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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 15 Analytical Methods 1 2 3 4 Development of the fingerprints for the quality evaluation of Cyathula 5 6 officinalis Kuan by LC-DAD-ESI-Q-TOF MS/MS coupled with 7 8 9 multivariate statistical analysis 10 11 12 13 a b, c d b, c b, c, 14 Qiao-Jia Fan , Jin-Liang Liu , Lei Sun , Shun-Lin Zheng , Ji-Chao Yuan ∗, Meng-Liang 15 16 Tian b, Fan-Lei Kong b, c 17 18 19 20 21 a College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China, Manuscript 22 23 b 24 College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China; 25 26 c Key Laboratory of Crop Ecophysiology and Farming System in Southwest China, Ministry of 27 28 29 P.R. China, Chengdu 611130, China; 30 31 d Chengdu Meteorological Bureau, Chendu 610071,China; Accepted 32 33 34 35 36 ABSTRACT 37 38 39 An LC-ESI-Q-TOF MS/MS method coupled with multivariate statistical analysis was 40 Methods 41 developed for chemical fingerprinting of Cyathula officinalis Kuan. Ten peaks were selected as 42 43 44 the common peaks, and cyasterone was used as a reference. The relative areas of common peaks 45 46 were used for hierarchical clustering analysis, principal component analysis and similarity 47 48 49 calculation. A total of 31 samples collected from different sources were classified into three 50 51 Analytical (principal component analysis) or four groups (hierarchical clustering analysis). The similarities of 52 53 54 31 batches of C. officinalis samples were between 0.653 and 0.999. The results obtained suggested 55 56 57 ∗ Corresponding author. Tel.: +86 13980074156. 58 E-mail address: [email protected] 59 60 1 Analytical Methods Page 2 of 15 1 2 3 4 that the chromatographic fingerprint technique could efficiently evaluate C. officinalis . 5 6 Keywords: Cyathula officinalis Kuan; quality evaluation; fingerprint; HPLC-ESI-Q-TOF MS/MS 7 8 9 1. Introduction 10 11 Cyathula officinalis Kuan is mainly distributed in Sichuan Province, China. It is a traditional 12 13 14 Chinese medicine (TCM), that has been widely used in China for many centuries. 15 16 Pharmacological and clinical studies indicated that the roots of C. officinalis are used for 17 18 1 19 removing blood stasis, restoring menstrual flow and inducing diuresis for treating stranguria . 20 21 To our knowledge, due to the degradation and hybridisation of varieties, planting limitations, Manuscript 22 23 24 changes in cultivation methods and other factors, led to the huge heteromorphosis in product 25 26 qualities 2-6. Exact evaluation of C. officinalis plays a significant role in guiding scientific 27 28 29 cultivation, breeding purification and rejuvenation. Therefore, development of an evaluation 30 31 method is necessary and valuable for quality control. Accepted 32 33 34 Quality control is now regarded as a core requirement for pharmaceuticals of international trade. 35 36 In 2000, the State Food and Drug Administration of China promulgated the regulation requiring 37 38 39 all injections made from herbal medicine related materials to be standardised by chromatographic 40 Methods 41 fingerprint 7-9. Fingerprint is a powerful tool for the analysis of multi-ingredient medicines, 42 43 44 emphasising on the systemic and comprehensive characterisation of samples. It has been widely 45 46 accepted as a useful means for the quality evaluation of herbal raw materials combined with 47 48 49 stoichiometry 4, 6, 10-19 . The commonly used statistical methods have hierarchical clustering analysis 50 51 Analytical (HCA) and principal component analysis (PCA). HCA is performed to classify samples on the 52 53 54 similarities of their chemical properties, and the results often give in the way of a tree diagram 55 56 which is called a dendrogram 20 . Any valid metric could be used as a measure of similarity between 57 58 59 60 2 Page 3 of 15 Analytical Methods 1 2 3 4 pairs of observations, and the pairwise distances between observations is the linkage criteria, 5 6 determines the choice of which clusters to merge of split 20 . PCA is a method for feature extraction 7 8 9 and dimensionality reduction, and to provide an overview of class separation, clustering and 10 11 outliers 20 . 