HPLC-LTQ-Orbitrap方法分析丹参的代谢轮廓

Metabolic profile of danshen in rats by HPLC-LTQ-Orbitrap mass spectrometry HPLC-LTQ-Orbitrap方法分析丹参的代谢轮廓 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:huangjm@bucm.edu.cn †E-mail:huangjm@bucm.edu.cn huangjm@bucm.edu.cn School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China Danshen, the dried root of Salvia miltiorrhiza Bunge (Lamiaceae), is one of the traditional Chinese medicines (TCMs) most commonly used for the treatment of cardiovascular and cerebrovascular diseases. However, little is known about the chemical and metabolic profiles of danshen in vitro or in vivo. In particular, more information is needed in relation to the 50% ethanol extracts usually used in danshen formulations such as Fufang Xueshuantong Capsules and Fufang Danshen tablets. High-performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer (HPLC-LTQ-Orbitrap) provides a sensitive and accurate method for analyzing the composition of samples. This method was used to determine the in vitro and in vivo chemical and metabolic profiles of danshen. Sixty-nine components of danshen extract and 118 components of danshen in rat plasma, urine, feces, and bile were unambiguously or tentatively identified. These results not only revealed the material composition of danshen, but also provided a comprehensive research approach for the identification of multi-constituents in TCMs. 灌胃给予丹参提取物后的生物样本中鉴定出共118个化合物,包括血浆中35个,尿液中63个,粪便中62个,以及胆汁中18个. Danshen Chemical profile Metabolic profile HPLC-LTQ-Orbitrap 丹参 化学轮廓 代谢轮廓 HPLC-LTQ-Orbitrap Introduction 1 Recently, high-performance liquid chromatography-mass spectrometry (HPLC-MS), especially for high-resolution mass spectrometry (HRMS), has become a powerful tool for detecting and identifying known and unknown metabolites of drugs owing to its high mass accuracy and high sensitivity (Liu et al., 2011 2011 ; Wang et al., 2011 2011 ; Liang et al., 2013 2013 ). MS/MS data provide abundant information for elucidating the structure of compounds. Thus, this method provides an effective and powerful tool for the identification of compounds in complex matrices, such as traditional Chinese medicines (TCMs) and bio-samples. The linear ion trap-Orbitrap mass spectrometer (LTQ-Orbitrap), an electrostatic Fourier-transform mass spectrometer, combines a high trapping capacity and MS n scanning function of the linear ion trap with accurate mass measurements to within 5 ppm (parts per million) and a resolving power of up to 100 000 (Cai et al., 2015 2015 ; Zhang et al., 2015 2015 ). Data-dependent MS/MS scanning can obtain more fragmentation information, improving the efficiency and accuracy of identification (Wang et al., 2016 2016 ). Danshen, the dried root of Chinese sage, Salvia miltiorrhiza Bunge (Lamiaceae), is one of the TCMs most commonly used in China and elsewhere, and is used either alone or in formulations. It has been widely used in the treatment of cardiovascular and cerebrovascular diseases, such as coronary artery disease (Ji et al., 2003 2003 ), myocardial infarction (Sun et al., 2005 2005 ), and stroke (Lam et al., 2003 2003 ). It has also been used to treat other conditions, such as renal diseases (Kang et al., 2004 2004 ) and diabetes (Belin et al., 2009 2009 ). Many formulations containing danshen, for instance the Fufang Danshen Dripping Pill and Fufang Xueshuantong Capsule, are now frequently used in the clinical treatment of cardiovascular diseases and eye diseases (Duan et al., 2013 2013 ; Yang et al., 2014 2014 ). There are two principal bioactive components in danshen: water-soluble phenolic acids and liposoluble tanshinones. The phenolic acids include danshensu, rosmarinic acid, lithospermic acid, salvianolic acid A, salvianolic acid B, and other salvianolic acids. The tanshinones include tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, 15,16-dihydrotanshinone I, and other tanshinones (Zhang et al., 2005 2005 ; Wu et al., 2006 2006 ). Previous in vivo studies have focused mainly on the water decoction of danshen (Zhao et al., 2015 2015 ) or its effective parts and components (Li et al., 2007 2007 ; Sun et al., 2007 2007 ). Danshen has often been used only as a component of ethanol extracts, especially in formulations, because of its complex composition and compatibility with other herbs. Also, there has been limited research on the excretion of danshen in feces and bile (Sun et al., 2007 2007 ). Therefore, comprehensive and systematic studies are needed of the chemical and metabolic profiles of danshen in vitro and in vivo. In the present study, we analyzed the chemical profile of a 50% ethanol extract of danshen, as such extracts are often used in its formulation. The metabolic profile of danshen was determined in bio-samples from rats. An HPLC-LTQ-Orbitrap method coupled with an extracted ion chromatogram (EIC) data-processing technique was applied to elucidate the chemical and metabolic profiles. A total of 69 components of danshen extract and 118 components of danshen in rat plasma, urine, feces, and bile were unambiguously or tentatively identified. The present study provides a basis for research on the quality control and pharmacology of danshen, and establishes a comprehensive and reliable method for identification of multi-components of TCMs both in vitro and in vivo. Materials and methods 1 Materials and reagents 2 Danshen crude drug was provided by the Guangdong Zhongsheng Pharmaceutical Co., Ltd. (Guangzhou, China) and