Rapid Analysis of the Chemical Compositions in Semiliquidambar cathayensis Roots by Ultra High-Performance Liquid Chromatography and Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Semiliquidambar cathayensis Chang was a traditional medicinal plant and used to treat rheumatism arthritis and rheumatic arthritis for centuries in China with no scientific validation, while only 15 components were reported. Thus, a rapid, efficient, and precise method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was applied in both positive- and negative-ion modes to rapidly analysis the main chemical compositions in S. cathayensis for the first time. Finally, a total of 85 chemical compositions, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified or tentatively characterized in the roots of S. cathayensis based on the accurate mass within 5 ppm error. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots.

1. Introduction

Semiliquidambar cathayensis Chang, is an epiphyllum tree belonging to the Hamamelidaceae family, native only to China, and grows in Jiangxi, Guangxi, Guangdong, Hainan and Guizhou [1]. Chinese people call the roots of S. cathayensis as Ban feng he (Chinese name 半枫荷), which have long been used in traditional Chinese medicine (TCM) for the treatment of rheumatism arthritis and rheumatic arthritis [2]. Modern pharmacological experiments have demonstrated that the crude extracts and/or fractions obtained from the roots of S. cathayensis have the effects of analgesia, anti-inflammatory, anti-hepatitis B virus, promoting blood circulation, and removing blood stasis [3,4,5,6]. Unfortunately, only 15 chemical compositions—including 7 terpenoids, 3 steroids, 3 tannins, and 2 fatty acids—were reported from the roots of S. cathayensis [7,8], which was a significant barrier for further pharmacological, metabolic and pharmacokinetic studies of this medicinal plant. Moreover, due to the indeterminate relationship between pharmacological activities, chemical components, the clinical application and quality control of S. cathayensis roots still faced big challenges. Therefore, a rapid and sensitive method to figure out the chemical components in the roots of S. cathayensis was urgently needed.

Conventional separation and identification processes were time and plant material consuming [9,10,11,12,13], whereas the use of a rapid, efficient, and prescise method focused on identification chemical components was very important for TCMs. Over the past decade, UHPLC coupled with high-resolution mass spectrometry (HRMS) has become the prime tool for investigating the chemical profiling of TCMs, because of its advantages on the peak capacity, resolution, separation time, and detection sensitivity, all of which are suitable for addressing the complicated characteristics of the constituents in TCMs [14,15,16,17]. Furthermore, quadrupole time-of-flight Q-TOF-MS/MS with powerful structural characterization can provide more specific and accurate mass measurements for both precursor and fragment ions. These features can greatly facilitate prediction of elemental compositions and fragmentation pathways [18,19,20].

In this study, a rapid, sensitive, and reliable approach based on UHPLC-Q-TOF-MS/MS method was established to determine the main chemical components in the roots of S. cathayensis for the first time, which will provide a basis for further study in vivo of S. cathayensis roots and the information of potential new drug structure for treating rheumatism arthritis and rheumatic arthritis.

2. Results and Discussion

2.1. Optimization of Chromatographic Separation

A series of parameters, including stationary and mobile phases, flow rate, and column temperature were investigated in order to obtain optimal chromatographic separation and analytical sensitivity for multiple constituents in the roots of S. cathayensis. A comparative study based on chromatographic selectivity and detection sensitivity revealed that the best performance was achieved with the BEH C18 column as the stationary phase and acetonitrile as the organic part of the mobile phase. Since alkaloid compounds generally exhibits better mass spectrometric responses in positive ionization mode, the addition of 0.1% formic acid into the aqueous part of the mobile phase was found to be beneficial to the subsequent positive electrospray ionization (ESI+) analysis. In addition to the optimization of the stationary and mobile phases, control of column temperature and flow rate were also optimized to improve selectivity and resolution. Finally, column temperature of 35 °C and the flow rate of 0.3 mL/min were suitable for the separation. The total ion chromatogram (TIC) of S. cathayensis roots extract in positive- and negative-ion modes were shown in Figure 1. Moreover, the tandem mass spectra of the main components were available in Supplementary Materials.

Figure 1

The total ion chromatograms of the S. cathayensis roots extract by UHPLC-Q-TOF-MS/MS in positive- and negative-ion modes.

