In Vivo Analysis and Spatial Profiling of Phytochemicals in Herbal

Feb 22, 2007 - Discovery and Synthesis, and Molecular Chinese Medicine Laboratory, Li Ka Shing Faculty of Medicine, The University of. Hong Kong, Hong...
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Anal. Chem. 2007, 79, 2745-2755

In Vivo Analysis and Spatial Profiling of Phytochemicals in Herbal Tissue by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Kwan-Ming Ng,*,†,‡ Zhitao Liang,§ Wei Lu,† Ho-Wai Tang,‡ Zhongzhen Zhao,§ Chi-Ming Che,† and Yung-Chi Cheng|

Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, and Molecular Chinese Medicine Laboratory, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, School of Chinese Medicine, The Baptist University of Hong Kong, Hong Kong, and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed for spatial profiling of phytochemicals and secondary metabolites in integrated herbal tissue without solvent extraction. Abundant alkaloid ions, including (+)-menisperine (m/z 356), magnoflorine (m/z 342), stepharanine (m/z 324), protonated sinomenine (m/z 330), protonated sinomendine (m/z 338), and a metabolite at m/z 314, could be directly desorbed from r-cyano-4-hydroxycinnamic acid- (CHCA-) coated stem tissue of Sinomenium acutum upon N2 laser (337 nm) ablation, while the ion signals desorbed from sinapinic acid- (SA-) coated and 2,5-dihydroxybenzoic acid- (DHB-) coated stem tissue were at least 10 times weaker. Solvent composition in the matrix solution could have significant effects on the ion intensity of the metabolites. Under optimized conditions that maximize the ion intensity and form homogeneous matrix crystals on the tissue surface, spatial distributions of the metabolites localized in different tissue regions, including cortex, phloem, xylem, rim, and pith, and their relative abundances could be semiquantitatively determined. The three metabolites detected at m/z 356, 342, and 314 showed specific distributions in the herbal samples collected from different growing areas, while others were not. By applying principal component analysis (PCA), the characteristic metabolites in specific tissue regions could be easily determined, allowing unambiguous differentiation of the herbal samples from different geographic locations. Chemical compositions of herbal materials are largely dependent on agricultural conditions, geographical location, harvest time, * To whom correspondence should be addressed. Fax: (852) 2857 1586. E-mail: [email protected]. † Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong. ‡ Molecular Chinese Medicine Laboratory, Li Ka Shing Faculty of Medicine, The University of Hong Kong. § The Baptist University of Hong Kong. | Yale University School of Medicine. 10.1021/ac062129i CCC: $37.00 Published on Web 02/22/2007

© 2007 American Chemical Society

and species.1-3 Variation of the chemical compositions can introduce uncertainty to the therapeutic effects of botanical medicines.4 Development of sensitive and specific methods for analyzing the original chemical compositions and differentiation of different herbal materials are especially important for quality control of herbal medicines.5 Gas chromatography (GC), highperformance liquid chromatography (HPLC), and capillary electrophoresis (CE) coupled with different detection systems have been used to determine chemical fingerprints of herbal medicines for assessing their chemical consistency.5 These methods are based on in vitro analysis of herbal extracts in solutions, and the chemical fingerprints could be affected by different extraction processes of the herbal materials. Metabolite profiling at tissue levels has been recently found to be specific to different species and growing conditions of plants.1,2,6,7 One of the main reasons is due to the variation of genetics in different species and/or different enzymatic activities responsible for formation/conversion of metabolites under different agricultural conditions.1,2,8,9 HPLC fingerprinting method cannot resolve the in vivo metabolite distribution directly. Some related research on histochemical analysis of herbal tissue based on physiochemical changes had been conducted to derive distributions and accumulations of active components in medicinal plants.10-12 Recently, fluorescent microscopic imaging and infrared (IR) spectroscopy based on different spectroscopic properties of (1) Smirnoff, N. In Environment and Plant Metabolism - Flexibility and Acclimation; Bios Scientific: Oxford, U.K., 1995. (2) Vickery, M. L.; Vickery, B. In Secondary Plant Metabolism; Macmillan Press: London, 1981. (3) Wijesekera, R. O. B. In The Medicinal Plant Industry, CRC Press: Boca Raton, FL, 1991. (4) Bent, S.; Ko, R. Am. J. Med. 2004, 116, 478-485. (5) Liang, Y. Z.; Xie, P.; Chan, K. J. Chromatogr. B 2004, 812, 53-70. (6) Fiehn, O. Plant Mol. Biol. 2002, 48, 155-171. (7) Krishnan, P.; Kruger, N. J.; Ratcliffe, R. G. J. Exp. Bot. 2005, 56, 255-265. (8) Yamazaki, Y.; Urano, A.; Sudo, H.; Kitajima, M.; Takayama, H.; Yamazaki, M.; Aimi, N.; Saito, K. Phytochemistry 2003, 62, 461-470. (9) Dra¨ger, B. Phytochemistry 2006, 67, 327-337. (10) Sacchetti, G.; Romagnoli, C.; Nicoletti, M.; Fabio, A. D.; Bruni, A.; Poli, F. Ann. Bot. 1999, 83, 87-92. (11) Ciccarelli, D.; Andreucci, A. C.; Pagni, A. M. Ann. Bot. 2001, 88, 637-644. (12) Hu, Z. H. Chin. Wild Plant Resour. 2005, 24 (1), 8-12.