was authenticated by Professor Jian-mei HUANG. Voucher specimens were deposited in the School of Chinese Materia Medica, Beijing University of Chinese Medicine, China. Eleven reference standards, including caffeic acid, protocatechuic aldehyde, protocatechuic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid, tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone, were purchased from the Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). Three reference standards of tanshinol B, danshenxinkun B, and tanshinone IIB were purchased from the Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Three authentic standards, namely salvianolic acids A, B, and C, were obtained from the School of Chinese Materia Medica, Beijing University of Chinese Medicine, China. HPLC-grade methanol and acetonitrile, and LC/MS-grade formic acid were purchased from Fisher Scientific (Fisher, Fair Lawn, NJ, USA). Instrumentation and analytical conditions 2 Chromatographic analysis was performed using a Thermo Accela 600 HPLC system (Thermo Scientific, Bremen, Germany) equipped with a binary pump and an autosampler. Samples were separated on a Waters XBridge-C18 column (5 μm, 150 mm×4.6 mm) at room temperature. A gradient elution of solvent acetonitrile (A) and water containing 0.1% formic acid (B) was applied according to the following program: 0–10 min, 5%–20% A; 10–25 min, 20%–30% A; 25–35 min, 30%–70% A; 35–60 min, 70% A. The flow rate was set at 1.0 ml/min. Sample solution (10 μl) was injected into the HPLC-MS/MS system. MS analysis was performed using an LTQ-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). The mass spectrometer was connected to the Accela HPLC system by an electrospray ionization (ESI) source and operated in both positive and negative ion modes. Compounds were detected by full scan mass analysis from m / z 100 to 1000 at a resolving power of 30 000 with data-dependent MS n ( n =3) analysis. The optimized source parameters in positive (and negative) mode were as follows: capillary temperature, 350 °C; sheath gas flow, 30 arbitrary unit (arb); auxiliary gas flow, 10 arb; source voltage, 4.0 kV; capillary voltage, 35 V; tube lens voltage, 110 V. The isolation width was 2 Da, and the normalized collision energy (CE) was 35%. Preparation of drugs 2 2.3.1 Preparation of danshen freeze-dried powder Danshen freeze-dried powder was prepared by refluxing the extract twice with 50% (v/v) ethanol (100 g/700 ml for 3 h the first time, and 100 g/500 ml for 2 h the second time) after soaking in 50% ethanol for 30 min. Each decoction was mixed, filtered, vacuum-evaporated, and freeze-dried. The yield of powdered extract was about 42.3% (w/w). 2.3.2 Preparation of danshen extract A total of 1.05 g danshen freeze-dried powder was accurately weighed and ultrasonicated with 30 ml of 50% ethanol for 30 min. The supernatants were filtered through a 0.22-μm membrane filter. The filtrates were collected and stored at 4 °C until HPLC-MS/MS analysis. 2.3.3 Preparation of danshen suspension Danshen freeze-dried powder was accurately weighed and suspended in deionized water to obtain a final concentration of 1.5 g/ml (crude drug) for intragastric administration. 2.3.4 Preparation of standard solutions Individual standard stock solutions of the seventeen standards were prepared by accurately weighing and then dissolving each standard in methanol, with concentrations ranging from 0.09 to 1.20 mg/ml. A working solution of each of the seventeen standards was obtained by diluting each stock solution with methanol to the desired concentration. Working solutions were stored at 4 °C before analysis. Animals and drug administration 2 Twelve male Sprague-Dawley rats, weighing (250±20) g, were purchased from the Si Bei Fu Experimental Animal Science and Technology Co., Ltd. (Beijing, China). The rats were divided into two groups: a control group ( n =3, one each for blank plasma, urine and feces, and bile) and a drug group ( n =9, 3 for dosed plasma, 3 for dosed urine and feces, and 3 for dosed bile). The rats were housed in a controlled environment (12-h light/12-h dark cycle, at consistent temperature and humidity) for three days before the experiment. Danshen was administered orally to the drug group once a day at a dose of 1 ml/100 g body weight for three days. An equal dose of deionized water was administered by oral gavage to the rats of the control group. Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, and all experimental protocols were reviewed and approved by the Institutional animal Experimentation Committee of Beijing University of Chinese Medicine. Biological sample collection 2 Before the last administration, the rats were deprived of food for 12 h. Blood samples (0.4 ml) were collected from the orbital vein and gathered into heparinized tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h, respectively. All blood samples were then centrifuged at 3000 g for 10 min to obtain plasma samples. Plasma samples from different rats and different time points in each group were then mixed in the same proportions to produce pooled plasma samples, which were stored at −80 °C until additional extraction and analysis. The urine and feces of rats in each group were collected over a 24-h period starting immediately after the last administration. The urine and feces samples in each group were combined separately and stored at −80 °C until additional extraction and analysis. Rats were fixed on a wooden