A Systematic Method for the Identification of Aporphine Alkaloid Constituents in Sabia schumanniana Diels Using UHPLC-Q-Exactive Orbitrap/Mass Spectrometry

Sabia schumanniana Diels (SSD) is a plant whose stems are used in traditional folk medicine for the treatment of lumbago and arthralgia. Previous studies have revealed chemical constituents of SSD, including triterpenoids and aporphine alkaloids. Aporphine alkaloids contain a variety of active components, which might facilitate the effective treatment of lumbago and arthralgia. However, only 5-oxoaporphine (fuseine) has been discovered in SSD to date. In this study, we sought to systematically identify the aporphine alkaloids in SSD. We established a fast and reliable method for the detection and identification of these aporphine alkaloids based on ultra-high-performance liquid chromatography (UHPLC)-Q-Exactive-Orbitrap/mass spectrometry combined with parallel reaction monitoring (PRM). We separated all of the analyzed samples using a Thermo Scientific Hypersil GOLD™ aQ C18 column (100 mm × 2.1 mm, 1.9 μm). Finally, we identified a total of 70 compounds by using data such as retention times and diagnostic ions. No fewer than 69 of these SSD aporphine alkaloids have been reported here for the first time. These findings may assist in future studies concerning this plant and will ultimately contribute to the research and development of new drugs.


Introduction
Sabia schumanniana Diels (SSD) is a deciduous climbing woody vine of the genus Sabia in the family Sabiaceae and is widely distributed in the Sichuan and Guizhou provinces of China. The stems of SSD are used in traditional folk medicine for the treatment of lumbago and arthralgia [1,2]. The main active constituents in the genus Sabia are alkaloids [3]; however, only triterpenoids and 5-oxoaporphine (fuseine) have been identified in Sabia schumanniana Diels before now [4]. Aporphine alkaloids are natural chemical compounds that are highly biologically active and play an important role in plants. In recent studies, aporphine alkaloids have been shown to exhibit potent anti-diabetic, anti-cancer [5], antiinflammatory [6], and antivirus properties [7]. Further studies on the aporphine alkaloid components of SSD are therefore warranted.
Ultra-high-performance liquid chromatography-Q-Exactive Orbitrap/mass spectrometry is a process which can be used for chemical constitution identification and offers high selectivity, high sensitivity, and high efficiency [8][9][10]. The fragment information obtained through MS combined with advanced post-processing technology data enables the determination of the diagnostic fragment ions and neutral losses. Typically, sample data acquisition involves a full scan with data-dependent MS 2 (full MS/dd-MS 2 ). However, MS 2 data cannot be detected in this mode if the relative abundance of MS 1 ions does not 2 of 16 reach a required level. As a result, any desired compounds that are present only in trace amounts are disregarded because of the limitations of the analytical method. Recently, this tool has been used to conveniently acquire MS 2 data using the parallel reaction monitoring (PRM) detection mode, which allows for the isolation of the targeted precursor ions and product fragment ions from the precursor and enables the detection of the resulting product ions based on the preset isolation window width and collision energy, eliminating most interference. By such means, researchers have achieved the accurate detection and quantification of confirmed and targeted fragments [11,12].
In this study, we systematically characterized SSD constituents using UHPLC-Q-Exactive Orbitrap MS combined with PRM. We putatively identified 70 aporphine alkaloids based on their precise mass measurement, chromatographic retention, MS n spectra analysis, and bibliographical data. No fewer than 69 of these aporphine alkaloids were identified in SSD for the first time in this study. These results may contribute to a better understanding of the medicinal effects of SSD and help to lay the groundwork for the future quality control of SSD-derived medicines in a clinical setting.

