Chemical Profiling of Lobelia chinensis with High-Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry (HPLC/Q-TOF MS) Reveals Absence of Lobeline in the Herb

Lobelia chinensis is a kind of herbal medicine widely distributed and used in Asia. The chemical components of this herb, however, have not been well studied until now. Lobeline, as an essential and famous bioactive compound in Lobelia genus, has been assumed to be present in L. chinensis. In order to ascertain its presence and, more importantly, proper use of this herb, chemical profiling this herb with highly sensitive and high-resolution analytical mass spectrometry was applied. In this study, high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) method was employed to systematically profile the chemical constituents of L. chinensis for the first time. Comparative chemical profiling study of L. chinensis and Lobelia inflata was also conducted to provide evidence whether lobeline is present or not. Piperidine alkaloids except for lobeline, alkaloid-lignan hybrids, flavonoids, polyacetylenes, nonanedioic acid, and some new phytochemicals were successfully identified in L. chinensis simultaneously. Comparing to the chemical profiles of L. inflata, lobeline was found to be absent in L. chinensis. All of the secondary metabolites in L. chinensis were determined with the HPLC/Q-TOF MS method. The absence of lobeline in L. chinensis was confirmed after this extensive study.


Introduction
Lobelia chinensis, also named as the Chinese Lobelia, is a plant belonging to the genus of Lobelia in Campanulaceae family. This plant is mainly distributed in East Asia, especially in China. The dried plant has been used as an herbal medicine by for long time [1]. In the famous traditional Chinese medicine book, namely, 'Compendium of Matera Medica,' L. chinensis was reported to promote urination, reduce edema, cleanse heat and toxicants. Traditionally, this herb is used to treat snakebites and to relieve edema according to the practices of Chinese folk medicines. This plant is included in the contemporary Chinese Pharmacopoeia and officially regarded as an herbal medicine [2].
Modern pharmacological study of L. chinensis revealed that this herb possesses some biological activities on cancer, bacteria, and viruses [3]. It is also reported to have inflammation modulatory Table 1. Ten batches of Lobelia chinensis samples collected from different areas of China.

Sample Extraction
All dried plant materials were pulverized and pass through a 50-mesh sieve prior to being extracted. Ten grams of sample powder was extracted with 20 mL methanol by sonication for 20 min and subjected to centrifugation at 4500 r.p.m. for 5 min. After centrifugation, the sample solution was collected and filtered with a 0.22 µm nylon syringe filter (Xiboshi, Tianjin, China) and the filtrate was used for subsequent analysis.
Mass spectrometry data was acquired in both positive and negative ionization modes with a mass range of 50-1000 Dalton. Instrumental parameters were optimized as follows: Voltage, 5500 V (positive) and −4500 V (negative); source gas 1 (nebulizer gas), 35 psi; source gas 2 (auxiliary gas), 60 psi; curtain gas, 30 psi; Turbo gas temperature, 500 °C; declustering potential, 80 V (positive) and −80 V (negative); focusing potential, 380 V (positive) and −380 V (negative); declustering potential 2, 10 V (positive) and −10 V (negative); collision gas, 3 psi. Nitrogen was used in all cases. For MS/MS analysis, the most abundantly charged ions from 150 to 700 above 100 counts threshold were selected for collision-induced dissociation (CID) using the information-dependent acquisition (IDA) method with automatic collision energy. This MS/MS analysis was conducted for each sample during HPLC/Q-TOF MS analysis. The instrument was calibrated using a solution of sodium bromate in both positive and negative modes prior to data collection.

