Analysis of the constituents in Semen Descurainiae by UPLC/Q TOF MS/MS

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1 Journal of Chinese Pharmaceutical Sciences 33 Analysis of the constituents in Semen Descurainiae by UPLC/Q TOF MS/MS Zuohuan Meng 1,2*, Li Qiao 1,2, Zongquan Wang 2, Zhensi Ma 2, Jiming Jia 2*, Qingxian Chang 2, Wenlie Li 2 1. Department of Collateral Theory, Combination of TCM with Western Medicine Academy of Hebei, Shijiazhuang 3, China 2. Yiling Medical Research Academy. CO, Shijiazhuang 3, China Abstract: A UPLC/Q TOF MS/MS method was developed for identifying the constituents in the Chinese herb Semen Descurainiae. A total of 14 compounds were identified or tentatively characterized based on their mass spectra, including 9 flavonoids, 4 fatty acids and 1 cardiac glycoside. Among them, quercetin 3 O β D [6 O sinnapoyl 2 O β D glucopyranosyl] glucopyranoside was identified in Semen Descurainiae for the first time. Keywords: Semen Descurainiae, UPLC/Q TOF MS/MS, Constituents, Separation, Identification CLC number: R917 Document code: A Article ID: 13 17(1) Introduction Semen Descurainiae came from the dry mature seeds of Deseurainia Sophia (L.) Webb. ex Prantl. The plant is cut in the summer, dried, and the seeds are collected [1]. It is mainly grown in the northeast of China, such as Hebei, Inner Mongolia, Shandong, Henan, Zhejiang, and Gansu Provinces. According to the theory of traditional Chinese medicine, Semen Descurainiae is used to quench lung fire, relieve dyspnea, promote dieresis and reduce edema. It has also been used in treatment of phlegm fluid accumulation, cough with excessive sputum, dyspnea, and general swelling [2]. In our previous reports, the quality control method relies on the traditional separation and identification techniques, i.e., HPLC fingerprint study and GC MS separation identification [3,4]. However, it is not sufficient to identify the chemical constituents only by HPLC fingerprint and GC MS. Recently, some studies using UPLC MS analysis on cardiac glycosides in Semen Descurainiae have been reported; for instance, four cardiac glycosides were identified [,6], and many flavonoids were detected by means of polyamide, silica gel, and Received: 1 1 1, Revised: 1 3 2, Accepted: Foundation items: National Basic Research Program of China (973 Program: Grant No. 12CB186), National Science Fund for Distinguished Young Scholars (Grant No ). * Corresponding author. Tel.: , E mail: jjm_41@163.com, @qq.com Sephadex LH column chromatography [7]. There is no systematical UPLC MS study on Semen Descurainiae. In the present study, a more rapid and accurate UPLC MS method was developed to analyze the main chemical compositions. Compound 9 was for the first time identified in Semen Descurainiae, which was reported to have been isolated from other cruciferous plants; especially compound 7 has been used in the treatment of hypoxia and ischemia diseases [8]. This study could provide valuable information for clarifying the efficacy and quality of Semen Descurainiae. Moreover, this method is highly sensitive and efficient, and is suitable for identifying the constituents in Semen Descurainiae. The results indicated that his method could be used for the quality control of Semen Descurainiae. 2. Experimental 2.1. Reagents and plant material HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA), distilled water was purchased from Watsons Food & Beverage Co., Ltd. (Lot No. 1319c, Guangzhou China), HPLC grade formic acid was purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Lot No. 114, Tianjin China), and the Standard of Semen Descurainiae (Lot No ) was purchased from National Institutes for Food and Drug Control (Beijing, China). Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

