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Thin layer chromatography combined with electrospray ionization mass spectrometry for characterizing herbal compounds
Accepted Manuscript Title: Thin Layer Chromatography Combined with Electrospray Ionization Mass Spectrometry for Characterizing Herbal Compounds Authors: Sy-Chyi Cheng, Suhail Muzaffar Bhat, Chi-Wei Lee, Jentaie Shiea PII: DOI: Reference:
S1387-3806(18)30230-6 https://doi.org/10.1016/j.ijms.2018.09.024 MASPEC 16021
To appear in:
International Journal of Mass Spectrometry
Received date: Revised date: Accepted date:
29-6-2018 13-9-2018 20-9-2018
Please cite this article as: Cheng S-Chyi, Bhat SM, Lee C-Wei, Shiea J, Thin Layer Chromatography Combined with Electrospray Ionization Mass Spectrometry for Characterizing Herbal Compounds, International Journal of Mass Spectrometry (2018), https://doi.org/10.1016/j.ijms.2018.09.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thin Layer Chromatography Combined with Electrospray Ionization Mass Spectrometry for Characterizing Herbal Compounds
Sy-Chyi Cheng1,*, Suhail Muzaffar Bhat1, Chi-Wei Lee2,3, and Jentaie Shiea1,4,5,6,* 1
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Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan 2 Institute of Medical Science and Technology, National Sun Yat-Sen University, Taiwan 3 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 4 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Rapid Screening Research Center for Toxicology and Biomedicine, National Sun Yat-Sen University, Kaohsiung, Taiwan
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*Corresponding author: Dr. Jentaie Shiea Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected] Dr. Sy-Chyi Cheng Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected]
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Graphical Abstract (TOC)
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Sawtooth TLC-ESI/MS was applied to characterize phytochemical compounds in medicinal
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Highlights
herbs
Highly toxic alkaloids were detected in Aconitum carmichaelii Debx
Ginsenosides — the active ingredients in Panax ginseng — were detected
Visualizable and non-visualizable analytes in TLC tracks were characterized
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Abstract
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Characterizing the active ingredients and toxins in herbal products has important health
implications in traditional Chinese medicine. Thin layer chromatography (TLC) is the most widely used technique for separating phytochemical compounds in plant extracts and has been coupled with mass spectrometry to provide sensitive and specific analysis. However, several analytical issues affect these methods, making them non-viable for long term and high-throughput analysis. We therefore developed a simple TLC/MS approach termed sawtooth thin layer chromatography2
electrospray ionization/mass spectrometry (sawtooth TLC-ESI/MS) to directly scan and characterize herbal compounds on developed TLC plates that were modified into sawtooth pieces to facilitate rapid analysis. We directly induced electrospray ionization at each tip along the sawtooth edges to avoid detaching gel particles during analysis and to characterize both visualizable and nonvisualizable analytes. Sawtooth TLC-ESI/MS was used to characterize the active ingredients and
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toxic compounds in Aconitum carmichaelii Debx and Panax ginseng root extracts. Sugar molecules and 16 alkaloids such as aconitine, mesaconitine, and hypaconitine were detected in the A.
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carmichaelii Debx extract, whereas 6 major ginsenosides were detected in the P. ginseng extract.
Keywords: Sawtooth TLC-ESI/MS; thin layer chromatography; medicinal herb; Aconitum
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carmichaelii Debx; Panax ginseng
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1. Introduction
Herbal plants, containing active ingredients and toxins, have versatile medicinal functions and
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have been used in medicinal therapy and primary healthcare for their antiemetic, antirheumatic, anti-
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hypertensive, anti-inflammatory, analgesic, and diuretic properties [1-3]. In traditional Chinese medicine, recognizing and using the proper amounts of medicinal herbs often relies on experience.
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Unfortunately, counterfeit herbs can be encountered in the marketplace due to the difficulties in distinguishing counterfeit and authentic herbs based on morphology. Raman spectroscopy has been
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used to differentiate between authentic and counterfeit herbs [4,5]; however, the technique lacks specificity and sensitivity, especially for analyzing mixtures of authentic and counterfeit herbs. As a
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result, it is necessary to accurately characterize the active and toxic ingredients in herbal plants for quality control and safe use. Gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) have been applied to characterize the compounds in herb extracts [6-10]. Although both techniques can identify phytochemical compounds with a high sensitivity and specificity, tedious sample preparation processes are required prior to chromatographic separation and mass spectrometric analysis, making these techniques non-ideal for 3
high-throughput and rapid analysis. On the other hand, thin layer chromatography (TLC) is cheap, simple, and rapid, and can simultaneously separate complex unpretreated samples [11-13]. It is widely and routinely used to characterize phytochemical compounds in plant extracts, tinctures, essential oils, and herbal products [13-15]. Developed TLC plates are examined under a light source or sprayed with chemical reagents
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to visualize analyte spots and obtain analyte separation profiles. Since TLC does not provide
information on analyte structure and molecular weight, further characterization of analyte spots using
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mass spectrometry is essential [16-18].
As a viable alternative to conventional MS methods, ambient mass spectrometry (AMS) allows
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direct and rapid analysis at atmospheric pressure and room temperature without tedious sample
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preparation [19-23]. Some AMS techniques have been applied to analyze herbal substances to assure
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their quality, efficacy, and safety [24]. For example, desorption electrospray ionization/mass spectrometry (DESI/MS) was used for the in situ detection of the active ingredients in Conium
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maculatum and Atropa belladonna tissues [25], while direct analysis in real time/mass spectrometry
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(DART/MS) was used to analyze plant extracts for rapid characterization of alkaloids, flavonoids, and ginsenosides in herbs [26]. With methods like tissue spray and leaf spray mass spectrometry,
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herbal leaves and slices were sharpened into tips at which ESI was induced to analyze compounds in these herbs [27-29]. Electrospray laser desorption ionization/mass spectrometry (ELDI/MS) was
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used to elucidate the distribution of triterpenoids on the surfaces of plant samples like Ganoder malucidum slices [30].
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Although the predominant compounds in herbs can be characterized and identified by AMS
without sample preparation, it is difficult to detect trace compounds due to the ion suppression effect. As a result, it is necessary to separate complex mixtures like herbal extracts before AMS analysis. Since both TLC and AMS are operated under ambient conditions, combining the two techniques for mixture analysis is easy and rational [13,31,32]. TLC-DESI/MS and TLC-DART/MS was used to characterize alkaloids in Hydrastis canadensis root extracts and identify markers in the fruits of 4
Evodia rutaecarpa and Schisandra chinensis, respectively [33,34]. However, TLC-AMS methods like TLC-DART/MS that use low thermal energy for analyte desorption are not ideal for characterizing thermally labile and nonvolatile compounds. Techniques like laser-induced acoustic desorption electrospray ionization/mass spectrometry (LIAD-ESI/MS) and plasma assisted multiwavelength laser desorption ionization/mass spectrometry (PAMLDI/MS) couple a laser beam
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with an atmospheric pressure ionization (API) source [35,36]. Both have been combined with TLC to analyze compounds in rosemary essential oil and Longjing tea [36,37]. Unfortunately, these TLC-
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AMS techniques inevitably detach TLC gel particles during analysis which can damage the vacuum pumps of the mass spectrometer over the long term.
