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Jiajia Liu1 Haimei Wu2 Feng Zheng1 Wenyuan Liu1,3 Feng Feng2 ∗ Ning Xie4 1 Department
of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, China 2 Department of Natural Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang, Nanjing, China 3 Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Tongjiaxiang, Nanjing, China 4 Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China Received April 3, 2014 Revised May 30, 2014 Accepted June 18, 2014
Research Article
Chemical constituents of Meconopsis horridula and their simultaneous quantification by high-performance liquid chromatography coupled with tandem mass spectrometry Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high-performance liquid chromatography with diode array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3-(kaempferol-8-yl)-2,3-epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high-performance liquid chromatography with tandem triple-quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine. Keywords: Chemical constituents / High-performance liquid chromatography / Meconopsis horridula / Simultaneous quantification / Tandem mass spectrometry DOI 10.1002/jssc.201400379
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The Meconopsis genus, known as the “Blue poppy,” includes a total of 54 species, of which 53 are distributed in the Sino-Himalayan region [1]. According to the Tibetan ancient medicinal literature and Flora of Tibet, Meconopsis species might have the capability of clearing heat antitoxicant, relieving cough and asthma, analgesia, anti-inflammation, and protecting liver [2–5]. The presence of alkaloids, flavonoids, and phenylpropanoids has also been substantiated in Meconopsis species by detection or isolation of members of these compound classes [6, 7]. Alkaloids and flavonoids are proved the major active components producing the pharmacological responses [8, 9]. Correspondence: Professor Wenyuan Liu, Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86-25-83271038
Abbreviations: DAD, diode array detector; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; M. horridula, Meconopsis horridula Hook.f.et Thoms C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Meconopsis horridula Hook.f. et Thoms (M. horridula), belonging to Meconopsis genus of the Papaveraceae family, is widely distributed in Qinghai and Tibet [10,11]. For hundreds of years, M. horridula has been used as a traditional Tibetan medicine (Chinese name Ci-Er-En) to clear away heat, relieve pain, mobilize static blood, and is honored as a good therapy for bruises [12]. Modern pharmacological studies revealed that M. horridula had effects of sedative, anti-inflammation, and anti-Shigella [13]. It is also included in some herbal combination remedies, such as Bawei Qinpi Pills, Ershiwuwei Luronghao Pills, and Ganluling Pills. Considering the increasing interest in this promising herbal medicine, the ability to investigate its complex constituents and carry out quantification is important. Until now, no analytical study on multiconstituents of M. horridula has been reported. Several chemical compounds isolated and elucidated by traditional chemical means from M. horridula, M. cambric, and M. robusta have been reported [14, 15]. Due to the complexity of the chemical constituents in M. horridula, routine HPLC cannot provide enough separation and information for structure identification on line. ∗ Additional corresponding author: Professor Feng Feng, E-mail: [email protected]
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Figure 1. Chemical structures of compounds identified in M. horridula.
Hence, in this study, a HPLC with diode array detection (DAD) and ESI Q-TOF-MS method has been developed. Based on the UV, high-resolution MS and the fragmentation data obtained, 40 constituents were identified or tentatively identified (Fig. 1). Among them, 17 compounds are reported in this herbal medicine for the first time. There is a novel compound isolated and confirmed. Besides, a rapid and sensitive HPLC–MS/MS method was established for the simultaneous determination of seven major constituents in M. horridula. The quantitative method was validated and quality parameters were established.
2 Materials and methods 2.1 Chemicals, reagents, and materials HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). HPLC-grade water was purified with a C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Millipore Mill Q-Plus system (Millipore, Bedford, MA, USA). Analytical grade formic acid and ammonium acetate were purchased from Nanjing Chemical Reagent (Nanjing, Jiangsu, China). A M. horridula sample was collected from Tibet in China and authenticated by Professor Feng Feng. A voucher specimen was deposited at the School of Traditional Chinese Medicine, China Pharmaceutical University. A reference standard of quercetin (40) was obtained from the National Institute for Food and Drug Control (Beijing, China). Reference standards of kaempferol-3-O-D-glucopyranoside (30), luteolin-7-O--D-glucopyranoside (32) and apigenin (35) were purchased from yuanye BioTechnology (Shanghai, China). Their purities were all over 98.0%. Reference standards of p-hydroxycinnamic acid (13), luteolin (33), and kaempferide (36) (purities all over 97.0%) were isolated and identified from M. horridula in our laboratory. www.jss-journal.com
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Figure 2. Total ion current chromatograms of M. horridula in positive mode (A) and negative mode (B).
2.2 Preparation of sample solutions The dried samples of M. horridula were ground into powder and passed through a 40 mesh screen. The fine powder (1.0 g) was accurately weighed and transferred to a 100 mL round-bottomed flask, and then 50 mL 80% methanol was added. After accurate weighing, the sample was heated under reflux for 30 min. When the mixture was cooled to room temperature, the same solvent was added for the complement of weightlessness. The solution was then centrifuged at 17 000 rpm for 10 min. Aliquots of the supernatant solution were filtered through a 0.45 m membrane filter before injection. 2.3 Preparation of standard solutions Seven stock solutions were prepared separately by dissolving appropriate amounts of each reference compound in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
methanol. Different volumes of each stock solution were transferred to a volumetric flask to make a mixed stock solution. This solution was further diluted with methanol to obtain seven serial working solutions. All solutions were stored at 4⬚C and filtered through a 0.45 m membrane filter before injection.
2.4 HPLC–DAD–Q-TOF-MS conditions for identification A 6520 Accurate-Mass LC–Q-TOF-MS device (Agilent Technologies, CA, USA) was used for constituent characterization. The Agilent 1200 SL series HPLC device consisted of a degasser, a thermostatted HiP-ALS autosampler, a binary pump Bin Pump SL, and a TCC SL column oven. Samples were separated on a Megres C18 column (250 × 4.6 mm, id, 5 m) from Jiangsu Hanbon Science & Technology (Nanjing, www.jss-journal.com
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Table 1. NMR data of compound 39 (DMSO-d6, ␦, J in Hz)
Position
13 C
2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6
117.5 80.0 191.4 163.5 97.0 167.5 95.2 160.5 98.8 123.7 128.3 114.9 158.7 114.9 128.3 147.5 136.1 176.7 163.9 94.8 164.9 105.3 151.3 106.1 121.4 130.2 115.3 159.6 115.3 130.2
1H
HMBC H-2 , H-6
5.94 (d, J = 2.1) H-6 5.91 (d, J = 2.1) H-8 H-6, H-8 H-3 , H-5 7.28 (d, J = 8.6) 6.78 (d, J = 8.6) 6.78 (d, J = 8.6) 7.28 (d, J = 8.6)
H-2 , H-3 , H-5 , H-6
H-2 , H-6
in the ratio of 63:37 v/v with isocratic elution. Mass spectrometric analysis was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (Thermo Finnigan, CA, USA) equipped with an ESI source. The triple quadrupole MS was set in selected reaction monitoring negative mode. The specific precursor ion and a unique product ion for p-hydroxycinnamic acid (13), kaempferol-3-O--Dglucopyranoside (30), luteolin-7-O--D-glucopy ranoside (32), luteolin (33), apigenin (35), kaempferide (36), and quercetin (40) were at m/z 163.0 → 118.9 (CE 15 eV), 446.9 → 284.9 (CE 28 eV), 446.8 → 254.9 (CE 40 eV), 284.8 → 132.9 (CE 38 eV), 269.0 → 117.0 (CE 38 eV), 298.9 → 283.8 (CE 20 eV), and 300.9 → 285.9 (CE 23 eV), respectively. The spray voltage was set at 4000 V for negative ion detection mode. The source collision-induced dissociation voltage was 10 V. Nitrogen sheath gas and auxiliary gas were set at 35 and 5 psi, respectively. Heated capillary temperature was 350⬚C. Data acquisition was performed by Xcalibur 1.3 software (Thermo Finnigan).