plate and anesthetized with ethylurethanm following the last administration. An abdominal incision was made and the common bile duct was cannulated with PE10 tubing (inside diameter (ID)=0.28 mm, San Diego, CA USA) for collection of the bile samples. Bile samples from each group were collected for 24 h and combined and stored at −80 °C until additional extraction and analysis. Biological sample pretreatment 2 An aliquot of 2 ml plasma for positive ion detection was suspended in 8 ml methanol. Another aliquot of 2 ml plasma for negative ion detection was suspended in 200 µl 10% (v/v) hydrochloric acid and 8 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000 g for 10 min. The supernatants were evaporated to dryness under nitrogen gas at room temperature, and the residues were dissolved in 200 µl 70% (v/v) methanol. After centrifugation at 12 000 g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis. Urine sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000 g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 600 µl 70% methanol. After centrifugation at 12 000 g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis. Bile sample (3 ml) was dissolved in 12 ml methanol, and then mixed by vortex for 3 min to precipitate protein, followed by centrifugation at 10 000 g for 10 min. The supernatant was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 1.5 ml 70% methanol. After centrifugation at 12 000 g for 10 min, 10 µl of the supernatant was injected into the HPLC-MS/MS system for analysis. Feces were dried at 37 °C and grinded into powder. Feces sample (1.5 g) was extracted with 30 ml 70% methanol in an ultrasonic bath for 30 min, followed by filtration. Filtrate (2 ml) was evaporated to dryness under nitrogen gas at room temperature, and the residue was dissolved in 400 µl 70% methanol. After centrifugation at 12 000 g for 10 min, a 10-µl aliquot of the supernatant was injected into the HPLC-MS/MS system for analysis. Data processing 2 Thermo Xcalibur 2.1 workstation (Thermo Fisher Scientific, Bremen, Germany) was used for data acquisition and processing. Metworks (Thermo Scientific, Bremen, Germany) was used for data-filtering and identification of possible metabolites. The maximum mass error between the measured and calculated values was 5 ppm. Results 1 Analysis of the chemical profile of danshen in vitro 2 The results from total ion chromatography (TIC) of the danshen extract and the reference standards in positive mode and negative mode are shown in Fig. 1 1 . Based on accurate mass measurements, MS/MS fragmentations, retention time, and reference data (Liu AH et al., 2007 2007 ; Liu M et al., 2007 2007 ; Su et al., 2015 2015 ), a total of 69 components of danshen, including 23 phenolic acids, 33 tanshinones, and 13 unknown compounds, were identified. Their accurate mass measurements, retention time, and HPLC-MS/MS data are shown in Table 1 1 . Among them, 16 compounds were unambiguously confirmed by comparison with reference standards. Their structures are shown in Fig. 2 2 . The numbering of compounds is consistent with that in Table 1 1 2 No. t R (min) Ion Theoretical mass ( m / z ) Experimental mass ( m / z ) Error (ppm) Formula [M−H] − /[M+H] + 2 MS/MS fragment Identification 11 Phenolic acids 2 1 4.83 [M−H] − 197.0444 197.0441 −1.6 C 9 H 9 O 5 MS 2 [197]: 179(100) MS 3 [179]: 135(100) 2 Danshensu a 2 2 5.70 [M−H] − 153.0182 153.0184 0.9 C 7 H 5 O 4 2 Protocatechuic acid a 2 3 7.58 [M−H] − 137.0233 137.0236 1.7 C 7 H 5 O 3 MS 2 [137]: 137(100) 2 Protocatechuic aldehyde a 2 4 9.16 [M−H] − 179.0339 179.0336 −1.3 C 9 H 7 O 4 MS 2 [179]: 135(100) 2 Caffeic acid a 2 5 10.08 [M−H] − 193.0495 193.0493 −1.2 C 10 H 9 O 4 MS 2 [193]: 178(100), 149(38), 134(81) MS 3 [178]: 134(100) 2 Ferulic acid/isoferulic acid a 2 6 14.42 [M−H] − 735.1556 735.1552 −0.5 C 36 H 31 O 17 MS 2 [735]: 537(100), 519(20) MS 3 [519]: 519(51), 357(73), 321(65), 297(100) 2 Hydrated salvianolic acid B 2 7 14.76 [M−H] − 537.1028 537.1028 0.1 C 27 H 21 O 12 MS 2 [537]: 339(100), 295(37) MS 3 [339]: 321(33), 295(100) 2 Salvianolic acid H/I 2 8 15.02 [M−H] − 735.1556 735.1553 −0.4 C 36 H 31 O 17 MS 2 [735]: 537(100), 519(49) MS 3 [537]: 519(51), 339(20), 321(70), 297(100) 2 Hydrated salvianolic acid B 2 9 16.74 [M−H] − 359.0761 359.0758 −1.0 C 18 H 15 O 8 MS 2 [359]: 197(24), 179(23), 161(100) MS 3 [161]: 161(38), 133(100) 2 Rosmarinic acid a 2 10 17.45 [M−H] − 493.1129 493.1129 0.0 C 26 H 21 O 10 MS 2 [493]: 295(100) MS 3 [295]: 280(13), 277(65), 159(100) 2 Salvianolic acid A isomer 2 11 19.07 [M−H] − 717.1450 717.1434 −2.2 C 36 H 29 O 16 MS 2 [717]: 519(100), 321(15) MS 3 [519]: 339(21), 321(100) 2 Salvianolic acid B a 2 12 20.89 [M−H] − 717.1450 717.1450 0.0 C 36 H 29 O 16 MS 2 [717]: 519(100), 321(18) MS 3 [519]: 339(21), 321(100) 2 Salvianolic acid E 2 13 21.32 [M−H] − 493.1129 493.1135 1.1 C 26 H 21 O 10 MS 2 [493]: 295(100) MS 3 [295]: 280(14), 277(66), 159(100) 2 Salvianolic acid A a 2 14 22.26 [M−H] − 731.1607 731.1598 −1.2 C 37 H 31 O 16 MS 2 [731]: 533(100) MS 3 [533]: 353(51), 335(100) 2 Methyl salvianolic acid B 2 15 23.68 [M−H] − 565.1341 565.1341 0.1 C 29 H 25 O 12 MS 2 [565]: 519(87), 367(87), 339(15), 321(100) 2 Dimethyl lithospermate 2 16 24.23 [M−H] − 491.0973 491.0982 1.9 C 26 H 19 O 10 MS 2 [491]: 311(60), 293(100) MS 3 [293]: 276(32), 275(11), 265(100), 249(89), 247(13) 2 Isosalvianolic acid C 2 17 24.44 [M−H] − 565.1341 565.1341 0.1 C 29 H 25 O 12 MS 2 [565]: 367(76), 339(17), 321(100) 2 