Analysis of Chemical Constituents of Melastoma dodecandrum Lour. by UPLC-ESI-Q-Exactive Focus-MS/MS

Abstract

The ethnic drug Melastoma dodecandrum Lour. (MDL) is widely distributed throughout South China, and is the major component of Gong Yan Ping Tablets/Capsules and Zi Di Ning Xue San. Although the pharmacological effects of MDL have been well documented, its chemical profile has not been fully determined. In this study, we have developed a rapid and sensitive UPLC-ESI-Q-Exactive Focus-MS/MS method to characterize the chemical constituents of MDL in the positive and negative ionization modes. A comparison of the chromatographic and spectrometric data obtained using this method with data from databases, the literature and reference standards allowed us to identify or tentatively characterize 109 compounds, including 26 fatty acids, 26 organic acids, 33 flavonoids, six tannins, 10 triterpenoids, two steroids and six other compounds. Notably, 55 of the compounds characterized in this study have never been detected before in this plant. The information obtained in this study therefore enriches our understanding of the chemical composition of MDL and could be used in quality control, pharmacological research and the development of drugs based on MDL. In addition, this study represents the first reported comprehensive analysis of the chemical constituents of MDL.

1. Introduction

Traditional Chinese medicine (TCM) has been used for thousands of years to treat a variety of different diseases. Based on its theoretical therapeutic efficacy and wide range of clinical applications, TCM has received considerable interest from healthcare professionals, as well as those working towards the identification of new therapeutic agents for commercialization. In contrast to the pharmacological characteristics of single agent drugs, multicomponent drugs can exhibit synergistic pharmacological effects, through a “network” approach, where multiple compounds interact with multiple targets, pharmacokinetic or physicochemical synergisms in vivo with interdependent activities to achieve an improved optimal effect [1,2,3]. It is therefore essential to evaluate the chemical composition of each TCM, so that this information can be used to support further studies, such as drug effect, toxicity and metabolism studies.

Melastoma dodecandrum Lour. (MDL) is extensively distributed throughout the southern provinces of China, including Guizhou, Fujian, Zhejiang, Jiangxi and Yunnan. This plant is widely used for its medicinal properties by the Yao, Miao and She people, as well as several other minority groups. Modern pharmacological studies have shown that MDL exhibits several biological effects, including antihypoglycemic, hemostatic, analgesic, anti-inflammatory, blood lipid reducing, antioxidant and liver protection properties [4]. MDL has been used to treat a variety of different ailments, including dysmenorrhea, postpartum abdominal pain, metrorrhagia, leucorrhea, hematochezia, dysentery, carbuncle swollen and boils. To date, only 76 compounds have been isolated from MDL, including organic acids, flavonoids, triterpenoids and steroids [5,6,7,8,9,10,11,12,13]. However, much of the chemical composition of MDL remains unknown, making it difficult to rationalize its bioactivity or evaluate the safety of this material as a therapeutic agent. There is therefore an urgent need to develop an analytical method capable of determining the chemical composition of MDL. With this in mind, the aim of the current study was to establish a rapid and sensitive method for identifying the constituents of MDL. In this study, we used a Q-Exactive Focus MS/MS method to obtain high-resolution mass spectra of the different components. This method was proven to be an advanced, accurate and reliable tool for the comprehensive identification of compounds belonging to a wide range of structural classes [14,15,16,17,18,19,20,21]. Using this method, we tentatively identified a total of 109 compounds, highlighting the efficiency and accuracy of this new technique.

2. Results and Discussion

Melastoma dodecandrum Lour. was analyzed in the positive and negative ionization modes using a Q Exactive Focus mass spectrometer, and the base peak chromatogram (BPC) chromatograms for both of these ESI modes are shown in Figure 1. Some of the compounds found in this study were identified based on a comparison of their analytical data (i.e., retention times and high-resolution mass spectra) with those of several reference standards. Thus compounds 10, 34, 35, 46, 68, 79, and 84 were unambiguously identified as gallic acid, luteolin, kaempferide, quercetin, oleanic acid, asiatic acid, and rutin, repectively. Moreover, the fragmentation patterns and pathways of the standards helped further confirm the structures of the derivatives of the reference compounds. Compounds without reference standards were identified by determining the elemental compositions of the precursor and product ions. The molecular formula and rational fragmentation patterns and pathways of these compounds were then identified based on a comparison of these data with chemical databases and the literature as described below in Section 3.5. In this way, we used a UPLC-ESI-Q-Exactive Focus-MS/MS method in combination with available standards, databases and literature data to characterize 109 compounds from MDL. Seven of these compounds were unambiguously identified based on a comparison with the corresponding reference standards. Data for all of these compounds are summarized in

Table 1. Tentative identification of the chemical constituents of Melastoma dodecandrum Lour. by UPLC-ESI-Q-Exactive Focus-MS/MS in negative and positive modes.