2.2. Identification of Main Constituents in S. Cathayensis Extract

A total of 85 chemical compositions, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified. The molecular formula was accurately assigned within mass error of 5 ppm. Then the fragment ions were used to further confirm the chemical structure. Information including compound name, retention time, formula, precursor ion, and fragment ions of the rest of these compositions can be found in Table 1; Table 2. The chemical structures of the main components in S. cathayensis roots extract are showed in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9. All the components were identified based on the existing literatures, which includes the database of Chinese medicine ingredients and the free chemical structure database, including ChemSpider, Massbank, and mzCloud. Furthermore, the fragmentation pathways of some compounds were proposed in order to facilitate structural identification.

Table 1

Compounds identified from the roots of S. cathayensis by UHPLC–Q-TOF-MS/MS in positive ion mode.

No.	tR (min)	Compounds	Molecular Formula	Molecular Weight	Measured Mass [M + H]	Error (ppm)	MS2	Ref.
Alkaloids
4	1.74	stachydrine	C7H13NO2	143.0946	144.1020	0.8	144.1004 a, 128.0647, 102.0585	[21]
6	4.06	gentianin	C10H9NO2	175.0633	176.0706	−0.2	176.0714 a, 148.0763, 133.0524, 130.0660, 103.0560, 120.0820, 117.0350	[22]
7	4.10	kalacolidine	C22H35NO5	393.2515	394.2593	1.4	394.2588, 376.2485 a	[23]
10	5.49	mesaconine	C24H39NO9	485.2625	486.2702	1.0	486.2697 a, 436.2332, 404.2070	[23]
11	5.54	16-β-hydroxycardiopetaline	C21H33NO4	363.2410	364.2486	1.0	364.2488 a, 346.2382, 328.2275	[24]
13	5.84	senbusine A	C23H37NO6	423.2621	424.2699	1.3	424.2701 a, 406.2591, 388.2487	[23]
16	6.05	carmichaeline	C22H35NO4	377.2566	378.2645	1.6	378.2640, 360.2532 a, 328.2271	[23]
18	6.10	isotalatizidine	C23H37NO5	407.2672	408.2749	1.2	390.2642 a, 358.2378	[23]
19	6.46	aconine	C25H41NO9	499.2781	500.2861	1.3	500.2863 a, 450.2495	[15]
20	6.56	songorine	C22H31NO3	357.2304	358.2381	1.2	358.2375, 340.2270 a	[23]
21	6.6	napelline	C22H33NO3	359.2460	360.2536	0.7	360.2536, 342.2430 a	[15]
22	6.74	hetisine	C20H27NO3	329.1991	330.2069	1.6	330.2062 a, 312.1954	[23]
25	7.38	hypaconine	C24H39NO8	469.2676	470.2751	0.6	470.2764 a, 438.2498, 406.2216	[24]
26	7.44	senbusine C	C24H39NO7	453.2727	454.2805	1.3	454.2786 a, 404.2427	[23]
28	7.91	neoline	C24H39NO6	437.2777	438.2854	0.8	438.2841 a, 420.2738, 388.2480, 356.2222, 154.1223	[23]
29	8.08	14-acetyl-karakoline	C24H37NO5	419.2672	420.2749	1.0	420.2757 a, 402.2653	[24]
35	9.18	talatisamine	C24H39NO5	421.2828	422.2906	1.2	390.2642 a, 358.2379	[24]
37	9.41	denudatine	C22H33NO2	343.2511	344.2589	1.4	344.2587 a, 326.2480	[23]
42	10.76	bullatine C	C26H41NO7	479.2883	480.2962	1.2	480.2980 a, 462.2858, 430.2587, 398.2295	[23]
43	11.22	chasmanine	C25H41NO6	451.2934	452.3012	1.2	452.3016 a, 420.2755, 388.2490	[23]
44	13.41	14-acetyl-talatisamine	C26H41NO6	463.2934	464.3011	1.0	464.3011 a, 432.2746	[23]
50	20.28	benzoylmesaconine	C31H43NO10	589.2887	590.2966	1.1	590.2941 a, 540.2579, 508.2323, 105.0341	[15]
54	21.85	benzoylaconine	C32H45NO10	603.3044	604.3122	0.9	604.3110 a, 554.2748	[15]
55	22.91	benzoylhypaconine	C31H43NO9	573.2938	574.3015	0.8	574.3017 a, 542.2755, 510.2495	[15]
58	24.20	benzoyl-3,13-deoxymesaconine	C31H43NO8	557.2989	558.3068	1.1	558.3055 a, 540.2955, 508.2697	[15]
59	24.25	10-hydroxy-mesaconitine	C33H45NO12	647.2942	648.3024	1.4	648.3002 a, 588.2791, 556.2540, 370.1653	[23]
60	24.27	benzoyldeoxyaconine	C32H45NO9	587.3094	588.3172	0.8	588.3174 a, 556.2918	[15]
61	25.21	pyrohypaconitine	C31H41NO8	555.2832	556.2910	0.9	556.2915 a, 524.2647, 492.2414, 452.2072, 402.2285, 238.1807, 192.1383	[15]
63	25.87	mesaconitine	C33H45NO11	631.2993	632.3070	0.7	632.3032 a, 572.2826, 540.2566, 512.2622, 508.2315, 354.1685, 105.0342	[15]
64	26.12	10-hydroxy-aconitine	C34H47NO12	661.3098	662.3180	1.4	662.3168 a, 602.2957, 570.2702, 384.1809	[15]
67	27.49	hypaconitine	C33H45NO10	615.3044	616.3125	1.5	616.3089 a, 556.2876, 524.2618, 496.2638, 342.2055, 338.1739, 105.0340	[15]
68	27.58	aconitine	C34H47NO11	645.3149	646.3232	1.5	646.3199 a, 586.2985, 554.2735, 526.2783, 522.2487, 368.1850, 105.0342	[23]
70	28.60	deoxyaconitine	C34H47NO10	629.3200	630.3281	1.3	630.3256 a, 570.3047, 538.2787, 510.2830, 478.2575, 356.2219, 352.1905	[15]
71	28.98	yunaconitine	C35H49NO11	659.3306	660.3386	1.2	660.3396 a, 572.2866, 540.2591, 354.1735	[24]
72	29.38	3,13-dideoxyaconitine	C34H47NO9	613.3251	614.3326	0.4	614.3302 a, 554.3091, 522.2835, 494.2880, 462.2620, 105.0345	[23]
Terpenoids
14	5.94	oxypaeoniflorin	C23H28O12	496.1581	497.1657	0.7	497.2676, 349.1575, 197.0831, 133.0687, 121.0297 a	[25]
31	8.56	paeoniflorin	C23H28O11	480.1632	481.1711	1.4	319.1245, 197.0808, 179.0691,151.0750, 133.0650, 105.0342 a	[26]
65	26.21	benzoylpaeoniflorin	C30H32O12	584.1894	585.1970	0.5	585.3271, 319.1195, 267.0885, 249.0785, 197.0807, 179.0705, 121.0666,105.0349 a	[26]
73	30.98	atractylenolide-1	C15H18O2	230.1307	231.1380	0.0	213.1257, 163.0778, 155.0848, 143.0931, 128.0610, 115.0541, 105.0712 a	[27]
79	36.18	3-oxo-olean-12-en-28-oic acid	C30H46O3	454.3447	455.3516	−0.8	455.3539, 437.3426, 247.1668, 233.1531, 229.1584, 197.1332, 189.1615 a	[28,29]