Dimethyl lithospermate 2 18 25.23 [M−H] − 565.1341 565.1345 0.7 C 29 H 25 O 12 MS 2 [565]: 519(88), 339(15), 321(100) MS 3 [321]: 293(31), 277(100), 249(76) 2 Dimethyl lithospermate 2 19 26.65 [M−H] − 565.1341 565.1345 0.7 C 29 H 25 O 12 MS 2 [565]: 367(100) 2 Dimethyl lithospermate 2 20 27.27 [M−H] − 491.0973 491.0977 0.9 C 26 H 19 O 10 MS 2 [491]: 311(22), 293(100) MS 3 [293]: 276(24), 275(18), 265(100), 249(21), 247(36) 2 Salvianolic acid C a 2 21 27.65 [M−H] − 745.1763 745.1760 −0.5 C 38 H 33 O 16 MS 2 [745]: 547(87), 519(100), 321(73) 2 Dimethyl salvianolic acid B 2 22 28.62 [M−H] − 745.1763 745.1762 −0.1 C 38 H 33 O 16 MS 2 [745]: 547(87), 519(100), 321(74) 2 Dimethyl salvianolic acid B 2 23 28.97 [M−H] − 313.0707 313.0709 0.8 C 17 H 13 O 6 MS 2 [313]: 269(36), 161(100) MS 3 [161]: 161(29), 133(100) 2 Salvianolic acid F 11 Tanshinones 2 24 33.73 [M−H] − 295.0965 295.0970 1.7 C 18 H 15 O 4 MS 2 [295]: 277(16), 267(27), 265(100) MS 3 [265]: 265(100) 2 Tanshinol B a 2 25 21.24 [M+H] + 313.1071 313.1056 −4.8 C 18 H 17 O 5 MS 2 [313]: 295(100), 267(22), 265(87) MS 3 [295]: 277(100), 267(40), 249(98) 2 Tanshindiol A/B/C 2 26 22.57 [M+H] + 313.1071 313.1056 −4.6 C 18 H 17 O 5 MS 2 [313]: 295(100), 267(7) MS 3 [295]: 267(100) 2 Tanshindiol A/B/C 2 27 26.14 [M+H] + 313.1071 313.1056 −4.7 C 18 H 17 O 5 MS 2 [313]: 295(100), 267(9) MS 3 [295]: 267(100) 2 Tanshindiol A/B/C 2 28 27.01 [M+H] + 293.0808 293.0797 −3.9 C 18 H 13 O 4 MS 2 [293]: 249(100) MS 3 [249]: 234(18), 221(24), 193(100), 178(39) 2 Hydroxyl tanshinone I 2 29 28.49 [M+H] + 313.1434 313.1422 −3.9 C 19 H 21 O 4 MS 2 [313]: 269(100) MS 3 [269]: 254(25), 251(16), 223(22), 199(100) 2 Hydroxyl cryptotanshinone 2 30 29.28 [M+H] + 283.0965 283.0954 −3.7 C 17 H 15 O 4 MS 2 [283]: 265(51), 255(27), 241(15), 237(100) MS 3 [237]: 222(11), 219(100), 209(87), 191(35), 181(25) 2 Dihydronortanshinone 2 31 30.79 [M+H] + 293.0808 293.0799 −3.3 C 18 H 13 O 4 MS 2 [293]: 249(100) MS 3 [249]: 234(18), 221(23), 193(100), 178(38) 2 Przewaquinone B 2 32 30.97 [M+H] + 295.0965 295.0953 −4.1 C 18 H 15 O 4 MS 2 [295]: 277(100), 267(44), 24(51) MS 3 [277]: 259(82), 249(100), 231(27) 2 Hydrated tanshinone I/trijuganone A 2 33 32.08 [M+H] + 295.0965 295.0951 −4.8 C 18 H 15 O 4 MS 2 [295]: 277(100), 249(11) MS 3 [277]: 249(100), 221(17) 2 Hydrated tanshinone I/trijuganone A 2 34 32.26 [M+H] + 313.1423 313.1423 −3.5 C 19 H 21 O 4 MS 2 [313]: 295(100), 267(73) MS 3 [295]: 277(100), 267(55), 249(52) 2 Hydroxyl cryptotanshinone 2 35 32.44 [M+H] + 301.1434 301.1424 −3.6 C 18 H 21 O 4 MS 2 [301]: 283(100) MS 3 [283]: 265(100), 255(26) 2 Salvianonol 2 36 32.61 [M+H] + 311.1278 311.1264 −4.6 C 19 H 19 O 4 MS 2 [311]: 293(100), 283(22), 267(80), 225(12) MS 3 [293]: 278(16), 275(100), 265(19), 251(80) 2 Tanshinone IIB a 2 37 32.77 [M+H] + 311.1278 311.1263 −4.6 C 19 H 19 O 4 MS 2 [311]: 293(14), 275(11), 267(100) MS 3 [267]: 252(100), 239(11), 225(63), 185(47) 2 Hydroxyl tanshinone IIA 2 38 33.46 [M+H] + 341.1384 341.1371 −3.8 C 20 H 21 O 5 MS 2 [341]: 281(100), 263(43) MS 3 [281]: 263(100), 235(19) 2 Methyl dihydrotanshinonate 2 39 33.69 [M+H] + 327.1214 327.1248 −3.9 C 19 H 19 O 5 MS 2 [327]: 309(100) MS 3 [309]: 265(100) 2 Hydroxyl tanshinone IIB 2 40 34.56 [M+H] + 281.1536 281.1524 −4.1 C 19 H 21 O 2 MS 2 [281]: 266(38), 263(50), 253(28), 239(100) MS 3 [239]: 224(22), 221(100), 193(54) 2 Dehydromiltirone 2 41 34.64 [M+H] + 293.1172 293.1159 −4.4 C 19 H 17 O 3 MS 2 [293]: 275(100), 265(11), 247(39) MS 3 [275]: 260(13), 247(100) 2 Dehydrotanshinone IIA 2 42 35.19 [M+H] + 279.1016 279.1004 −4.3 C 18 H 15 O 3 MS 2 [279]: 261(100), 233(5) MS 3 [261]: 233(100) 2 Dihydrotanshinone I a 2 43 35.71 [M+H] + 315.1591 315.1576 −4.7 C 19 H 23 O 4 MS 2 [315]: 297(100) MS 3 [297]: 279(100), 251(57) 2 Neocryptotanshinone 2 44 35.84 [M+H] + 281.1172 281.1160 −4.2 C 18 H 17 O 3 MS 2 [281]: 263(100), 253(7), 235(71) MS 3 [263]: 248(16), 245(10), 235(100) 2 Danshenxinkun B a 2 45 35.98 [M+H] + 339.1227 339.1219 −2.3 C 20 H 19 O 5 MS 2 [339]: 279(100) MS 3 [279]: 261(100) 2 Methyl tanshinonate 2 46 36.24 [M+H] + 295.1329 295.1319 −3.3 C 19 H 19 O 3 MS 2 [295]: 277(100), 267(15), 249(47) MS 3 [277]: 249(100) 2 Dehydrocryptotanshinone 2 47 37.34 [M+H] + 297.1485 297.1474 −3.7 C 19 H 21 O 3 MS 2 [297]: 279(100), 251(81) MS 3 [279]: 251(100) 2 Cryptotanshinone a 2 48 37.62 [M+H] + 277.0859 277.0850 −3.5 C 18 H 13 O 3 MS 2 [277]: 249(100), 231(13) 2 Tanshinone I a 2 49 38.84 [M+H] + 279.1016 279.1003 −4.7 C 18 H 15 O 3 MS 2 [279]: 261(100), 233(6) MS 3 [261]: 233(100), 205(3) 2 Dihydrotanshinone I 2 50 38.98 [M+H] + 293.1172 293.1166 −2.2 C 19 H 17 O 3 MS 2 [293]: 275(100), 265(11), 247(38) MS3[275] : 260(13), 247(100) 2 Dehydrotanshinone IIA 2 51 40.05 [M+H] + 281.1536 281.1524 −4.1 C 19 H 21 O 2 MS 2 [281]: 266(17), 263(43), 253(82), 221(100) MS 3 [221]: 206(17), 193(100) 2 Dehydromiltirone 2 52 40.28 [M+H] + 293.1172 293.1159 −4.5 C 19 H 17 O 3 MS 2 [293]: 275(100), 265(11), 247(37) MS 3 [275]: 260(13), 247(100) 2 Dehydrotanshinone IIA 2 53 41.17 [M+H] + 295.1329 295.1317 −4.0 C 19 H 19 O 3 MS 2 [295]: 277(100), 249(14) MS 3 [277]: 249(100) 2 Tanshinone IIA a 2 54 41.54 [M+H] + 281.1536 281.1526 −3.7 C 19 H 21 O 2 MS 2 [281]: 266(18), 263(43), 253(93), 221(100) MS 3 [221]: 206(14), 193(100) 2 Dehydromiltirone 2 55 42.34 [M+H] + 283.1693 283.1682 −3.6 C 19 H 23 O 2 MS 2 [283]: 265(100), 241(47), 223(63) MS 3 [265]: 237(64), 223(100) 2 Miltirone 2 56 45.75 [M+H] + 557.1959 557.1942 −3.1 C 36 H 