2 34 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), UPLC and MS conditions 3. Results UPLC for analysis of Semen Descurainiae, a Shimazhu A Infinity Series system was coupled with hybrid quadrupole time of flight tandem mass spectrometer (LCMS Quadruple TOF, AB Sciex, Foster City, CA) which equipped with an LC ADXR binary pump, a SIL AC autosampler, and a CTO ACXR column oven. Chromatographic separation was performed on C 18 reversed phase LC column (PerkinElmer, 2.7 μm particles, 2.1 mm 1 mm). The mobile phase consisted of (A) water acetic acid (1:1, v/v) and (B) acetonitrile using a gradient program of % % (B) in 18 min, % 1% (B) in min, 1% (B) for 2 min. The flow rate was.4 ml/min and column temperature was maintained at ºC. Q TOF MS A hybrid quadrupole time of flight mass spectrometer equipped with ESI source was used. The conditions of MS/MS detector were as follows: ion spray voltage,. kv/ 4. kv; ion source temperature, C; declustering potential (DP), V/ V; collision energy (CE), 4 ev/ ev. Nitrogen was used as the nebulizer and the auxiliary gas, and the nebulizer gas (gas 1), the heater gas (gas 2) and the curtain gas were set to, and 3 psi. The complete scan was performed in the ESI positive ion mode and ESI negative mode within the m/z 1 1 amu mass range. The strongest fragment ions over 1 cps were scanned within the above mentioned mass range. The collision voltage difference was 1 ev/ ev, and dynamic background subtraction was open. An automatic calibration system (CDS) was then used for automatic tuning and calibration of the MS and MS/MS. Data acquisition and processing analysis were conducted using the Analyst TF 1.6 software and PeakView 1.2 software. The full scan UPLC/Q TOF MS/MS chromatogram and the chromatogram produced using in source collision induced dissociation were acquired, respectively [9] Preparation of sample solution Accurately weigh. g of Semen Descurainiae medicinal plants into a 1 ml centrifuge tube, and add. ml methanol water (7:3, v/v). The herb was soaked overnight, underwent ultrasonic extraction for 1. h, and centrifuged at 4 r/min for min. The extraction solution was filtered with. μm membrane and the injection volume was 1 μl Components identified by LC MS The Semen Descurainiae medicinal material reference standard was qualitatively analyzed. The total ion currents (TIC) of (+) ESI/MS and ( ) ESI MS are shown in Figure Analysis of Semen Descurainiae chemical composition Using the literature data and mass spectrometry information and through calculating accuracy mass and isotopic fit value of the molecular ion cluster, 14 peaks were identified by elucidating the mass spectrum produced via in source collision induced dissociation. They included a cardiac glycoside, nine flavonoid glycosides and four organic acids (Tables 1 and 2). Positive ion mode can detect one cardiac glycoside and seven flavonoid glycosides, and negative ion mode can detect nine flavonoid glycosides and four organic acids. The Semen Descurainiae UPLC MS chemical fingerprint in positive and negative ion