Sample Extraction
All dried plant materials were pulverized and pass through a 50-mesh sieve prior to being extracted. Ten grams of sample powder was extracted with 20 mL methanol by sonication for 20 min and subjected to centrifugation at 4500 r.p.m. for 5 min. After centrifugation, the sample solution was collected and filtered with a 0.22 µm nylon syringe filter (Xiboshi, Tianjin, China) and the filtrate was used for subsequent analysis.
Mass spectrometry data was acquired in both positive and negative ionization modes with a mass range of 50-1000 Dalton. Instrumental parameters were optimized as follows: Voltage, 5500 V (positive) and −4500 V (negative); source gas 1 (nebulizer gas), 35 psi; source gas 2 (auxiliary gas), 60 psi; curtain gas, 30 psi; Turbo gas temperature, 500 • C; declustering potential, 80 V (positive) and −80 V (negative); focusing potential, 380 V (positive) and −380 V (negative); declustering potential 2, 10 V (positive) and −10 V (negative); collision gas, 3 psi. Nitrogen was used in all cases. For MS/MS analysis, the most abundantly charged ions from 150 to 700 above 100 counts threshold were selected for collision-induced dissociation (CID) using the information-dependent acquisition (IDA) method with automatic collision energy. This MS/MS analysis was conducted for each sample during HPLC/Q-TOF MS analysis. The instrument was calibrated using a solution of sodium bromate in both positive and negative modes prior to data collection. For accurate mass measurements, the solution of leucine-enkephalin ([M + H] + 556.2771 and [M − H] − 554.2615) at a concentration of 0.5 µM was used for internal calibration in both positive and negative ionization modes. The calibration solution was continuously spiked into post-column sample solution using a PEEK tee fitting (0.020", Vertical, Thailand) by a syringe pump (Harvard Apparatus 11 Plus, Holliston, MA, USA) at a flow rate of 5 µL·min −1 during full analysis. An Analyst QS 2.0 software was employed for data acquiring and processing.

HPLC/Q-TOF MS Analysis of the Chemical Constituents of L. Chinensis
Being a powerful tool for analysis of secondary plant metabolites, an HPLC system coupled with a high-resolution Q-TOF mass spectrometer was employed for the chemical profiling the L. chinensis (LC-01) extract. Based on the required retention time, accurate masses and MS/MS spectra, and comparison of these data with previous references or available chemical standards, individual chemical components ( Figure 2) in the total ion current (TIC) chromatograms of L. chinensis were assigned and listed as shown in Table 2 and Figure 3. concentration of 0.5 µM was used for internal calibration in both positive and negative ionization modes. The calibration solution was continuously spiked into post-column sample solution using a PEEK tee fitting (0.020", Vertical, Thailand) by a syringe pump (Harvard Apparatus 11 Plus, Holliston, MA, USA) at a flow rate of 5 µL•min −1 during full analysis. An Analyst QS 2.0 software was employed for data acquiring and processing.