29 O 6 MS 2 [557]: 539(28), 529(100), 511(48) MS 3 [529]: 511(100), 501(81), 483(46) 2 Neoprzewaquinone A 11 Others 2 Un1 9.62 [M−H] − 509.2229 509.2230 0.2 C 22 H 37 O 13 MS 2 [509]: 463(100) MS 3 [463]: 331(100), 161(27) 2 Unknown 2 Un2 10.91 [M−H] − 571.1082 571.1080 −0.5 C 27 H 23 O 14 MS 2 [571]: 527(21), 483(100), 439(73) 2 Unknown 2 Un3 11.73 [M−H] − 627.4044 627.4059 2.4 C 41 H 55 O 5 MS 2 [627]: 610(14), 581(70), 564(100) 2 Unknown 2 Un4 12.96 [M−H] − 723.5042 723.5013 −3.9 C 41 H 71 O 10 MS 2 [723]: 678(100) MS 3 [678]: 659(100), 451(25), 338(25) 2 Unknown 2 Un5 14.07 [M−H] − 836.5856 836.5854 −0.2 C 44 H 84 O 14 MS 2 [836]: 791(100) MS 3 [791]: 773(100), 565(28) 2 Unknown 2 Un6 16.21 [M−H] − 717.1450 717.1452 0.3 C 36 H 29 O 16 MS 2 [717]: 519(100), 321(16) MS 3 [519]: 339(22), 321(100) 2 Unknown 2 Un7 28.14 [M−H] − 341.1020 341.1023 0.9 C 19 H 17 O 6 MS 2 [341]: 297(100), 253(14) MS 3 [297]: 253(100) 2 Unknown 2 Un8 28.33 [M−H] − 671.1395 671.1402 1.0 C 35 H 27 O 14 MS 2 [671]: 473(100) MS 3 [473]: 429(100), 321(100) 2 Unknown 2 Un9 24.40 [M+H] + 327.1227 327.1212 −4.6 C 19 H 19 O 5 MS 2 [327]: 283(100), 265(8) MS 3 [283]: 265(29), 254(100) 2 Unknown 2 Un10 25.27 [M+H] + 369.0969 369.0955 −3.7 C 20 H 17 O 7 MS 2 [369]: 323(100), 295(77) MS 3 [323]: 295(100) 2 Unknown 2 Un11 34.38 [M+H] + 297.1485 297.1471 −4.7 C 19 H 21 O 3 MS 2 [297]: 253(100) MS 3 [253]: 238(33), 211(100) 2 Unknown 2 Un12 42.22 [M+H] + 283.1693 283.1677 −5.4 C 19 H 23 O 2 MS 2 [283]: 265(100), 241(47), 223(63) MS 3 [265]: 237(20), 223(100) 2 Unknown 2 Un13 50.83 [M+H] + 587.2064 587.2059 −0.9 C 37 H 31 O 7 MS 2 [587]: 569(100), 541(24) MS 3 [569]: 551(100), 541(71) 2 Unknown Analysis of the metabolic profile of danshen in vivo 2 For the identification of original components in bio-samples, the extract ion chromatograms (EICs) combined with the accurate mass measurements, MS/MS fragmentations, and retention time were compared with those of blank samples. For the identification of possible metabolites in bio-samples, firstly, all of the possible metabolic pathways of one component were input in Metworks; secondly, all of the possible metabolites proposed by the software were summarized in an Excel table; thirdly, the EICs, mass measurements, and MS/MS fragmentations of each metabolite were compared with those of blank samples. As a result, 118 components were unambiguously or tentatively identified, including 38 original components and 80 transformative components (Table 2 2 ). Among these components, 7 phenolic acids and 28 tanshinones were identified in rat plasma; 17 phenolic acids and 46 tanshinones were tentatively identified in rat urine; 25 phenolic acids and 37 tanshinones were identified in rat feces; and 1 phenolic acid and 17 tanshinones were identified in rat bile. Confirmed by reference standards; Original components in danshen extract. "×":detected;"√":undetected No. t R (min) Ion Theoretical mass ( m / z ) Experimental mass ( m / z ) Formula [M−H] − /[M+H] + Error (ppm) MS/MS fragment Identification Plasma Urine Feces Bile 13 Phenolic acids 1 2.11 [M−H] − 277.0013 277.0020 C 9 H 9 O 8 S 2.8 MS 2 [277]: 259(57), 215(35), 197(100) Sulfate danshensu × × √ × 2 4.26 [M−H] − 277.0013 277.0010 C 9 H 9 O 8 S −0.8 MS 2 [277]: 215(40), 197(100) MS 3 [196]: 179(100) Sulfate danshensu × √ × × 3 4.77 [M−H] − 197.0444 197.0446 C 9 H 9 O 5 0.6 MS 2 [197]: 179(100) MS 3 [179]: 135(100) Danshensu a,b √ √ √ × 4 7.35 [M−H] − 211.0601 211.0603 C 10 H 11 O 5 0.9 MS 2 [211]: 193(100), 165(23) MS 3 [193]: 149(29), 134(100) Methyl danshensu √ √ √ √ 5 7.47 [M−H] − 179.0339 179.0344 C 9 H 7 O 4 3.1 MS 2 [179]: 135(100) MS 3 [135]: 135(100) Demethyl ferulic acid × √ × × 6 7.52 [M−H] − 137.0233 137.0236 C 7 H 5 O 3 1.7 MS 2 [137]: 137(100) Protocatechuic aldehyde a,b × × √ × 7 7.62 [M−H] − 181.0495 181.0502 C 9 H 9 O 4 3.7 MS 2 [181]: 163(100) MS 3 [162]: 119(100) Dihydro caffeic acid × √ √ × 8 8.29 [M−H] − 179.0339 179.0341 C 9 H 7 O 4 1.2 MS 2 [179]: 135(100) MS 3 [135]: 135(100) Acetylated protocatechuic aldehyde × √ × × 9 8.59 [M−H] − 165.0546 165.0550 C 9 H 9 O 3 2.4 MS 2 [165]: 121(100) MS 3 [121]: 121(100) Decarbonyl ferulic acid × √ × × 10 9.38 [M−H] − 361.0918 361.0910 C 18 H 17 O 8 −2.2 MS 2 [361]: 317(39), 273(41), 239(100), 221(68) MS 3 [239]: 195(100), 151(19) Dihydro rosmarinic acid × × √ × 11 9.86 [M−H] − 193.0495 193.0502 C 10 H 9 O 4 3.5 Ferulic acid/isoferulic acid a,b × × √ × 12 12.03 [M−H] − 343.0812 343.0806 C 18 H 15 O 7 −1.8 MS 2 [343]: 299(100) MS 3 [299]: 255(100) Hydroxyl and methyl salvianolic acid F × × √ × 13 13.62 [M−H] − 535.1082 535.1072 C 24 H 23 O 14 −1.9 MS 2 [535]: 359(100) MS 3 [359]: 197(23), 179(20), 161(100) Rosmarinic acid glucuronide conjugate × √ × × 14 13.99 [M−H] − 539.1184 539.1169 C 27 H 23 O 12 −2.7 MS 2 [539]: 399(100), 297(67) MS 3 [297]: 219(100), 201(70) Dihydro salvianolic acid H/I × √ √ × 15 14.67 [M−H] − 537.1028 537.1025 C 27 H 21 O 12 −0.4 MS 2 [537]: 339(100), 295(38) Salvianolic acid H/I b × × √ × 16 15.03 [M−H] − 735.1556 735.1539 C 36 H 31 O 17 −2.3 Hydrated salvianolic acid B b × × √ × 17 15.17 [M−H] − 343.0812 343.0806 C 18 H 15 O 7 −1.7 MS 2 [343]: 255(100) MS 3 [255]: 237(91), 148(100) Hydroxyl and methyl salvianolic acid F × × √ × 18 16.55 [M−H] − 555.1133 555.1125 C 27 H 23 O 13 −1.5 MS 2 [555]: 375(100), 357(39) MS 3 [375]: 331(100), 269(60) Hydrated salvianolic acid H/I × × √ × 19 16.71 [M−H] − 359.0761 359.0761 C 18 H 15 O 8 −0.2 MS 2 [359]: 271(100) MS 3 [271]: 149(100), 135(46), 121(74) Rosmarinic acid a,b √ × × × 20 17.01 [M−H] − 523.1235 523.1225 C 27 H 23 O 11 −1.9 MS 2 [523]: 505(53), 281(100) MS 3 [281]: 263(26), 174(100) Demethyl and hydroxyl salvianolic acid A × × √ × 21 17.14 [M−H] − 315.0863 315.0863 C 17 H 15 O 6 0.0 MS 2 [315]: 297(18), 285(100), 267(12) Dihydro salvianolic acid F × √ × × 22 17.46 [M−H] − 493.1129 493.1130 C 26 H 21 O 10 0.1 MS 2 [493]: 295(100) MS 3 [295]: 277(56), 159(100) Iso salvianolic acid A b √ √ √ × 23 19.02 [M−H] − 717.1450 717.1439 C 36 H 29 O 16 −1.5 MS 2 [717]: 519(100), 321(17) MS 3 [519]: 339(22), 321(100) Salvianolic acid B a,b × √ √ × 24 20.40 [M−H] − 763.1869 763.1854 C 38 H 35 O 17 −1.9 MS 2 [763]: 565(100), 520(57), 321(15) Hydrated dimethyl salvianolic acid B × × √ × 25 20.80 [M−H] − 717.1450 717.1441 C 36 H 29 O 16 −1.3 MS 2 [717]: 519(100), 321(19) MS 3 [519]: 339(23), 321(100) Salvianolic acid E b × √ √ × 26 21.26 [M−H] − 493.1129 493.1129 C 26 H 21 O 10 −0.1 MS 2 [493]: 295(100) MS 3 [295]: 277(59), 159(100) Salvianolic acid A a,b √ √ √ × 27 21.70 [M−H] − 551.1184 551.1177 C 28 H 23 O 12 −1.3 MS 2 [551]: 519(43), 371(100), 353(51), 339(73) Methyl salvianolic acid H/I × × √ × 28 22.20 [M−H] − 731.1607 731.1597 C 37 H 31 O 16 −1.4 MS 2 [731]: 533(100) MS 3 [533]: 353(49), 335(100) Methyl salvianolic acid B b √ √ √ × 29 24.15 [M−H] − 491.0973 491.0971 C 26 H 19 O 10 −0.3 MS 2 [491]: 293(100) MS 3 [293]: 276(33), 264(100), 249(91) Iso salvianolic acid C b × × √ × 30 24.35 [M−H] − 565.1341 565.1334 C 29 H 25 O 12 −1.1 MS 2 [565]: 519(84), 367(78), 321(100) Dimethyl lithospermate b × × √ × 31 25.16 [M−H] − 565.1341 565.1331 C 29 H 25 O 12 −1.7 MS 2 [565]: 519(87), 367(79), 321(100) Dimethyl lithospermate b √ √ √ × 32 26.60 [M−H] − 565.1341 565.1331 C 29 H 25 O 12 −1.8 Dimethyl lithospermate b × √ × × 33 27.21 [M−H] − 491.0973 491.0972 C 26 H 19 O 10 −0.1 MS 2 [491]: 293(100) Salvianolic acid C a,b × × √ × 13 Tanshinones 34 9.08 [M+H] + 345.0969 345.0976 C 18 H 17 O 7 2.0 MS 2 [345]: 327(100), 281(53) MS 3 [327]: 309(14), 281(100) Demethyl and trihydroxyl tanshinone IIB × × √ × 35 12.06 [M+H] + 517.1704 517.1683 C 26 H 29 O 11 −2.1 Methyl dihydrotanshinonate glucuronide conjugate × √ × × 36 12.77 [M+H] + 343.0812 343.0814 C 18 H 15 O 7 0.5 MS 2 [343]: 325(92), 297(100) MS 3 [297]: 279(100), 255(56) Demethyl and carboxylated tanshindiol A/B/C × × √ × 37 13.06 [M+H] + 329.1020 329.1023 C 18 H 17 O 6 1.1 MS 2 [329]: 311(100), 265(58) MS 3 [311]: 283(25), 265(100) Demethyl and two hydroxyl tanshinone IIB × × √ × 38 13.37 [M+H] + 620.1909 620.1917 C 28 H 34 O 11 N 3 S 1.3 MS 2 [620]: 602(55), 584(32), 455(100) Tanshindiol A/B/C glutathione conjugate × × × √ 39 13.66 [M+H] + 343.0812 343.0814 C 18 H 15 O 7 0.5 MS 2 [343]: 325(95), 297(100) MS 3 [297]: 279(100), 255(60) Demethyl and carboxylated tanshindiol A/B/C × × √ × 40 14.04 [M+H] + 471.1286 471.1286 C 24 H 23 O 10 0.1 MS 2 [471]: 295(57), 277(23), 267(100) MS 3 [267]: 249(50), 237(100) Hydroxyl and glucuronidated dihydrotanshinone I × √ × × 41 14.25 [M−H] − 487.1235 487.1232 C 24 H 23 O 11 −0.6 MS 2 [487]: 311(100) MS 3 [311]: 283(30), 281(100) Hydroxyl and glucuronidated tanshinol B × √ × × 42 14.64 [M+H] + 473.1442 473.1440 C 24 H 25 O 10 −0.5 MS 2 [473]: 297(80), 279(100), 251(39) MS 3 [279]: 261(100), 251(100) Hydroxyl and glucuronidated danshenxinkun B × √ × × 43 16.60 [M+H] + 327.0863 327.0866 C 18 H 15 O 6 0.7 MS 2 [327]: 309(100), 281(67), 265(12) MS 3 [309]: 291(13), 281(100) Hydroxyl and dehydro tanshindiol A/B/C × × √ × 44 18.89 [M+H] + 345.1333 345.1338 C 19 H 21 O 6 1.5 MS 2 [345]: 327(100), 309(23) MS 3 [327]: 309(100), 281(19) Hydrated and hydroxyl tanshinone IIB × √ × × 45 19.32 [M+H] + 301.1434 301.1433 C 18 H 21 O 4 −0.4 MS 2 [301]: 283(100) Demethyl neocryptotanshinone √ × × × 46 19.43 [M+H] + 301.1434 301.1439 C 18 H 21 O 4 1.6 MS 2 [301]: 283(100) MS 3 [283]: 265(100), 255(26) Demethyl and two hydroxyl miltirone × √ × × 47 19.81 [M−H] − 313.0707 313.0708 C 17 H 13 O 6 0.4 MS 2 [313]: 269(27), 252(100) Demethyl and two hydroxyl tanshinol B × × √ × 48 20.40 [M+H] + 355.1176 355.1157 C 20 H 19 O 6 −2.0 MS 2 [355]: 337(100), 309(45) Hydroxyl methyltanshinonate × √ × × 49 20.47 [M+H] + 489.1391 489.1397 C 24 H 25 O 11 1.1 MS 2 [489]: 313(100), 295(57) Tanshindiol A/B/C glucuronide conjugate √ × × × 50 21.17 [M+H] + 313.1071 313.1077 C 18 H 17 O 5 2.0 MS 2 [313]: 295(100), 265(78) Tanshindiol A/B/C b √ × √ × 51 21.54 [M+H] + 339.1227 339.1209 C 20 H 19 O 5 −1.8 MS 2 [339]: 321(100) Methyl tanshinonate × √ × × 52 22.00 [M+H] + 297.1121 297.1129 C 18 H 17 O 4 2.4 MS 2 [297]: 279(100), 251(49), 237(36) Hydrated dihydrotanshinone I × √ × × 53 22.49 [M+H] + 313.1071 313.1077 C 18 H 17 O 5 2.0 MS 2 [313]: 295(100), 267(7) MS 3 [295]: 267(100) Tanshindiol A/B/C b √ √ √ × 54 22.98 [M+H] + 309.0758 309.0763 C 18 H 13 O 5 1.8 MS 2 [309]: 291(18), 265(20), 235(100) MS 3 [235]: 207(18), 179(100) Dihydroxyl tanshinone I × √ × × 55 23.60 [M+H] + 295.0601 295.0603 C 17 H 11 O 5 0.6 MS 2 [295]: 267(100) MS 3 [267]: 239(100) Demethyl and two hydroxyl tanshinone I × × √ × 56 23.87 [M+H] + 299.1278 299.1281 C 18 H 19 O 4 0.5 MS 2 [299]: 281(42), 271(49), 253(100) Demethyl and hydroxyl cryptotanshinone × √ × × 57 23.89 [M−H] − 311.0914 311.0915 C 18 H 15 O 5 0.3 MS 2 [311]: 283(32), 281(100) MS 3 [281]: 253(100) Hydroxyl tanshinol B × √ × × 58 24.85 [M+H] + 299.1278 299.1284 C 18 H 19 O 4 1.1 MS 2 [299]: 281(19), 271(44), 253(100) Demethyl and hydroxyl