detection mode are shown in Figure Identification of Semen Descurainiae components by negative ion fragmentation The MS 2 spectrum of compounds 1 13 in negative ion mode are shown in Figure 3, and the composition analysis is as follows. For peak 1, the mass spectrum showed a quasi molecular ion peak at m/z 787. Literature [7] reported that quercetin 3 O β D glucopyranosyl 7 O β gentiobioside has a relative molecular mass of 788, therefore showing a molecular ion [M H] of m/z 787. MS 2 analysis was carried out on the m/z 787 ion, showing 787 m/z [M H], m/z 62 [M H glc], m/z 463 [M H 2glc], and m/z 31 [M H 3glc] ions. By comparing with literature data, it was determined to be quercetin 3 O β D glucopyranosyl 7 O β gentiobioside with a chemical composition of C 33 H O 22. For peak, mass spectrum showed an accurate molecular ion at m/z 639, literature [7] reported that it should be isorhamnetin 7 O β gentiobioside/isorhamnetin 3,7 di O β D Gluco pyranosyl and its molecular mass is 6. m/z 639 would be its [M H] molecular ion. MS 2 Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

3 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), Intensity (%) (of 1.1e7) (A) Intensity (%) (of 9.e6) (B) Figure 1. Total ion chromatograms of positive ions (A) and negative ions (B) in Semen Descurainiae. Intensity 1.8e 1.7e 1.6e 1.e 1.4e 1.3e 1.2e 1.1e 1.e 9.e4 8.e4 7.e4 6.e4.e4 4.e4 3.e4 2.e4 1.e4.e (A) Intensity 3.e 2.8e 2.6e 2.4e 2.2e 2.e 1.8e 1.6e 1.4e 1.2e 1.e 8.e4 6.e4 4.e4 2.e4.e (B) Figure 2. (A) Extracted chromatogram of compounds 1, 2, 4,, 7 9, 14 in positive ion mode; (B) The extracted chromatogram of compounds 1 13 in negative ion mode. Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

4 36 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), Table 1. The identified components by negative ion mode NO T (min) Measured [M H] Error Fomular Identification C 33H O 22 Quercetin 3 O β D glucopyranosyl 7 O β gentiobioside C 27H 3O 17 Quercetin 7 O β gentiobioside C 33H O 21 Kaempferol 3 O β D glucopyranosyl 7 O β gentiobioside C 34H 42O 22 Isorhamnetin 3 O β D glucopyranosyl 7 O β gentiobioside C 28H 32O 17 Isorhamnetin 7 O β gentiobioside/isorhamnetin 3,7 di O β D glucopyranosyl C 44H O 2 Kaempferol 3 O β D glucopyranosyl 7 O [(2 O trans sinnapoyl) β Dglucopyranosyl(1 6)] β D glucopyranoside C 21H O 12 Quercetin 3 O β D glucopyranoside C 22H 22O 12 Isorhamnetin 3 O β D glucopyranoside C 38H O 21 Quercetin 3 O β D [6 O sinnapoyl 2 O β D glucopyranosyl] glucopyranoside C 18H 3O 2 Linplenic acid C 18H 32O 2 Linoleic acid C 16H 32O 2 Palmitic acid C 18H 34O 2 Oleic acid Table 2. The identified components by positive ion mode NO T (min) Measured [M+H] Error Fomular Identification C 33H O 22 Quercetin 3 O β D glucopyranosyl 7 O β gentiobioside C 27H 3O 17 Quercetin 7 O β gentiobioside C 34H 42O 22 Isorhamnetin 3 O β D glucopyranosyl 7 O β gentiobioside C 28H 32O 17 Isorhamnetin 7 O β gentiobioside/isorhamnetin 3,7 di O β D glucopyranosyl C 21H O 12 Quercetin 3 O β D glucopyranoside C 22H 22O 12 Isorhamnetin 3 O β D glucopyranoside C 38H O 21 Quercetin 3 O β D [6 O sinnapoyl 2 O β D glucopyranosyl] glucopyranoside C 23H 32O 6 Strophanthidine Intensity (%) (of 168.) Intensity (%) (of 1171.) Intensity (%) (of 649.) Intensity (%) (of 1171.) Figure 3. MS 2 spectra of compounds 1 13 in negative ion mode. Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

