identified in B. smithii, B. smithii var. parvifolium, B. bicaule and B. angustissimum. However, no polyacetylenes (1-9) were detected in the authentic Chaihu samples and the other Bupleurum species. The results indicated that the toxic B. longiradiatum could readily be distinguished from other medicinal Bupleurum species based on the polyacetylene profiles, and the guidelines and quality control of polyacetylenes for Chaihu are useful. The acute toxicity of the ethanol extract of B. longiradiatum and its fractions was also investigated.     

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography-triple quadrupole mass spectrometry with novel shell-type column

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae.     

Comparative pharmacokinetics of prim-O-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of Radix Saposhnikoviae extract, cimifugin monomer solution and prim-O-glucosylcimifugin monomer solution to rats

A sensitive and reliable liquid chromatography–mass spectrometry method has been developed and validated for simultaneous determination of cimifugin and prim‐O‐glucosylcimifugin in rat plasma after oral administration of Radix Saposhnikoviae (RS) extract, prim‐O‐glucosylcimifugin monomer solution and cimifugin monomer solution. Plasma samples were pretreated by protein precipitation with acetonitrile containing the internal standards puerarin and daidzein. LC separation was achieved on a Zorbax SB‐C₁₈ column (150 × 4.6 mm i.d., 5 µm) with 0.1% formic acid in water and methanol by isocratic elution. The detection was carried out in select‐ion‐monitoring mode with a positive electrospray ionization interface. The fully validated method was successfully applied to the pharmacokinetic study of the analytes in rats. A bimodal phenomenon appeared in the concentration–time curve of prim‐O‐glucosylcimifugin and cimifugin after oral administration of RS extract. Prim‐O‐glucosylcimifugin mainly transformed to cimifugin when it was absorbed into blood. Both absorption and elimination of cimifugin after oral administration of RS were longer than after administration of single cimifugin. The pharmacokinetic parameters (AUC_0–t, AUC_0–∞ and t_1/2) of prim‐O‐glucosylcimifugin and cimifugin by giving cimifugin monomer solution, prim‐O‐glucosylcimifugin monomer solution and RS extract had significant differences (P < 0.05). Copyright © 2012 John Wiley & Sons, Ltd.     

Covalent immobilization of tyrosinase on magnetic multi-walled carbon nanotubes for inhibitor screening

The inhibition of tyrosinase is considered to be a common therapeutic strategy for some hyperpigmentation disorders. Screening of tyrosinase inhibitors is of great significance to the treatment of pigmentation diseases. In this study, tyrosinase was covalently immobilized on magnetic multi-walled carbon nanotubes for the first time, and the immobilized tyrosinase was applied for ligand fishing of tyrosinase inhibitors from complex medicinal plants. The immobilized tyrosinase was characterized by transmission electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, vibrating sample magnetometry, and thermo-gravimetric analyzer, which indicated that tyrosinase was immobilized onto magnetic multi-walled carbon nanotubes. The immobilized tyrosinase showed better thermal stability and reusability than the free one. The ligand was fished out from Radix Paeoniae Alba and identified as 1,2,3,4,6-pentagalloylglucose by ultra-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry. 1,2,3,4,6-pentagalloylglucose was found to be a tyrosinase inhibitor with similar half maximal inhibitory concentration values of 57.13 ± 0.91 μM compared to kojic acid (41.96 ± 0.78 μM). This work not only established a new method for screening tyrosinase inhibitors but also holds considerable potential for exploring the new medicinal value of medicinal plants.