No.	Tentative Compound	tR (min)	Molecular Formula	Measured m/z	m/z Error in ppm	MS/MS (m/z)	Type of Compounds
1	Malic acid	0.99	C4H6O5	133.01422 [M−H]−	−0.19	115.00355 [M − H − H2O]− 71.0138 [C3H3O2]− 89.02435 [M − H − CO2]−	A
2	Salicylic acid	9.34	C7H6O3	137.02438 [M − H]−	−0.25	93.03443 [M – H − CO2]−	B
3	m-Salicylic acid	10.00	C7H6O3	137.02437 [M − H]−	−0.36	93.03445 [M – H − CO2]−	B
4	Citramalic acid	1.41	C5H8O5	147.02991 [M − H]−	0.06	129.01924 [M – H − H2O]− 101.02427 [M – H − HCOOH]− 85.02937 [C4H5O2]−	A
5	Protocatechuic acid	5.55	C7H6O4	153.01932 [M − H]−	−0.09	109.02937 [M – H − CO2]−	B
6	Gentisic acid	9.51	C7H6O4	153.01932 [M − H]−	−0.09	109.02943 [M − H − CO2]− 108.02159 [C6H4O2]−	B
7	Pimelic acid	13.87	C7H12O4	159.0663 [M − H]−	0.11	115.07629 [M – H − CO2]− 97.06577 [C6H9O]− 141.0556 [M − H − H2O]−	A
8	Coumaric acid	13.94	C9H8O3	163.04005 [M − H]−	−0.08	119.05009 [M − H − CO2]−	B
9 #	Vanillic acid	11.72	C8H8O4	167.03494 [M − H]−	−0.23	152.0114 [M − H − CH3]− 123.045 [M − H − CO2]− 108.02161 [C6H4O2]−	B
10 *,#	Gallic acid	2.23	C7H6O5	169.01416 [M − H]−	−0.51	125.02429 [M − H − CO2]− 97.0294 [C5H5O2]− 81.03452 69.03456 [C4H5O]−	B
11	Shikimic acid	1.15	C7H10O5	173.04547 [M − H]−	−0.44	155.03479 [M − H − H2O]− 137.02423 [M – H − 2H2O]− 111.04502 [M − H − H2O − CO2]− 93.03445 [C6H5O]− 73.02941 [C3H5O2]−	B
12	2-Isopropylmalic acid	11	C7H12O5	175.06120 [M − H]−	0.03	157.05048 [M − H − H2O]− 115.03992 [C5H7O3]− 113.06075 [C6H9O2]− 85.0658 [C5H9O]−	B
13	Glucose	0.89	C6H12O6	179.05605 [M − H]−	−0.37	59.01379 [C2H3O2]− 71.01382 [C3H3O2]− 89.02422 [C3H5O3]− 101.02422 [C4H5O3]−	F
14	2-Hydroxy-3-(2-hydroxyphenyl)propanoic acid	9.78	C9H10O4	181.05064 [M − H]−	0.06	163.03993 [M − H − H2O]− 135.04512 [M – H − HCOOH]− 119.0501 [M − H − H2O − CO2]−	B
15 #	Methyl gallate	10.88	C8H8O5	183.02988 [M − H]−	−0.11	168.00624 [M − H − CH3]− 140.0114 [M − H − CH3 − CO]− 124.01643 [C6H4O3]−	B
16	Citric acid	0.99	C6H8O7	191.01971 [M − H]−	−0.06	111.00864 [M − H − H2O – COOH − OH]− 87.00864 [C3H3O3]− 129.01915 [M − H − H2O − CO2]− 85.02939	A
17 #	Ferulic acid	18.04	C10H10O4	193.05052 [M − H]−	−0.57	178.02702 [M − H − CH3]− 149.06059 [M − H − CO2]− 134.03719 [M − H − CH3 − CO2]−	B
18 #	Vanillylmandelic acid	14.47	C9H10O5	197.04573 [M − H]−	0.93	153.05563 [M − H − CH3]− 138.03203 [M − H − CH3-CO2]− 121.0294 [M − H − CH3 − CO2 − OH]−	B
19	Sebacic acid	20.14	C10H18O4	201.11327 [M − H]−	0.17	183.10249 [M − H − H2O]− 139.11273 [M − H − H2O − CO2]−	A
20	1-Oxo-1,2,4-butanetricarboxylic acid	1.48	C7H8O7	203.01970 [M − H]−	−0.13	141.01923 [M − H − H2O − CO2]− 97.02934 [M − H − H2O − 2CO2]− 69.03453 [C4H5O]−	A
21	Undecanedioic acid	22.12	C11H20O4	215.12874 [M − H]−	−0.67	197.11826 [M − H − H2O]− 153.12842 [M − H − H2O − CO2]−	A
22	2-Hydroxysebacic acid	16.5	C10H18O5	217.10825 [M − H]−	0.46	199.09734 [M − H − H2O]− 171.10257 [M – H − HCOOH]− 155.10768 [M − H − H2O − CO2]−	A