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The combination of thin layer chromatography and electrospray ionization/mass spectrometry
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(TLC-ESI/MS) allows for the characterization of volatile and non-volatile compounds with minimal
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detachment of TLC gel particles [38]. Developed analyte spots were excised from aluminum-based TLC plates to form a V-shaped piece [39,40]. An organic solvent was applied on the surface of the
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V-shaped TLC piece to extract and elute analytes toward the tip, and a high voltage was
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subsequently applied to the aluminum sheet to induce an ESI plume at the tip. Although such TLCESI/MS approaches can characterize the compounds in a mixture, it was laborious to visualize the
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analyte spots and cut the V-shaped pieces. In addition, characterizing non-visualizable analytes was impossible without further sample pretreatment with chemical reagents. Alternatively, a liquid
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junction surface sampling probe was developed to scan TLC plate surfaces to directly characterize the analytes on the plates [41]. However, the long analytical time made this technique non-ideal for
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high-throughput analysis. To overcome the shortcomings of the aforementioned TLC/MS techniques, we developed
sawtooth thin layer chromatography-electrospray ionization/mass spectrometry (sawtooth TLCESI/MS). The TLC separation of the complex mixtures of herb extracts attenuated the ion suppression effect, while the ESI/MS ionization of analytes directly on TLC plates avoided the detachment of gel particles while enabling sensitive and specific detection of visualizable and non5
visualizable compounds. Analysis was rapid and simple, making this technique applicable for highthroughput studies. Sawtooth TLC-ESI/MS was used to characterize phytochemical compounds in P. ginseng and A. carmichaelii Debx root extracts.
2. Experimental
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2.1 Materials Methanol (MeOH), 1-butanol, and aluminum-based silica gel 60 F254 TLC plates were purchased
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from Merck (Darmstadt, Germany). Ammonium hydroxide (NH4OH), acetic acid (AA), and toluene were obtained from J. T. Baker (Phillipsburg, NJ, U.S.A.). Ethyl acetate (EA) was obtained from Macron Fine Chemicals (Center Valley, PA, U.S.A.). A PURELAB Pulse water purifier (ELGA
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LabWater, Marlow, U.K.) was used to produce deionized water. The processed roots of Panax
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ginseng and Aconitum carmichaelii Debx (Chuan Wu herb) were obtained from a local pharmacy for
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traditional Chinese medicines. The herbal slices were ground and extracted as follows: 100 mg of the
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ground powder was added to 1 mL of 70% MeOH, after which the mixture was ultrasonicated at
collected for analysis.
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2.2 TLC Separation
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room temperature for 30 min and centrifuged at 6,000 rpm for 10 min. The suspension was then
Sample solutions were deposited on silica TLC plates using a TLC sample applicator
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(LINOMAT 5, CAMAG, Switzerland) at a dosage speed of 80 nL/s. The A. carmichaelii Debx root extract (4 uL) was deposited as a band of 7 mm long and 0.7 mm wide on each TLC plate; analytes were subsequently separated in a mobile phase of 1-butanol-toluene-EA-MeOH-NH4OH
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(40:28:5:3:6, v/v/v/v/v). The P. ginseng root extract (6 uL) was deposited as a band (6 mm) on each TLC plate; analytes were then separated in a mobile phase of 1-butanol-AA-water solution (7:1:2, v/v/v). After it was air-dried, the developed TLC plate was placed in a documentation system (TLC Visualizer 2, CAMAG) and photographed by a CCD camera, wherein the plate was illuminated with white light, 254 nm, and 366 nm UV light. 6
2.3 Sawtooth TLC-ESI-MS System To characterize both visible and non-visible analyte spots on the TLC track, a pair of stainless steel zigzag cut scissors (Nikken, Japan) with a cutting teeth distance of 3 mm was used to cut the TLC track in half, dividing the developed TLC plate into two sawtooth-shaped pieces, each with multiple triangular tips on one side (Figures 1a-b). The internal angle of each triangular tip was 100°
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and the distance between two triangular tips was 3 mm. A sawtooth TLC plate piece was placed on an XYZ stage and clipped with an alligator clip on the side that was opposite the serrated side
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(Figure 1c). The piece was positioned in front of the MS analyzer so that a triangular sawtooth tip was pointed directly at the MS inlet; the inlet was sheathed by a ceramic tube (1.5 cm long) to
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prevent discharging. The tip was placed 1.5 mm away from the ceramic tube. A high DC voltage of +5 kV was applied to the aluminum sheet of the sawtooth TLC plate through the alligator clip, and 1
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μL of an ESI solvent (100% MeOH for the analysis of ginseng, EA-MeOH solvent [50:50, v/v] for the analysis of A. carmichaelii) was deposited using a micropipette at the center of the triangle being
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analyzed. The function of the methanol solvent was to extract and carry analytes toward the tip for
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ESI ionization. A quadrupole time-of-flight mass analyzer (Q-TOF, micrOTOF-QII, Bruker Daltonics, MA, U.S.A.) with an MS inlet potential of -500 V, drying gas temperature of 200 °C,
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drying gas flow rate of 1.1 L/min, and scan speed of 2 Hz was operated in the positive ion mode. The collision energy was set between 8–35 eV for MS/MS analysis. The ion signal lasted for
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approximately 15 s. After the mass spectrum from the first triangular tip was obtained, the XYZ stage was moved to align the adjacent triangular tip with the MS inlet for the next analysis. The
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analytical process was repeated until all triangular tips on one sawtooth TLC piece were analyzed and then repeated for the other piece.
3. Results and discussion 3.1 TLC analysis of A. carmichaelii The roots and tubers of the Aconitum plant species (also known as aconite) contains toxic 7
alkaloids which can induce respiratory paralysis and cardiac arrhythmias [42,43]. However, the aconite plant provides anti-rheumatic, anti-inflammatory, and analgesic effects in small doses, making it a widely used plant in traditional Chinese medicine [43]. For the purpose of internal use, the aconite roots and tubers are generally processed through soaking and boiling processes to decrease the toxic alkaloid content. However, improper or insufficient processing temperature, time,
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and steps of aconite will increase the risk of poisoning. Before sawtooth TLC-ESI/MS analysis, the A. carmichaelii extract was first characterized using infusion ESI/MS to understand the chemical
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profile of the processed A. carmichaelii root (Figure 2a). Although the A. carmichaelii root has been processed, highly toxic compounds including aconitine (m/z 646.3221), mesaconitine (m/z
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632.3064), and hypaconitine (m/z 616.3111) and other alkaloids (i.e. m/z 400–700 for aconitum
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alkaloids and m/z 800–900 for lipo-alkaloids) were still detected in the extract. Sawtooth TLC-
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ESI/MS was then utilized to separate and analyze highly toxic alkaloids and other compounds in an A. carmichaelii extract. The extract was first separated on a silica TLC plate. After air-dried, the
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photograph of developed TLC plate was recorded by a documentation system. Figures 2b–d display
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the TLC profile of the A. carmichaelii root extract under white light, 254 nm UV light, and 366 nm UV light, respectively. Without using a chemical reagent, only a few bands at lower Rf values (0 to
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0.2) were observed on the TLC track, demonstrating the inability of TLC to characterize nonvisualizable analytes while showing the necessity of MS to detect such analytes.