a base peak.

Table 2

Compounds identified from the roots of S. cathayensis by UHPLC–Q-TOF-MS/MS in negative ion mode

No.	tR (min)	Compounds	Molecular Formula	Molecular Weight	Measured Mass [M − H]−	Error (ppm)	MS2	Ref.
Flavonoids
24	7.33	puerarin	C21H20O9	416.1107	415.1015	−4.6	415.0970, 295.0614, 277.0490, 267.0653 a	[30]
33	8.85	scoparin	C22H22O11	462.1162	461.1069	−4.4	415.0991 a, 252.0361	[30]
34	9.17	isorhamnetin-3-O-neohespeidoside	C28H32O16	624.1690	623.1602	−2.5	623.1609, 461.1022 a, 417.1203, 315.0710, 153.0226, 145.0338	[31]
36	9.38	kaempferol-3-O-glucorhamnoside	C27H30O15	594.1585	593.1485	−4.6	593.1010, 547.1243, 430.0954, 275.0347,112.9889 a	[32]
39	9.44	methyl hesperidin	C29H36O15	624.2054	623.1963	−2.9	623.0455, 577.0923, 534.0988, 461.1451, 410.0366, 315.1067, 145.0319 a	[33]
17	6.06	catechin	C15H14O6	290.0790	289.0715	−1.1	221.0899, 205.0532, 203.0700 a, 187.0372, 159.0452, 125.0280, 123.0486	[34]
46	17.01	naringin	C27H32O14	580.1792	579.1716	−0.6	579.1721, 459.1144, 313.0736, 271.0617 a, 177.0209, 151.0048	[33]
48	19.58	hesperidin	C28H34O15	610.1898	609.1815	−1.6	609.1826, 301.0706 a, 286.0481, 242.0583	[35]
56	23.57	5,8-dihydroxy-6,7-dimethoxyflavone	C17H14O6	314.0790	313.0709	−2.9	297.0313, 283.0226, 266.0197, 255.0309 a, 227.0318, 211.0393, 185.0235, 183.0456	[33]
57	24.10	juglanin	C20H18O10	418.0900	417.0814	−3.1	161.0578, 135.0527, 129.0226 a	[20]
62	25.32	naringenin	C15H12O5	272.0685	271.0609	−1.1	151.0030, 119.0509 a, 117.0421	[33]
66	26.53	hesperetin	C16H14O6	302.0790	301.0713	−1.7	258.0578 a, 134.0383	[33]
Terpenoids
76	33.74	2α,3β-dihydroxyolean-12-en-28-oic-acid	C30H48O4	472.3553	471.3466	−3.0	471.3494 a, 451.0162, 411.0302, 389.2158, 330.9965, 264.9917	[28,29]
81	36.45	oleanic acid	C30H48O3	456.3604	455.3512	−4.2	455.3495 a, 409.2443, 391.2341, 373.2227, 355.2079	[28,29]
Phenylpropanoids
15	6.01	ferulic acid	C10H10O4	194.0579	193.0510	2.0	134.0379 a	[36]
22	7.10	fraxin	C16H18O10	370.0900	369.0814	−3.5	223.0462 a, 205.0350, 129.0210, 125.0241	[32]
32	8.76	3-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid	C9H10O5	198.0528	197.0456	0.2	162.8375 a, 160.8401, 138.0358, 123.0085	[37]
38	9.44	acteoside	C29H36O15	624.2054	623.1963	−2.9	623.0455, 577.0923, 461.1451, 315.1067, 145.0319 a	[30]
52	21.42	bergaptol	C11H6O4	202.0266	201.0192	−0.8	228.9172 a, 166.8855, 147.8874, 117.0436	[38]
Fatty Acids