cryptotanshinone √ √ × × 59 25.78 [M−H] − 325.0707 325.0706 C 18 H 13 O 6 −0.3 MS 2 [325]: 297(31), 295(100), 268(30) MS 3 [294]: 267(100) Demethyl and carboxylated tanshinol B × √ × × 60 26.06 [M+H] + 313.1071 313.1074 C 18 H 17 O 5 1.3 MS 2 [313]: 295(100) Tanshindiol A/B/C b √ √ √ × 61 26.21 [M+H] + 345.1333 345.1341 C 19 H 21 O 6 0.5 MS 2 [345]: 327(100) MS 3 [327]: 299(100), 281(13) Demethyl and carboxylated neocryptotanshinone × √ × × 62 28.01 [M−H] − 297.1121 297.1124 C 18 H 17 O 4 0.9 MS 2 [297]: 253(100), 239(74), 221(26) MS 3 [253]: 238(100) Dihydro tanshinol B × √ × × 63 28.04 [M+H] + 343.1176 343.1184 C 19 H 19 O 6 0.1 MS 2 [343]: 325(100) Dihydroxyl tanshinone IIB × √ √ × 64 28.27 [M+H] + 372.1806 372.1811 C 21 H 26 O 5 N 1.7 MS 2 [372]: 354(100), 311(55), 283(60) MS 3 [354]: 326(100), 311(59) Neocryptotanshinone glycine conjugate × √ √ × 65 28.44 [M+H] + 313.1434 313.1437 C 19 H 21 O 4 −0.5 MS 2 [313]: 269(100) MS 3 [269]: 254(25), 223(21), 199(100), 171(74) Hydroxyl cryptotanshinone b √ √ √ × 66 28.70 [M−H] − 471.1286 471.1286 C 24 H 23 O 10 0.0 MS 2 [471]: 295(100) Tanshinol B glucuronide conjugate × √ × × 67 28.73 [M+H] + 331.1540 331.1545 C 19 H 23 O 5 0.7 MS 2 [331]: 313(100), 295(38) Hydroxyl and methyl salvianonol √ × × × 68 28.80 [M+H] + 459.1286 459.1287 C 23 H 23 O 10 1.5 MS 2 [459]: 283(100) MS 3 [283]: 265(25), 237(100) Dihydronortanshinone glucuronide conjugate × √ × × 69 28.99 [M−H] − 471.1286 471.1286 C 24 H 23 O 10 0.1 MS 2 [471]: 295(100) Tanshinol B glucuronide conjugate × √ × × 70 29.00 [M+H] + 618.2116 618.2119 C 29 H 36 O 10 N 3 S −0.4 MS 2 [618]: 471(44), 309(100) Tanshinone IIB glutathione conjugate × × × √ 71 29.01 [M+H] + 327.1227 327.1231 C 19 H 19 O 5 1.6 MS 2 [327]: 309(24), 299(22), 281(100), 263(57) MS 3 [281]: 263(100), 235(36) Hydroxyl tanshinone IIB × √ × × 72 29.41 [M+H] + 309.1121 309.1121 C 19 H 17 O 4 −2.0 MS 2 [309]: 265(100) MS 3 [265]: 247(51), 223(100), 195(18) Hydroxyl and dehydro tanshinone IIA √ × × × 73 29.54 [M+H] + 343.1176 343.1185 C 19 H 19 O 6 1.1 MS 2 [343]: 325(100) Dihydroxyl tanshinone IIB × √ × × 74 30.06 [M+H] + 313.1434 313.1438 C 19 H 21 O 4 1.1 MS 2 [313]: 269(35), 251(100) MS 3 [251]: 223(100) Hydroxyl cryptotanshinone × √ × × 75 30.44 [M+H] + 445.1857 445.1864 C 24 H 29 O 8 −1.8 MS 2 [445]: 269(100) MS 3 [269]: 254(100), 239(14) Decarbonylated and glucuronidated cryptotanshinone × × × √ 76 30.63 [M+H] + 293.0808 293.0813 C 18 H 13 O 4 2.4 MS 2 [293]: 275(22), 263(100), 249(23) MS 3 [263]: 235(100) Hydroxyl tanshinone I √ √ √ × 77 31.05 [M+H] + 295.0965 295.0966 C 18 H 15 O 4 1.8 MS 2 [295]: 251(100) MS 3 [251]: 223(97), 195(95), 169(100) Hydrated tanshinone I b √ √ × × 78 31.08 [M+H] + 297.1121 297.1124 C 18 H 17 O 4 0.6 MS 2 [297]: 279(100), 261(36) MS 3 [279]: 261(100) Demethyl and hydroxyl tanshinone IIA √ √ √ × 79 31.30 [M+H] + 285.1485 285.1489 C 18 H 21 O 3 1.0 MS 2 [285]: 243(100), 229(6) MS 3 [243]: 228(71), 225(92), 1815(100) Demethyl and hydroxyl miltirone √ √ × × 80 31.32 [M−H] − 311.0914 311.0912 C 18 H 15 O 5 −0.7 MS 2 [311]: 267(100), 223(29) Hydroxyl tanshinol B × √ × × 81 31.36 [M+H] + 473.1806 473.1806 C 25 H 29 O 9 2.3 MS 2 [473]: 297(36), 269(100) MS 3 [269]: 241(100), 213(92), 199(87) Cryptotanshinone glucuronide conjugate × × √ × 82 31.61 [M+H] + 339.1227 339.1210 C 20 H 19 O 5 1.2 MS 2 [339]: 321(100), 295(21) Methyl tanshinonate × √ × × 83 32.13 [M+H] + 459.2014 459.2012 C 25 H 31 O 8 2.4 MS 2 [459]: 283(100) Miltirone glucuronide conjugate × × × √ 84 32.36 [M+H] + 381.1333 381.1318 C 22 H 21 O 6 2.2 MS 2 [381]: 363(60), 337(100) Acetylated methyltanshinone √ × × × 85 32.65 [M+H] + 311.1278 311.1282 C 19 H 19 O 4 1.4 MS 2 [311]: 293(40), 275(37), 267(100) MS 3 [267]: 252(100) Tanshinone IIB a,b √ √ √ √ 86 32.84 [M+H] + 287.1642 287.1645 C 18 H 23 O 3 0.8 MS 2 [287]: 269(100) MS 3 [269]: 251(58), 241(100), 213(86) Decarbonylated neocryptotanshinone √ × × × 87 33.16 [M+H] + 299.1642 299.1646 C 19 H 23 O 3 1.4 MS 2 [299]: 281(100), 255(68), 253(62) Hydroxyl miltirone × √ × × 88 33.24 [M+H] + 267.1016 267.1022 C 17 H 15 O 3 0.3 MS 2 [267]: 249(100), 221(22) Decarbonylated hydrated tanshinone I √ × × × 89 33.31 [M+H] + 457.1493 457.1496 C 24 H 25 O 9 0.5 MS 2 [457]: 281(100), 263(73), 261(41) MS 3 [281]: 263(100) Danshenxinkun B glucuronide conjugate × × × √ 90 33.59 [M+H] + 341.1384 341.1388 C 20 H 21 O 5 1.2 MS 2 [341]: 281(100), 263(42) MS 3 [281]: 263(100), 235(18) Methyl dihydrotanshinonate b × × √ × 91 33.67 [M+H] + 279.1016 279.1021 C 18 H 15 O 3 −0.2 MS 2 [279]: 261(100) MS 3 [261]: 233(100), 205(13) Dihydro tanshinone I √ × × √ 92 33.69 [M−H] − 295.0965 295.0968 C 18 H 15 O 4 1.2 MS 2 [295]: 277(17), 267(28), 265(100), 238(27) MS 3 [265]: 237(100) Tanshinol B a,b √ √ √ √ 93 33.73 [M+H] + 309.1121 309.1120 C 19 H 17 O 4 2.5 MS 2 [309]: 265(100) MS 3 [265]: 247(55), 223(100) Hydroxyl and dehydro tanshinone IIA √ √ × × 94 34.29 [M+H] + 297.1485 297.1489 C 19 H 21 O 3 0.3 MS 2 [297]: 253(100) MS 3 [253]: 238(33), 225(24), 211(100), 209(16) Hydroxyl dehydromiltirone √ × × × 95 34.61 [M+H] + 293.1172 293.1174 C 19 H 17 O 3 1.5 MS 2 [293]: 275(100), 247(39) MS 3 [275]: 247(100) Dehydrotanshinone IIA b × × √ × 96 34.63 [M+H] + 297.1485 297.1490 C 19 H 21 O 3 1.6 MS 2 [297]: 279(100), 251(81) MS 3 [279]: 251(100), 237(67) Dihydro tanshinone IIA × × × √ 97 34.72 [M+H] + 311.1278 311.1284 C 19 H 19 O 4 1.8 MS 2 [311]: 283(100) MS 3 [283]: 265(100), 237(17) Hydroxyl tanshinone IIA √ × × × 98 35.14 [M+H] + 