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3.2 Sawtooth TLC-ESI/MS analysis of the A. carmichaelii extract Figure 2e shows the developed TLC plate with a track length of 5.3 cm after it was cut into two
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sawtooth pieces with 17 triangular tips on each piece. The tips in the right piece were labeled with even numbers (#2 to #34), whereas the tips in the left piece were labeled with odd numbers (#1 to #33). The sawtooth plate piece was then positioned on an XYZ stage and in front of the mass analyzer to characterize the phytochemical compounds present in the triangles. The volume of the extraction solvent (EA-MeOH) deposited at the center of the triangular tip should be well-controlled to prevent excessive solvent spread that would carry analytes to adjacent tips. We used an optimal 8
volume of 1 μL of extraction solvent for the analysis of all tips. The mass spectrum was acquired as a high voltage (+ 5 kV) and EA-MeOH solution was applied to the triangular tip aligned to the MS inlet. Figure 3 displays the sawtooth TLC-ESI mass spectra for the 34 triangular tips from the two TLC pieces. Although most of the analyte bands were not visualizable under UV light, ion signals for protonated aconitum alkaloids [M+H]+ and lipo-alkaloids [M+H]+ were detected, including those
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for 14-benzoylmesaconine-8-nonadecenoate (m/z 868.5383), 14-benzoylhypaconine-8-linoleate (m/z 836.5267), aconitine (m/z 646.3220), deoxyaconitine (m/z 630.3263), hypaconitine (m/z 616.3117),
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mesaconitine (m/z 632.3038), 10-hydroxymesaconitine (m/z 648.2991), talatisamine (m/z 422.2891), benzoylaconine (m/z 604.3107), benzoylmesaconine (m/z 590.2965), neoline (m/z 438.2830),
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isotalatizidine/talatizidin (m/z 408.2738), benzoyldeoxyaconine (m/z 588.3075), benzoylhypaconine
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(m/z 574.3039), fuziline (m/z 454.2807), and senbusine A/B (m/z 424.2703) (Table 1). The MS/MS
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information for aconitum alkaloids and lipo-alkaloids has been reported in previous works [44-46]; therefore, the detected analyte ions were further characterized using MS/MS to compare fragment
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ion patterns with those reported in previous publications. In addition to alkaloids, ion signals for
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sodiated sugars were also detected at tips #1 and #2 (i.e. the original position of the sample band); the detected sugars included disaccharides (m/z 365.1074, [M+Na]+), dimeric disaccharides (m/z
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707.2189, [2M+Na]+), and trisaccharides (m/z 527.1624, [M+Na]+), which were detected using sawtooth TLC-ESI/MS but not seen in the ESI mass spectrum (Figures 1a and 2). Furthermore, a
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series of background ions with a mass difference of 74 Da (i.e. m/z 467.1084, 541.1259, 615.1447, and 689.1593) was detected in the mass spectra, which may be from silica gel on TLC plate.
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3.3 TLC analysis of Panax Ginseng Ginseng (Panax ginseng) is an important herb in traditional Chinese medicine. It is considered
that ginsenosides are the major active ingredients in ginseng, conferring anti-fatigue, antiinflammatory, anti-tumor, and antioxidant benefits as well as boosting the immune system. The ESI mass spectrum of the ginseng root extract revealed that sugars and ginsenosides were detected as potassiated molecules [M+K]+ instead as protonated molecules [M+H]+ (Figure 4a). Similar results 9
were also reported in other study where the ginseng tissue was sharpened to generate an ESI plume at the tip [29]. In this study, the ginseng root extract was separated on a silica gel TLC plate first, it was then subjected for sawtooth TLC-ESI/MS analysis. Figures 4b–d display the TLC profile of the ginseng root extract under white light and 254 and 366 nm UV light. Although a TLC track was observed under 366 UV light, most of the analyte bands were not clearly resolved, again showing the
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need for MS analysis to characterize non-visualizable analytes. 3.4 Sawtooth TLC-ESI/MS analysis of P. Ginseng extract
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Figure 4e shows the developed TLC plate with a track length of ca. 5 cm after it was cut into two sawtooth pieces with 16 triangular tips on each piece. Figure 5 displays the sawtooth TLC-ESI mass
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spectra for 32 triangular tips from the two TLC pieces. Since trace amounts of sodium were present in the stationary phase, sugars and sugar derivatives such as ginsenosides were detected as sodiated
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ions. The compounds such as protopanaxadiol (m/z 499.2334, [M+K]+) and disaccharide (m/z
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365.1047, [M+Na]+; m/z 707.2207, [2M+Na]+) and a series of ion signals with a mass difference of
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342 Da (i.e., m/z 729.2010, 1071.3182, and 1413.4367) were detected at or near the original position
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of the sample band. The tandem mass spectrum of m/z 729 showed the main fragment ion signal at m/z 365. Therefore, the ions were assigned as disaccharide-related cluster ions (i.e., m/z 729.2010,
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[2M-H+2Na]+; m/z 1071.3182, [3M-H+2Na]+; 1413.4367, [4M-H+2Na]+; M: disaccharide). Furthermore, ginsenoside-related compounds observed at m/z 1233.6217, 1131.5905, 1263.6296,
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1101.5816, 969.5387, 985.5128, 823.4815, 807.4871, 1239.6580, and 754.5445 were clearly detected at different triangles on the TLC pieces.
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Due to a lack of analyte standards and the MS/MS information for sodiated ginsenosides was not comprehensively reported in the literature [47,48], the ginsenoside isomers we detected in this study could only be identified based on their molecular ion information: m/z 1233.6217 for Ra1 or Ra2, m/z 1131.5905 for Rb1, m/z 1101.5816 for Rb2 or Rc, m/z 969.5387 and 985.5128 (i.e. [M+Na]+ and [M+K]+) for Rd or Re, m/z 823.4815 for Rf or Rg1, and m/z 807.4871 for Rg2 or Rg3 (Table 2). The number of ginseng-related ions detected using sawtooth TLC-ESI/MS was much more than that 10
detected using infusion ESI/MS by simply comparing the spectra for the two techniques. The ions detected in sawtooth TLC-ESI/MS reflect the detailed chemical composition of ginseng and can be used for quality control assessments as well as distinguishing authentic from counterfeit ginseng. In this study, the detection of positive analyte ions on the TLC plate was demonstrated. The detection of
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negative ions can be achieved simply by switching the polarity of the mass analyzer. 4. Conclusion
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Sawtooth TLC-ESI/MS was utilized to combine the analytical advantages of TLC and ESI/MS for the separation and detection of phytochemical compounds in unpretreated herbal extracts. Both visible and non-visible analyte spots were successfully detected from developed TLC plates that
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were modified to induce ESI directly on the plates. Active ingredients (such as sugars and
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ginsenosides) and highly toxic alkaloids (such as aconitine, mesaconitine, and hypaconitine) were
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detected in the root extracts of Panax ginseng and Aconitum carmichaelii Debx, respectively. Our
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results demonstrate that sawtooth TLC-ESI/MS is a simple, rapid, and useful technique for
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identifying compounds of interest in herbs for quality control evaluations and safe medicinal use.