23	Glucoheptonic acid	0.82	C7H14O8	225.06168 [M − H]−	0.38	179.05602 [C6H11O6]− 161.04546 [C6H9O5]− 87.00864 [C3H3O3]−	A
24	Traumatic Acid	22.97	C12H20O4	227.12894 [M − H]−	0.24	183.13876 [M − H − CO2]− 165.12823 [M − H − H2O − CO2]−	A
25	1-O-galloyl-glycerol	8.17	C10H12O7	243.05095 [M − H]−	−0.32	169.01408 [M − H − CO2 − 2CH3]− 125.02431 [M – H − 2CO2 − 2CH3]−	B
26	Oxododecanedioic acid	18.35	C12H20O5	243.12381 [M − H]−	0.05	225.1131 [M − H − H2O]− 207.10254 [M − H − H2O]− 181.1234 [C11H17O2]−	A
27	Palmitic acid	39.09	C16H32O2	255.23297 [M − H]−	0.07	237.06160 [M − H − H2O]−	A
28	Abscisic acid	19.75	C15H20O4	263.12885 [M − H]−	−0.12	219.13887 [M − H − CO2]− 204.11528 [M − H − CO2 − CH3]− 151.07634 [C9H11O2]−	B
29 #	Apigenin	19.79	C15H10O5	269.04559 [M − H]−	0.17	117.03447 [C8H5O]− 151.00354 [C7H3O4]− 107.01373 [C6H3O2]−	C
30 #	Naringenin	21.35	C15H12O5	271.06122 [M − H]−	0.08	177.01917 [C9H5O4]− 151.00352 [C7H3O4]− 119.05009 [C8H7O]−	C
31	Hydroxyhexadecanoic acid	36.77	C16H32O3	271.22797 [M − H]−	0.36	225.22221 [M – H − HCOOH]−	A
32	Oleic acid	39.49	C18H34O2	281.24860 [M − H]−	−0.01	237.06163 [M − H − CO2]−	A
33	Stearic acid	41.08	C18H36O2	283.26425 [M − H]−	−0.01	265.14810 [M − H − H2O]− 237.06181 [M – H − HCOOH]−	A
34 *,#	Kaempferol	19.95	C15H10O6	285.04047 [M − H]−	0.02	257.04535 [M − H − CO]− 241.05112 [M − H − CO2]− 151.00352 [C7H3O4]− 133.02942 [C8H5O2]−	C
35 *,#	Luteolin	21.79	C15H10O6	285.04062 [M − H]−	0.56	239.03464 [M − H − CO − H2O]− 185.06078 [C12H9O2]− 159.04491 [C10H7O2]− 93.03454 [C6H5O]−	C
36	Hexadecanedioic acid	30.38	C16H30O4	285.20709 [M − H]−	−0.14	267.19635 [M − H − H2O]− 223.20653 [M − H − H2O − CO2]−	A
37	3,5-Dihydroxy-hexadecanoic acid	23.84	C16H32O4	287.22278 [M − H]−	−0.01	269.21246 [M − H − H2O]− 241.21735 [M − H − H2O − CO2]−	A
38	Epicatechin	11.49	C15H14O6	289.07193 [M − H]−	0.58	245.0816 [M − H − CO2]− 203.07123 [C12H11O3]− 137.02423 [C7H5O3]− 109.02934 [C6H5O2]−	C
39	Catechin	13.27	C15H14O6	289.07184 [M − H]−	0.27	245.0089 [M – H − C3H8]− 217.01398 [M − H − C3H8 − CO]− 189.01923 [M − H − C3H8 − 2CO]− 173.0242 [C10H5O3]− 145.0294 [M – H − C3H8 − 2CO − CO2]−	C
40	4,9-Dihydroxy-6,7-dimethoxynaphtho(2,3-d)-1,3-dioxole-5,8-dione	14.56	C13H10O8	293.03040 [M − H]−	0.36	249.04028 [M − H − CO2]− 225.11308 [C12H17O4]− 162.0321 [M − H − CO2 − CH3 − CO]−	F
41	9-Hode	32.28	C18H32O3	295.22791 [M − H]−	0.12	277.21716 [M − H − H2O]− 171.1026 [C9H15O3]−	A
42	Ricinoleic acid	32.63	C18H34O3	297.24353 [M − H]−	0.0	183.13885 [C11H19O2]−	A
43	2-Glucopyranosyloxybenzoic acid	9.95	C13H16O8	299.07730 [M − H]−	0.2	137.02425 [M − H − Glc]− 93.03445 [M − H − glc − CO2]−	B
44	Hydroxystearic acid	39.09	C18H36O3	299.25919 [M − H]−	0.06	253.25348 [M − H − HCOOH]− 225.22246 [C15H29O]−	A