H-6
2.6 Validation of method
6.69 (s) H-6
H-6 H-3 , H-5 8.14 (d, J = 8.8) 6.93 (d, J = 8.8)
H-2 , H-3 , H-5 , H-6
6.93 (d, J = 8.8) 8.14 (d, J = 8.8)
Jiangsu, China). The mobile phase consisted of methanol (A) and 0.5% formic acid (containing 5 mM ammonium acetate) (B). The gradient elution program was as follows: 0–15 min, 5–12% A; 15–18 min, 12–14% A; 18–48 min, 14–25% A; 48– 50 min, 25–31% A; 50–100 min, 31–38% A; 100–104 min, 38–50% A; 104–130 min, 50–100% A. The flow rate was 1.0 mL/min. The column temperature was set at 30⬚C. Aliquots of 10 L were injected in sequence for analysis. The eluent from the column was introduced into the Q-TOFMS equipped via an ESI source. The optimized MS operating conditions were as follows: negative- and positive-ionization mode, scan spectra from m/z 100–1700, drying gas flow rate of 8.0 L/min, drying gas temperature of 325⬚C, nebulizer pressure of 40 psi, capillary voltage of 4000 V, skimmer of 65 V, fragmentor voltage of 120 V, and collision energy of 35 V. Data were processed using MassHunter Workstation Data Acquisition Software Ver. A.01.00 (Agilent Technologies).
2.5 HPLC–MS/MS conditions for simultaneous quantification HPLC parameters were as described in Section 2.4, except the mobile phase consisted of methanol and 0.1% v/v formic acid C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The calibration curve was constructed by plotting the peak area versus the corresponding concentration. The LOD and LOQ were determined at S/N ratio of 3 and 10, respectively. Working solution was diluted to a certain concentration to detect the LOD and LOQ. Intraday and interday variations were determined for the precision of the established method. For intraday variability test, working solutions of three concentration levels (high, middle, and low) were injected in triplicate in a single day, while for interday variability, the same was performed for three consecutive days. The stability of sample solution was evaluated at room temperature by analyzing the same sample at 0, 2, 4, 8, 12, and 24 h. A recovery test was performed to assess the accuracy. Three concentration levels of reference substances (approximately equivalent to 80, 100, and 120% levels of target compounds) were added with two parallels at each level and analyzed in the same way [16].
3 Results and discussion 3.1 Optimization of sample preparation and HPLC conditions To achieve an effective extraction of analyte, several factors were optimized, including extraction method (ultrasonication, hot reflux, soaking at room temperature, and Soxhlet extraction), extraction solvents (30, 50, 80, and 100% methanol), extraction time (10, 20, 30, 45, 60, or 90 min), and sampleto-solvent ratio (1:10, 1:30, 1:50, and 1:70). The optimized preparation method was to extract 1.0 g powder with 50 mL of 80% methanol in a heated reflux device for 30 min. HPLC parameters were also investigated. Different C18 columns (Megres C18 , Intertsil ODS-SP, and Symmetry C18 ), different column temperatures (25, 30 and 35⬚C), and www.jss-journal.com
tR (min)
5.675 7.317 28.061 30.059 36.233 36.959 38.943 41.467 42.667
52.384 53.505 55.419 55.536 55.979 58.964 60.173 63.013 64.381 65.028 68.093
68.979
71.021
Peak no.
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
21
22
330
272, 338
278 275 290, 318 282, 308 278 280 295 288 268, 352 265, 348 292
220, 280 223, 280 275 280 250, 310 320 280 290, 320 275
UV max (nm)
356.1498[M+H]+
324.1245[M+H]+
370.1644[M+H]+ 342.1700[M+H]+ 328.1542[M+H]+ 163.0398[M−H]− 326.1384[M+H]+ 147.1739[M]+ 396.1434[M]+ 354.1335[M+H]+ 356.1397[M+H]+ 625.1382[M−H]− 340.1549[M+H]+
191.0196[M−H]− 147.0291[M−H]− 341.0879[M−H]− 381.2386[M+H]+ 342.1701[M]+ 325.0929[M−H]− 177.0197[M−H]− 179.0339[M−H]− 328.1532[M+H]+
Experiment mass (m/z)
324.1600; 204.1002; 190.0851 296.1052; 285.1127; 280.1070 178.0853; 163.0614; 151.0748 119.04898 311.1159; 295.0986; 178.0850; 163.0623; 119.0477 131.0487; 103.0541 381.1134; 366.1341; 335.1158; 202.1145 275.0696; 206.0802; 188.0701; 149.0588 354.1122; 340.1228; 189.0694 301.0276; 271.0246 309.1121; 297.1129; 278.0943; 263.0707; 251.1054; 192.0835; 149.0592 309.1093; 294.0953; 276.0935; 266.1113; 176.0707; 149.0600 354.1233; 340.1157; 295.0971
111.0078 103.0168 179.0344 364.2026; 247.0855 297.1095; 282.0874; 265.0857; 237.0893 117.0338; 145.0293 132.0186; 105.0366 135.0448 297.1104; 282.0874
MS/MS fragment ions (m/z)
Table 2. Compounds identified in Meconopsis horridula Hook.f.et Thoms
−1.43
−4.47
−0.07 1.01 0.34 1.57 0.01 0.56 1.98 0.21 1.30 −1.12 −3.87
0.83 3.23 −0.21 1.95 −0.15 0.94 −1.81 3.54 3.39
Error (ppm)
C20 H21 NO5
C19 H17 NO4
C21 H23 NO5 C20 H23 NO4 C19 H21 NO4 C9 H8 O3 C19 H19 NO4 C9 H9 NO C22 H22 NO6 C20 H19 NO5 C20 H21 NO5 C27 H30 O17 C20 H21 NO4
C6 H8 O7 C9 H8 O2 C15 H18 O9 C21 H18 NO6 C20 H24 NO4 C15 H18 O8 C9 H6 O4 C9 H8 O4 C19 H21 NO4
Formula
Amurensinine N-oxide B
Stylopine
Citric acid Cinnamic acid Caffeic acid glucoside Simplicifolianine Magnoflorine p-Hydroxycinnamic acid esters 6,7-Dihydroxy-coumarin Caffeic acid 6-Methoxy-17-methyl-2,3[methylenebis(oxy)]morphinan-5-en-7-one Cryptopine O-Methylflavlnantine Scoulerine p-Hydroxycinnamic acid Amurine Cinnamamide Alborine Protopine Amurensinine N-oxide A Quercetin3-O-gentiobioside Sinactine
Identification
[36]
[29]
[30] [29] [36] [18] [29]
[30]
[29] [30] [29]
[38] [30]
[37] [37] [39] [27] [30]
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tR (min)
74.371 74.695
74.852
84.438 85.147 89.378
91.435
102.288
102.887
109.048
113.434 115.389 116.562 117.052 117.161 121.252 125.718
130.741
Peak no.