Acknowledgement
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This work was partially supported by the Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.
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[27] J.I. Zhang, X. Li, Z. Ouyang, R.G. Cooks, Direct analysis of steviol glycosides from Stevia leaves by ambient ionization mass spectrometry performed on whole leaves, Analyst 137 (2012) 3091–3098. [28] B. Hu, Y.H. Lai, P.K. So, H.W. Chen, Z.P. Yao, Direct ionization of biological tissue for mass spectrometric analysis, Analyst 137 (2012) 3613–3619. [29] S.L. Chan, M.Y. Wong, H.W. Tang, C.M. Che, K.M. Ng, Tissue-spray ionization mass spectrometry for raw herb analysis, Rapid Commun. Mass Spectrom. 25(19) (2011) 2837–2843 [30] M.Z. Huang, S.S. Jhang, J. Shiea, Electrospray laser desorption ionization (ELDI) mass spectrometry for molecular imaging of small molecules on tissues, Methods Mol. Biol. 1203 (2015) 107–116. [31] S.C. Cheng, M.Z. Huang, J. Shiea, Thin layer chromatography/mass spectrometry, J. Chromatogr. A, 1218 (19) (2011) 2700–2711. [32] G. Morlock, W. Schwack, Coupling of planar chromatography to mass spectrometry, TrACTrends Anal. Chem. 29 (10) (2010) 1157–1171.
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[33] G.J.V. Berkel, B.A. Tomkins, V. Kertesz, Thin-layer chromatography/desorption electrospray
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ionization mass spectrometry: investigation of goldenseal alkaloids, Anal. Chem. 79 (2007) 2778–2789. [34] H.J. Kim, E.H. Jee, K.S. Ahn, H.S. Choi, Y.P. Jang, Identification of marker compounds in herbal drugs on TLC with DART-MS, Arch. Pharm. Res. 33 (2010) 1355–1359. [35] S.C. Cheng, T.L. Cheng, H.C. Chang, J. Shiea, Using laser-induced acoustic desorption/electrospray ionization mass spectrometry to characterize small organic and large biological compounds in the solid state and in solution under ambient conditions, Anal. Chem. 81 (3) (2009) 868–874. [36] J. Zhang, Z. Zhou, J. Yang, W. Zhang, Y. Bai, H. Liu, Thin layer chromatography/plasma assisted multiwavelength laser desorption ionization mass spectrometry for facile separation and
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selective identification of low molecular weight compounds, Anal. Chem. 84 (3) (2012) 1496– 1503. [37] S.C. Cheng, M.Z. Huang, J. Shiea, Thin-layer chromatography/laser-induced acoustic
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desorption/electrospray ionization mass spectrometry, Anal. Chem. 81 (22) (2009) 9274–9281. [38] F.L. Hsu, C.H. Chen, C.H. Yuan, J. Shiea, Interfaces to connect thin-layer chromatography with electrospray ionization mass spectrometry, Anal. Chem. 75 (10) (2003) 2493–2498. [39] M. Himmelsbach, M. Waser, C.W. Klampfl, Thin layer chromatography-spray mass spectrometry: a method for easy identification of synthesis products and UV filters from TLC aluminum foils, Anal. Bioanal. Chem. 406 (15) (2014) 3647–3656. [40] B. Hu, G.Z. Xin, P.K. So, Z.P. Yao, Thin layer chromatography coupled with electrospray ionization mass spectrometry for direct analysis of raw samples, J. Chromatogr. A. 1415
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(2015)155–160. [41] G.J. Van Berkel, A.D. Sanchez, J.M. Quirke, Thin-layer chromatography and electrospray mass spectrometry coupled using a surface sampling probe, Anal. Chem. 74 (24) (2002) 6216–6223. [42] T.Y. Chan, Aconite poisoning, Clin. Toxicol. (Phila). 47 (4) (2009) 279–285. [43] E. Nyirimigabo, Y. Xu, Y. Li, Y. Wang, K. Agyemang, Y. Zhang, A review on phytochemistry, pharmacology and toxicology studies of Aconitum, J. Pharm. Pharmacol. 67 (1) (2015) 1–19. [44] G. Yan, H. Sun, W. Sun, L. Zhao, X. Meng, X. Wang, Rapid and global detection and characterization of aconitum alkaloids in Yin Chen Si Ni Tang, a traditional Chinese medical formula, by ultra performance liquid chromatography-high resolution mass spectrometry and automated data analysis, J. Pharm. Biomed. Anal. 53 (3) (2010) 421–431. [45] D. Csupor, E.M. Wenzig, I. Zupkó, K. Wölkart, J. Hohmann, R. Bauer, Qualitative and quantitative analysis of aconitine-type and lipo-alkaloids of Aconitum carmichaelii roots, J. Chromatogr. A. 1216 (11) (2009) 2079–2086. [46] N. Zhang, Y. Song, Q. Song, S. Shi, Q. Zhang, Y. Zhao, J. Li, P. Tu, Qualitative and quantitative assessments of aconiti lateralis radix praeparata using high-performance liquid
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chromatography coupled with diode array detection and hybrid ion trap-time-of-flight mass
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spectrometry, J. Chromatogr. Sci. 54 (6) (2016) 888–901. [47] M. Cui, F. Song, Z. Liu, S. Liu, Metal ion adducts in the structural analysis of ginsenosides by electrospray ionization with multi-stage mass spectrometry, Rapid Commun. Mass Spectrom. 15 (8) (2001) 586–595. [48] S. Liu, M. Cui, Z. Liu, F. Song, W. Mo, Structural analysis of saponins from medicinal herbs using electrospray ionization tandem mass spectrometry, J. Am. Soc. Mass Spectrom. 15 (2) (2004) 133–41.
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Figure legends Figure 1. Photographs of a silica TLC plate (a) before and (b) after being cut by zigzag scissors. (c) Photograph of the sawtooth TLC-ESI/MS setup.
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Figure 2. (a) ESI mass spectra for the A. carmichaelii root extract. Photographs of the TLC profile of A. carmichaelii root extract obtained under (b) white light and (c) 254 nm UV and (d, e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
Figure 3. Sawtooth TLC-ESI mass spectra for phytochemical compounds on a developed TLC plate.
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The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 1e from which the mass spectrum was obtained. A series of background ions with a mass difference of 74 Da (i.e. m/z 467, 541, 615, and 689) were originated from the silica stationary
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phase.
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Figure 4. (a) ESI mass spectrum for the P. ginseng root extract. Photographs of the TLC profile of P. ginseng root extract obtained under (b) white light and (c) 254 nm UV and (d & e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors. Figure 5. Sawtooth TLC-ESI mass spectra for ginsenosides and sugars on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth
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piece in Figure 3e from which the mass spectrum was obtained.
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Figure 1. Photographs of a silica TLC plate (a) before and (b) after being cut by zigzag scissors. (c) Photograph of the sawtooth TLC-ESI/MS setup.