45 #	Ellagic acid	15.56	C14H6O8	300.99899 [M − H]−	0	283.99619 [M − H − OH]− 245.009 [C12H5O6]− 229.01402 [M − H − CO2 − CO]− 201.01927 [M − H − CO2 − 2CO]− 185.02431 [C11H5O3]−	D
46 *,#	Quercetin	19.95	C15H10O7	301.03534 [M − H]−	−0.12	178.99843 [C8H3O5]− 151.00351 [M – H − C6H6 − CO2 − CO]− 107.01373 [M – H − C6H6 − 2CO2 − CO]−	C
47	Gallocatechin	11.12	C15H14O7	305.06683 [M − H]−	0.52	261.0766 [M − H − CO2]− 219.06612 [C12H11O4]− 167.03488 [C8H7O4]− 137.02432 [C7H5O3]− 125.02433 [C6H5O3]−	C
48	Eicosanoic acid	42.84	C20H40O2	311.29562 [M − H]−	0.22	293.17941 [M − H2O]−	A
49	Glucovanillin	10.72	C14H18O8	313.09308 [M − H]−	0.6	161.04539 [C6H9O5]− 113.02431 [C5H5O3]− 101.02427 [C4H5O3]− 71.01381 [C3H3O2]−	F
50	Octadecanedioic acid	29.15	C18H34O4	313.23849 [M − H]−	0.2	295.2272 [M − H − H2O]−	A
51 #	2,3,8-Trihydroxy-7-methoxychromeno[5,4,3-cde]chromene-5,10-dione	21.54	C15H8O8	315.01489 [M − H]−	0.80	300.99841 [C14H5O8]− 269.10269 161.04578 [C6H9O5]− 71.01388 [C3H3O2]−	D
52	Dihydroxystearic acid	30.79	C18H36O4	315.25403 [M − H]−	−0.17	297.24344 [M − H − H2O]− 201.11363 [M − H − C8H18]−	A
53	Digallate	11.39	C14H10O9	321.02530 [M − H]−	0.29	169.01404 [C7H5O5]− 125.02428 [C6H5O3]−	B
54	2,3-Di-O-methylellagic acid	20.25	C16H10O8	329.03040 [M − H]−	0.32	314.00681 [M − H − CH3]− 298.98325 [C14H3O8]− 270.98834 [M − H − 2CH3 − CO]−	D
55	Woodorien	9.46	C14H18O9	329.08795 [M − H]−	0.44	167.03482 [M − H − Glc]− 152.01135 [M − H − Glc − CH3]− 121.0294 [M − H – Glc − HCOOH]− 108.02159 [M − H − Glc − CH3 − CO2]−	B
56	Galloylglucose	3.25	C13H16O10	331.06729 [M − H]−	0.67	271.04575 [C11H11O8]− 211.02464 [C9H7O6]− 169.01405 [M − H − Glc]− 125.02432 [C6H5O3]−	B
57	Caffeic acid-3-glucoside	11.08	C15H18O9	341.08798 [M − H]−	0.52	305.06638 [M – H − 2H2O]− 281.06647 [C13H13O7]− 251.05588 [C12H11O6]− 221.04532 [C11H9O5]− 179.03485 [M − H − Glc]− 135.04509 [M − H − Glc − CO2]−	B
58	2-Hydroxy-3,7,8-trimethoxychromeno[5,4,3-cde]chromene-5,10-dione	23.33	C17H12O8	343.04590 [M − H]−	−0.13	328.0224 [M − H − CH3]− 312.99899 [M – H − 2CH3]− 297.97522 [M − H − 3CH3]− 269.98053 [M − H − 3CH3 − CO]−	D
59	Theogallin	6.68	C14H16O10	343.06720 [M − H]−	0.38	169.01408 [C7H5O5]− 125.02427 [C6H5O3]−	B
60	Chlorogenic acid	9.88	C16H18O9	353.08813 [M − H]−	0.93	191.05602 [C7H11O6]− 179.0349 [C9H7O4]− 135.04512 [C8H7O2]−	B
61 #	Vitexin	15.74	C21H20O10	431.09833 [M − H]−	−0.1	341.06631 [C18H13O7]− 311.05603 [C17H11O6]− 283.06088 [C16H11O5]−	C
62 #	9,10-Dihydro-10-(4-hydroxyphenyl)-pyrano[2,3-h]epicatechin-8-one	19.9	C24H20O8	435.10876 [M − H]−	0.51	341.06641 [M − H − Phenol]− 217.01392 [C11H5O5]− 189.01918 [C10H5O4]− 177.01915 [C9H5O4]−	C
63	Epicatechin monogallate	15.92	C22H18O10	441.08286 [M − H]−	0.31	289.07166 [C15H13O6]− 169.0141 [C7H5O5]− 125.02431 [C6H5O3]−	C
64	Astragalin	15.06	C21H20O11	447.09344 [M − H]−	0.36	357.06146 [C18H13O8]− 327.05087 [C17H11O7]− 299.05569 [C16H11O6]−	C