HPLC/Q-TOF MS Analysis of the Chemical Constituents of L. Chinensis
Being a powerful tool for analysis of secondary plant metabolites, an HPLC system coupled with a high-resolution Q-TOF mass spectrometer was employed for the chemical profiling the L. chinensis (LC-01) extract. Based on the required retention time, accurate masses and MS/MS spectra, and comparison of these data with previous references or available chemical standards, individual chemical components ( Figure 2) in the total ion current (TIC) chromatograms of L. chinensis were assigned and listed as shown in Table 2 and Figure 3.    In the TIC chromatogram of L. chinensis in positive ionization mode, Peaks 1−10, 12 and 13 were assigned as piperidine alkaloids. According to the reported fragmentation behaviors of 2,6-disubstituted N-methylpiperidine alkaloids where cleaving α to N is preferred, the cleavage of one side chain, or both side chains with hydrogen transfer to leave singly or doubly unsaturated N-methylpiperidine, or the cleavage of N-methyl group is the characteristic behavior of 2,6-disubstituted N-methylpiperidine alkaloids in their MS/MS spectra [10,21,22].  [14]. Peaks 5 and 7, giving [M + H] + at m/z 242.2132 and 242.2106, respectively, were identified as two stereoisomers of 8,10-diethyllobelionol [15]. Almost the same fragmentation ions could be obtained as given in Table 2. These fragmentation In the TIC chromatogram of L. chinensis in positive ionization mode, Peaks 1−10, 12 and 13 were assigned as piperidine alkaloids. According to the reported fragmentation behaviors of 2,6-disubstituted N-methylpiperidine alkaloids where cleaving α to N is preferred, the cleavage of one side chain, or both side chains with hydrogen transfer to leave singly or doubly unsaturated N-methylpiperidine, or the cleavage of N-methyl group is the characteristic behavior of 2,6-disubstituted N-methylpiperidine alkaloids in their MS/MS spectra [10,21,22].  [14]. Peaks 5 and 7, giving [M + H] + at m/z 242.2132 and 242.2106, respectively, were identified as two stereoisomers of 8,10-diethyllobelionol [15]. Almost the same fragmentation ions could be obtained as given in Table 2 only Peak 20 providing m/z at 508.2336 was assumed to be a stereoisomer of alkaloid-lignan hybrid lobechinenoid according to the accurate mass measurement result. This compound was first isolated as a mixture of two diastereomeric pairs of enantiomers lobechinenoids A-D from the aerial parts of L. chinensis [18]. The fragment at m/z 478 indicated the loss of a methyloxyl group. Peak 16 at m/z 494.2177, which was 14 Da (CH 2 ) less than the protonated molecular ion of lobechinenoid, was tentatively identified to be a stereoisomer of demethyllobechinenoid for the first time. Peak 11 at m/z 656.2699 and Peak 14 at m/z 670.2863 were 162 Da (C 6 H 10 O 5 ) more than the protonated molecular ions of demethyllobechinenoid and lobechinenoid respectively, which indicated the addition of a glucose molecule. Therefore, Peaks 11 and 14 were tentatively identified as lobechinenoid glucoside and demethyllobechinenoid glucoside respectively. These two glycosides were not reported in previous research [4][5][6]8,[14][15][16]18]. Therefore, this was also the first detection of these two glycosides in L. chinensis.  [4][5][6]. The unidentified Peak 15 showed similar fragments as lobetyolinin and lobetyolin at m/z 59, 71, 89, 113 and 119, so it was suspected as an analogue of lobetyolinin and lobetyolin which was not reported before.
Peak 18, giving [M − H] − at m/z 187.0957, was identified as nonanedioic acid according to its MS/MS spectrum and accurate mass [17]. Peaks 19 and 24 were identified as flavonoids linarin and diosmin respectively according to reference standards and previous reports [4,5].  [19]. This is the first time that this compound was found in L. chinensis.
To summarize, piperidine alkaloids, alkaloid-lignan hybrids, involving their corresponding glucosides, flavonoids, polyacetylenes, and one organic acid were identified from L. chinensis. Phytochemicals, 8-ethyl-10-propyllobelionol, 8-propyl-10-ethyllobelionol, demethyllobechinenoid, together with two alkaloid glucosides, lobechinenoid glucoside, and demethyllobechinenoid glucoside, were identified as new compounds in this study. A flavonoid compound, 3'-methoxyl-linarin, was detected in L. chinensis for the first time. The bioactivities of some of the detected phytochemicals were studied and demonstrated in previous research. L. chinensis alkaloids were testified to have anti-growth activity on cancer cells [23,24]. The detected diosmin and its aglycone form diosmetin were proved as natural dietary agonists of the aryl hydrocarbon receptor (AhR) causing a strong increase in cytochrome P450 1A1 transcription and activity [25]. Flavonoid compound linarin was evaluated as a better anti-inflammatory agent in mice and rats comparing with pectolinarin [26].