279.1016 279.1020 C 18 H 15 O 3 1.4 MS 2 [279]: 261(100), 233(5) MS 3 [261]: 233(100), 215(7), 205(14) Dihydrotanshinone I b × √ √ √ 99 35.36 [M+H] + 293.0808 293.0815 C 18 H 13 O 4 2.2 MS 2 [293]: 249(100) MS 3 [249]: 221(26), 193(100), 178(52) Hydroxyl hydrotanshinone I × × × √ 100 35.69 [M+H] + 315.1591 315.1594 C 19 H 23 O 4 1.0 MS 2 [315]: 297(100) MS 3 [297]: 279(100), 268(13), 254(18), 251(58) Hydrated cryptotanshinone √ × × √ 101 35.83 [M+H] + 281.1172 281.1178 C 18 H 17 O 3 2.2 MS 2 [281]: 263(100), 235(72) MS 3 [263]: 235(100) Danshenxinkun B a b × √ √ × 102 35.96 [M+H] + 339.1227 339.1235 C 20 H 19 O 5 2.5 MS 2 [339]: 279(100) MS 3 [279]: 261(100) Methyl tanshinonate b b × × √ √ 103 36.22 [M+H] + 295.1329 295.1331 C 19 H 19 O 3 0.9 MS 2 [295]: 277(100), 249(40) MS 3 [277]: 262(53), 249(100), 235(38) Dehydrocryptotanshinone b b × × √ × 104 36.59 [M+H] + 309.1121 309.1124 C 19 H 17 O 4 0.8 MS 2 [309]: 265(100) MS 3 [265]: 247(52), 223(100) Hydroxyl and methyl dihydrotanshinone I × × × √ 105 36.67 [M+H] + 301.1798 301.1802 C 19 H 25 O 3 1.4 MS 2 [301]: 271(100) MS 3 [271]: 256(100) Hydrated miltirone × × √ × 106 36.96 [M+H] + 313.1434 313.1439 C 19 H 21 O 4 1.5 MS 2 [313]: 295(100), 277(31), 271(33), 267(25) MS 3 [295]: 277(100), 253(27), 249(23) Hydroxyl cryptotanshinone √ × × × 107 37.31 [M+H] + 297.1485 297.1488 C 19 H 21 O 3 1.0 MS 2 [297]: 279(100), 251(81) MS 3 [279]: 251(100), 237(73) Cryptotanshinone a a b b × √ √ × 108 37.58 [M+H] + 277.0859 277.0863 C 18 H 13 O 3 1.4 MS 2 [277]: 249(100), 231(13) MS 3 [249]: 234(18), 221(89), 193(100), 178(30) Tanshinone I a a b b √ √ √ × 109 38.82 [M+H] + 279.1016 279.1017 C 18 H 15 O 3 0.6 MS 2 [279]: 261(100) MS 3 [261]: 233(100) Dihydrotanshinone I a a b b × √ √ √ 110 38.97 [M+H] + 293.1172 293.1178 C 19 H 17 O 3 2.1 MS 2 [293]: 275(100), 247(40) MS 3 [275]: 247(100) Dehydrotanshinone IIA b b √ √ √ √ 111 40.01 [M+H] + 281.1536 281.1540 C 19 H 21 O 2 1.4 Dehydromiltirone b b × × √ × 112 40.23 [M+H] + 293.1172 293.1175 C 19 H 17 O 3 1.0 Dehydrotanshinone IIA b b √ √ √ × 113 40.58 [M−H] − 277.0859 277.0865 C 18 H 13 O 3 2.1 MS 2 [277]: 249(100), 221(53) Dehydrated tanshinol B × × √ × 114 41.15 [M+H] + 295.1329 295.1331 C 19 H 19 O 3 0.9 MS 2 [295]: 277(100), 249(14) MS 3 [277]: 262(29), 249(100) Tanshinone IIA a a b b √ √ √ √ 115 41.27 [M−H] − 277.0859 277.0863 C 18 H 13 O 3 1.4 MS 2 [277]: 249(100), 221(60) Dehydrated tanshinol B × × √ × 116 42.32 [M+H] + 283.1693 283.1694 C 19 H 23 O 2 0.4 MS 2 [283]: 265(100), 241(47), 223(63) MS 3 [265]: 237(62), 223(100) Miltirone b b × √ √ × 117 42.70 [M+H] + 269.1536 269.1537 C 18 H 21 O 2 0.4 MS 2 [269]: 254(100) MS 3 [254]: 239(100) Demethyl miltirone × × √ × 118 47.15 [M+H] + 299.1642 299.1646 C 19 H 23 O 3 1.3 MS 2 [299]: 281(100), 256(52), 253(49) Dihydro cryptotanshinone × × √ × 2 Total 35 63 62 18 Discussion 1 To better identify the metabolites of danshen in vivo after oral administration, the original components of danshen were identified by HPLC-MS/MS in both negative and positive modes. According to the literature (Wei et al., 2007 2007 ; Lv et al., 2010 2010 ), the responses of phenolic acids are more sensitive to negative mode, while those of tanshinones are more sensitive to positive mode. In this study, the phenolic acids were detected in negative mode, and exhibited their parent ions as [M−H] − ; the tanshinones were detected in positive mode, and exhibited their parent ions of [M+H] + and/or [M+Na] + . Although 10% hydrochloric acid was added to rat plasma to increase the recovery ratios of phenolic acids, few phenolic acids were detected. This may have been because of the low bioavailability and transformation of phenolic acids in vivo (Gao et al., 2009 2009 ; Sun et al., 2013 2013 ). Under our experimental conditions, very few phenolic acids and their metabolites were detected in rat bile, except methyl danshensu. Compared with plasma and bile, many more metabolites were detected and unambiguously identified in rat urine and feces. This suggests that urine and feces might be the major route for elimination of danshen after oral administration. From the analysis of metabolites, we found that hydroxylation (36 out of 118), methylation/demethylation (35 out of 118), glucuronidation (14 out of 118), hydration/dehydration (8 out of 118), and hydrogenation/dehydrogenation (11 out of 118) might be the main metabolic pathways of danshen in vivo. The metabolic pathway for phenolic acids was mainly methylation/demethylation (11 out of 33), while tanshinones mostly showed hydroxylation (31 out of 85) and methylation/demethylation (19 out of 85). Hydrogenation, sulfation, acetylation, and glutathione conjugation were found to be the possible metabolic pathways of danshen. This research provided a comprehensive in vitro chemical profile and in vivo metabolic profile of danshen after oral administration, which could be useful in research on the quality control and pharmacology of danshen. 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The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time-of-flight mass spectrometry Compliance with ethics guidelines Huan-huan PANG, Mei-fang JIANG, Qin-hui WANG, Xiao-ye WANG, Wei GAO, Zhi-hao TIAN, and Jian-mei HUANG declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1700105 Project supported by the Ministry of Science and Technology of China (No. 2011ZX09201-201-22) 2017-02-25 2017-06-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 19