65 #	Kaempferol-3-glucoside	16.47	C21H20O11	447.09354 [M − H]−	0.56	285.03983 [M − H − Glc]−	C
66 #	Luteolin-7-glucoside	17.4	C21H20O11	447.09357 [M − H]−	0.63	284.0325 [M − H − Glc]− 255.02986 [C14H7O5]− 227.03485 [C13H7O4]−	C
67 #	Ursolic acid	35.96	C30H48O3	455.35315 [M − H]−	0.18	407.33292 [C29H43O]−	E
68 *,#	Oleanic acid	36.51	C30H48O3	455.35333 [M − H]−	0.57	407.33292 [C29H43O]−	E
69 #	Quercetin-3-alloside	16.21	C21H20O12	463.08856 [M − H]−	0.78	300.02731 [M − H − Gal]− 271.02469 [C14H7O6]− 255.02979 [C14H7O5]−	C
70	Nigranoic acid	32.15	C30H46O4	469.33249 [M − H]−	0.33	423.327 [C29H43O2]−	B
71 #	4-O-(6″-O-p-Coumaroyl-glucopyranosyl)-p-coumaric acid	18.64	C24H24O10	471.12994 [M − H]−	0.58	307.08206 [C15H15O7]− 163.03993 [C9H7O3]− 119.05008 [C8H7O]−	B
72	Corosolic acid	31.81	C30H48O4	471.34802 [M − H]−	0.08	453.33932 [C30H45O3]−	E
73 #	7-Hydroxy-3,8-dimethoxy-5,10-dioxo-5,10-dihydro-chromeno[5,4,3-cde]chromen-2-yl 6-deoxymannopyranoside	18.83	C22H20O12	475.08853 [M − H]−	0.7	460.06451 [M − H − CH3]− 328.02231 [M – H − Rha]− 312.99887 [M − H − Rha − CH3]− 269.98038 [C13H2O7]−	D
74	Isorhamnetin-7-O-glucopyranoside	17.62	C22H22O12	477.10416 [M − H]−	0.64	314.04306 [M − H − Glc]− 271.02448 [M − H − Glc − CH3 − CO]− 243.02959	C
75	Isomyricitrin	14.78	C21H20O13	479.08301 [M − H]−	−0.22	316.02216 [M − H − Glc]− 271.02454 [C14H7O6]−	C
76	1,6-Bis-O-galloyl-glucose	11.52	C20H20O14	483.07819 [M − H]−	0.33	465.10410 [M − H − H2O]− 439.08768 [M − H − CO2]−	B
77	Quillaic acid	26.36	C30H46O5	485.32706 [M − H]−	−0.39	407.29538 [M − H − CH3 − H2O]− 241.10255	E
78 #	2-O-(E)-caffeoyl-1-O-p-(E)-coumaroylglucopyrannose	12.86	C24H24O11	487.12473 [M − H]−	0.29	323.07706 [C15H15O8]− 161.02429 [C9H5O3]− 119.05011 [C8H7O]−	B
79 *,#	Asiatic acid	26.85	C30H48O5	487.34271 [M − H]−	−0.38	469.33224 [M − H − H2O]−	E
80	Oenin	18.16	C23H24O12	491.11981 [M − H]−	0.64	313.03531 [M − H − Glc − CH]−3 299.01956 [C15H7O7]− 285.04028 [M − H − Glc − CH3 − CO]−	C
81	Medicagenic acid	24.38	C30H46O6	501.32227 [M − H]−	0.21	455.31689 [M − H − HCOOH]−	E
82	2″-O-Galloylisovitexin	17.72	C28H24O14	583.10974 [M − H]−	0.71	431.09842 [C21H19O10]− 341.06635 [C18H13O7]− 311.056 [C17H11O6]− 283.061 [C16H11O5]−	C
83	Nicotiflorin	17.02	C27H30O15	593.15167 [M − H]−	0.81	285.03989 [C15H9O6]− 255.02983 [C14H7O5]− 227.03491 [C13H7O4]−	C
84 *	Rutin	15.9	C27H30O16	609.14630 [M − H]−	0.32	300.02737 [C15H8O7]− 271.02469 [C14H7O6]− 255.02977 [C14H7O5]−	C
85	2′-O-Galloylhyperin	15.56	C28H24O16	615.09943 [M − H]−	0.44	463.08804 [C21H19O12]− 300.02734 [C15H8O7]− 271.02454 [C14H7O6]− 255.02972 [C14H7O5]−	C
86	3-O-trans-p-Coumaroylmaslinic acid	34.83	C39H54O6	617.38501 [M − H]−	0.4	145.02936 [C9H5O2]−	E
87	Delphinidin-3-caffeoylglucoside	18.25	C30H26O15	625.12048 [M − H]−	0.94	463.08832 [C21H19O12]− 300.02731 [C15H8O7]− 271.02472 [C14H7O6]− 255.02985 [C14H7O5]−	C