5	2.05	citric acid	C6H8O7	192.0270	191.0199	0.7	146.9074, 111.0110 a	[36]
69	28.24	trihydroxy-octadecaenoic acid	C18H34O5	330.2406	329.2326	−1.5	329.2354, 229.1443, 211.1346, 183.1371, 171.1026 a, 139.1137	[27]
74	32.9	dihydroxy-octadecatrienoic acid	C18H30O4	310.2144	309.2069	−0.9	291.1995 a, 199.8548, 179.1442, 110.0373	[27]
77	34.13	hydroxy-octadecatrienoic acid	C18H30O3	294.2195	293.2121	−0.4	293.2080 a, 199.8526, 149.0939, 125.1018	[28]
78	36.16	linolenic acid	C18H30O2	278.2246	277.2171	−0.7	134.8951 a	[28]
80	36.22	stearic acid	C18H36O2	284.2715	283.2640	−0.9	283.2633, 199.8512 a	[28]
83	37.05	linoleic acid	C18H32O2	280.2402	279.2329	−0.2	279.2319 a, 261.2194	[28]
84	38.09	palmitic acid	C16H32O2	256.2402	255.2332	0.9	256.2333, 255.2327 a, 114.9333	[28]
85	38.20	oleic acid	C18H34O2	282.2559	281.2484	−0.9	281.2489 a	[28]
Cyclic Peptides
27	7.59	cyclo trileucyl (or isoleucyl)	C18H33N3O3	339.2522	384.2488 b	−1.3	135.0456 a	[28]
40	10.07	cyclo tetraleucyl (or isoleucyl)	C24H44N4O4	452.3363	497.3328 b	−1.1	497.1555, 451.3294 a, 433.3159, 337.2669, 224.1758, 137.0247	[28]
45	13.48	cyclo pentaleucyl (or isoleucyl)	C30H55N5O5	565.4203	610.4168 b	−1.0	564.4112 a, 546.4021, 225.1592	[28]
47	17.66	cyclo hexaleucyl (or isoleucyl)	C36H66N6O6	678.5044	723.5020 b	0.7	677.4961 a	[28]
49	19.86	cyclo hetaleucyl (or isoleucyl)	C42H77N7O7	791.5885	836.5862 b	0.8	790.5791 a	[28]
51	21.0	cyclo octaleucyl (or isoleucyl)	C48H88N8O8	904.6725	949.6704 b	0.9	946.6691, 903.6636 a	[28]
53	21.78	cyclo nonaleucyl (or isoleucyl)	C54H99N9O9	1017.7566	1062.7546 b	0.9	1062.7547, 1016.7472 a	[28]
Others
1	0.35	glucogallin	C13H16O10	332.0744	331.0662	−2.7	169.0124, 125.0257 a	[39]
2	0.69	gallic acid	C7H6O5	170.0215	169.0149	3.6	124.0178 a	[40]
3	1.46	sucrose	C12H22O11	342.1162	341.1078	−3.5	221.0641, 179.0592, 161.0419, 119.0379, 113.0253 a	[41]
8	4.29	piscidic acid	C11H12O7	256.0583	255.0508	−0.7	218.8641, 180.9830, 165.0550 a, 118.9815	[42]
9	5.37	vanillin	C8H8O3	152.0473	151.0410	4.1	105.0368 a	[43]
12	5.59	protocatechuic acid pentoside	C12H14O8	286.0689	285.0611	−1.6	152.0117, 108.0243 a	[44]
30	8.37	vanillic acid	C8H8O4	168.0423	167.0354	2.2	108.0206 a	[36]
41	10.65	paeonol	C9H10O3	166.0630	165.0564	3.8	147.0476, 119.0505, 117.0379 a, 103.0575, 101.0392	[31]
75	33.10	dibutyl phthalate	C16H22O4	278.1518	277.1444	−0.3	147.0072 a	[45]
82	36.52	dimethisterone	C23H32O2	340.2402	339.2326	−1.1	339.2317, 163.1140 a	[45]