12 13 14 Previous studies have reported that the major active ingredients of C. officinalis are cyasterone, 15 16 rhapontisterone, daidzin, sergostesone, isocyasterone, sengosterone, polysaccharides and so on 1, 21, 17 18 22 19 . Therefore, evaluation of raw materials using a few compounds as a quality compound is 20 21 insufficient. The present study aimed at developing the LC-ESI-Q-TOF MS/MS characteristic Manuscript 22 23 24 fingerprint of C. officinalis and then the fingerprint model could accurately reflect the quality and 25 26 guarantee clinical efficacy. 27 28 29 2. Experimental 30 31 2.1 Material and Chemicals Accepted 32 33 34 Thirty-one samples of raw herbs of C. officinalis from different areas of China were collected 35 36 (Table 1). The samples were authenticated by Professor Ji-chao Yuan (College of Agronomy, 37 38 39 Sichuan Agricultural University, Chengdu, China). The samples were dried at 60 °C and ground to 40 Methods 41 a fine powder. Cyasterone was obtained from National Institutes for Food and Drug Control, 42 43 44 Beijing, China, and used as a standard. Methanol (HPLC grade) was purchased from Fisher 45 46 Scientific, Inc. Pure water was obtained from a Millipore Milli Q-Plus system (Millipore, Bedford, 47 48 49 MA, USA). 50 51 Analytical Table 1 Raw material used in this experiment 52 53 b No. Sample code Sources growing year Harvesting time Similarity 54 55 1 s1 Baoxing, Ya'an City 2 2008.12 0.89 56 2 s2 Baoxing, Ya'an City 2 2007.12 0.91 57 3 s3 Baoxing, Ya'an City 3 2007.12 0.87 58 59 60 3 Analytical Methods Page 4 of 15 1 2 3 4 s4 Baoxing, Ya'an City 2 2007.12 0.91 4 5 s5 Baoxing, Ya'an City 2 2007.12 0.89 5 6 6 s6 Baoxing, Ya'an City 3 2007.12 0.97 7 7 s7 Baoxing, Ya'an City 3 2007.12 0.96 8 8 s8 Jinkouhe, Leshan City 2 2008.01 0.93 9 9 s9 Jinkouhe, Leshan City 3 2008.01 0.94 10 11 10 s10 Jinkouhe, Leshan City 4 2008.01 0.94 12 11 s11 Tianquan, Ya'an City 3 2007.12 0.87 13 12 s12 Tianquan, Ya'an City 3 2007.12 0.97 14 13 s13 Tianquan, Ya'an City 2 2007.12 0.94 15 16 14 s14 Tianquan, Ya'an City 2 2007.12 0.90 17 15 s15 Tianquan, Ya'an City 2 2007.12 0.94 18 16 s16 Tianquan, Ya'an City 3 2007.12 0.94 19 17 s17 Tianquan, Ya'an City 3 2008.12 0.84 20 21 18 s18 Tianquan, Ya'an City 3 2008.12 0.79 Manuscript 22 19 s19 Tianquan, Ya'an City 3 2008.12 0.81 23 20 s20 Tianquan, Ya'an City 3 2008.12 0.80 24 21 s21 Yingjing, Ya'an City 3 2008.11 0.88 25 26 22 s22 Yingjing, Ya'an City 3 2008.11 0.90 27 23 s23 Yingjing, Ya'an City 3 2008.11 0.82 28 24 s24 Yingjing, Ya'an City 3 2008.11 0.87 29 25 s25 Yingjing, Ya'an City 3 2008.11 0.88 30 26 s26 Yingjing, Ya'an City 3 2008.11 0.88 31 Accepted 32 27 s27 Yingjing, Ya'an City 3 2008.11 0.89 33 28 s28 a Unknown Unknown Unknown 0.92 34 29 s29 a Baoxing, Ya'an City Unknown Unknown 0.90 35 a 36 30 s30 Baoxing, Ya'an City Unknown Unknown 0.90 37 31 s31 a Baoxing, Ya'an City Unknown Unknown 0.92 38 a This batch was bought from medicinal materials market, so the original area, the growing year or harvesting time 39 were not clear. 40 Methods 41 b Similarities of each chromatogram to the corresponding representative standard fingerprint of each areas by 42 Similarity Evaluation System. 43 44 2.2 LC-MS conditions 45 46 The liquid chromatography equipment was a Shimadzu LC-20A series binary pump and 47 48 49 Shimadzu PDA detector. Analysis was carried out on a Diamonsil C18 column (250 mm × 4.6 mm, 50 51 Analytical 5 µm). The mobile phase was methanol (A) and pure water (B) with the following gradient 52 53 54 program: 0-5 min, linear gradient 5%-46% A; 5-32 min, linear gradient 46%-66% A; 32-40 min, 55 56 57 66%-100% A; 30 °C and flow-rate of 0.9 ml/min.