88	3-O-trans-p-Coumaroyltormentic acid	31.21	C39H54O7	633.37982 [M − H]−	0.23	163.04001 [C9H7O3]− 145.02939 [C9H5O2]−	E
89	1,3,6-Tri-O-galloylglucose	14.4	C27H24O18	635.08948 [M − H]−	0.77	465.06714 [C20H17O13]− 211.02463 [C9H7O6]− 169.01404 [C7H5O5]− 125.02427 [C6H5O3]−	B
90	(3,5,9)-3-Hydroxy-27-{[(2E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoyl]oxy}olean-12-en-28-oic acid	35.86	C40H56O7	647.39587 [M − H]−	0.84	632.37152 [C37H52O7]− 453.33835 [C30H45O3]− 175.03993 [C10H7O3]− 133.02946 [C8H5O2]−	E
91	3-O-trans-Feruloyleuscaphic acid	32.02	C40H56O8	663.39069 [M − H]−	0.67	648.36658 [C39H52O8]− 175.03989 [C10H7O3]− 160.01643 [C9H4O3]− 132.02156 [C8H4O2]−	E
92 #	4′-Hydroxyacetophenone	12.32	C8H8O2	137.05969 [M + H]+	−0.01	122.03635 [M + H − CH3]+ 109.06508 [M + H − CO]+	F
93	Dihydroxyacetophenone	13.7	C8H8O3	153.05461 [M + H]+	−0.06	125.05981 [M + H − CO]+ 111.04433 [C6H7O2]+	F
94	N-Lauryldiethanolamine	37.04	C16H35O2N	256.26334 [M + H]+	−0.6	144.1382 [C8H18ON]+ 116.1072 [C6H14ON]+	F
95	Licanic acid	26.99	C18H28O3	293.21085 [M + H]+	−0.94	275.20044 [M + H − H2O]+ 257.19003 [M + H − 2H2O]+	A
96	Kamlolenic acid	33.51	C18H30O3	295.22678 [M + H]+	0.06	277.21609 [M + H − H2O]+ 259.20532 [M + H − 2H2O]+ 231.21051 [M + H − 3H2O]+	A
97	Diosmetin	21.99	C16H12O6	301.0705 [M + H]+	−0.56	286.04681 [M + H − CO3]+ 258.05197 [M + H − CH3 − CO]+	C
98 #	Sitosterol	39.21	C29H50O	397.38269 [M + H − H2O]+	−0.47	243.21089 [C18H27]+ 175.14799 [C13H19]+ 147.11684 [C11H15]+	G
99 #	Stigmasterol	38.08	C29H48O	395.36725 [M + H − H2O]+	0.05	241.1945 [C18H25]+ 199.14772 [C15H19]+ 173.13248 [C13H17]+	G
100	Apigenin-7-O-glucoside	17.83	C21H20O10	433.1127 [M + H]+	−0.51	271.05975 [C15H11O5]+ 153.01814 [C7H5O4]+	C
101 #	Quercetin-3-arabinoside	16.62	C20H18O11	435.09235 [M + H]+	0.36	303.04959 [C15H11O7]+ 153.01819 [C7H5O4]+	C
102	Luteolin-7-galactoside	12.52	C21H20O11	449.10745 [M + H]+	−0.86	287.05453 [M − H − Glc]+ 269.044 [M + H − Glc]+	C
103	Pelargonidin-3-O-(6-caffeoyl-glucoside)	19.91	C30H26O13	595.14459 [M + H]+	−0.04	433.11255 [M − H − C6H6 − 3CO]+ 313.07028 [C17H13O6]+ 163.03888 [C9H7O3]+	C
104	Tiliroside	20.42	C30H26O13	595.14435 [M + H]+	−0.45	287.0546 [C15H11O6]+ 147.04393 [C9H7O2]+ 119.04929 [C8H7O]+	C
105	Kaempferol-3-(6″-galloylgalactoside)	16.8	C28H24O15	601.11914 [M + H]+	0.57	287.05457 [C15H11O6]+ 153.01813 [C7H5O4]+ 137.0233 [C7H5O3]+	C
106	Quercetin-3-O-(6″-O-p-coumaroyl)-glucopyranoside	19.41	C30H26O14	611.13922 [M + H]+	−0.51	147.04388 [C9H7O2]+ 303.04953 [C15H11O7]+	C
107	2′-O-Galloylhyperin	15.51	C28H24O16	617.11322 [M + H]+	−0.89	153.01813 [C7H5O4]+ 303.0495 [C15H11O7]+	C
108	Quercetin-3-(6″-caffeoylgalactoside)	18.29	C30H26O15	627.13409 [M + H]+	−0.56	163.03873 [C9H7O3]+ 303.04941 [C15H11O7]+	C
109 #	Casuarinin	12.61	C41H28O26	937.09332 [M + H]+	−0.89	153.01817 [C7H5O4]+ 171.04417 [C11H7O2]+ 277.03397 [C13H9O7]+ 345.02377 [C16H9O9]+	D