23 24
25
26 27 28
29
30
31
32
33 34 35 36 37 38 39
40
Table 2. Continued
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
305.0625; 292.0957; 277.0714; 262.0881; 234.0874 285.0395; 255.0303
301.0275; 271.0300; 151.0030;178.9949
301.0354; 271.0191; 255.0266 301.0365; 271.0255; 255.0283 285.0412; 155.0302
147.0434; 119.0484 285.0335 329.1076 285.0410 151.0030; 133.0297 257.0445; 229.0480; 185.0558; 169.0650; 151.2254 243.0645; 153.0166; 119.0482 284.0342; 256.0369; 227.0358; 185.0199; 151.0021 313.0338; 299.0208; 271.0240; 243.0295 284.0323; 255.0304 437.3220; 367.3660; 296.2934 285.1502 187.0761; 147.0452; 135.0448
625.1419[M−H]−
463.0874[M−H]− 463.0883[M−H]− 609.1457[M−H]−
284.1279[M+H]+ 447.0940[M−H]− 491.1198[M−H]− 447.0926[M−H]− 285.0423[M−H]− 285.0411[M−H]− 271.0567[M+H]+ 299.0565[M−H]− 329.0669[M−H]− 463.1026[M−H]− 571.4950[M+H]+ 303.2526 [M+H]+ 301.1451 [M−H]−
MS/MS fragment ions (m/z)
320.0915[M]+ 609.1462[M−H]−
Experiment mass (m/z)
0.96 −0.01
−1.77 −2.11 2.74 −1.52 −0.81 0.59 −0.93
1.6
1.12
−1.54
2.31
−0.11 −0.11 0.71
1.59
−1.33 −0.01
Error (ppm)
C15 H10 O7
C15 H10 O6 C15 H10 O6 C15 H10 O5 C16 H12 O6 C17 H14 O7 C25 H20 O9 C30 H18 O12
C21 H20 O11
C23 H24 O12
C21 H20 O11
C17 H17 NO3
C21 H20 O12 C21 H20 O12 C27 H30 O16
C27 H30 O17
C19 H14 NO4 C27 H30 O16
Formula
Coptisine Quercetin-3-O-␣-Lrhamnopyranosyl (1→2)--D-glucopyranoside Quercetin-3-O-[-Dgalactopyranosyl (1→2)]--D-glucopyranoside Hyperoside Isoquercetin Kaempferol-3-O-[-Dglucopyranosyl(1→2)]--Dglucopyranoside N-p-hydroxyl-transcoumaroyltyramine Kaempferol-3-O--Dglucopyranoside Tricin-7-O--Dglucopyranoside Luteolin-7-O--Dglucopyranoside Luteolin Kaempferol Apigenin Kaempferide Tricin Hydnocarpin 3-(Kaempferol-8-yl)-2,3epoxyflavanone Quercetin
Identification
[39]
[38] [26] [16]
[35] [36] [37]
[34]
[33]
[32]
[34]
[18] [18] [18]
[18]
[29] [18]
[Literature]
J. Liu et al.
282 283
266, 348 270 268, 335 270, 348 272 278 288
272, 328
268, 352
265, 350
272, 352
355 368 265, 350
270, 328
278 268, 312
UV max (nm)
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Figure 3. The characteristic MS/MS fragment pathways of six protoberberine isoquinoline alkaloids (4, 12, 16, 20, 21, and 23) (A), and the two protopine isoquinoline alkaloids (10, 17) (B).
different detection wavelengths (230, 254, 284, 320, and 360 nm) were compared. The results illustrated that a Megres C18 column at 30⬚C with detection wavelength of 284 nm can achieve the best separation efficiency for the multi-ingredient determination in this herb. Furthermore, the mobile phase was optimized with different systems (acetonitrile and methanol), a variety of modifiers (formic acid, acetic acid, and ammonium acetate) and various sample-to-solvent ratios. As a result, a mixture of methanol and water was used. In order to achieve a better ionization of analytes and good repeatability of chromatogram, ammonium acetate was added in mobile phase. Finally, mobile phase consisting of methanol (A) and 0.5% formic acid (containing 5 mM ammonium acetate) (B) was employed. Due to the complexity of sample, a detailed gradient program (as mentioned Section 2.4) was employed. The total ion current chromatogram of M. horridula test solution is shown in Fig. 2.
3.2 Identification of chemical constituents 3.2.1 Isolation and structure elucidation The isolation and structure elucidation on the chemical constituents of M. horridula had been performed in advanced in our lab. As a result, seven flavonoids and one phenylpropanoid, namely, p-hydroxycinnamic acid (13), kaempferol-3-O--D-glucopyranoside (30), luteolin-7-O--D-glucopyranoside (32), luteolin (33), apigenin (35), kaempferide (36), 3-(kaempferol-8-yl)2,3-epoxyflavanone(39), and quercetin (40) were isolated and identified. It is worth mentioning that 3-(kaempferol8-yl)-2,3-epoxyflavanone (39) is a novel naturally occurring biflavone. This compound was obtained as a yellow powder of C30 H18 O12 ([M+H]+ at m/z 571.4950). 1 H and 13 C NMR spectra presented signals for four aromatic rings, and suggested that the compound had a biflavone skeleton. Combining with analyses of heteronuclear single quantum C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
correlation (HSQC) and heteronuclear multiple bond correlation (HMBC), compound 39 is confirmed to be 3-(kaempferol-8-yl)-2,3-epoxyflavanone [17]. Its NMR data is shown in Table 1. HSQC and HMBC data are shown in the Supporting Information. 3.2.2 Identification of constituents by HPLC–DAD–Q-TOF-MS A total of 40 constituents (Fig. 1) were identified or tentatively identified, including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids. Among them, 17 compounds (1, 2, 3, 4, 6, 7, 8, 12, 13, 19, 20, 21, 24, 25, 26, 27, and 39) were identified in this herbal medicine for the first time. Their UV and MS data are shown in Table 2. Compounds 6, 7, 13, 15, 17, 19, 24, 25, 28, 30, 31, 32, 33, 34, 35, 36, 37, 39, and 40 were unambiguously identified by comparing MS/MS data and retention time with those of the reference standards. Other compounds were tentatively inferred by their fragmentation pathways and chromatographic behavior. Specific MS fragmentation data of the 40 compounds are shown in the Supporting Information. 3.2.2.1 Identification of flavonoids Compounds 26 and 27 yielded the same quasimolecular ions at m/z 463 and corresponding product ions at m/z 301, 271, 255, and 179 due to loss of one glucosyl group from quercetin. Moreover, compound 26 was eluted earlier in HPLC system than 27 [18]. Hence, compounds 26 and 27 were identified as hyperoside and isoquercetin, respectively. Compound 38 was supposed as hydnocarpin by comparison with the report of Meconopsis (M. quintuplinervia) [19]. 3.2.2.2 Identification of alkaloids Alkaloids showed ions corresponding to [M+H]+ in positive mode and fragments of [M+H–18]+ , [M+H–28]+ , and [M+H–16]+ due to the loss of H2 O, CO, and CH4 , respectively. Compounds 4, 12, 16, 20, 21, and 23 showed skeleton www.jss-journal.com