5 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), Intensity (%) (of 491.) Intensity (%) (of 312.) Intensity (%) (of 68.) Intensity (%) (of 634.) Intensity (%) (of 13.) Intensity (%) (of 2341.) Intensity (%) (of 1641.) Intensity (%) (of 1.) Intensity (%) (of 296.) Figure 3. Continued. Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

6 38 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), analysis was carried out on the m/z 639 ion, and fragment molecular mass of linplenic acid is 278, and m/z 277 would peaks of m/z 639 [M H], m/z 477 [M H glc], and 317 be its [M H] molecular ion. MS 2 analysis was carried out [M H 2glc] were observed. By comparing with literature on m/z 277, and m/z 277 [M H] ion was observed. It was data, it was determined to be isorhamnetin 7 O β identified as linplenic acid by literature comparison, and [7,1,12] gentiobioside/isorhamnetin 3,7 di O β D glucopyranosyl its chemical composition is C 18 H 3 O 2. with a chemical composition of C 28 H 32 O 17. For peak 11, the mass spectrum showed an accurate For peak 6, the mass spectrum showed a quasi molecular ion of m/z 279. The chemical composition molecular ion peak of m/z 977. Literature [7] reported that the could be C 18 H 32 O 2 as calculated by Formula Finder. MS 2 molecular mass of kaempferol 3 O β D glucopyranosyl 7 spectra showed m/z 279 [M H], m/z 261 [M H H 2 O], O [(2 O trans sinnapoyl) β D glucopyranosyl (1 6)] m/z 177 [M H 2 octene], and m/z 13 [M H CH 2 COOHβ D glucopyranoside is 978, and m/z 977 would be its 1 hexene] fragment ions. By comparing with literature [M H] molecular ion peak. MS 2 analysis was carried data, it was identified as linoleic acid [7]. out on the m/z 977 ion, and m/z 977 [M H], m/z 81 Using the same method, by comparing the accurate [M H glc], and 447 [M H glc sinnapyglucosyl] fragment molecular ion and fragmentation patterns, the peaks 2, ions were observed. By comparing with literature data, it 3, 4, 7, 8, 12, and 13 were identified as quercetin 7 Owas identified as kaempferol 3 O β D Glucopyranosyl β gentiobioside, kaempferol 3 O β D glucopyranosyl 7 O [(2 O trans sinnapoyl) β D glucopyranosyl(1 6)] 7 O β gentiobioside, isorhamnetin 3 O β D glucopyranosylβ D glucopyranoside with a chemical composition of 7 O β gentiobiosi de, quercetin 3 O β D glucopyranoside, C 44 H O 2. isorhamnetin 3 O β D glucopyranoside, palmitic acid For peak 9, the mass spectrum showed an accurate and oleic acid [,7,8,13]. molecular ion peak of m/z 831. Literature [1] reported that the molecular mass of quercetin 3 O β D [6 O 3.4. Identification of Semen Descurainiae components sinnapoyl 2 O β D glucopyranosyl] glucopyranoside is by positive ion fragmentation 832, and m/z 831 would be its [M H] molecular ion. MS 2 analysis was carried out on m/z 831, and m/z 831 The mass spectrum of Peak 14 showed an accurate [M H], m/z 669 [M H 6 O sinnapoyl], m/z 463 [M H molecular ion of m/z. The MS 2 spectrum is shown 6 O sinnapoyl 2 O β D glucopyranosyl], and m/z 31 in Figure 4. Literature [7,13] reported that strophanthidine [M H 3 O β D [6 O sinnapoyl 2 O β D glucopyranosyl] is present in Semen Descurainiae, and its molecular glucopyranoside] fragment ions were observed. By mass is 4. Therefore, m/z would be its [M+H] + comparing with literature data, it was identified as quercetin molecular ion. MS 2 analysis was carried out on m/z 3 O β D [6 O sinnapoyl 2 O β D glucopyranosyl], and m/z [M+H] +, m/z 369 [M+H 2H 2 