a base peak, b measured mass [M + HCOO]−.

Figure 2

Chemical structures of alkaloids from S. cathayensis roots.

Figure 3

Tandem mass spectra and its fragmentation of mesaconitine in positive ion mode.

Figure 4

Tandem mass spectra and its fragmentation of bullatine C in positive ion mode.

Figure 5

Chemical structures of flavonoids from S. cathayensis roots.

Figure 6

Tandem mass spectra and its fragmentation of naringin in negative ion mode.

Figure 7

Chemical structures of terpenoids from S. cathayensis roots.

Figure 8

Tandem mass spectra and its fragmentation of oleanolic acid in negative ion mode.

Figure 9

Chemical structures of phenylpropanoids from S. cathayensis roots.

2.2.1. Alkaloids

A total of 35 alkaloids (Figure 2) in the roots of S. cathayensis extract were indentified in positive ion mode, all ones except compounds 4 and 6 were diterpenoid alkaloids, which can be classified into C18-, C19-, and C20- diterpenoid alkaloids according to skeleton carbons [46]. In this study, compounds 7, 20, 21, 22, and 37 were C20-diterpenoid alkaloids, while others were C19-diterpenoid alkaloids, which were belonging to aconitum alkaloids. Moreover, aconitum alkaloids include diester-diterpenoid, monoester-diterpenoid, amine-diterpenoid, and other alkaloids [25]. In tandem mass spectrum of aconitum alkaloids commonly observe the neutral losses of H2O, MeOH, AcOH, or BzOH. In diester-diterpenoid alkaloids, the hydroxyl groups of C8 and C14 in these compounds are combined with acetic acid and benzoic acid to form esters, respectively. The neutral loss of 28 Da corresponding to eliminate one molecule of CO or C2H4 was the feature loss. Meanwhile, the order of eliminations of benzyl, acetyl, carboxyl, ethyl, or methyl and methoxy was also investigated. In monoester-diterpenoid alkaloids, only hydroxyl group of C14 binds with benzoic acid to form esters. Then, hydroxyl at C1 position was the most active site. Unlike them, hydroxyl at C15 position could not be eliminated even at a high fragment or voltage in amine diterpenoid alkaloids [15].

Compounds 59, 63, 64, 67, 68, 70, 71, and 72 were diester-diterpenoid alkaloids. Among them, 63, 67, and 68 showed [M + H]+ ion at m/z 632.3032, 616.3089 and 646.3199. They have similar fragmentation pathways, including [M + H − HAc]+, [M + H − HAc − CH3OH]+, [M + H − HAc − CH3OH − CO]+, [M + H − HAc − 2CH3OH − CO]+, [M + H − HAc − 2CH3OH]+, and [M + H − HAc − 3CH3OH − C6H5COOH]+, which were identified as mesaconitine, hypaconitine, and aconitine, respectively. The possible fragmentation mechanism of mesaconitine is depicted in Figure 3.