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5.56 101.32 4.20 4.81 22.58 0.9/5 4.32 3.41 0.9991
4.53 100.87 3.71 2.69 30.01 1.2/3 4.85 2.22 0.9990
6.71 97.29 4.27 0.89 149.11 4/10 5.23 4.64 0.9975
6.53 105.24 4.92 2.72 134.84 4/10 5.14 0.9995 0.40–4..04
0.69
107.17 4.29 1.61 14.03 2/10 4.69 0.9964 0.10–1.00
3.45
5.18 102.98 4.84 2.20 630.69 6/24 4.33 2.42 0.9959 2.38–23.8
3.82 2.06 54.48 5/16 5.28 3.70
0.09–0.85 Quercetin
0.12–1.16 Kaempferide
0.41–4.08 Apigenin
Kaempferol-3-O-D-glucopyranoside Luteolin-7-O--Dglucopyranoside Luteolin
0.16–1.61
y = 86486x + 1162.9 y = 30487x + 16621 y = 262658x + 3194.3 y = 136424 x −696.36 y = 114165x + 5687 y = 576434x −3599.3 y = 18533x − 347.89 P-hydroxycinnamic acid
0.9971
Content (g /g)
RSD (%)
4.17
106.99
RSD (%) Mean
Recovery (%; n = 9) Stability RSD (%) M. horridula LOD /LOQ (ng/mL)
Intraday precision (RSD%) Interday precision (RSD%) R2 Linear range (g/mL) Regressive equation
3.2.2.3 Identification of phenylpropanoids The characteristic ions for phenolic compounds were the product ions due to the loss of CO2 , CO, and H2 O groups from their quasimolecular ions [29,30]. Compound 3 exhibited the [M–H]− ion at m/z 341, which is 162 Da heavier than that of caffeic acid. Therefore, compound 3 was identified as caffeic acid glucoside [31]. Compound 6 yielded a [M−H]− ion at m/z 325, [M−H–Glc]− ion at m/z 117 and [M−H–Glc–CO]− ion
Analyte
cracking of C-7, C-8 and C-13, C-14, which was the characteristic of protoberberine isoquinoline alkaloids [20] (Fig. 3A). Compounds 10 and 17 showed the cracking of C-7, C-8 and C13, C-14, which was the representative pathway of protopine isoquinoline alkaloids [20] (Fig. 3B). Compounds 12, 16, 21, and 23 presented a loss of C9 H8 O2 , which arises from a RDA fragmentation. Compound 4 generated quasimolecular ion [M+H]+ at m/z 381 and product ions at m/z 364, 336, and 247 due to serious losses of OH, CO, and C8 H5 O2 . Compound 4 was supposed as simplicifolianine [5]. Compound 12 yielded predominant [M+H]+ at m/z 328 and a major product ion at m/z, which may be explained by a retro-Diels–Alder (RDA) fragmentation. Compound 16 generated an obvious quasimolecular ion [M+H]+ at m/z 396. The dominant product ion at m/z 202 was assigned to [M-C11 H14 O3 ]+ by RDA loss. Series losses of CH3 , CH3 O, and C11 H14 O3 produced three ions at m/z 381, 366, 335, and 202, respectively. Similarly, compound 20 exhibited [M+H]+ ion at m/z 340 and showed series characteristic ions at m/z 309 [M+H–CH3 O]+ , 297 [M+H– H2 O–CH3 ]+ , 192 [M+H–C9 H8 O2 ]+ , and 149 [C9 H8 O2 +H]+ . As reported [20–22], compounds 12, 16, 20, 21, and 23 were tentatively assigned as scoulerine, alborine, sinactine, stylopine, and coptisine, respectively. Compound 5 generated a singly charged ion at m/z 342.1701 [M]+ and five major product ions at m/z 297.1095[M–(CH3 )2 NH]+ , 282.0874[M–(CH3 )2 NH– CH3 ]+ , 265.0857 [M–(CH3 )2 NH–CH3 –OH]+ , 237.0893 [M–(CH3 )2 NH–CH3 –OH–CO]+ , 222.0667 [M–(CH3 )2 NH– 2CH3 –OH–CO]+ . Compound 14 was deduced as amurine by their quasimolecular ions and corresponding product ions produced by loss of C9 H8 O2 . With reference to to reports [22–25], compounds 5 and 14 were tentatively identified as magnoflorine and amurine. Compound 29 exhibited [M+H]+ ion at m/z 284, [M+H–137]+ ion at m/z 147 and [M+H–137–CO]+ ion at m/z 119, and therefore was assigned as N-p-hydroxyl-trans-coumaroyltyramine [26]. Two epimeric N-oxides, amurensinine N-oxide A (18) and amurensinine N-oxide B (22), were identified. The two isomers both showed [M+H]+ ion at m/z 356 and [M+H–2]+ ion at m/z 354 due to dehydrogenation. They both showed product ions of [M+H–16]+ , which was the characteristic for N-oxides, and amurensinine skeleton ions at m/z 354, 340, 295, 206, 189 [27]. The two isomers can be distinguished by the relative response intensity of characteristic ions. The product ion at m/z 189 [M+H–C10 H16 O2 ]+ was stronger for 18 while 295 [M+H–C2 H7 NO]+ was stronger for 22 [28].
3.70
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Table 3. Quantification of seven components by HPLC–MS/MS
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Figure 4. Chromatograms for detection of seven compounds (13, 30, 32, 33, 35, 36, and 40).
at m/z 145. Similarly, compound 7 generated a [M−H]− ion at m/z 177, [M−H–CO2 ]− ion at m/z 132 and [M−H–CO2 – CO]− ion at m/z 105. Compound 15 produced the [M]+ ion at m/z 147, [M−NH2 ]+ ion at m/z 131 and [M−NH2 –CO]+ ion at m/z 103. Compounds 6, 7, and 15 were identified as p-hydroxycinnamic acid esters, 6,7-dihydroxycoumarin and cinnamamide, respectively.
and 149.11 g/g, respectively, followed by p-hydroxycinnamic acid (13), kaempferide (36), quercetin (40), and luteolin-7-O-D-glucopyranoside (32) with contents of 54.48, 30.01, 22.58, and 14.03 g/g, respectively. The total content of flavonol glycosides was higher than that of flavonols.