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Figure 2. (a) ESI mass spectra for the A. carmichaelii root extract. Photographs of the TLC profile of A. carmichaelii root extract obtained under (b) white light and (c) 254 nm UV and (d, e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
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Figure 3. Sawtooth TLC-ESI mass spectra for phytochemical compounds on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 1e from which the mass spectrum was obtained. A series of background ions with a mass difference of 74 Da (i.e. m/z 467, 541, 615, and 689) were originated from the silica stationary phase. 19
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Figure 4. (a) ESI mass spectrum for the P. ginseng root extract. Photographs of the TLC profile of P. ginseng root extract obtained under (b) white light and (c) 254 nm UV and (d & e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
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Figure 5. Sawtooth TLC-ESI mass spectra for ginsenosides and sugars on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 3e from which the mass spectrum was obtained.
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Table 1. Alkaloids detected in the A. carmichaelii Debx extract by sawtooth TLC-ESI/MS.
Formula
Rf a
14-benzoylhypaconine-8-
C49H73NO10
0.99
836.5267
aconitine
C34H47NO11
0.99
646.3220
deoxyaconitine
C34H47NO10
0.99
630.3263
hypaconitine
C33H45NO10
0.99
616.3117
14-benzoylmesaconine-8-
C50H77NO11
0.96
868.5383
mesaconitine
C33H45NO11
0.96
talatisamine
C24H39NO5
0.92
10-hydroxymesaconitine
C33H45NO12
0.91
benzoylaconine
C32H45NO10
0.84
benzoylmesaconine
C31H43NO10
0.76
590.2965
neoline
C24H39NO6
0.73
438.2830
isotalatizidine/talatizidin
C23H37NO5
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Measured
Compound name
0.69
408.2738
benzoyldeoxyaconine
C32H45NO9
0.66
588.3075
C31H43NO9
0.54
574.3039
C24H39NO7
0.43
454.2807
C23H37NO6
0.38
424.2703
m/z [M+H]+
fuziline
A
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senbusine A/B
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benzoylhypaconine
a
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nonadecenoate
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linoleate
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The Rf values were calculated base on the following formula: [(distance from start line to the triangle where analyte was detected)/(distance from start line to solvent front)].
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Table 2. Ginsenosides detected in the P. ginseng root extract by sawtooth TLC-ESI/MS.
Measured
Compound name
Formula
Rf a
Rg2/Rg3
C42H72O13
0.72
807.4871
Rf/Rg1
C42H72O14
0.59
823.4815
Rd/Re
C48H82O18
0.43
969.5387
Rb2/Rc
C53H90O22
0.29
1101.5816
Rb1
C54H92O23
0.25
1131.5905
Ra1/Ra2
C58H98O26
0.20
1233.6217
a
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m/z [M+Na]+
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The Rf values were calculated base on the following formula: [(distance from start line to the triangle where analyte was detected)/(distance from start line to solvent front)].
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S1387-3806(18)30230-6 https://doi.org/10.1016/j.ijms.2018.09.024 MASPEC 16021
To appear in:
International Journal of Mass Spectrometry
Received date: Revised date: Accepted date:
29-6-2018 13-9-2018 20-9-2018
Please cite this article as: Cheng S-Chyi, Bhat SM, Lee C-Wei, Shiea J, Thin Layer Chromatography Combined with Electrospray Ionization Mass Spectrometry for Characterizing Herbal Compounds, International Journal of Mass Spectrometry (2018), https://doi.org/10.1016/j.ijms.2018.09.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Thin Layer Chromatography Combined with Electrospray Ionization Mass Spectrometry for Characterizing Herbal Compounds
Sy-Chyi Cheng1,*, Suhail Muzaffar Bhat1, Chi-Wei Lee2,3, and Jentaie Shiea1,4,5,6,* 1
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Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan 2 Institute of Medical Science and Technology, National Sun Yat-Sen University, Taiwan 3 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 4 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Rapid Screening Research Center for Toxicology and Biomedicine, National Sun Yat-Sen University, Kaohsiung, Taiwan
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*Corresponding author: Dr. Jentaie Shiea Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected] Dr. Sy-Chyi Cheng Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected]
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Graphical Abstract (TOC)
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Sawtooth TLC-ESI/MS was applied to characterize phytochemical compounds in medicinal
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Highlights
herbs
Highly toxic alkaloids were detected in Aconitum carmichaelii Debx
Ginsenosides — the active ingredients in Panax ginseng — were detected
Visualizable and non-visualizable analytes in TLC tracks were characterized
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Abstract
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Characterizing the active ingredients and toxins in herbal products has important health
implications in traditional Chinese medicine. Thin layer chromatography (TLC) is the most widely used technique for separating phytochemical compounds in plant extracts and has been coupled with mass spectrometry to provide sensitive and specific analysis. However, several analytical issues affect these methods, making them non-viable for long term and high-throughput analysis. We therefore developed a simple TLC/MS approach termed sawtooth thin layer chromatography2
electrospray ionization/mass spectrometry (sawtooth TLC-ESI/MS) to directly scan and characterize herbal compounds on developed TLC plates that were modified into sawtooth pieces to facilitate rapid analysis. We directly induced electrospray ionization at each tip along the sawtooth edges to avoid detaching gel particles during analysis and to characterize both visualizable and nonvisualizable analytes. Sawtooth TLC-ESI/MS was used to characterize the active ingredients and
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toxic compounds in Aconitum carmichaelii Debx and Panax ginseng root extracts. Sugar molecules and 16 alkaloids such as aconitine, mesaconitine, and hypaconitine were detected in the A.
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carmichaelii Debx extract, whereas 6 major ginsenosides were detected in the P. ginseng extract.
Keywords: Sawtooth TLC-ESI/MS; thin layer chromatography; medicinal herb; Aconitum
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carmichaelii Debx; Panax ginseng
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1. Introduction
Herbal plants, containing active ingredients and toxins, have versatile medicinal functions and
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have been used in medicinal therapy and primary healthcare for their antiemetic, antirheumatic, anti-
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hypertensive, anti-inflammatory, analgesic, and diuretic properties [1-3]. In traditional Chinese medicine, recognizing and using the proper amounts of medicinal herbs often relies on experience.
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Unfortunately, counterfeit herbs can be encountered in the marketplace due to the difficulties in distinguishing counterfeit and authentic herbs based on morphology. Raman spectroscopy has been
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used to differentiate between authentic and counterfeit herbs [4,5]; however, the technique lacks specificity and sensitivity, especially for analyzing mixtures of authentic and counterfeit herbs. As a
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result, it is necessary to accurately characterize the active and toxic ingredients in herbal plants for quality control and safe use. Gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) have been applied to characterize the compounds in herb extracts [6-10]. Although both techniques can identify phytochemical compounds with a high sensitivity and specificity, tedious sample preparation processes are required prior to chromatographic separation and mass spectrometric analysis, making these techniques non-ideal for 3
high-throughput and rapid analysis. On the other hand, thin layer chromatography (TLC) is cheap, simple, and rapid, and can simultaneously separate complex unpretreated samples [11-13]. It is widely and routinely used to characterize phytochemical compounds in plant extracts, tinctures, essential oils, and herbal products [13-15]. Developed TLC plates are examined under a light source or sprayed with chemical reagents
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to visualize analyte spots and obtain analyte separation profiles. Since TLC does not provide
information on analyte structure and molecular weight, further characterization of analyte spots using
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mass spectrometry is essential [16-18].