Investigation of Lobeline in L. Chinensis
As the most significant kind of piperidine alkaloid in the Lobelia genus, especially in L. inflata, lobeline showed remarkable biological activities on the central nervous system (CNS) [9,27]. Lobeline was often regarded as the bioactive secondary metabolite of L. chinensis also [1,4]. However, in our study on the chemical profiling of L. chinensis using sensitive HPLC/Q-TOF MS, twelve stereochemically different piperidine alkaloids were detected without well-known lobeline. To investigate the presence of lobeline, 10 batches of L. chinensis together with one batch of L. inflata were simultaneously analyzed with HPLC/Q-TOF MS.
The final results are shown in Figure 4. The 10 batches of L. chinensis samples tested showed very similar TIC chromatograms which were totally different from L. inflata. Lobeline was easily identified in the TIC chromatogram of L. inflata with a retention time at 27.0 min by the retention time and MS/MS spectrum of the reference standard and previous reports [10,22]. The fragmentation behavior and corresponding MS/MS spectrum of lobeline are shown in Figure 5. In MS/MS spectrum, neutral loss of a side chain induced the fragments at m/z 216 or 218, and successive losses of one water moiety and the ketone side chain leaded to fragments at m/z 320 and 200 respectively. Two characteristic fragments at m/z 98 and 96 belonging to unsaturated N-methlypiperidines were also obtained. However, in the TIC chromatograms of all 10 batches of L. chinensis samples collected from different regions of China, no peak belonging to (−)-lobeline with a retention time at 27.0 min could be detected. This result demonstrated that lobeline was absent in the Lobelia plant L. chinensis.
Two characteristic fragments at m/z 98 and 96 belonging to unsaturated N-methlypiperidines were also obtained. However, in the TIC chromatograms of all 10 batches of L. chinensis samples collected from different regions of China, no peak belonging to (−)-lobeline with a retention time at 27.0 min could be detected. This result demonstrated that lobeline was absent in the Lobelia plant L. chinensis.

Conclusions
In conclusion, as a powerful tool for rapid identification of chemical constituents in complex materials, such as herbs, HPLC/Q-TOF MS was applied for the determination of the secondary metabolites of L. chinensis in this study. By comparing the acquired retention time, accurate masses and MS/MS spectra with reference standards and previous references, 12 stereochemically different piperidine alkaloids, four alkaloid-lignan hybrid analogues, four flavonoids, two polyacetylenes, and an organic acid nonanedioic acid were simultaneously discovered in L. chinensis. Among these phytochemicals, two types of piperidine alkaloids, 8-ethyl-10-propyllobelionol and 8-propyl-10-ethyllobelionol, and three piperidine alkaloid analogues, demethyllobechinenoid, demethyllobechinenoid glucoside, and lobechinenoid glucoside, were detected for the first time. As the most significant piperidine alkaloids detected in Lobelia plant L. inflata, lobeline was found to be absent in all 10 batches of the L. chinensis collected from different regions of China. This is the first systematic study on the chemical constituents of L. chinensis using sensitive HPLC/Q-TOF MS. The comparative investigation of L. chinensis and L. inflata provided direct and solid evidence to prove the absence of lobeline in L. chinensis. This research will be helpful for the quality control and further study of L. chinensis as a valuable herbal medicine.

Conclusions
In conclusion, as a powerful tool for rapid identification of chemical constituents in complex materials, such as herbs, HPLC/Q-TOF MS was applied for the determination of the secondary metabolites of L. chinensis in this study. By comparing the acquired retention time, accurate masses and MS/MS spectra with reference standards and previous references, 12 stereochemically different piperidine alkaloids, four alkaloid-lignan hybrid analogues, four flavonoids, two polyacetylenes, and an organic acid nonanedioic acid were simultaneously discovered in L. chinensis. Among these phytochemicals, two types of piperidine alkaloids, 8-ethyl-10-propyllobelionol and 8-propyl-10-ethyllobelionol, and three piperidine alkaloid analogues, demethyllobechinenoid, demethyllobechinenoid glucoside, and lobechinenoid glucoside, were detected for the first time. As the most significant piperidine alkaloids detected in Lobelia plant L. inflata, lobeline was found to be absent in all 10 batches of the L. chinensis collected from different regions of China. This is the first systematic study on the chemical constituents of L. chinensis using sensitive HPLC/Q-TOF MS. The comparative investigation of L. chinensis and L. inflata provided direct and solid evidence to prove the absence of lobeline in L. chinensis. This research will be helpful for the quality control and further study of L. chinensis as a valuable herbal medicine.