* These compound were unambiguously identified by the use of authentic reference compounds. ^# These compound were isolated from Melastoma dodecandrum Lour. according to the literature [5,6,7,8,9,10,11,12,13]. Glc, glucopyranosyl, Rha, rhamnopyranosyl. A, fatty acid; B, organic acid; C, flavonoid; D, tannin; E, pentacyclic triterpene; F, others; G, steroid.

.

2.1. Fragmentation Pattern of Main Compounds

2.1.1. Flavonoids

Flavonoids are 2-phenylchromone systems that consist of two benzene rings (A and B) connected by a pyran ring (ring C), which is fused to the A ring. Flavonoids can be classified into several subclasses, including flavones, flavonols, flavanones, flavanonols, anthocyanidins, chalcones, isoflavonoids and flavan-3-ols, depending on the nature of the substituents attached to the different rings. Based on the results of accurate molecular mass measurements and the MS² fragmentation pathways of the different materials [16,22,23], we characterized a total of 33 different flavonoids (aglycones) in MDL, including eight flavones, one flavanone, five flavan-3-ols, two anthocyanidins and 17 flavonols. The structures of the 33 flavonoids are shown below (Figure 2).

Two different fragmentation patterns and pathways were observed for the flavonoids (I and II, shown in Figure 3). The retro-Diels–Alder (RDA) fragmentation (I) would result in the formation of A 1, 3 and B 1, 3 as the main fragment ions of the flavonoid moiety (aglycones) because of the X 1, 3 cleavage of the C ring. The substituent groups on the parent compounds were determined based on the compositions of the A 1, 3 and B 1, 3 fragments. The fragmentation of the parent compound according to Pattern I resulted in high-intensity fragment ions, whereas the fragmentation according to pattern II result in low-intensity fragment ions. Furthermore, the main fragmentation patterns of flavonoids (glycosides) typically consist of fragments associated with deglycosylation, demethylation and decarboxylation yielding [M − H − Glc]⁻, [M − H − CH₃]⁻ and [M − H − CO₂]⁻ ions, respectively. The structures of the fragments resulting from the RDA fragmentation are shown in

Table 2. RDA fragmentation pathways of compound 29, 30, 46, and 63.

Compound No.	Mass Spectra of MS/MS	RDA Fragmentation Pathway
29
30
46
63

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2.1.2. Pentacyclic Triterpenes

Pentacyclic triterpenes are wildly distributed in Nature and consist of five rings, which are typically referred to as the A, B, C, D and E rings. Pentacyclic triterpenes can be divided into several different categories, depending on the nature of their E ring. In this study, we characterized two different types of pentacyclic triterpenes, including ursane- and oleanane-type pentacyclic triterpenes. The endo-double bond in pentacyclic triterpenes can readily undergo a RDA reaction during MS analysis (shown in Figure 4). Dehydration and decarboxylation are also observed as common fragmentation pathways in these systems during MS analysis [21,22,23]. Depending on the different substituents attached to their endo-double bonds and their accurate mass measurements, we were able to fully characterize all of the pentacyclic triterpenes found in MDL. As shown in Figure 5, we characterized a total of nine pentacyclic triterpenes using high-resolution MS² mass spectrometry, including four ursane-type and five oleanane-type compounds pentacyclic triterpenes.

2.1.3. Tannins

The main structural features of tannins include their gallic acid ester moieties (or polymer) and glucose core (or other polyols). The MS² spectra of the tannins revealed fragment ions with m/z values of 1091, 939, 769, 617, 599, 447 and 277. The m/z differences between these fragment ions were 152 and 170 atomic mass units (amu), which indicated that the fragments ions were produced by the successive removal of O-galloylhyperin and gallic acid anions. The fragment ion observed with an m/z value of 169 was characteristic of the fragment ions derived from O-galloylhyperin, whereas the fragment ion observed with an m/z value of 125 was attributed to the loss of CO₂ from gallic acid [17]. A total of six other tannins were characterized in this way using high-resolution MS² mass spectrometry. The structure of tannins characterized were shown in Figure 6.

Taking compound 109 as a representative example, the analysis of this compound in the positive ionization mode (t_R = 12.61 min) gave an [M + H]⁺ ion with an m/z value of 937.09332, indicating a molecular formula of C₄₁H₂₈O₂₆ (Δ = −0.89 ppm) (Table 2). The MS² spectrum of compound 109 contained five fragment ions with m/z values of 345.02377, 277.03397, 231.02875, 171.04417 and 153.01817. The fragment pathway for this compound is shown in Figure 7. Based on these results, compound 109 was characterized as casuarinin.

2.1.4. Organic Acids

Compound 4 (t_R = 1.41 min) was analyzed in the negative ionization mode and gave an [M − H]⁻ ion with an m/z value of 147.02991, which indicated a molecular formula of C₅H₈O₅ (Δ = 0.06 ppm) (Table 1). The MS² spectrum of compound 4 gave three fragment ions with m/z values of 129.01924 [M − H − H₂O]⁻, 101.02427 [M – H − HCOOH]⁻ and 85.02937 [M − H − H₂O − CO₂]⁻. This fragmentation process was used to confirm the identities of the other organic acids, resulting in the characterization of 26 compounds as organic acids.

3. Materials and Methods

3.1. Chemicals and Reagents

Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (Waltham, MA, USA). Distilled water was purchased from Watson’s Food & Beverage Co., Ltd. (Guangzhou, China). Formic acid (MS grade) was purchased from Fisher Scientific (Waltham, MA, USA). Reference standards of oleanolic acid (94.9%, batch No. 110709-201206), kaempferol (93.2%, batch No. 110861-201209), luteolin (100%, batch No. 111520-200504) and quercetin (97.4%, batch No. 100081-200907) were purchased from the National Institutes for Food and Drug Control (Beijing, China). A reference standard of asiatic acid (99%, batch No. 20150901) was purchased from CRM/RM center of China (Beijing, China). Reference standards of rutin (98%, batch No. 153-18-4) and gallic acid (98.5%, batch No. 149-91-7) were purchased from Chengdu-PUSH Bio-Technology Co., Ltd. (Chengdou, Sichuan, China).

