HPLC Characteristic Chromatograms of Belladonnae Herba and Multi-Component Content Determination
Objective To establish the high-performance liquid chromatography(HPLC)characteristic chromatograms of Belladonnae Herba and a method for the content determination of seven main components in this medicinal herb.Methods The HPLC method was adopted,the chromatographic column was the Ultimate® XB-C18 column(250 mm×4.6 mm,5 µm),the mobile phase was acetonitrile-0.05% formic acid aqueous solution(gradient elution),the flow rate was 0.8 mL/min,the detection wavelength was 290 nm,the column temperature was 30℃,and the injection volume was 10 µL.With hyoscyamine sulfate as a reference,the HPLC fingerprints of 16 batches of medicinal herb samples were established,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Version)was used for similarity evaluation.The cluster analysis(CA)and partial least square-discriminant analysis(PLS-DA)were performed,and the main differential components were identified based on variable importance in projection(VIP)values.The contents of seven main components were determined by the above chromatographic conditions.Results A total of 13 common peaks were marked in the fingerprints of 16 batches of medicinal herb samples,with the similarity in the range of 0.939 to 0.993.The CA and PLS-DA showed that 16 batches of medicinal herb samples were clustered into three categories,and 13 differential components were screened based on the VIP values,among which seven were known.The linear ranges of scopolin,chlorogenic acid,scopolamine hydrobromide,hyoscyamine sulfate,scopoletin,rutin and trifolin were 5.04-151.18 µg/mL,5.11-153.41 µg/mL,4.63-138.79 µg/mL,5.91-177.22 µg/mL,5.03-150.90 µg/mL,5.00-149.98 µg/mL,5.97-179.16 µg/mL(R2>0.999 0,n=6),respectively.The RSDsof precision,stability and repeatability tests were all lower than 2.0% .The average recovery rates of the above components were 100.82%,102.22%,101.55%,101.98%,101.50%,100.34%,100.17%,with the RSDs of 2.00%,1.37%,2.53%,1.11%,2.73%,2.14%,1.17% respectively(n=9).The contents of the above seven components were in the range of 0.32 to 0.81 mg/g,0.68 to 1.01 mg/g,0.33 to 0.46 mg/g,1.57 to 1.99 mg/g,0.37 to 0.64 mg/g,0.35 to 0.79 mg/g,0.74 to 1.90 mg/g,respectively.Conclusion The established characteristic chromatograms,chemometric methods and multi-component content determination method can indentify some differential components of Belladonnae Herba,which provides a reference for the quality control of this medicinal herb.
Belladonnae HerbaHPLCcharacteristic chromatogramchemometricscontent determinationquality control
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