Results and Discussion
From the experimental data for our SSD sample and a summarized fragmentation pattern, we identified a total of 70 aporphine alkaloids. Tables 1 and S1 give the chromatographic and mass data for these detected constituents and includes retention times (tR), experimental masses, and the discrepancies between the theoretical and experimental masses (in ppm), in addition to molecular formulas for all the aporphine alkaloids as well as MS/MS fragment ions. Figure 1 illustrates the high-resolution extracted ion chromatogram from the SSD extract in the positive ion mode. All compounds are numbered according to their order of elution.   determination of the diagnostic fragment ions and neutral losses. Typically, sample data acquisition involves a full scan with data-dependent MS 2 (full MS/dd-MS 2 ). However, MS 2 data cannot be detected in this mode if the relative abundance of MS 1 ions does not reach a required level. As a result, any desired compounds that are present only in trace amounts are disregarded because of the limitations of the analytical method. Recently, this tool has been used to conveniently acquire MS 2 data using the parallel reaction monitoring (PRM) detection mode, which allows for the isolation of the targeted precursor ions and product fragment ions from the precursor and enables the detection of the resulting product ions based on the preset isolation window width and collision energy, eliminating most interference. By such means, researchers have achieved the accurate detection and quantification of confirmed and targeted fragments [11,12].
In this study, we systematically characterized SSD constituents using UHPLC-Q-Exactive Orbitrap MS combined with PRM. We putatively identified 70 aporphine alkaloids based on their precise mass measurement, chromatographic retention, MS n spectra analysis, and bibliographical data. No fewer than 69 of these aporphine alkaloids were identified in SSD for the first time in this study. These results may contribute to a better understanding of the medicinal effects of SSD and help to lay the groundwork for the future quality control of SSD-derived medicines in a clinical setting.

Results and Discussion
From the experimental data for our SSD sample and a summarized fragmentation pattern, we identified a total of 70 aporphine alkaloids. Table 1 and table 1S give the chromatographic and mass data for these detected constituents and includes retention times (tR), experimental masses, and the discrepancies between the theoretical and experimental masses (in ppm), in addition to molecular formulas for all the aporphine alkaloids as well as MS/MS fragment ions. Figure 1 illustrates the high-resolution extracted ion chromatogram from the SSD extract in the positive ion mode. All compounds are numbered according to their order of elution.

Establishment of the Analytical Method
For this study, we established an analytical strategy based on utilizing UHPLC-Q-Exactive Orbitrap MS combined with parallel reaction monitoring (PRM) to identify diagnostic fragment ions (DFIs) and neutral losses (NLs) in order to comprehensively screen for and detect the aporphine alkaloids present in SSD. First, we injected SSD samples into a UHPLC-Q-Exactive Orbitrap MS to obtain full mass raw data via use of the full-mass scanning mode. Second, we predicted the potential chemical compounds using Compound Discoverer 3.0 and Metabolite Workflow. We determined parameters in line with [13]. The drug was set to magnoflorine, while roemerine and the added group were assigned to a list of substituents including -CH 3 , -OH, -OCH 3 , C=O, and -OCH 2 O-. Third, we collected fragmentation ions using UHPLC-Q-Exactive Orbitrap MS based on the parallel reaction monitoring mode activated by inclusion ions from the list described above. Finally, we performed an accurate full-scan mass spectrometry and MS 2 . We also extracted the retention time information and incorporated relevant database and literature data. By such means, we obtained our SSD identification results.

Identifification and Analysis of Aporphine Alkaloids in SSD
We used UHPLC-Q-Exactive Orbitrap MS to examine the fragmentation patterns of four reference standards in positive mode in order to establish the neutral loss and the diagnoses for fragmentation ions. Figure 2A shows the proposed fragmentation pathway for magnoflorine. This generated a fragment ion at m/z 297.1123 (C 18 H 17 O 4 + ) via the neutral loss at m/z 45 [C 2 H 7 N] when the isoquinoline ring was opened and the amino group along with two methyl groups were removed, this being an essential characteristic of aporphine alkaloids [14,15]. We then obtained a product ion at m/z 265.0852 (C 17 H 13 O 3 + ) by the precursor-ion neutral loss of CH 3 OH. The presence of a fragment ion at m/z 282.0877 (C 17 H 14 O 4 + ) manifested the parallel loss of CH 3 . The base peak for the fragment ions was obtained at m/z 265.0852, and the loss of CO was obtained at m/z 237.0910 (C 16 H 13 O 2 + ). Figure 2B shows the proposed fragmentation pathway for lirinidine. The neutral loss of CH 3 NH 2 and the production of the ion at m/z 251.1067 (C 17 H 14 O 2 + ), in addition to the consequent neutral loss of CH 3 OH and CO, yielded fragment ions at m/z 219.0806 (C 16 H 10 O + ) and 191.0856 (C 15 H 10 + ). Figure 2D shows the proposed fragmentation pathway for roemerine. This yielded a fragment ion at m/z 249.0912 (C 17 H 13 O 2 + ) because of the characteristic elimination of CH 3 NH 2 , and also involved the expulsion of CH 3 O, which produced a fragment ion at m/z 219.0805 (C 16 H 10 O + ). The consequent neutral loss of CO generated a fragment ion at m/z 191.0856 (C 15 H 10 + ). Figure 2C shows the proposed fragmentation pathway for N-nornuciferine.