Moreover, compounds 59 and 64 gave [M + H]+ ion at m/z 648.3002 and 662.3168, 16 Da greater than that of mesaconitine and aconitine, respectively. As C10 position was commonly substitued by hydroxyl in aconitum alkaloids, they were presumed as 10-OH mesaconitine and 10-OH aconitine, respectively. However, compounds 70 and 72 gave [M + H]+ ion at m/z 630.3256 and 614.3302, 16 Da and 32 Da less than aconitine, respectively. Their tandem mass spectra were smililar to aconitine, and some fragment ions of compounds 70 and 72 were 16 Da and 32 Da less than the fragment ions of aconitine, respectively. They were reported as deoxyaconitine and 3,13-dideoxyaconitine, respectively. Compound 71 showed [M + H]+ ion at m/z 660.3396, 14 Da greater than that of aconitine. Moreover, as C14 position was commonly substitued by methoxybenzoyl in aconitum alkaloids, 71 was presumed as yunaconitine [24,47].

Compounds 29, 42, 44, 50, 54, 55, 58, 60, and 61 are monoester-diterpenoid alkaloids. Compound 29 gave fragment ions at m/z 420.2757 and 402.2653 were corresponding to [M + H]+ and [M + H − H2O]+, respectively, and it was identified as 14-acetyl-karakoline by comparing with the literature [25]. Compound 42 gave fragment ions at m/z 480.2980, 462.2858, 430.2587, and 398.2295 in the positive mode were corresponding to [M + H]+, [M + H − H2O]+, [M + H − H2O − CH3OH]+, and [M + H − H2O − 2CH3OH]+, respectively, and it was tentatively identified as bullatine C (Figure 4). The [M + H]+ ion at m/z 464.3011 of compound 44 also gave the fragment ion at m/z 432.2746 [M + H − CH3OH]+, and identified as 14-acetyl-talatisamine. Compounds 50, 54, 55, and 60 were 42 Da less than mesaconitine, aconitine, hypaconitine, and deoxyaconitine, respectively. As C8 position was substitued by hydroxyl group instead of acetyl group. Therefore, they could be considered as benzoylmesaconine, benzoylaconine, benzoylhypaconine, and benzoyldeoxyaconine, respectively. Moreover, compound 58 was 32 Da less than that of benzoylmesaconine, and was identified as benzoyl-3,13-deoxymesaconine. Meanwhile, compound 61 was 60 Da less than that of hypaconitine, and was presumed as pyrohypaconitine, attributing to one molecule of acetic acid eliminated from hypaconitine.

Compounds 10, 11, 13, 16, 18, 19, 25, 26, 28, 35, and 43 are amine diterpenoid alkaloids. Among them, 10, 16, 19, 25, and 35 were 104 Da less than benzoylmesaconine, 14-acetyl-karakoline, benzoylaconine, benzoylhypaconine, and 14-acetyl-talatisamine; and considered as mesaconine, karacoline, aconine, hypaconine, and talatisamine, respectively, because C14 position was substitued by hydroxyl group instead of benzoyl group. Moveover, 11, 13, 18, 26, 28, and 43 could be considered as 16-β-hydroxycardiopetaline, senbusine A, isotalatizidine, senbusine C, neoline, and chasmanine, respectively, based on their molecular weight and tandem fragment patterns.

Compounds 7, 20, 21, 22 and 37 were C20-diterpenoid alkaloids, which gave [M + H]+ ions at m/z 394.2588, 358.2375, 360.2536, 330.2062 and 344.2587, respectively. Thus, they were respectively identified as kalacolidine, songorine, napelline, hetisine, and denudatine by comparing with the literatures [15,48].

The [M + H]+ ion of compound 4 was shown at m/z 144.1004. Its MS2 fragment ions at m/z 128.0647 and 102.0585 exhibited the loss of CH4 and continuous loss of two CH4, and it was presumed as stachydrine [22]. For compound 6, the positive mode MS spectrum showed the parent ion at m/z 176.0714 [M + H]+, and MS2 spectrum showed the fragment ions at m/z 148.0763 [M + H − C2H4]+, 130.0660 [M + H − COOH]+, and 120.0820 [M + H − C2H4 − CO]+. Compared with literature data, compound 6 was identified as gentianin [23].