4 Concluding remarks 3.3 Simultaneous quantification of seven components by HPLC–MS/MS As shown in Table 3, all the analytes showed a good linear regression (R2 > 0.996), which presented that this method was precise and sensitive for the quantitative evaluation. LODs for seven compounds were estimated to be 0.9–6 ng/mL, and the LOQs were 3–24 ng/mL. The intraday and interday precisions (RSDs) were 0.21–5.33 and 2.57–5.94%, respectively. The repeatability and stability of the RSDs were all
J. Sep. Sci. 2014, 37, 2513–2522
Jiajia Liu1 Haimei Wu2 Feng Zheng1 Wenyuan Liu1,3 Feng Feng2 ∗ Ning Xie4 1 Department
of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, China 2 Department of Natural Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang, Nanjing, China 3 Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Tongjiaxiang, Nanjing, China 4 Jiangxi Qingfeng Pharmaceutical Corporation, Ganzhou, China Received April 3, 2014 Revised May 30, 2014 Accepted June 18, 2014
Research Article
Chemical constituents of Meconopsis horridula and their simultaneous quantification by high-performance liquid chromatography coupled with tandem mass spectrometry Meconopsis horridula Hook.f. Thoms has been used as a traditional Tibetan medicine to clear away heat, relieve pain, and mobilize static blood. In this study, a reliable method based on high-performance liquid chromatography with diode array detection and electrospray ionization quadrupole time-of-flight tandem mass spectrometry was established for the identification of components in this herb. A total of 40 compounds (including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids) were identified or tentatively identified. Among them, 17 components were identified in the herb for the first time. Compound 39 appears to be a novel compound, which is confirmed as 3-(kaempferol-8-yl)-2,3-epoxyflavanone by NMR spectroscopy and mass spectrometry. Moreover, seven major constituents were simultaneously quantified by the developed high-performance liquid chromatography with tandem triple-quadrupole mass spectrometry method. The quantitative method was validated and quality parameters were established. The study provides a comprehensive approach for understanding this herbal medicine. Keywords: Chemical constituents / High-performance liquid chromatography / Meconopsis horridula / Simultaneous quantification / Tandem mass spectrometry DOI 10.1002/jssc.201400379
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The Meconopsis genus, known as the “Blue poppy,” includes a total of 54 species, of which 53 are distributed in the Sino-Himalayan region [1]. According to the Tibetan ancient medicinal literature and Flora of Tibet, Meconopsis species might have the capability of clearing heat antitoxicant, relieving cough and asthma, analgesia, anti-inflammation, and protecting liver [2–5]. The presence of alkaloids, flavonoids, and phenylpropanoids has also been substantiated in Meconopsis species by detection or isolation of members of these compound classes [6, 7]. Alkaloids and flavonoids are proved the major active components producing the pharmacological responses [8, 9]. Correspondence: Professor Wenyuan Liu, Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China E-mail: [email protected] Fax: +86-25-83271038
Abbreviations: DAD, diode array detector; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; M. horridula, Meconopsis horridula Hook.f.et Thoms C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Meconopsis horridula Hook.f. et Thoms (M. horridula), belonging to Meconopsis genus of the Papaveraceae family, is widely distributed in Qinghai and Tibet [10,11]. For hundreds of years, M. horridula has been used as a traditional Tibetan medicine (Chinese name Ci-Er-En) to clear away heat, relieve pain, mobilize static blood, and is honored as a good therapy for bruises [12]. Modern pharmacological studies revealed that M. horridula had effects of sedative, anti-inflammation, and anti-Shigella [13]. It is also included in some herbal combination remedies, such as Bawei Qinpi Pills, Ershiwuwei Luronghao Pills, and Ganluling Pills. Considering the increasing interest in this promising herbal medicine, the ability to investigate its complex constituents and carry out quantification is important. Until now, no analytical study on multiconstituents of M. horridula has been reported. Several chemical compounds isolated and elucidated by traditional chemical means from M. horridula, M. cambric, and M. robusta have been reported [14, 15]. Due to the complexity of the chemical constituents in M. horridula, routine HPLC cannot provide enough separation and information for structure identification on line. ∗ Additional corresponding author: Professor Feng Feng, E-mail: [email protected]
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Figure 1. Chemical structures of compounds identified in M. horridula.
Hence, in this study, a HPLC with diode array detection (DAD) and ESI Q-TOF-MS method has been developed. Based on the UV, high-resolution MS and the fragmentation data obtained, 40 constituents were identified or tentatively identified (Fig. 1). Among them, 17 compounds are reported in this herbal medicine for the first time. There is a novel compound isolated and confirmed. Besides, a rapid and sensitive HPLC–MS/MS method was established for the simultaneous determination of seven major constituents in M. horridula. The quantitative method was validated and quality parameters were established.
2 Materials and methods 2.1 Chemicals, reagents, and materials HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). HPLC-grade water was purified with a C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Millipore Mill Q-Plus system (Millipore, Bedford, MA, USA). Analytical grade formic acid and ammonium acetate were purchased from Nanjing Chemical Reagent (Nanjing, Jiangsu, China). A M. horridula sample was collected from Tibet in China and authenticated by Professor Feng Feng. A voucher specimen was deposited at the School of Traditional Chinese Medicine, China Pharmaceutical University. A reference standard of quercetin (40) was obtained from the National Institute for Food and Drug Control (Beijing, China). Reference standards of kaempferol-3-O-D-glucopyranoside (30), luteolin-7-O--D-glucopyranoside (32) and apigenin (35) were purchased from yuanye BioTechnology (Shanghai, China). Their purities were all over 98.0%. Reference standards of p-hydroxycinnamic acid (13), luteolin (33), and kaempferide (36) (purities all over 97.0%) were isolated and identified from M. horridula in our laboratory. www.jss-journal.com
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Figure 2. Total ion current chromatograms of M. horridula in positive mode (A) and negative mode (B).
2.2 Preparation of sample solutions The dried samples of M. horridula were ground into powder and passed through a 40 mesh screen. The fine powder (1.0 g) was accurately weighed and transferred to a 100 mL round-bottomed flask, and then 50 mL 80% methanol was added. After accurate weighing, the sample was heated under reflux for 30 min. When the mixture was cooled to room temperature, the same solvent was added for the complement of weightlessness. The solution was then centrifuged at 17 000 rpm for 10 min. Aliquots of the supernatant solution were filtered through a 0.45 m membrane filter before injection. 2.3 Preparation of standard solutions Seven stock solutions were prepared separately by dissolving appropriate amounts of each reference compound in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
methanol. Different volumes of each stock solution were transferred to a volumetric flask to make a mixed stock solution. This solution was further diluted with methanol to obtain seven serial working solutions. All solutions were stored at 4⬚C and filtered through a 0.45 m membrane filter before injection.
2.4 HPLC–DAD–Q-TOF-MS conditions for identification A 6520 Accurate-Mass LC–Q-TOF-MS device (Agilent Technologies, CA, USA) was used for constituent characterization. The Agilent 1200 SL series HPLC device consisted of a degasser, a thermostatted HiP-ALS autosampler, a binary pump Bin Pump SL, and a TCC SL column oven. Samples were separated on a Megres C18 column (250 × 4.6 mm, id, 5 m) from Jiangsu Hanbon Science & Technology (Nanjing, www.jss-journal.com
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Table 1. NMR data of compound 39 (DMSO-d6, ␦, J in Hz)
Position
13 C
2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6
117.5 80.0 191.4 163.5 97.0 167.5 95.2 160.5 98.8 123.7 128.3 114.9 158.7 114.9 128.3 147.5 136.1 176.7 163.9 94.8 164.9 105.3 151.3 106.1 121.4 130.2 115.3 159.6 115.3 130.2
1H
HMBC H-2 , H-6
5.94 (d, J = 2.1) H-6 5.91 (d, J = 2.1) H-8 H-6, H-8 H-3 , H-5 7.28 (d, J = 8.6) 6.78 (d, J = 8.6) 6.78 (d, J = 8.6) 7.28 (d, J = 8.6)
H-2 , H-3 , H-5 , H-6
H-2 , H-6
in the ratio of 63:37 v/v with isocratic elution. Mass spectrometric analysis was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer (Thermo Finnigan, CA, USA) equipped with an ESI source. The triple quadrupole MS was set in selected reaction monitoring negative mode. The specific precursor ion and a unique product ion for p-hydroxycinnamic acid (13), kaempferol-3-O--Dglucopyranoside (30), luteolin-7-O--D-glucopy ranoside (32), luteolin (33), apigenin (35), kaempferide (36), and quercetin (40) were at m/z 163.0 → 118.9 (CE 15 eV), 446.9 → 284.9 (CE 28 eV), 446.8 → 254.9 (CE 40 eV), 284.8 → 132.9 (CE 38 eV), 269.0 → 117.0 (CE 38 eV), 298.9 → 283.8 (CE 20 eV), and 300.9 → 285.9 (CE 23 eV), respectively. The spray voltage was set at 4000 V for negative ion detection mode. The source collision-induced dissociation voltage was 10 V. Nitrogen sheath gas and auxiliary gas were set at 35 and 5 psi, respectively. Heated capillary temperature was 350⬚C. Data acquisition was performed by Xcalibur 1.3 software (Thermo Finnigan).