Fragmentation Pattern for Quaternary Aporphine Alkaloids
We accurately identified Compound 27 as magnoflorine by comparing the retention time and the MS and MS 2 spectra with the reference-standard data. We also found that Compounds 19 and 42 were eluted at 6.70 and 8.97 min, respectively, and they possessed the same MS 1 at 342.1670 [M] + . They also exhibited five distinct fragment ion peaks at m/z 58.0658, 237.0905, 265.0852, 282.0877, and 297.1123. We identified these as magnoflorine isomers.
Compound 1 had a molecular formula of C 20 H 24 NO 5 and a retention time of 3.32 min. This compound produced the precursor ion at 358.1649 [M] + and four fragment ion peaks at m/z 58.0660, 227.0703, 255.0667, and 287.0917 in the positive ion mode. Based on secondary fragmentation data, we identified Compound 1 as C 6a -hydroxylation of magnoflorine [16].
For Compounds 3, 11, 21, and 29, we determined a molecular design based on the structure of magnoflorine with one methoxy group removed, giving a molecular formula of C 19 H 22 NO 3 . We obtained the precursor ion at m/z 312.1594 [M] + and observed four characteristic fragment ion peaks at m/z 58.0660, 217.0650, 207.0808, and 267.1018 in the positive ion mode. We tentatively identified these four compounds as C 2 -O-demethylation of magnoflorine isomers [16].
The isomeric Compounds 4, 8, 15, and 35 exhibited identical fragment ions and molecular ions. The precursor ion at m/z 358.1649 [M] + was formed using the chemical formula C 20 H 24 NO 5 . We observed five characteristic fragment ion peaks at m/z 58.0659, 253.0863, 281.0809, 285.0740, and 313.1071 in the positive ion mode. We identified these compounds as isomers of trilobinine [17].
Compounds 17 and 40 had a molecular formula of C 20 H 26 NO 3 and produced a precursor ion at m/z 328.1907 [M] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0659, 251.1067, 253.1226, and 283.1328. On the basis of the above information, we tentatively identified these compounds as isomers of N-ring-opening C 1 -dehydroxylation of magnoflorine [16].
Compound 16 had a chemical formula of C 20 H 26 NO 4 , was eluted at 6.22 min, and produced a precursor ion at m/z 344.1856 [M] + in the positive ion mode. We observed five characteristic fragment ion peaks at m/z 58.0659, 137.0598, 143.0493, 175.0754, and 299.1278. On the basis of the above MS and previous findings in the literature, we identified this compound as zizyphusine+ 2H [18].
For Compounds 20, 34, and 46, we designed a molecular structure from dihydroxylation of magnoflorine, giving it the molecular formula of C 20 H 24 NO 6 , and produced a precursor ion at m/z 374.1599 [M] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 58.0659, 297.0758, and 329.1022. We identified these compounds as isomers of di-hydroxylation of magnoflorine [19].
Compounds 22 and 26 had the chemical formula of C 21 H 28 NO 4 and generated a precursor ion at m/z 358.20128 [M] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 58.0660, 281.0813, and 313.1446. We compared these data with previous findings in the literature and identified these compounds as isomers of pareirarinea [20].  [16].
Compounds 36, 43, and 49 were obtained form a molecular design in which one of the hydroxyl groups that is in magnoflorine becomes methoxy. These compounds had a molecular formula of C 21 [22].
Compound 41 had a molecular formula of C 22 H 26 NO 6 and a retention time of 8.83 min; it produced a precursor ion at m/z 400.1755 [M] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0660, 295.0961, 323.0918, and 355.1180. We identified this compound as C 10 -OCH 3 -hydroxylation and C 11 -O-acetylation of magnoflorine [16].
Compounds 44 and 61 had a molecular design based on the structure of magnoflorine with one hydroxyl group and one methoxy group removed, giving a molecular formula of C 19 H 22 NO 2 , and it produced a precursor ion at m/z 296.1645 [M] + . We observed six characteristic fragment ion peaks at m/z 58.0660, 219.0807, 220.0842, 221.0957, 236.0826, and 251.1068 in the positive ion mode. On the basis of the above information, we identified Compounds 44 and 61 as isomers of C 1 -demethoxy-C 2 -dehydrox of magnoflorine [16].
Compound 48 had a molecular formula of C 22 [16].
The molecular design of Compound 52 was based on the structure of magnoflorine, from which one adjacent hydroxyl group and one methoxy group were removed, and one adjacent hydroxyl group and one methoxy group were changed to dioxolane, giving a molecular formula of C 19 H 20 NO 2 . This compound had a retention time of 13.07 min and produced a precursor ion at m/z 294.1489 [M] + . We observed four characteristic fragment ion peaks at m/z 58.0659, 191.0862, 219.0805, and 249.0911. On the basis of the above molecular design and fragmentation information, we identified Compound 52 as roemrefidine [23].
Compound 56 had a molecular formula of C 20 H 22 NO 5 and a retention time of 13.53 min, and it produced a precursor ion at m/z 356.1492 [M] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0659, 251.0703, 279.1028, and 311.0918. On the basis of the above fragmentation patterns, we tentatively identified Compound 56 as C 5 -methylene to ketone of magnoflorine [16].
For Compounds 59 and 64, we obtained a molecular design based on the structure of magnoflorine, with an adjacent hydroxyl group in which one methoxy group was changed to 1,3-dioxolane and from which one methoxy group was removed, giving a molecular formula of C 19 H 20 NO 3 . These compounds had retention times of 14.11 and 17.08 min, respectively, and produced a precursor ion at m/z 310.1438 [M] + . We observed five characteristic fragment ions at m/z 58.0659, 177.0555, 205.0648, 233.0598, and 265.0859. On the basis of the above information, we identified Compounds 59 and 64 as isomers of C 1 -demethoxy -C 2 -dehydrox-C 10 , C 11 -ethyl epoxide of magnoflorine [16].

