ABSTRACT
Objective:To establish the high performance liquid chromatography （HPLC） fingerprint of Citri Sarcodactylis Fructus， and to search for makers to characterize the quality difference of Citri Sarcodactylis Fructus from different origins coupled with chemometrics. Method:The analysis was performed on a Thermo Hypersil GOLD C<sub>18</sub> column （4.6 mm×250 mm， 5 μm） with mobile phase consisted of acetonitrile-0.05% phosphoric acid solution for gradient elution， and the detection wavelength was set at 254 nm. A total of 31 batches of samples were analyzed to establish the HPLC fingerprint of Citri Sarcodactylis Fructus. Similarity evaluation was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System （2012 edition） to confirm the common peaks， which were identified by comparison of reference substances. On the basis， chemometrics methods were used to analyze and evaluate the quality of Citri Sarcodactylis Fructus from different origins. At the same time， 3 batches of 5 species of decoction pieces from the genus <italic>Citrus</italic> in the family Rutaceae， including Citri Sarcodactylis Fructus， Aurantii Fructus Immaturus， Aurantii Fructus， Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium， were randomly collected for evaluating the effectiveness and reliability of the established HPLC fingerprint of Citri Sarcodactylis Fructus. Result:HPLC fingerprint of Citri Sarcodactylis Fructus was established and 22 common peaks were identified. And seven common peaks among them were identified as 6，7-dimethoxycoumarin， diosmin， hesperidin， byakangelicin， 5，7-dimethoxycoumarin， bergapten and oxypeucedanin. Except for 2 batches of samples， the similarities of fingerprints between other 29 batches of samples were >0.9. The 31 batches of Citri Sarcodactylis Fructus were basically divided into 3 groups by cluster analysis and principal component analysis， which were consistent with the classification of three different producing areas. Eight differential markers were screened by orthogonal partial least squares discriminant analysis and four of them （5，7-dimethoxycoumarin， bergapten， 6，7-dimethoxycoumarin and diosmin） were identified by reference substances. Similarity evaluation of 5 species of decoction pieces from genus <italic>Citrus</italic> in the family Rutaceae was carried out by taking the reference fingerprint of Citri Sarcodactylis Fructus as treference chromatogram， similarity of Citri Sarcodactylis Fructus decoction pieces was 0.892-0.977， and the similarities of the other 4 kinds of decoction pieces were 0.215-0.517. Conclusion:The established fingerprint method is reasonable， effective and accurate for quality control of Citri Sarcodactylis Fructus， the characterization information is more comprehensive combined with chemometrics.
ABSTRACT
Objective::To analyze and identify the chemical constituents of Citri Sarcodactylis Fructus by an ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbirap HRMS) method. Method::Compounds were separated on Thermo Scientific Accucore™ C18 column (3 mm×100 mm, 2.6 μm). The mobile phase was 0.1% formic acid solution and 0.1% formic acetonitrile solution. The flow rate was 0.3 mL·min-1, and the column temperature was set at 30 ℃. HRMS was performed using an electrospray ion source (ESI), and scanned in a positive ion mode by means of full scan/data dependent secondary scan (Full MS/dd-MS2). Compound Discoverer 3.0 software that could be linked to mzCloud network database and local high-resolution mass spectrometry database of traditional Chinese medicine components was used to analyze the data, based on accurate molecular mass, retention behaviors and characteristic ion fragmentation of the compounds, as well as literature information and relevant reference materials. Result::Totally 54 chemical constituents in Citri Sarcodactylis Fructus were identified, including 16 flavonoids, 17 coumarins, 3 limonoids, 6 nucleosides and nucleobases, 2 organic acids, 3 aromatic aldehydes, 1 amino acid and 6 other components. Conclusion::The established UPLC-Q-Orbitrap HRMS method can be used to effectively and rapidly identify main chemical constituents of Citri Sarcodactylis Fructus. The findings provide a methodological reference and theoretical foundation for defining the pharmacodynamic material base and optimizing quality control index of Citri Sarcodactylis Fructus, which is of guiding significance for further development and utilization of the resources.