3.2. Plant Materials and Sample Preparation

The whole plants of MDL were collected from Yunnan Province in China and authenticated by Professor Yaojun Yang (Beijing University of Chinese Medicine, Beijing, China). Voucher specimens (DN001) of the plant were deposited at the authors’ laboratory. The samples were dried and powdered, before being sieved through a 40-mesh sieve. A sample of the powder (approximately 2.0 g) was suspended in 25 mL of methanol, and the resulting mixture was subjected to ultrasonic treatment for 30 min before being cooled to room temperature. Methanol was added to compensate for the lost weight and the resulting mixture was filtered through a 0.22-μm PTFE syringe filter. The filtrate was collected and subjected to centrifugation (13,000 rpm, 10 min). The supernatant was then transferred to an autosampler vial for analysis by UPLC-ESI-Q-Exactive Focus-MS/MS.

3.3. UPLC-ESI-Q-Exactive Focus-MS/MS Analysis

UPLC analysis was performed on a Thermo Scientific Ultimate 3000 system (Sunnyvale, CA, USA) equipped with a binary solvent delivery manager and a sample manger. Chromatographic separations were performed on a Thermo Scientific Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 μm). The column temperature was maintained at 40 °C. A mobile phase consisting of 0.1% formic acid in water (A) and acetonitrile (B) was used to elute the column according to the optimized gradient program, as follows: 98% A from 0 to 5 min; 98%–80% A from 5 to 15 min; 80%–40% A from 15 to 30 min; 40%–2% A from 30 to 40 min; 2% A from 40 to 47 min; 2%–98% A from 47 to 47.1 min; 98% A from 47.1 to 50 min. The flow rate was set at 0.3 mL/min. An injection volume of 5 μL was used for the reference compounds and then analytical samples. For MS detection, the operating parameters were as follows: spray voltage, +3500 V/–3200 V; atomization temp, 350 °C; sheath gas pressure, 35 arb; aux gas pressure, 10 arb; capillary temperature, 320 °C; S-lens RF, 60 V; resolution, MS full scan 70,000 FWHM, MS/MS full scan 15,000 FWHM; scan range, m/z 100–1500 for MS; m/z 30–1500 for MS/MS; scanning mode, fullscan-ddms2.

3.4. Optimization of Analytical Conditions

To obtain better chromatographic separation and mass spectrometric detection, we evaluated three different mobile phase systems, including aqueous methanol, aqueous acetonitrile and aqueous acetonitrile-formic acid solutions. The aqueous acetonitrile solution resulted in the best separation of the major components of MDL. Furthermore, the addition of 0.1% formic acid to this mobile phase resulted in a considerable improvement in the symmetry properties of most of the chromatographic peaks. We also varied the flow rate (0.25, 0.3, and 0.35 mL/min), column temperature (30, 35, and 40 °C) and injection volume (2, 3, and 5 μL) during method development. The results of these optimization experiments established the following conditions for the chromatographic separation of the different components of MDL: mobile phase, aqueous acetonitrile containing 0.1% formic; flow rate, 0.3 mL/min; column temperature, 40 °C; and injection volume, 5 μL.

3.5. Structure Analysis Procedure

In the positive and negative scan mode, based on the high-accuracy precursor ions and product ions obtained from Q-Exactive Focus-MS/MS, the elemental compositions were calculated when the maximum tolerance of mass error for all the precursor ions and product ions was set at 1.5 ppm, which can satisfy the requirements for positive identification. Based on the elemental compositions of the precursors, the most rational molecular formula was sought in different chemical databases such as the Spectral Database for Organic Compounds SDBS (http://sdbs.db.aist.go.jp), m/z cloud (https://www.mzcloud.org) and ChemSpider (http://www.chemspider.com). Meanwhile by searching literature sources, such as PubMed of the U.S. National Library Medicine and the National Institutes of Health, Scifinder Scholar of the American Chemical Society, Science Direct of Elsevier and Chinese National Knowledge Infrastructure (CNKI) of Tsinghua University, all components reported in the literatures on MDL and plants of the same family were summarized in a Microsoft Office Excel table to establish an in-house library [5,6,7,8,9,10,11,12,13] for searching the most rational molecular formula. When several matching compounds with the same formula were found, the fragmentation patterns and pathways of the compounds were analyzed and then validated by Mass Frontier 7.0 (Thermo Scientific) for positive identification.

4. Conclusions

A new UPLC-ESI-Q-Exactive Focus-MS/MS method was developed to analyze the chemical constituents of MDL based on their mass spectral fragmentation patterns. This new method resulted in the characterization of 109 compounds. The results of this study therefore provide an important reference to improve our understanding of the composition of MDL. We found that flavonoids are the main components of MDL, especially the flavonols, which possess a wide range of interesting pharmacological activities, such as anticancer, antibacterial, and antiviral activities. In terms of their structural characteristics, the triterpenoids found in MDL were ursane- and oleanane-type systems. Several tannins and steroids were also found in MDL. In addition to the fatty acids found in MDL, we found 55 other compounds that have never been reported in MDL besides fatty acids. Further studies pertaining to the chemical constituents in Melastoma dodecandrum Lour. are currently underway in our laboratory. Moreover, The study shows that, with the application of the UPLC-ESI-Q-Exactive Focus-MS/MS to characterizing the constituents of MDL, this method offers a rapid, sensitive and high throughput methodology for the identification of constituents of TCM prescriptions and herbal medicines.