2.2.2. Flavonoids

Flavonoids were a kind of basic 2-phenyl chromogenic ketones, which exist widely in nature and were important natural organic compounds. Two flavonoids (17 and 56) and 10 flavonoid glycosides in the roots of S. cathayensis extract were indentified in negative ion mode (Figure 5). For flavonoids, small molecules and radicals like CH3 (15 Da), H2O (18 Da) and CO (28 Da) were feature loss. The main MS/MS behavior of aglycones described previously was retro Diels-Alder (RDA) fragmentation pathway. RDA fragments of m/z 135 and 119 were the feature fragments in negative ion mode [20,49]. Taking compound 17 as an example, it had [M − H]− ion at m/z 289.0715. It yielded fragments at m/z 205.0532, 203.0700, 187.0372, and 125.0280 by loss of 2(C2H2O), 2(C2H3O), 2(C2H2O)-H2O, and C9H8O3 moieties. It was consistent with literature and identified as catechin [35].

Moreover, the loss of a glucuronic acid (176 Da) and hexose residue (glucose 162 Da, rhamnose 146 Da) were often seen in flavonoid glycosides. Compounds 24, 33, 34, 36, 39, 46, 48, 56, 57, 62, and 66 were considered as puerarin, scoparin, isorhamnetin-3-O-neohespeidoside, kaempferol-3-O-glucorhamnoside, methyl hesperidin, naringin, hesperidin, 5,8-dihydroxy-6,7-dimethoxyflavone, juglanin, naringenin, and hesperetin, respectively [31,32,33,34,36]. Take compound 46 as an example (Figure 6), it had [M − H]− ion at m/z 579.17156 in the spectrum. Four main fragment ions at m/z 459.1144, 271.0617, 177.0209, and 151.0048 were obviously observed. Among them, the most abundant fragment ion m/z 271.0617 was suggested by the loss of rutinose residue [M − H − 146 − 162]−. Fragment ions at m/z 459.1144 and 151.0048 were glycoside and aglycone by RDA. The fragment information at m/z 177.0209 was detected as aglycone without C ring. Compared to the MS spectra data and references [34,36] compound 46 was tentatively identified as naringin.

2.2.3. Terpenoids

Terpenoids were a class of structures derived from methylglutaric acid (MVA) and have two or more isoprene units (C5) on the basic carbon shelf. Seven terpenoids were identified in this study, including three monoterpenes (14, 31, and 65), one sesquiterpene (73) and three triterpenes (76, 79, and 81) (Figure 7). Monoterpenes usually detected the neutral losses of a benzoic acid at m/z 121 or glucosyl group at m/z 165, and aglycone ions at m/z 195 or 197, or their fragmentations of losing H2O and CO [15]. In that case, fragmentation behaviors showed that compound 14, 31, and 65 were oxypaeoniflorin, paeoniflorin, and benzoylpaeoniflorin, respectively [26,27].

Compound 73 had [M + H]+ ion at m/z 231.13795, and its fragments were at m/z 163.0778 [M + H − C5H8]+, 155.0848 [M + H − HCOOH − C2H6]+, 143.0931 [M + H − HCOOH − C3H6]+, and 105.0712 [M + H − HCOOH − C3H6 − C4H2]+. Its fragmentation process was the same as the literature [28] and identified as atractylenolide-1.

It was reported that triterpenes (76, 79, and 81) had similar tandem fragment patterns [29,30]. Taking compound 81 as an example, it had [M − H]− ion at m/z 455.3495, and its fragments were at m/z 409.2443 [M − H − CH2O2]− and 391.2341 [M − H − CH2O2 − H2O]−. Its fragmentation process was the same as the literature [29,30], and identified as oleanolic acid (Figure 8).

2.2.4. Phenylpropanoids

Phenylpropanoids were structures containing one or more C6-C3 units, which were widely distributed in medicinal plants. A total of five phenylpropanoids in the roots of S. cathayensis extract were indentified in negative ion mode (Figure 9). Compounds 15, 22, 32, 38, and 52 were considered as ferulic acid, fraxin, 3-(3,4-dihydroxyphenyl)-2-hydroxy-propanoic acid, acteoside, and bergaptol, respectively [31,33,37,38,39].

Taking compound 38 as an example, it had [M − H]− ion at m/z 623.0455, and its fragments were at m/z 461.1451 [M − H − C9H6O3]– and 315.1067 [M −H − C9H6O3 − C6H10O4]−. Its fragmentation process was the same as the literature [31] and identified as acteoside.

2.2.5. Fatty Acids

Fatty acids found in medicinal plants vary in length chains from 12 to 22 carbon atoms, of which 16–20 carbon atoms are the most common fatty acids in nature. It was reported that these compositions have a wide range of biological activities, including stabilizing cell membranes, maintaining the balance of cytokines and lipoproteins, and fighting cardiovascular diseases. In this study, nine fatty acids with long aliphatic hydrocarbon chains and a carboxyl group at one end (compounds 5, 69, 74, 77, 78, 80, 83, 84, and 85) were identified based on the existing literatures [37,45,46,50], and in the relevant databases, such as ChemSpider, Massbank, and mzCloud.