As a viable alternative to conventional MS methods, ambient mass spectrometry (AMS) allows
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direct and rapid analysis at atmospheric pressure and room temperature without tedious sample
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preparation [19-23]. Some AMS techniques have been applied to analyze herbal substances to assure
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their quality, efficacy, and safety [24]. For example, desorption electrospray ionization/mass spectrometry (DESI/MS) was used for the in situ detection of the active ingredients in Conium
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maculatum and Atropa belladonna tissues [25], while direct analysis in real time/mass spectrometry
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(DART/MS) was used to analyze plant extracts for rapid characterization of alkaloids, flavonoids, and ginsenosides in herbs [26]. With methods like tissue spray and leaf spray mass spectrometry,
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herbal leaves and slices were sharpened into tips at which ESI was induced to analyze compounds in these herbs [27-29]. Electrospray laser desorption ionization/mass spectrometry (ELDI/MS) was
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used to elucidate the distribution of triterpenoids on the surfaces of plant samples like Ganoder malucidum slices [30].
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Although the predominant compounds in herbs can be characterized and identified by AMS
without sample preparation, it is difficult to detect trace compounds due to the ion suppression effect. As a result, it is necessary to separate complex mixtures like herbal extracts before AMS analysis. Since both TLC and AMS are operated under ambient conditions, combining the two techniques for mixture analysis is easy and rational [13,31,32]. TLC-DESI/MS and TLC-DART/MS was used to characterize alkaloids in Hydrastis canadensis root extracts and identify markers in the fruits of 4
Evodia rutaecarpa and Schisandra chinensis, respectively [33,34]. However, TLC-AMS methods like TLC-DART/MS that use low thermal energy for analyte desorption are not ideal for characterizing thermally labile and nonvolatile compounds. Techniques like laser-induced acoustic desorption electrospray ionization/mass spectrometry (LIAD-ESI/MS) and plasma assisted multiwavelength laser desorption ionization/mass spectrometry (PAMLDI/MS) couple a laser beam
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with an atmospheric pressure ionization (API) source [35,36]. Both have been combined with TLC to analyze compounds in rosemary essential oil and Longjing tea [36,37]. Unfortunately, these TLC-
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AMS techniques inevitably detach TLC gel particles during analysis which can damage the vacuum pumps of the mass spectrometer over the long term.
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The combination of thin layer chromatography and electrospray ionization/mass spectrometry
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(TLC-ESI/MS) allows for the characterization of volatile and non-volatile compounds with minimal
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detachment of TLC gel particles [38]. Developed analyte spots were excised from aluminum-based TLC plates to form a V-shaped piece [39,40]. An organic solvent was applied on the surface of the
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V-shaped TLC piece to extract and elute analytes toward the tip, and a high voltage was
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subsequently applied to the aluminum sheet to induce an ESI plume at the tip. Although such TLCESI/MS approaches can characterize the compounds in a mixture, it was laborious to visualize the
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analyte spots and cut the V-shaped pieces. In addition, characterizing non-visualizable analytes was impossible without further sample pretreatment with chemical reagents. Alternatively, a liquid
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junction surface sampling probe was developed to scan TLC plate surfaces to directly characterize the analytes on the plates [41]. However, the long analytical time made this technique non-ideal for
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high-throughput analysis. To overcome the shortcomings of the aforementioned TLC/MS techniques, we developed
sawtooth thin layer chromatography-electrospray ionization/mass spectrometry (sawtooth TLCESI/MS). The TLC separation of the complex mixtures of herb extracts attenuated the ion suppression effect, while the ESI/MS ionization of analytes directly on TLC plates avoided the detachment of gel particles while enabling sensitive and specific detection of visualizable and non5
visualizable compounds. Analysis was rapid and simple, making this technique applicable for highthroughput studies. Sawtooth TLC-ESI/MS was used to characterize phytochemical compounds in P. ginseng and A. carmichaelii Debx root extracts.
2. Experimental
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2.1 Materials Methanol (MeOH), 1-butanol, and aluminum-based silica gel 60 F254 TLC plates were purchased
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from Merck (Darmstadt, Germany). Ammonium hydroxide (NH4OH), acetic acid (AA), and toluene were obtained from J. T. Baker (Phillipsburg, NJ, U.S.A.). Ethyl acetate (EA) was obtained from Macron Fine Chemicals (Center Valley, PA, U.S.A.). A PURELAB Pulse water purifier (ELGA
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LabWater, Marlow, U.K.) was used to produce deionized water. The processed roots of Panax
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ginseng and Aconitum carmichaelii Debx (Chuan Wu herb) were obtained from a local pharmacy for
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traditional Chinese medicines. The herbal slices were ground and extracted as follows: 100 mg of the
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ground powder was added to 1 mL of 70% MeOH, after which the mixture was ultrasonicated at
collected for analysis.
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2.2 TLC Separation
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room temperature for 30 min and centrifuged at 6,000 rpm for 10 min. The suspension was then
Sample solutions were deposited on silica TLC plates using a TLC sample applicator
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(LINOMAT 5, CAMAG, Switzerland) at a dosage speed of 80 nL/s. The A. carmichaelii Debx root extract (4 uL) was deposited as a band of 7 mm long and 0.7 mm wide on each TLC plate; analytes were subsequently separated in a mobile phase of 1-butanol-toluene-EA-MeOH-NH4OH
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(40:28:5:3:6, v/v/v/v/v). The P. ginseng root extract (6 uL) was deposited as a band (6 mm) on each TLC plate; analytes were then separated in a mobile phase of 1-butanol-AA-water solution (7:1:2, v/v/v). After it was air-dried, the developed TLC plate was placed in a documentation system (TLC Visualizer 2, CAMAG) and photographed by a CCD camera, wherein the plate was illuminated with white light, 254 nm, and 366 nm UV light. 6
2.3 Sawtooth TLC-ESI-MS System To characterize both visible and non-visible analyte spots on the TLC track, a pair of stainless steel zigzag cut scissors (Nikken, Japan) with a cutting teeth distance of 3 mm was used to cut the TLC track in half, dividing the developed TLC plate into two sawtooth-shaped pieces, each with multiple triangular tips on one side (Figures 1a-b). The internal angle of each triangular tip was 100°
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and the distance between two triangular tips was 3 mm. A sawtooth TLC plate piece was placed on an XYZ stage and clipped with an alligator clip on the side that was opposite the serrated side
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(Figure 1c). The piece was positioned in front of the MS analyzer so that a triangular sawtooth tip was pointed directly at the MS inlet; the inlet was sheathed by a ceramic tube (1.5 cm long) to
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prevent discharging. The tip was placed 1.5 mm away from the ceramic tube. A high DC voltage of +5 kV was applied to the aluminum sheet of the sawtooth TLC plate through the alligator clip, and 1
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μL of an ESI solvent (100% MeOH for the analysis of ginseng, EA-MeOH solvent [50:50, v/v] for the analysis of A. carmichaelii) was deposited using a micropipette at the center of the triangle being
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analyzed. The function of the methanol solvent was to extract and carry analytes toward the tip for
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ESI ionization. A quadrupole time-of-flight mass analyzer (Q-TOF, micrOTOF-QII, Bruker Daltonics, MA, U.S.A.) with an MS inlet potential of -500 V, drying gas temperature of 200 °C,
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drying gas flow rate of 1.1 L/min, and scan speed of 2 Hz was operated in the positive ion mode. The collision energy was set between 8–35 eV for MS/MS analysis. The ion signal lasted for
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approximately 15 s. After the mass spectrum from the first triangular tip was obtained, the XYZ stage was moved to align the adjacent triangular tip with the MS inlet for the next analysis. The
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analytical process was repeated until all triangular tips on one sawtooth TLC piece were analyzed and then repeated for the other piece.
3. Results and discussion 3.1 TLC analysis of A. carmichaelii The roots and tubers of the Aconitum plant species (also known as aconite) contains toxic 7
alkaloids which can induce respiratory paralysis and cardiac arrhythmias [42,43]. However, the aconite plant provides anti-rheumatic, anti-inflammatory, and analgesic effects in small doses, making it a widely used plant in traditional Chinese medicine [43]. For the purpose of internal use, the aconite roots and tubers are generally processed through soaking and boiling processes to decrease the toxic alkaloid content. However, improper or insufficient processing temperature, time,
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and steps of aconite will increase the risk of poisoning. Before sawtooth TLC-ESI/MS analysis, the A. carmichaelii extract was first characterized using infusion ESI/MS to understand the chemical
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profile of the processed A. carmichaelii root (Figure 2a). Although the A. carmichaelii root has been processed, highly toxic compounds including aconitine (m/z 646.3221), mesaconitine (m/z
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632.3064), and hypaconitine (m/z 616.3111) and other alkaloids (i.e. m/z 400–700 for aconitum
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alkaloids and m/z 800–900 for lipo-alkaloids) were still detected in the extract. Sawtooth TLC-
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ESI/MS was then utilized to separate and analyze highly toxic alkaloids and other compounds in an A. carmichaelii extract. The extract was first separated on a silica TLC plate. After air-dried, the
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photograph of developed TLC plate was recorded by a documentation system. Figures 2b–d display
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the TLC profile of the A. carmichaelii root extract under white light, 254 nm UV light, and 366 nm UV light, respectively. Without using a chemical reagent, only a few bands at lower Rf values (0 to
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0.2) were observed on the TLC track, demonstrating the inability of TLC to characterize nonvisualizable analytes while showing the necessity of MS to detect such analytes.
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3.2 Sawtooth TLC-ESI/MS analysis of the A. carmichaelii extract Figure 2e shows the developed TLC plate with a track length of 5.3 cm after it was cut into two
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sawtooth pieces with 17 triangular tips on each piece. The tips in the right piece were labeled with even numbers (#2 to #34), whereas the tips in the left piece were labeled with odd numbers (#1 to #33). The sawtooth plate piece was then positioned on an XYZ stage and in front of the mass analyzer to characterize the phytochemical compounds present in the triangles. The volume of the extraction solvent (EA-MeOH) deposited at the center of the triangular tip should be well-controlled to prevent excessive solvent spread that would carry analytes to adjacent tips. We used an optimal 8
volume of 1 μL of extraction solvent for the analysis of all tips. The mass spectrum was acquired as a high voltage (+ 5 kV) and EA-MeOH solution was applied to the triangular tip aligned to the MS inlet. Figure 3 displays the sawtooth TLC-ESI mass spectra for the 34 triangular tips from the two TLC pieces. Although most of the analyte bands were not visualizable under UV light, ion signals for protonated aconitum alkaloids [M+H]+ and lipo-alkaloids [M+H]+ were detected, including those
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for 14-benzoylmesaconine-8-nonadecenoate (m/z 868.5383), 14-benzoylhypaconine-8-linoleate (m/z 836.5267), aconitine (m/z 646.3220), deoxyaconitine (m/z 630.3263), hypaconitine (m/z 616.3117),
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mesaconitine (m/z 632.3038), 10-hydroxymesaconitine (m/z 648.2991), talatisamine (m/z 422.2891), benzoylaconine (m/z 604.3107), benzoylmesaconine (m/z 590.2965), neoline (m/z 438.2830),
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isotalatizidine/talatizidin (m/z 408.2738), benzoyldeoxyaconine (m/z 588.3075), benzoylhypaconine
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(m/z 574.3039), fuziline (m/z 454.2807), and senbusine A/B (m/z 424.2703) (Table 1). The MS/MS
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information for aconitum alkaloids and lipo-alkaloids has been reported in previous works [44-46]; therefore, the detected analyte ions were further characterized using MS/MS to compare fragment
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ion patterns with those reported in previous publications. In addition to alkaloids, ion signals for
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sodiated sugars were also detected at tips #1 and #2 (i.e. the original position of the sample band); the detected sugars included disaccharides (m/z 365.1074, [M+Na]+), dimeric disaccharides (m/z
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707.2189, [2M+Na]+), and trisaccharides (m/z 527.1624, [M+Na]+), which were detected using sawtooth TLC-ESI/MS but not seen in the ESI mass spectrum (Figures 1a and 2). Furthermore, a
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series of background ions with a mass difference of 74 Da (i.e. m/z 467.1084, 541.1259, 615.1447, and 689.1593) was detected in the mass spectra, which may be from silica gel on TLC plate.
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3.3 TLC analysis of Panax Ginseng Ginseng (Panax ginseng) is an important herb in traditional Chinese medicine. It is considered
that ginsenosides are the major active ingredients in ginseng, conferring anti-fatigue, antiinflammatory, anti-tumor, and antioxidant benefits as well as boosting the immune system. The ESI mass spectrum of the ginseng root extract revealed that sugars and ginsenosides were detected as potassiated molecules [M+K]+ instead as protonated molecules [M+H]+ (Figure 4a). Similar results 9
were also reported in other study where the ginseng tissue was sharpened to generate an ESI plume at the tip [29]. In this study, the ginseng root extract was separated on a silica gel TLC plate first, it was then subjected for sawtooth TLC-ESI/MS analysis. Figures 4b–d display the TLC profile of the ginseng root extract under white light and 254 and 366 nm UV light. Although a TLC track was observed under 366 UV light, most of the analyte bands were not clearly resolved, again showing the
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need for MS analysis to characterize non-visualizable analytes. 3.4 Sawtooth TLC-ESI/MS analysis of P. Ginseng extract
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Figure 4e shows the developed TLC plate with a track length of ca. 5 cm after it was cut into two sawtooth pieces with 16 triangular tips on each piece. Figure 5 displays the sawtooth TLC-ESI mass
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spectra for 32 triangular tips from the two TLC pieces. Since trace amounts of sodium were present in the stationary phase, sugars and sugar derivatives such as ginsenosides were detected as sodiated
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ions. The compounds such as protopanaxadiol (m/z 499.2334, [M+K]+) and disaccharide (m/z
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365.1047, [M+Na]+; m/z 707.2207, [2M+Na]+) and a series of ion signals with a mass difference of
M
342 Da (i.e., m/z 729.2010, 1071.3182, and 1413.4367) were detected at or near the original position
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of the sample band. The tandem mass spectrum of m/z 729 showed the main fragment ion signal at m/z 365. Therefore, the ions were assigned as disaccharide-related cluster ions (i.e., m/z 729.2010,
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[2M-H+2Na]+; m/z 1071.3182, [3M-H+2Na]+; 1413.4367, [4M-H+2Na]+; M: disaccharide). Furthermore, ginsenoside-related compounds observed at m/z 1233.6217, 1131.5905, 1263.6296,
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1101.5816, 969.5387, 985.5128, 823.4815, 807.4871, 1239.6580, and 754.5445 were clearly detected at different triangles on the TLC pieces.
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Due to a lack of analyte standards and the MS/MS information for sodiated ginsenosides was not comprehensively reported in the literature [47,48], the ginsenoside isomers we detected in this study could only be identified based on their molecular ion information: m/z 1233.6217 for Ra1 or Ra2, m/z 1131.5905 for Rb1, m/z 1101.5816 for Rb2 or Rc, m/z 969.5387 and 985.5128 (i.e. [M+Na]+ and [M+K]+) for Rd or Re, m/z 823.4815 for Rf or Rg1, and m/z 807.4871 for Rg2 or Rg3 (Table 2). The number of ginseng-related ions detected using sawtooth TLC-ESI/MS was much more than that 10
detected using infusion ESI/MS by simply comparing the spectra for the two techniques. The ions detected in sawtooth TLC-ESI/MS reflect the detailed chemical composition of ginseng and can be used for quality control assessments as well as distinguishing authentic from counterfeit ginseng. In this study, the detection of positive analyte ions on the TLC plate was demonstrated. The detection of
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negative ions can be achieved simply by switching the polarity of the mass analyzer. 4. Conclusion
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Sawtooth TLC-ESI/MS was utilized to combine the analytical advantages of TLC and ESI/MS for the separation and detection of phytochemical compounds in unpretreated herbal extracts. Both visible and non-visible analyte spots were successfully detected from developed TLC plates that
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were modified to induce ESI directly on the plates. Active ingredients (such as sugars and
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ginsenosides) and highly toxic alkaloids (such as aconitine, mesaconitine, and hypaconitine) were
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detected in the root extracts of Panax ginseng and Aconitum carmichaelii Debx, respectively. Our
M
results demonstrate that sawtooth TLC-ESI/MS is a simple, rapid, and useful technique for
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identifying compounds of interest in herbs for quality control evaluations and safe medicinal use.
Acknowledgement
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This work was partially supported by the Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.
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Figure legends Figure 1. Photographs of a silica TLC plate (a) before and (b) after being cut by zigzag scissors. (c) Photograph of the sawtooth TLC-ESI/MS setup.
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Figure 2. (a) ESI mass spectra for the A. carmichaelii root extract. Photographs of the TLC profile of A. carmichaelii root extract obtained under (b) white light and (c) 254 nm UV and (d, e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
Figure 3. Sawtooth TLC-ESI mass spectra for phytochemical compounds on a developed TLC plate.
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The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 1e from which the mass spectrum was obtained. A series of background ions with a mass difference of 74 Da (i.e. m/z 467, 541, 615, and 689) were originated from the silica stationary
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phase.
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Figure 4. (a) ESI mass spectrum for the P. ginseng root extract. Photographs of the TLC profile of P. ginseng root extract obtained under (b) white light and (c) 254 nm UV and (d & e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors. Figure 5. Sawtooth TLC-ESI mass spectra for ginsenosides and sugars on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth
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piece in Figure 3e from which the mass spectrum was obtained.
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Figure 1. Photographs of a silica TLC plate (a) before and (b) after being cut by zigzag scissors. (c) Photograph of the sawtooth TLC-ESI/MS setup.
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Figure 2. (a) ESI mass spectra for the A. carmichaelii root extract. Photographs of the TLC profile of A. carmichaelii root extract obtained under (b) white light and (c) 254 nm UV and (d, e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
18
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Figure 3. Sawtooth TLC-ESI mass spectra for phytochemical compounds on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 1e from which the mass spectrum was obtained. A series of background ions with a mass difference of 74 Da (i.e. m/z 467, 541, 615, and 689) were originated from the silica stationary phase. 19
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Figure 4. (a) ESI mass spectrum for the P. ginseng root extract. Photographs of the TLC profile of P. ginseng root extract obtained under (b) white light and (c) 254 nm UV and (d & e) 366 nm UV light. (e) Photograph of the developed TLC plate after being cut by zigzag scissors.
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Figure 5. Sawtooth TLC-ESI mass spectra for ginsenosides and sugars on a developed TLC plate. The number in the top right corner of each mass spectrum represents the tip position on the sawtooth piece in Figure 3e from which the mass spectrum was obtained.
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Table 1. Alkaloids detected in the A. carmichaelii Debx extract by sawtooth TLC-ESI/MS.
Formula
Rf a
14-benzoylhypaconine-8-
C49H73NO10
0.99
836.5267
aconitine
C34H47NO11
0.99
646.3220
deoxyaconitine
C34H47NO10
0.99
630.3263
hypaconitine
C33H45NO10
0.99
616.3117
14-benzoylmesaconine-8-
C50H77NO11
0.96
868.5383
mesaconitine
C33H45NO11
0.96
talatisamine
C24H39NO5
0.92
10-hydroxymesaconitine
C33H45NO12
0.91
benzoylaconine
C32H45NO10
0.84
benzoylmesaconine
C31H43NO10
0.76
590.2965
neoline
C24H39NO6
0.73
438.2830
isotalatizidine/talatizidin
C23H37NO5
N
Measured
Compound name
0.69
408.2738
benzoyldeoxyaconine
C32H45NO9
0.66
588.3075
C31H43NO9
0.54
574.3039
C24H39NO7
0.43
454.2807
C23H37NO6
0.38
424.2703
m/z [M+H]+
fuziline
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senbusine A/B
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benzoylhypaconine
a
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nonadecenoate
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linoleate
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The Rf values were calculated base on the following formula: [(distance from start line to the triangle where analyte was detected)/(distance from start line to solvent front)].
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Table 2. Ginsenosides detected in the P. ginseng root extract by sawtooth TLC-ESI/MS.
Measured
Compound name
Formula
Rf a
Rg2/Rg3
C42H72O13
0.72
807.4871
Rf/Rg1
C42H72O14
0.59
823.4815
Rd/Re
C48H82O18
0.43
969.5387
Rb2/Rc
C53H90O22
0.29
1101.5816
Rb1
C54H92O23
0.25
1131.5905
Ra1/Ra2
C58H98O26
0.20
1233.6217
a
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m/z [M+Na]+
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M
A
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The Rf values were calculated base on the following formula: [(distance from start line to the triangle where analyte was detected)/(distance from start line to solvent front)].
23