O] +, m/z glucopyranoside with a chemical composition of 31 [M+H 3H 2 O] +, and m/z 323 [M+H 3H 2 O CO] + C 38 H O 21. fragment ions were observed. By comparing with For peak 1, the mass spectrum showed an accurate literature data, it was identified as strophanthidine with molecular ion of m/z 277. Literature [7] reported that the a chemical composition of C 23 H 32 O Intensity (%) (of 123.) Figure 4. MS 2 spectrum of compound 14 in positive ion mode Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University

7 Meng, Z.H. et al. / J. Chin. Pharm. Sci. 1, 24 (), Discussion and conclusion In the present study, a rapid and sensitive method using UPLC ESI MS/MS has been developed for the analysis of the components in Semen Descurainiae. Electrospray ionization mass spectrometry (ESI MS) is a soft ionization technique that forms mainly molecular ion peaks, and the fragment ions from multi stage tandem mass spectrometry can provide rich structural information of the compounds. Fourteen ingredients which are mainly flavonoid glycosides and organic acids have been determined by LC MS/MS in negative and positive scanning modes. This method identified components without reference standard. Moreover, enhanced selectivity and sensitivity, and rapid analysis times allowed the UPLC MS/MS method as an effective and reliable technique for qualitative analysis of complex samples such as TCM (traditional Chinese medicine) [14]. The trace ingredients in Semen Descurainiae could be detected; for example, peaks 9 and 12 were too low to be detected by UV. Some unknown components were also detected. It gave a new direction for the phytochemical research on Semen Descurainiae, and these unknown ingredients should be investigated in future study of Semen Descurainiae [1]. Acknowledgements This study was financially supported by the National No. 12CB186); and the National Science Fund for Distinguished Young Scholars (Grant No ). References [1] Chinese pharmacopoeia [1]. [S]. 1. [2] Zhou, X.D.; Tang, L,Y.; Zhou, G.H.; Kou, Z.Z.; Wang, T.; Wang, C.Z. Chin. J. Chin. Mater. Med. 14, 24, [3] Wang, A.Q.; Wang, X.K.; Li, J.L.; Zhou, Y.X. J. Med. 3,, [4] Wang, X.F.; Dong, Y.F.; Liu, H.L. Shandong J. Tradit. Chin. Med., 24, [] Chen, M.Q.; Li, Z.; Wang, Y.W. Acta Pharm. Sin. 1981, 16, [6] C.A. 1974:81:16639 [7] Wang, A.Q. Ph. M. thesis, Beijing university of Chinese medicine. 3. [8] Zhou, Y.Q.; Wang, X.R. CN: , 7. [9] Zhu, H.M. Ph. D. thesis, Hebei Medical University. 14. [1] Zhao, H.Y. Ph. M. thesis, Beijing university of Chinese medicine.. [11] Lee, J.Y.; Kim, N.S.; Kim, H.J. Aral Pharm. Res. 13, 36, 36. [12] Xiao, P.G. Modern Chinese Materia Mediea. Vol 2. 2nd ed. Beijing: Chemical Industry Press. 2, [13] Jacobs, W.A. J. Biol. Chem. 1922, 4, 23. [14] Wu, Y.Y.; Wang, L.; Liu, G.X.; Xu, F.; Shang, M.Y.; Cai, S.Q. J. Chin. Pharm. Sin. 14, 23, [1] Liu, J.H.; Wang, X.; Cai, S.Q.; Komatsu. K.; Namba. T. Basic Research Program of China (973 Program: Grant J. Chin. Pharm. Sin. 4, 13, 南葶苈子化学成分的 UPLC/Q TOF MS/MS 分析孟作环 1,2*, 乔莉 1,2, 王宗权 2, 马振嗣 2, 贾继明 2*, 常青鲜 2, 李文列 2 1. 河北省中西医结合医药研究院, 河北石家庄 3 2. 河北以岭医药研究院有限公司, 河北石家庄 3 摘要 : 对南葶苈子标准品建立了 UPLC/Q TOF MS/MS 方法, 根据质谱特征指认了 14 个化学成分, 包括 9 个黄酮类, 4 个脂肪酸类和 1 个强心苷类, 其中槲皮素 3 O β D [6 O 芥子酰基 2 O β D 吡喃葡萄糖基 ] 吡喃葡萄糖苷为南葶苈子首次报道 关键词 : 南葶苈子 ; UPLC/Q TOF MS/MS; 化学成分 ; 分离 ; 鉴定 Copyright 1 Journal of Chinese Pharmaceutical Sciences, School of Pharmaceutical Sciences, Peking University