2.2.6. Cyclic Peptides

Compounds 27, 40, 45, 47, 49, 51, and 53 had similar fragmentation behaviors, and showed [M + HCOO]− ions at m/z 384.2488, 497.3328, 610.4168, 723.5020, 836.5862, 949.6704, and 1062.7547, respectively. According to reference mass spectra and fragmentation spectra reported in the literatures [46,51,52], a total of seven cyclic peptides were identified with 3–9 leucyl (or isoleucyl) groups in the roots of S. cathayensis.

2.2.7. Others

Other 10 compounds 1–3, 8, 9, 12, 30, 41, 75, and 82 were considered as glucogallin, gallic acid, sucrose, piscidic acid, vanillin, protocatechuic acid pentoside, vanillic acid, paeonol, dibutyl phthalate, and dimethisterone, respectively [32,37,40,41,42,43,44,45,46].

3. Experimental Section

3.1. Chemicals and Reagents

Acetonitrile and formic acid (LC-MS grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Deionized water was purified by a Milli-Q ultrapure water system (Merck Millipore, Milford, MA, USA). All other regents used of at least analytical grade.

3.2. Plant Material and Extraction

The roots of S. cathayensis were collected in the town of Longsheng, Guilin City, Guangxi, China, in October 2016. A botanical voucher specimen of this plant (no. SC20161022) was deposited at authors’ laboratory and was identified by one of the authors Ronghua Liu. 1.0 g aliquots of the roots of S. cathayensis powders were weighed and transferred into a 100-mL conical flask. 50 mL of 50% aqueous ethanol solution was added, and then extracted with a reflux twice for 60 min. Then, the fluid was filtrated and concentrated under reduced pressure in a rotary evaporator. Subsequently, the concentrated extract was lyophilized and dissolved in ACN. The solution was filtered through a 0.22-μm PTFE membrane before submitting for instrumental analysis.

3.3. UHPLC-Q-TOF-MS/MS

The UHPLC analysis were carried out on a Shimadzu System (Kyoto, Japan), equipped with a LC-3AD solvent delivery system, a SIL-30ACXR auto-sampler, a CTO-30AC column oven, a DGU-20A3 degasser and a CBM-20A controller. Chromatographic separation was conducted on a ACQUITY UPLC®BEH C18 (100 × 2.1 mm, 1.7 μm) keeping at 35 °C. 0.1% aqueous formic acid (v/v, A) and acetonitrile (B) were used as the mobile phase. The gradient elution with the flow rate of 0.3 mL/min was performed as follows: 0–5 min 5–15% B; 5–15 min 15–18% B; 15–25 min 18–35% B; 25–35 min 35–95% B; 35–37 min 95–95% B; 37–37.1 min 95–5% B; 37.1–40.0 min 5–5% B. The sample inject volume was 3 μL.

UHPLC-Q-TOF-MS/MS detection was conducted on a Triple TOFTM 5600+ system with a Duo Spray source in both positive and negative electrospray ion mode (AB SCIEX, Foster City, CA, USA). The MS analysis was carried out by the ESI source in both positive- and negative-ion modes. The parameters were set as follows: ion spray voltage, −5500 V; ion source temperature, 500 °C; curtain gas, 40 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), 50 psi; and decluster potential (DP), -100 V. Mass ranges were set at 100–1500 Da for the TOF-MS scan and 100–1500 Da for the TOF MS/MS experiments. In the IDA-MS/MS experiment, the collision energy (CE) was set at 45 eV, and the collision energy spread (CES) was (±) 15 eV for the UHPLC-Q-TOF-MS/MS detection. The most intensive five ions from each TOF-MS scan were selected as MS/MS fragmentation. LC-MS/MS data were analyzed using PeakView®1.2 software (AB SCIEX, Foster City, CA, USA).

4. Conclusions

In this study, a rapid, efficient, and precise UHPLC-Q-TOF-MS/MS approach was developed for the separation and identification of the main compositions in the roots of S. cathayensis for the first time. By the virtue of high resolution and high separation speed of UHPLC, and accurate MS data of Q-TOF-MS, a total of 85 components, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified by comparisons of their retention times, accurate masses, fragment ions, related literatures. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide a scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots.