High-performance thin-layer chromatography

Research Article Received: 20 March 2015

Revised: 7 June 2015

Accepted: 8 June 2015

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2015, 29, 1530–1534 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7246

High-performance thin-layer chromatography/desorption electrospray ionization mass spectrometry imaging of the crude extract from the peels of Citrus aurantium L. (Rutaceae) Bianca S. Bagatela1,2, Andrey P. Lopes1,2, Elaine C. Cabral1, Fábio F. Perazzo2 and Demian R. Ifa1* 1

Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, Ontario, Canada Department of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, Diadema, São Paulo, Brazil

2

RATIONALE: Citrus aurantium L. is a plant belonging to the Rutaceae family, whose extracts are extensively used in

weight management products and as thermogenic agents. Here we present two methodologies to analyse the extracts obtained from the peels of Citrus aurantium L. that usually require multiple sample preparation and detection steps. METHODS: Polar compounds of the crude extract from the peels of Citrus aurantium L. (Rutaceae) were investigated by direct infusion electrospray ionization mass spectrometry (ESI-MS) and high-performance thin-layer chromatography (HPTLC) coupled to desorption electrospray ionization mass spectrometry (DESI-MS). ESI-MS was performed in both positive and negative ion modes. Molecular imaging of the HPTLC plates was used for the direct analysis of the phytocompounds present in the crude extract from the peels of Citrus aurantium L. by DESI-MS imaging. RESULTS: Characteristic mass spectra with many diagnostic ions were obtained from the extract analysis, allowing a fast and reliable identification of these species. Tandem mass spectrometry (MS/MS) was employed to confirm the identity of specific metabolites. CONCLUSIONS: HPTLC/DESI-MS imaging is a relatively fast, versatile, and efficient technique for natural product analysis, since many more ions are observed than with the direct infusion ESI-MS. The MS/MS technique provided information about the component structures, revealing the presence of important bioactive components. The application of DESI-MS imaging may contribute to the improvement identification and characterization of pharmacologically active compounds in phytochemistry. Copyright © 2015 John Wiley & Sons, Ltd.

Citrus aurantium L. is a plant belonging to the Rutaceae family, whose extracts are extensively used in weight management products and as thermogenic agents.[1] The extract of Citrus aurantium L. is widely known as ’bitter orange’ extract, a product derived from the peels of the Seville orange. These extracts have been used for centuries in traditional Chinese medicine.[2] Bitter orange extract is used in weight management products, as mentioned, due to its alleged effects on metabolic processes, including an increase in basal metabolic rate and lipolysis, besides appetite suppression.[3] Liquid chromatography coupled to mass spectrometry (LC/MS) is the gold standard technique used for the chemical characterization of the compounds found in plant extracts including Citrus aurantium.[4–10] However, direct electrospray ionization (ESI) fingerprinting has been demonstrated to provide a very fast and versatile methodology for the screening of polar compounds from natural products.[11–13]

1530

* Correspondence to: D. R. Ifa, Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, Ontario, M3J 1P3, Canada. E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2015, 29, 1530–1534

In this technique, chromatographic separation is not performed and the complex sample is directly delivered to MS analysis. Applications of this technique include the analysis of essentials oils,[14] differentiation of levels of ripeness of fruits, as well as their post-harvesting process,[15] and quality control of vegetable oils.[16] Thin-layer chromatography (TLC) is a robust, rapid, inexpensive, and powerful technique. Generally, after TLC analysis, the compounds are scraped off the TLC plate and extracted using a solvent. The compounds are then ionized and analyzed by mass spectrometry. This procedure is time consuming, and the reproducibility and recovery of the compounds are sometimes poor, mainly due to spot overlap or loss of the sample. The possibility of analyzing small molecules by coupling TLC with desorption electrospray ionization mass spectrometry (DESI-MS) has been previously investigated.[17–23] In DESI, a solvent is electrosprayed onto the surface where it forms a thin film; secondary scattered droplets are generated as subsequently arriving primary droplets splash into the film. As the analyte-containing droplets are carried through the atmosphere into the vacuum interface, the solvent evaporates generating ionized molecules that can be mass-analyzed.[24]

Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of ’bitter orange’ extracts by HPTLC/DESI-MSI We present the analysis of complex phytoconstituents based on both direct infusion ESI and high-performance thin-layer chromatography (HPTLC) separation followed by interrogation of the compounds on the HPTLC plates using DESI-MS imaging. This work was motivated by the desire to develop a methodology for the analysis of the extracts obtained from natural products that usually can require multiple sample preparation and detection steps.

EXPERIMENTAL Sample preparation Peels from Citrus aurantium L. (Rutaceae) were air-dried, crushed and extracted with ethanol:water (1:3 v/v) by maceration for 14 days. The macerated material was filtered in a Whatman Qualitative filter paper (Grade no. 1; Whatman, Maidstone, UK) and then concentrated and dried in a rotary evaporator (BUCHI Corp., New Castle, DE, USA) to produce the crude extract. Samples of the crude extracts (10 mg) were dissolved in 1 mL of methanol by vortex mixing using a Mini Vortexer MV1 (VWR Scientific Products, Mississauga, ON, Canada), for 1 min. The samples were then centrifuged at 11,000 rpm using a model 5417 C/R centrifuge (Eppendorf Canada, Mississauga, ON, Canada), for 1 min. Afterwards, 10 μL of each sample were diluted in 990 μL methanol with 0.1% acetic acid for analysis in the positive ion mode. For the negative ion mode experiments, 10 μL of each solution were diluted in 990 μL of a solution of methanol/water (1:1) with 0.1%

ammonium hydroxide. All reagents and solvents used in this work were purchased from Sigma-Aldrich (Oakville, ON, Canada). ESI-MS fingerprinting ESI-MS fingerprinting was performed in both positive and negative ion modes using a LTQ linear ion trap (Thermo Fisher Scientific, San Jose, CA, USA) mass spectrometer. The samples were directly infused by means of a syringe pump (Harvard Apparatus, Holliston, MA, USA) at a flow rate of 10 μL/min. The mass spectrometer was operated at a capillary voltage of ±3200 V depending on the ion mode selected. The inlet temperature was maintained at 150 °C. Full scan spectra were acquired in the range of m/z 100 to 1000 and accumulated for 60 s. MS/MS spectra were obtained for the selected ions (see Table 1) using collision energies ranging from 10 to 30 arbitrary units. Helium was used as the collision gas at a pressure of 40 psi. High-performance thin-layer chromatography Separation by HPTLC was performed on 5 × 5 cm Nano-Silica XHL HPTLC plates (glass backed, 200 μm stationary phase; Sorbent Technologies, Norcross, GA, USA). Aliquots containing 0.2 μg of the extract were applied separately to the HPTLC plates in triplicate. The plates were developed in one dimension with a solvent system containing ethyl acetate, acetic acid, formic acid, and water (100:5:5:13 v/v/v/v). The plates were air-dried, and some of them were sprayed with vanillin to allow the visualization of the spots. All plates were then subjected to mass spectrometric analysis.

Table 1. Phytocompounds from the crude extract of Citrus aurantium L. (Rutaceae)* ESI-MS Compound A B C D E F G G H I J K L M N O P Q R S

Phytocompound

M.W.

(+)

(-)

DESI-MSI (+)

Tyramine N-methyltyramine Hordenine Synephrine Isomeranzin Hesperitin/Quercetin Tangeritin Auranetin Nobiletin Isovitexin 3’-Methoxyisovitexin Rhoifolin Naringin Neodiosmin Hesperidin Diosmetin-6,8-di-C-glucoside 7-O-6"-malonylnaringin Melitidin Rhoifolin 4’-glucoside Brutieridin

137 151 165 167 260 302 372 372 402 432 462 578 580 608 610 624 666 724 740 754

✓ ✓ ✓ ✓ -

✓ ✓ ✓ ✓ -

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

Reference [7] [7] [7] [4,7] [10] [4,9] [4,9,10] [4,9] [4,9,10] [9] [9] [10] [4,9,10] [10] [4,9] [9,10] [9] [10] [10] [10]



wileyonlinelibrary.com/journal/rcm

1531

*For the ESI-MS experiments, the precursor ion and the characteristic product ions were observed by MS/MS experiments, and are consistent with previous reports. For DESI-MSI experiments, the assignments of the compounds are tentative, based only on the nominal mass of the precursor ion observed.

B. S. Bagatela et al. Desorption electrospray ionization mass spectrometry (DESI-MS) DESI-MS was performed in the positive ion mode using the Thermo LTQ linear ion trap mass spectrometer equipped with the custom-built, automated DESI ion source, which was described in detail previously.[25,26] The experiments were conducted in full scan mode; methanol was the spray solvent employed. The flow rate of the solvent was set at 5 μL/min. The DESI source was operated such that the incoming droplets were sprayed from the emitter placed 2 mm from the surface. The incident angle was set at 52° to the surface plane, and the collection angle was 10°. Imaging of HPTLC plates In order to image the HPTLC plates, mass spectra were acquired with the automatic gain control (AGC) turned off. Imaging experiments were performed by continuously scanning the surface in the x-direction at a surface velocity of 250 μm/s while acquiring mass spectra every 0.5 s in full scan mode over the range m/z 100-1000 and stepping in the ydirection at the end of each line, with the cycle being restarted until the selected area had been covered. The procedure for mapping the compounds separated on the HPTLC plate resulted in the collection of 4800 mass spectra on an array of 320 x 15 pixels. Under these conditions a lateral spatial resolution of 125 μm was achieved. More detailed information about the instrumentation can be found elsewhere.[25,26] Labwritten software was used to convert the Xcalibur™ mass spectral files (.raw) into a format compatible with BioMap (freeware[27]) which was the software used to process the mass spectral data to generate spatially accurate ion images.[25] Different color templates and appropriate maximum and minimum intensity values in the color bar were selected to increase the contrast in the images.[28]

Figure 2. Full scan ESI(–) mass spectrum of the crude extract of Citrus aurantium L. (Rutaceae). A few components from the extract can be seen: synephrine (D), naringin (L) and hesperidin (N).

RESULTS AND DISCUSSION ESI-MS fingerprinting ESI-MS fingerprinting was performed in both positive and negative ion modes (Figs. 1 and 2). A few components of the extract could be identified in the positive ion mode such as synephrine, tangeritin, nobiletin, and isovitexin (Fig. 1 and Table 1). In the negative ion mode, it was possible to detect synephrine, naringin and hesperidin (Fig. 2 and Table 1). Tandem mass spectrometry (MS/MS) was employed for the characterization of the compounds. For instance, in the positive ion mode, tangeritin, [M+H]+, m/z 373, showed product ions at m/z 358 and 343, resulting from consecutive losses of CH.3 (Fig. 3).[9] In negative ion mode, naringin, [M–H] , m/z 579, showed product ions at m/z 459 and 271, the former resulting from the loss of the two sugar rings of the compound[9] (Fig. 4). All the MS/MS mass spectra acquired were in agreement with previously reported data.[4,7,9,10] HPTLC/DESI-MSI

1532

High-performance thin-layer chromatography images were acquired by desorption electrospray ionization mass spectrometry of the crude extract in the positive ion mode


Figure 1. Full scan ESI(+) mass spectrum of the crude extract of Citrus aurantium L. (Rutaceae). A few components from the extract can be seen: synephrine (D), tangeritin (G), nobiletin (H), and isovitexin (I).

(Fig. 5). Note that the spectra from the direct ESI and the DESI-MS (Figs. 1 and 5, respectively) are different. This difference results mainly from the different extract concentration analyzed and also the different solvent flow rates employed by the two techniques. The HPTLC plates were then developed in one dimension. This procedure enabled the separation of the phytocompounds according to their different polarities, as reported in Table 1, with [M+H]+ ions being observed in positive ion mode and [M–H] ions in negative ion mode. Especially for the polar phytocompounds, the flow rate of the sprayed solvent was a critical parameter to obtain efficient desorption, whereas the substances from the HPTLC plate surface were properly released and, consequently, successful ionization was achieved. Low flow rates from 1 to 3 μL/min, as usually applied for DESI-MSI assays, did not allow the desorption of some phytocompounds and provided weak signals. Therefore, a flow rate of 5 μL/min was necessary to produce adequate signal intensity, especially for the more



Analysis of ’bitter orange’ extracts by HPTLC/DESI-MSI

Figure 3. ESI(+)-MS/MS spectrum of tangeritin (G) acquired during the direct injection of the crude extract of Citrus aurantium L. (Rutaceae).

Figure 6. DESI(+) ion images of the HPTLC spots. A: m/z 138; B: m/z 152; C: m/z 166; D: m/z 168; E: m/z 261; F: m/z 303; G: m/z 373; H: m/z 403; I: m/z 433; J: m/z 463; K: m/z 579; L: m/z 581; M: m/z 609; N: m/z 611; O: m/z 625; P: m/z 667; Q: m/z 725; R: m/z 741; S: m/z 755. See Table 1 for the legends (A–S).

polar compounds. Finally, flow rates higher than 5 μL/min produced spot sizes which were too large and thus not suitable for our imaging experiments. The images of each HPTLC spot are shown in Fig. 6. The visualization of the spots using UV light could not show the spot distribution accurately; however, the ion images generated by the mass spectral data processed demonstrated precisely the localization of each phytocompound. This reinforces that DESI-MS coupled to HPTLC is a versatile and efficient technique for natural product analysis, since many more ions were observed than with direct infusion ESI-MS or DESI-MS.

CONCLUSIONS Figure 4. ESI(–)-MS/MS spectrum of naringin (L) acquired during the direct injection of the crude extract of Citrus aurantium L. (Rutaceae).

We report the application of ESI-MS fingerprinting and HPTLC coupled to DESI-MS imaging for the analysis of crude extracts obtained from Citrus aurantium. Qualitative analysis of the extract can be done by both ESI-MS and DESI-MS. However, a separation step such as HPTLC allows the identification of more compounds in the extract. This relatively fast and reliable identification can be used to evaluate the authenticity of herbal extracts as well as for quality control purposes. Quantitation of the components in the extract is not possible without the use of internal standards and calibration curves. The application of DESI-MS imaging for the quantitation has been previously demonstrated, but this feature has not been extensively explored.[29] A method for the quantitative analysis of the extract, applicable to the development of herbal medicines, will be further developed and validated using HPLC and DESIMS imaging.

Acknowledgements


We thank the Brazilian National Council for Scientific and Technological Development (CNPq) and the Natural Science and Engineering Research Council of Canada (NSERC) for financial support.



1533

Figure 5. Full scan DESI(+) mass spectrum of the crude extract of Citrus aurantium L. (Rutaceae). Synephrine (D) is the only component from the extract that can be seen.

B. S. Bagatela et al.

REFERENCES [1] S. J. Stohs, H. G. Preuss, M. Shara. A review of the human clinical studies involving Citrus aurantium (bitter orange) extract and its primary protoalkaloid p-synephrine. Int. J. Med. Sci. 2012, 9, 527. [2] Y. Lu, C. Zhang, P. Bucheli, D. Wei. Citrus flavonoids in fruit and traditional Chinese medicinal food ingredients in China. Plant Foods Hum. Nutr. 2006, 61, 57. [3] S. Bent, A. Padula, J. Neuhaus. Safety and efficacy of citrus aurantium for weight loss. Am. J. Cardiol. 2004, 94, 1359. [4] X.-g. He, L.-z. Lian, L.-z. Lin, M. W. Bernart. Highperformance liquid chromatography–electrospray mass spectrometry in phytochemical analysis of sour orange (Citrus aurantium L.). J. Chromatogr. A 1997, 791, 127. [5] L. Mattoli, F. Cangi, A. Maidecchi, C. Ghiara, M. Tubaro, P. Traldi. A rapid liquid chromatography electrospray ionization mass spectrometry(n) method for evaluation of synephrine in Citrus aurantium L. samples. J. Agric. Food Chem. 2005, 53, 9860. [6] L. Ding, X. Luo, F. Tang, J. Yuan, Q. Liu, S. Yao. Simultaneous determination of flavonoid and alkaloid compounds in Citrus herbs by high-performance liquid chromatography-photodiode array detection-electrospray mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 857, 202. [7] B. C. Nelson, K. Putzbach, K. E. Sharpless, L. C. Sander. Mass spectrometric determination of the predominant adrenergic protoalkaloids in bitter orange (Citrus aurantium). J. Agric. Food Chem. 2007, 55, 9769. [8] M. Scordino, L. Sabatino, P. Traulo, M. Gargano, V. Panto, G. Gagliano. HPLC-PDA/ESI-MS/MS detection of polymethoxylated flavones in highly degraded citrus juice: a quality control case study. Eur. Food Res. Technol. 2011, 232, 275. [9] Z. Lin, H. Wang, Y. Xu, J. Dong, Y. Hashi, S. Chen. Identification of antioxidants in Fructus aurantii and its quality evaluation using a new on-line combination of analytical techniques. Food Chem. 2012, 134, 1181. [10] T. Mencherini, L. Campone, A. L. Piccinelli, M. G. Mesa, D. M. Sanchez, R. P. Aquino, L. Rastrelli. HPLC-PDA-MS and NMR characterization of a hydroalcoholic extract of Citrus aurantium L. var. amara peel with antiedematogenic activity. J. Agric. Food Chem. 2013, 61, 1686. [11] M. Beckmann, D. Parker, D. P. Enot, E. Duval, J. Draper. High-throughput, nontargeted metabolite fingerprinting using nominal mass flow injection electrospray mass spectrometry. Nat. Protocols 2008, 3, 486. [12] J. Draper, A. J. Lloyd, R. Goodacre, M. Beckmann. Flow infusion electrospray ionisation mass spectrometry for high throughput, non-targeted metabolite fingerprinting: a review. Metabolomics 2013, 9, S4. [13] A. Sawaya, D. M. Tomazela, I. B. S. Cunha, V. S. Bankova, M. C. Marcucci, A. R. Custodio, M. N. Eberlin. Electrospray ionization mass spectrometry fingerprinting of propolis. Analyst 2004, 129, 739. [14] J. K. S. Moller, R. R. Catharino, M. N. Eberlin. Electrospray ionization mass spectrometry fingerprinting of essential oils: Spices from the Labiatae family. Food Chem. 2007, 100, 1283.

[15] H. Chen, Y. Sun, A. Wortmann, H. Gu, R. Zenobi. Differentiation of maturity and quality of fruit using noninvasive extractive electrospray ionization quadrupole time-of-flight mass spectrometry. Anal. Chem. 2007, 79, 1447. [16] E. C. Cabral, L. Sevart, H. M. Spindola, M. B. Coelho, I. M. Sousa, N. C. Queiroz, M. A. Foglio, M. N. Eberlin, J. M. Riveros. Pterodon pubescens oil: characterisation, certification of origin and quality control via mass spectrometry fingerprinting analysis. Phytochem. Anal. 2013, 24, 184. [17] G. Paglia, D. R. Ifa, C. Wu, G. Corso, R. G. Cooks. Desorption electrospray ionization mass spectrometry analysis of lipids after two-dimensional high-performance thin-layer chromatography partial separation. Anal. Chem. 2010, 82, 1744. [18] S. R. Ellis, J. R. Hughes, T. W. Mitchell, M. I. H. Panhuis, S. J. Blanksby. Using ambient ozone for assignment of double bond position in unsaturated lipids. Analyst 2012, 137, 1100. [19] E. L. Harry, J. C. Reynolds, A. W. Bristow, I. D. Wilson, C. S. Creaser. Direct analysis of pharmaceutical formulations from non-bonded reversed-phase thin-layer chromatography plates by desorption electrospray ionisation ion mobility mass spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 2597. [20] J. H. Kennedy, J. M. Wiseman. Direct analysis of Salvia divinorum leaves for salvinorin A by thin layer chromatography and desorption electrospray ionization multi-stage tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 1305. [21] S. P. Pasilis, V. Kertesz, G. J. Van Berkel, M. Schulz, S. Schorcht. HPTLC/DESI-MS imaging of tryptic protein digests separated in two dimensions. J. Mass Spectrom. 2008, 43, 1627. [22] J. A. Seng, S. R. Ellis, J. R. Hughes, A. T. Maccarone, R. J. Truscott, S. J. Blanksby, T. W. Mitchell. Characterisation of sphingolipids in the human lens by thin layer chromatography-desorption electrospray ionisation mass spectrometry. Biochim. Biophys. Acta 2014, 1841, 1285. [23] G. J. Van Berkel, M. J. Ford, M. A. Deibel. Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization. Anal. Chem. 2005, 77, 1207. [24] A. B. Costa, R. G. Cooks. Simulated splashes: Elucidating the mechanism of desorption electrospray ionization mass spectrometry. Chem. Phys. Lett. 2008, 464, 1. [25] D. R. Ifa, J. M. Wiseman, Q. Y. Song, R. G. Cooks. Development of capabilities for imaging mass spectrometry under ambient conditions with desorption electrospray ionization (DESI). Int. J. Mass Spectrom. 2007, 259, 8. [26] N. E. Manicke, T. Kistler, D. R. Ifa, R. G. Cooks, Z. Ouyang. High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 2009, 20, 321. [27] Available: http://www.maldi-msi.org/. [28] J. M. Wiseman, D. R. Ifa, A. Venter, R. G. Cooks. Ambient molecular imaging by desorption electrospray ionization mass spectrometry. Nat. Protocols 2008, 3, 517. [29] C. Wu, A. L. Dill, L. S. Eberlin, R. G. Cooks, D. R. Ifa. Mass spectrometry imaging under ambient conditions. Mass Spectrom. Rev. 2013, 32, 218.

1534 wileyonlinelibrary.com/journal/rcm