H-6
2.6 Validation of method
6.69 (s) H-6
H-6 H-3 , H-5 8.14 (d, J = 8.8) 6.93 (d, J = 8.8)
H-2 , H-3 , H-5 , H-6
6.93 (d, J = 8.8) 8.14 (d, J = 8.8)
Jiangsu, China). The mobile phase consisted of methanol (A) and 0.5% formic acid (containing 5 mM ammonium acetate) (B). The gradient elution program was as follows: 0–15 min, 5–12% A; 15–18 min, 12–14% A; 18–48 min, 14–25% A; 48– 50 min, 25–31% A; 50–100 min, 31–38% A; 100–104 min, 38–50% A; 104–130 min, 50–100% A. The flow rate was 1.0 mL/min. The column temperature was set at 30⬚C. Aliquots of 10 L were injected in sequence for analysis. The eluent from the column was introduced into the Q-TOFMS equipped via an ESI source. The optimized MS operating conditions were as follows: negative- and positive-ionization mode, scan spectra from m/z 100–1700, drying gas flow rate of 8.0 L/min, drying gas temperature of 325⬚C, nebulizer pressure of 40 psi, capillary voltage of 4000 V, skimmer of 65 V, fragmentor voltage of 120 V, and collision energy of 35 V. Data were processed using MassHunter Workstation Data Acquisition Software Ver. A.01.00 (Agilent Technologies).
2.5 HPLC–MS/MS conditions for simultaneous quantification HPLC parameters were as described in Section 2.4, except the mobile phase consisted of methanol and 0.1% v/v formic acid C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The calibration curve was constructed by plotting the peak area versus the corresponding concentration. The LOD and LOQ were determined at S/N ratio of 3 and 10, respectively. Working solution was diluted to a certain concentration to detect the LOD and LOQ. Intraday and interday variations were determined for the precision of the established method. For intraday variability test, working solutions of three concentration levels (high, middle, and low) were injected in triplicate in a single day, while for interday variability, the same was performed for three consecutive days. The stability of sample solution was evaluated at room temperature by analyzing the same sample at 0, 2, 4, 8, 12, and 24 h. A recovery test was performed to assess the accuracy. Three concentration levels of reference substances (approximately equivalent to 80, 100, and 120% levels of target compounds) were added with two parallels at each level and analyzed in the same way [16].
3 Results and discussion 3.1 Optimization of sample preparation and HPLC conditions To achieve an effective extraction of analyte, several factors were optimized, including extraction method (ultrasonication, hot reflux, soaking at room temperature, and Soxhlet extraction), extraction solvents (30, 50, 80, and 100% methanol), extraction time (10, 20, 30, 45, 60, or 90 min), and sampleto-solvent ratio (1:10, 1:30, 1:50, and 1:70). The optimized preparation method was to extract 1.0 g powder with 50 mL of 80% methanol in a heated reflux device for 30 min. HPLC parameters were also investigated. Different C18 columns (Megres C18 , Intertsil ODS-SP, and Symmetry C18 ), different column temperatures (25, 30 and 35⬚C), and www.jss-journal.com
tR (min)
5.675 7.317 28.061 30.059 36.233 36.959 38.943 41.467 42.667
52.384 53.505 55.419 55.536 55.979 58.964 60.173 63.013 64.381 65.028 68.093
68.979
71.021
Peak no.
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
21
22
330
272, 338
278 275 290, 318 282, 308 278 280 295 288 268, 352 265, 348 292
220, 280 223, 280 275 280 250, 310 320 280 290, 320 275
UV max (nm)
356.1498[M+H]+
324.1245[M+H]+
370.1644[M+H]+ 342.1700[M+H]+ 328.1542[M+H]+ 163.0398[M−H]− 326.1384[M+H]+ 147.1739[M]+ 396.1434[M]+ 354.1335[M+H]+ 356.1397[M+H]+ 625.1382[M−H]− 340.1549[M+H]+
191.0196[M−H]− 147.0291[M−H]− 341.0879[M−H]− 381.2386[M+H]+ 342.1701[M]+ 325.0929[M−H]− 177.0197[M−H]− 179.0339[M−H]− 328.1532[M+H]+
Experiment mass (m/z)
324.1600; 204.1002; 190.0851 296.1052; 285.1127; 280.1070 178.0853; 163.0614; 151.0748 119.04898 311.1159; 295.0986; 178.0850; 163.0623; 119.0477 131.0487; 103.0541 381.1134; 366.1341; 335.1158; 202.1145 275.0696; 206.0802; 188.0701; 149.0588 354.1122; 340.1228; 189.0694 301.0276; 271.0246 309.1121; 297.1129; 278.0943; 263.0707; 251.1054; 192.0835; 149.0592 309.1093; 294.0953; 276.0935; 266.1113; 176.0707; 149.0600 354.1233; 340.1157; 295.0971
111.0078 103.0168 179.0344 364.2026; 247.0855 297.1095; 282.0874; 265.0857; 237.0893 117.0338; 145.0293 132.0186; 105.0366 135.0448 297.1104; 282.0874
MS/MS fragment ions (m/z)
Table 2. Compounds identified in Meconopsis horridula Hook.f.et Thoms
−1.43
−4.47
−0.07 1.01 0.34 1.57 0.01 0.56 1.98 0.21 1.30 −1.12 −3.87
0.83 3.23 −0.21 1.95 −0.15 0.94 −1.81 3.54 3.39
Error (ppm)
C20 H21 NO5
C19 H17 NO4
C21 H23 NO5 C20 H23 NO4 C19 H21 NO4 C9 H8 O3 C19 H19 NO4 C9 H9 NO C22 H22 NO6 C20 H19 NO5 C20 H21 NO5 C27 H30 O17 C20 H21 NO4
C6 H8 O7 C9 H8 O2 C15 H18 O9 C21 H18 NO6 C20 H24 NO4 C15 H18 O8 C9 H6 O4 C9 H8 O4 C19 H21 NO4
Formula
Amurensinine N-oxide B
Stylopine
Citric acid Cinnamic acid Caffeic acid glucoside Simplicifolianine Magnoflorine p-Hydroxycinnamic acid esters 6,7-Dihydroxy-coumarin Caffeic acid 6-Methoxy-17-methyl-2,3[methylenebis(oxy)]morphinan-5-en-7-one Cryptopine O-Methylflavlnantine Scoulerine p-Hydroxycinnamic acid Amurine Cinnamamide Alborine Protopine Amurensinine N-oxide A Quercetin3-O-gentiobioside Sinactine
Identification
[36]
[29]
[30] [29] [36] [18] [29]
[30]
[29] [30] [29]
[38] [30]
[37] [37] [39] [27] [30]
[Literature]
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tR (min)
74.371 74.695
74.852
84.438 85.147 89.378
91.435
102.288
102.887
109.048
113.434 115.389 116.562 117.052 117.161 121.252 125.718
130.741
Peak no.