Fragmentation Pattern of Tertiary Aporphine Alkaloid
We definitively identified Compound 45 as lirinidine by comparing its retention time and MS and MS 2 spectra with reference standard data. Furthermore, Compounds 23 and 31 were eluted at 7.38 and 7.72 min, respectively, and exhibited the same MS 1 at m/z 282.1489 [M+H] + . We observed five distinct fragment ion peaks at m/z 58.0660(90), 191.0855(5), 219.0806(23), 237.0911(100), and 251.1063, as with lirinidine. We therefore identified Compounds 23 and 31 as lirinidine isomers.
We precisely identified Compound 53 as roemerine by comparing its retention time and its MS and MS 2 spectra with those in the reference standard data.
Compound 5 had a molecular formula of C 18 H 15 NO 2 , was eluted at 5.33 min, and produced a precursor ion at m/z 278.1175 [M+H] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 107.0497, 246.0928, and 262.0858. We therefore identified this compound as dehydroroemerine [24].
For Compounds 6, 12, 18, and 25, the molecular design was based on the structure of lirinidine with an additional set of adjacent hydroxyls and methoxy groups, giving a molecular formula of C 19 H 22 NO 4 . These were eluted at 5.40, 5.88, 6.40, and 7.53 min, respectively, and they produced a precursor ion at m/z 328.1543 [M+H] + in the positive ion mode. In addition, we observed five characteristic fragment ion peaks at m/z 58.0660, 177.0551, 222.1118, 265.0862, and 283.0967. On the basis of the above fragment characteristics, we identified these compounds as isomers of bolidine [25].
Compound 10 had a molecular formula of C 18 H 19 NO 4 , was eluted at 5.81 min, and produced a precursor ion at m/z 298.1437 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0659, 192.1022, 254.0953, and 283.1197. We identified Compound 10 as apoglaziovine [26].
Compounds 28, 47, and 62 had a molecular formula of C 19 H 19 NO 2 and produced a precursor ion at m/z 294.1488 [M+H] + in the positive ion mode. We observed six characteristic fragment ion peaks at m/z 58.0658, 217.0650, 236.0831, 250.0946, 263.1286, and 279.1256. On the basis of the above fragmentation patterns, we tentatively identified these compounds as dehydronuciferine isomers [27].
We produced Compound 30 by adding two adjacent methoxy groups and N-methyl to the structure of roemerine, giving a molecular formula of C 21 H 24 NO 4 . This compound exhibited a retention time of 7.69 min and produced a precursor ion at m/z 354.1670 [M+H] + . We observed three fragment ion peaks at m/z 58.0660, 251.1074, and 309.1119 in the positive ion mode. On the basis of these results, and previously reported findings in the literature [28], we identified Compound 30 as N-methylnantenine.
Compounds 32, 37, and 58 had a chemical formula of C 19 H 21 NO 3 and produced a precursor ion at m/z 358.20128 [M+H] + in the positive ion mode. We observed five fragment ion peaks at m/z 58.0659, 217.0650, 267.1016, 280.1064, and 294.1487. On the basis of this information, we identified these compounds as isomers of isothebaine [29].
Compound 38 had a molecular formula of C 20 H 21 NO 4 and exhibited a retention time of 8.66 min. This compound produced a precursor ion at m/z 340.1543 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0660, 220.0526, 264.0755, and 309.1354. Hence, Compound 38 was tentatively identified as crebanine [26].
Compounds 55, 65 and 67 had a molecular formula of C 19 H 17 NO 4 and produced a precursor ion at m/z 324.1230 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0659, 177.0554, 263.0940, and 293.1054. We identified these compounds as isomers of neolitsine [14].
Compounds 54, 68, and 69 had a molecular formula of C 18 H 13 NO 3 and produced a precursor ion at m/z 292.0968 [M+H] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 248.0712, 264.1024, and 277.1039. The product ion at m/z 277.1039 [M+H−NH] + was obtained via the neutral loss of NH. We ascribed the loss of this fragment to NH serving as a different substituent for nitrogen. On the basis of the above fragmentation patterns, we tentatively identified these compounds as lysicamine isomers [30].
Compound 60 had a molecular formula of C 19 H 19 NO 3 , exhibited a retention time of 14.22 min, and produced a precursor ion at m/z 310.1437 [M+H] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 58.0660, 279.1008, and 264.0792. On the basis of the above fragmentation patterns, we tentatively identified Compound 60 as stephanine [31].
Compound 63 had a molecular formula of C 18 H 15 NO 3 , exhibited a retention time of 15.99 min, and produced a precursor ion at m/z 294.1124 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 58.0658, 239.0951, 257.1901, and 262.0863. On the basis of the above fragmentation patterns, we tentatively identified Compound 63 as N-formyl-annonain [32].
Compound 66 had a molecular formula of C 19 H 17 NO 3 , exhibited a retention time of 18.00 min, and produced a precursor ion at m/z 308.1281 [M+H] + in the positive ion mode. We observed three characteristic fragment ion peaks at m/z 191.0859, 219.0806, and 249.0914. The product ion at m/z 219.0806 [M+ H−C 2 H 5 NO] + was obtained via the neutral loss of C 2 H 5 NO. We ascribed the loss of this fragment to NHCOCH 3 serving as a different substituent for nitrogen. Based on the secondary fragmentation data and mass spectral fragmentation behavior, we identified Compound 66 as N-acetylanonaine [33].

Fragmentation Pattern of Secondary Aporphine Substituted
We unambiguously identified Compound 50 as N-nornuciferine by comparing its retention time and its MS and MS 2 spectra with the reference standard data. Compounds 2 and 13 had a molecular formula of C 25 H 31 NO 9 , and they produced a precursor ion at m/z 490.2072 [M+H] + in the positive ion mode. We observed five characteristic fragment ion peaks at m/z 192.1019, 237.0901, 265.0861, 297.1122, and 328.1544. On the basis of the above fragments, we identified Compounds 2 and 13 as isomers of 11-glc-norisocorydine [18].
Compound 9 had a molecular formula of C 18 H 19 NO 4, was eluted at 5.77 min, and produced a precursor ion at m/z 314.1386 [M+H] + in the positive ion mode. We observed six characteristic fragment ions at m/z 58.0660, 165.0913, 205.0658, 237.0910, 265.0861, and 297.1124. We identified this compound as laurolitsine [34].
Compound 51 had a molecular formula of C 17 H 15 NO 2 , exhibited a retention time of 12.80 min, and produced a precursor ion at m/z 266.1176 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 131.0494, 191.0855, 219.0804, and 249.0912 in the positive ion mode. On the basis ofn the above information, we identified Compound 51 as anonaine [33].
Compound 57 had a molecular formula of C 18 H 17 NO 4 , was eluted at 13.83 min, and produced a precursor ion at m/z 312.1230 [M+H] + in the positive ion mode. We observed five characteristic fragment ion peaks at m/z 58.0659, 264.1164, 265.0865, 280.1095, and 295.1328. We identified Compound 57 as nandigerine [21].
Compound 70 had a molecular formula of C 18 H 17 NO 4 , was eluted at 19.08 min, and produced a precursor ion at m/z 338.13860 [M+H] + in the positive ion mode. We observed four characteristic fragment ion peaks at m/z 279.1258, 307.1201 308.1265, and 323.1153. We identified Compound 70 as sinomendine [35].