23 24
25
26 27 28
29
30
31
32
33 34 35 36 37 38 39
40
Table 2. Continued
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305.0625; 292.0957; 277.0714; 262.0881; 234.0874 285.0395; 255.0303
301.0275; 271.0300; 151.0030;178.9949
301.0354; 271.0191; 255.0266 301.0365; 271.0255; 255.0283 285.0412; 155.0302
147.0434; 119.0484 285.0335 329.1076 285.0410 151.0030; 133.0297 257.0445; 229.0480; 185.0558; 169.0650; 151.2254 243.0645; 153.0166; 119.0482 284.0342; 256.0369; 227.0358; 185.0199; 151.0021 313.0338; 299.0208; 271.0240; 243.0295 284.0323; 255.0304 437.3220; 367.3660; 296.2934 285.1502 187.0761; 147.0452; 135.0448
625.1419[M−H]−
463.0874[M−H]− 463.0883[M−H]− 609.1457[M−H]−
284.1279[M+H]+ 447.0940[M−H]− 491.1198[M−H]− 447.0926[M−H]− 285.0423[M−H]− 285.0411[M−H]− 271.0567[M+H]+ 299.0565[M−H]− 329.0669[M−H]− 463.1026[M−H]− 571.4950[M+H]+ 303.2526 [M+H]+ 301.1451 [M−H]−
MS/MS fragment ions (m/z)
320.0915[M]+ 609.1462[M−H]−
Experiment mass (m/z)
0.96 −0.01
−1.77 −2.11 2.74 −1.52 −0.81 0.59 −0.93
1.6
1.12
−1.54
2.31
−0.11 −0.11 0.71
1.59
−1.33 −0.01
Error (ppm)
C15 H10 O7
C15 H10 O6 C15 H10 O6 C15 H10 O5 C16 H12 O6 C17 H14 O7 C25 H20 O9 C30 H18 O12
C21 H20 O11
C23 H24 O12
C21 H20 O11
C17 H17 NO3
C21 H20 O12 C21 H20 O12 C27 H30 O16
C27 H30 O17
C19 H14 NO4 C27 H30 O16
Formula
Coptisine Quercetin-3-O-␣-Lrhamnopyranosyl (1→2)--D-glucopyranoside Quercetin-3-O-[-Dgalactopyranosyl (1→2)]--D-glucopyranoside Hyperoside Isoquercetin Kaempferol-3-O-[-Dglucopyranosyl(1→2)]--Dglucopyranoside N-p-hydroxyl-transcoumaroyltyramine Kaempferol-3-O--Dglucopyranoside Tricin-7-O--Dglucopyranoside Luteolin-7-O--Dglucopyranoside Luteolin Kaempferol Apigenin Kaempferide Tricin Hydnocarpin 3-(Kaempferol-8-yl)-2,3epoxyflavanone Quercetin
Identification
[39]
[38] [26] [16]
[35] [36] [37]
[34]
[33]
[32]
[34]
[18] [18] [18]
[18]
[29] [18]
[Literature]
J. Liu et al.
282 283
266, 348 270 268, 335 270, 348 272 278 288
272, 328
268, 352
265, 350
272, 352
355 368 265, 350
270, 328
278 268, 312
UV max (nm)
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Figure 3. The characteristic MS/MS fragment pathways of six protoberberine isoquinoline alkaloids (4, 12, 16, 20, 21, and 23) (A), and the two protopine isoquinoline alkaloids (10, 17) (B).
different detection wavelengths (230, 254, 284, 320, and 360 nm) were compared. The results illustrated that a Megres C18 column at 30⬚C with detection wavelength of 284 nm can achieve the best separation efficiency for the multi-ingredient determination in this herb. Furthermore, the mobile phase was optimized with different systems (acetonitrile and methanol), a variety of modifiers (formic acid, acetic acid, and ammonium acetate) and various sample-to-solvent ratios. As a result, a mixture of methanol and water was used. In order to achieve a better ionization of analytes and good repeatability of chromatogram, ammonium acetate was added in mobile phase. Finally, mobile phase consisting of methanol (A) and 0.5% formic acid (containing 5 mM ammonium acetate) (B) was employed. Due to the complexity of sample, a detailed gradient program (as mentioned Section 2.4) was employed. The total ion current chromatogram of M. horridula test solution is shown in Fig. 2.
3.2 Identification of chemical constituents 3.2.1 Isolation and structure elucidation The isolation and structure elucidation on the chemical constituents of M. horridula had been performed in advanced in our lab. As a result, seven flavonoids and one phenylpropanoid, namely, p-hydroxycinnamic acid (13), kaempferol-3-O--D-glucopyranoside (30), luteolin-7-O--D-glucopyranoside (32), luteolin (33), apigenin (35), kaempferide (36), 3-(kaempferol-8-yl)2,3-epoxyflavanone(39), and quercetin (40) were isolated and identified. It is worth mentioning that 3-(kaempferol8-yl)-2,3-epoxyflavanone (39) is a novel naturally occurring biflavone. This compound was obtained as a yellow powder of C30 H18 O12 ([M+H]+ at m/z 571.4950). 1 H and 13 C NMR spectra presented signals for four aromatic rings, and suggested that the compound had a biflavone skeleton. Combining with analyses of heteronuclear single quantum C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
correlation (HSQC) and heteronuclear multiple bond correlation (HMBC), compound 39 is confirmed to be 3-(kaempferol-8-yl)-2,3-epoxyflavanone [17]. Its NMR data is shown in Table 1. HSQC and HMBC data are shown in the Supporting Information. 3.2.2 Identification of constituents by HPLC–DAD–Q-TOF-MS A total of 40 constituents (Fig. 1) were identified or tentatively identified, including 17 flavonoids, 15 alkaloids, and eight phenylpropanoids. Among them, 17 compounds (1, 2, 3, 4, 6, 7, 8, 12, 13, 19, 20, 21, 24, 25, 26, 27, and 39) were identified in this herbal medicine for the first time. Their UV and MS data are shown in Table 2. Compounds 6, 7, 13, 15, 17, 19, 24, 25, 28, 30, 31, 32, 33, 34, 35, 36, 37, 39, and 40 were unambiguously identified by comparing MS/MS data and retention time with those of the reference standards. Other compounds were tentatively inferred by their fragmentation pathways and chromatographic behavior. Specific MS fragmentation data of the 40 compounds are shown in the Supporting Information. 3.2.2.1 Identification of flavonoids Compounds 26 and 27 yielded the same quasimolecular ions at m/z 463 and corresponding product ions at m/z 301, 271, 255, and 179 due to loss of one glucosyl group from quercetin. Moreover, compound 26 was eluted earlier in HPLC system than 27 [18]. Hence, compounds 26 and 27 were identified as hyperoside and isoquercetin, respectively. Compound 38 was supposed as hydnocarpin by comparison with the report of Meconopsis (M. quintuplinervia) [19]. 3.2.2.2 Identification of alkaloids Alkaloids showed ions corresponding to [M+H]+ in positive mode and fragments of [M+H–18]+ , [M+H–28]+ , and [M+H–16]+ due to the loss of H2 O, CO, and CH4 , respectively. Compounds 4, 12, 16, 20, 21, and 23 showed skeleton www.jss-journal.com
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5.56 101.32 4.20 4.81 22.58 0.9/5 4.32 3.41 0.9991
4.53 100.87 3.71 2.69 30.01 1.2/3 4.85 2.22 0.9990
6.71 97.29 4.27 0.89 149.11 4/10 5.23 4.64 0.9975
6.53 105.24 4.92 2.72 134.84 4/10 5.14 0.9995 0.40–4..04
0.69
107.17 4.29 1.61 14.03 2/10 4.69 0.9964 0.10–1.00
3.45
5.18 102.98 4.84 2.20 630.69 6/24 4.33 2.42 0.9959 2.38–23.8
3.82 2.06 54.48 5/16 5.28 3.70
0.09–0.85 Quercetin
0.12–1.16 Kaempferide
0.41–4.08 Apigenin
Kaempferol-3-O-D-glucopyranoside Luteolin-7-O--Dglucopyranoside Luteolin
0.16–1.61
y = 86486x + 1162.9 y = 30487x + 16621 y = 262658x + 3194.3 y = 136424 x −696.36 y = 114165x + 5687 y = 576434x −3599.3 y = 18533x − 347.89 P-hydroxycinnamic acid
0.9971
Content (g /g)
RSD (%)
4.17
106.99
RSD (%) Mean
Recovery (%; n = 9) Stability RSD (%) M. horridula LOD /LOQ (ng/mL)
Intraday precision (RSD%) Interday precision (RSD%) R2 Linear range (g/mL) Regressive equation
3.2.2.3 Identification of phenylpropanoids The characteristic ions for phenolic compounds were the product ions due to the loss of CO2 , CO, and H2 O groups from their quasimolecular ions [29,30]. Compound 3 exhibited the [M–H]− ion at m/z 341, which is 162 Da heavier than that of caffeic acid. Therefore, compound 3 was identified as caffeic acid glucoside [31]. Compound 6 yielded a [M−H]− ion at m/z 325, [M−H–Glc]− ion at m/z 117 and [M−H–Glc–CO]− ion
Analyte
cracking of C-7, C-8 and C-13, C-14, which was the characteristic of protoberberine isoquinoline alkaloids [20] (Fig. 3A). Compounds 10 and 17 showed the cracking of C-7, C-8 and C13, C-14, which was the representative pathway of protopine isoquinoline alkaloids [20] (Fig. 3B). Compounds 12, 16, 21, and 23 presented a loss of C9 H8 O2 , which arises from a RDA fragmentation. Compound 4 generated quasimolecular ion [M+H]+ at m/z 381 and product ions at m/z 364, 336, and 247 due to serious losses of OH, CO, and C8 H5 O2 . Compound 4 was supposed as simplicifolianine [5]. Compound 12 yielded predominant [M+H]+ at m/z 328 and a major product ion at m/z, which may be explained by a retro-Diels–Alder (RDA) fragmentation. Compound 16 generated an obvious quasimolecular ion [M+H]+ at m/z 396. The dominant product ion at m/z 202 was assigned to [M-C11 H14 O3 ]+ by RDA loss. Series losses of CH3 , CH3 O, and C11 H14 O3 produced three ions at m/z 381, 366, 335, and 202, respectively. Similarly, compound 20 exhibited [M+H]+ ion at m/z 340 and showed series characteristic ions at m/z 309 [M+H–CH3 O]+ , 297 [M+H– H2 O–CH3 ]+ , 192 [M+H–C9 H8 O2 ]+ , and 149 [C9 H8 O2 +H]+ . As reported [20–22], compounds 12, 16, 20, 21, and 23 were tentatively assigned as scoulerine, alborine, sinactine, stylopine, and coptisine, respectively. Compound 5 generated a singly charged ion at m/z 342.1701 [M]+ and five major product ions at m/z 297.1095[M–(CH3 )2 NH]+ , 282.0874[M–(CH3 )2 NH– CH3 ]+ , 265.0857 [M–(CH3 )2 NH–CH3 –OH]+ , 237.0893 [M–(CH3 )2 NH–CH3 –OH–CO]+ , 222.0667 [M–(CH3 )2 NH– 2CH3 –OH–CO]+ . Compound 14 was deduced as amurine by their quasimolecular ions and corresponding product ions produced by loss of C9 H8 O2 . With reference to to reports [22–25], compounds 5 and 14 were tentatively identified as magnoflorine and amurine. Compound 29 exhibited [M+H]+ ion at m/z 284, [M+H–137]+ ion at m/z 147 and [M+H–137–CO]+ ion at m/z 119, and therefore was assigned as N-p-hydroxyl-trans-coumaroyltyramine [26]. Two epimeric N-oxides, amurensinine N-oxide A (18) and amurensinine N-oxide B (22), were identified. The two isomers both showed [M+H]+ ion at m/z 356 and [M+H–2]+ ion at m/z 354 due to dehydrogenation. They both showed product ions of [M+H–16]+ , which was the characteristic for N-oxides, and amurensinine skeleton ions at m/z 354, 340, 295, 206, 189 [27]. The two isomers can be distinguished by the relative response intensity of characteristic ions. The product ion at m/z 189 [M+H–C10 H16 O2 ]+ was stronger for 18 while 295 [M+H–C2 H7 NO]+ was stronger for 22 [28].
3.70
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J. Liu et al.
Table 3. Quantification of seven components by HPLC–MS/MS
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J. Sep. Sci. 2014, 37, 2513–2522
Liquid Chromatography
2521
Figure 4. Chromatograms for detection of seven compounds (13, 30, 32, 33, 35, 36, and 40).
at m/z 145. Similarly, compound 7 generated a [M−H]− ion at m/z 177, [M−H–CO2 ]− ion at m/z 132 and [M−H–CO2 – CO]− ion at m/z 105. Compound 15 produced the [M]+ ion at m/z 147, [M−NH2 ]+ ion at m/z 131 and [M−NH2 –CO]+ ion at m/z 103. Compounds 6, 7, and 15 were identified as p-hydroxycinnamic acid esters, 6,7-dihydroxycoumarin and cinnamamide, respectively.
and 149.11 g/g, respectively, followed by p-hydroxycinnamic acid (13), kaempferide (36), quercetin (40), and luteolin-7-O-D-glucopyranoside (32) with contents of 54.48, 30.01, 22.58, and 14.03 g/g, respectively. The total content of flavonol glycosides was higher than that of flavonols.
4 Concluding remarks 3.3 Simultaneous quantification of seven components by HPLC–MS/MS As shown in Table 3, all the analytes showed a good linear regression (R2 > 0.996), which presented that this method was precise and sensitive for the quantitative evaluation. LODs for seven compounds were estimated to be 0.9–6 ng/mL, and the LOQs were 3–24 ng/mL. The intraday and interday precisions (RSDs) were 0.21–5.33 and 2.57–5.94%, respectively. The repeatability and stability of the RSDs were all