Pharmacological Activity of Aporphine Alkaloids in SSD
Natural aporphine alkaloids exhibit a wide range of biological properties, including antioxidant, antiplatelet-aggregation, anticonvulsant, antispasmodic, anti-cancer, antimalarial, antiprotozoal, anti-poliovirus, anticytotoxicity, and anti-Parkinson effects. Natural products and their synthetic derivatives from the mainstay of research can be made into new medications for a wide range of disorders [36].
Aporphine alkaloids are widely distributed in various medicinal plants and are the active ingredients in many traditional Chinese medicines. Magnoflorine is one of the most important pharmacologically active compounds in the quaternary aporphine alkaloid, with reported anti-diabetic and anti-inflammatory effects [37]. Lirinidine is a tertiary aporphine alkaloid which greatly inhibits the production of collagen and arachidonic acid and reduces the aggregation of platelet-activating factor-induced platelets [38]. Among the secondary aporphine alkaloids, norisocorydine can help regulate transporters in the small intestine [39] and N-nornuciferine exhibits anti-inflammatory effects [40].

Chemicals and Materials
We obtained acetonitrile and LC grade methanol from MACKIN Company. We acquired MS grade formic acid from Thermo Fisher Scientific Co., Ltd. (New Jersey, NJ, USA). We obtained purified water from Guangzhou Watsons Food & Beverage Co., Ltd.

Standard and Solution Preparation
We pulverized an SSD stem and accurately weighed 1 g of sample powder. We transferred this to a flask containing 10 mL of 70% aqueous methanol (v/v) and performed ultrasonic extraction for 60 min at room temperature. We obtained supernatant after filtrating (nylon needle filter, 0.45 µm) and centrifuging at 13,523 g for 20 min at 10 • C.
We prepared reference-standard stock solutions of magnoflorine, lirinidine, N-nornuciferine, and roemerine at concentrations of 0.1 mg/mL with methanol. These were stored at 4 • C.
All samples were examined in the positive mode using the following tune approach. We used full-scan mode to produce high-resolution mass spectra with a resolution of 70 000 and a mass range of m/z 120-1000. PRM parameters were set as follows: the resolution was 35,000; the isolation window was 3.0 m/z; and the NEC (normalized collision energy) was set to 35, with 5.0 × e 4 of automatic gain control (AGC) target. We processed data using Xcalibur™ version 4.1 (Thermo Fisher Scientific, California, CA, USA) and Compound Discovery version 3.0 (Thermo Fisher Scientific, California, CA, USA). ESI source parameters were set as follows: the spray voltage was 3.5 kV; flow rates of 30 and 10 (arbitrary units) were used for the sheath gas and auxiliary gas, respectively; nitrogen was ≥99.99%; capillary temperature and the heater temperature were set to 320 • C and 350 • C, respectively; the S-lens RF level was 50.

Data Processing
We used the Thermo Xcalibur software version 4.1 and Compound Discover software version 3.0 (Thermo Fisher Scientific, California, CA, USA) to process all the raw data, including full-scan MS and MS 2 data. We set the minimum peak intensity to 10,000 and calculated detailed chemical formula parameters from accurate masses for all the parent and fragment ions of selected peaks using a formula predictor, as follows: the maximum element counts were C30, H60, O20, and N10; the MS and MS 2 mass tolerances were set to 5 and 10 ppm, respectively.

Conclusions
Using UHPLC Q-Exactive MS, we established an effective method to fully identify the aporphine alkaloids in SSD. We identified a total of 70 aporphine alkaloid constituents in SSD based on their chromatographic retention, MS and MS 2 , and bibliographic data. Sixty-nine of these are here reported as constituents of SSD for the first time. Some of these compounds have previously been shown to exhibit good pharmacological properties, including anti-cancer and anti-diabetic effects. Our findings lay the groundwork for more in-depth investigations of the pharmacodynamic substance basis for SSD.