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certain chemical groups within herbal tissue have also been applied for the analysis of herbal materials.13-15 However, all these methods are not specific enough to resolve molecular identities of the phytochemicals. While nuclear magnetic resonance (NMR) spectroscopy has been successfully applied for in vivo analysis of plant tissue to localize metabolites at tissue and even cellular levels, the relatively low sensitivity of detection (at the milligram level) has limited its application mainly to abundant metabolites.6,7 Development of a molecule-resolved method for in vivo analysis of phytochemicals/secondary metabolites and determination of their spatial distributions within herbal tissue can provide additional chemical information for quality control. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed as a useful tool for direct analysis and for imaging the distributions of peptides/proteins, drugs, and their metabolites in intact mammalian tissues.16-21 Its application has been widened to marine natural products, saccharides, phenolic antioxidants, terpenoids, and tannins.22-26 Recently, the technique had also been used to analyze externally applied agrochemicals in plant tissue27 and to map the distributions of amino acids, sugars, and their phosphorylated metabolites within plant tissue directly.28 Most recently, the method has also been applied for direct analysis of alkaloids in plants.29 However, its application for spatial profiling of phytochemicals within herbal tissue remains to be explored. The merits of MALDI are its high sensitivity of detection and soft ionization nature. It can desorb/ionize analytes from solid samples at picomole or even femtomole levels as intact molecular ions or protonated species with minimal fragmentations.30,31 When coupled to a time-of-flight (TOF) mass analyzer operated in the reflectron mode, elemental compositions of analyte ions determined at high mass accuracy (within 10 ppm for the ions smaller (13) Liang, Z. T.; Jiang, Z. H.; Leung, K. S. Y.; Peng, Y.; Zhao, Z. Z. Microsc. Res. Tech. 2006, 69, 277-282. (14) Sun, S. Q.; Zhou, Q.; Qin, Z. Atlas of Two-Dimensional Correlation Infrared Spectroscopy for Traditional Chinese Medicine Identification; Chemical Industry Press: Beijing, 2003. (15) Yap, K. Y. L.; Chan, S. Y.; Chan, Y. W.; Lim, C. S. Assay Drug Dev. Technol. 2005, 3, 683-699. (16) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (17) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (18) Rubakhin, S. S.; Garden, R. W.; Fuller, R. R.; Sweedler, J. V. Nat. Biotechnol. 2000, 18, 172-175. (19) Reyzer, M. L.; Hsieh, Y.; Ng, K.; Korfmacher, W. A.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 1081-1092. (20) Rubakhin, S. S.; Jurchen, J. C.; Monroe, E. B.; Sweedler, J. V. Drug Discovery Today 2005, 10, 823-837. (21) Khatib-Shahidi, S.; Andersson, M.; Herman, J. L.; Gillespie, T. A.; Caprioli, R. M. Anal. Chem. 2006, 78, 6448-6456. (22) Manning, T. J.; Rhodes, E.; Land, M.; Parkman, R.; Sumner, B.; Lam, T. T.; Marshall, A. G.; Philips, D. Nat. Prod. Res. 2006, 20, 611-628. (23) Okada, H.; Fukushi, E.; Yamamori, A.; Kawazoe, N.; Onodera, S.; Kawabata, J.; Shiomi, N. Carbohydr. Res. 2006, 341, 925-929. (24) Cai, Y. Z.; Xing, J.; Sun, M.; Zhan, Z. Q.; Corke, H. J. Agric. Food Chem. 2005, 53, 9940-9948. (25) Scalarone, D.; Duursma, M. C.; Boon, J. J.; Chiantore, O. J. Mass Spectrom. 2005, 40, 1527-1535. (26) Ishida, Y.; Kitagawa, K.; Goto, K.; Ohtani, H. Rapid Commun. Mass Spectrom. 2005, 19, 706-710. (27) Mullen, A. K.; Clench, M. R.; Crosland, S.; Sharples, K. R. Rapid Commun. Mass Spectrom. 2005, 19, 2507-2516. (28) Burrell, M. M.; Earnshaw, C. J.; Clench, M. R. J. Exp. Bot. Advance Access published October 4, 2006; http://dx.doi.org/10.1093/jxb/er1139. (29) Wu, W.; Liang, Z. T.; Zhao, Z. Z.; Cai, Z. W. J. Mass Spectrom. 2007, 42, 58-69. (30) Zenobi, R.; Knochenmuss, R. Mass Spectrom. Rev. 1998, 17, 337-366. (31) Gabelica, V.; Schulz, E.; Karas, M. J. Mass Spectrom. 2004, 39, 579-593.

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than 1500) can be obtained.32 However, desorption/ionization efficiency of analytes in the MALDI process is dependent on the correct choice of matrixes.30,31,33 Previous studies also showed that the heterogeneity of the matrix crystal layer leading to the “hot spot” phenomenon could significantly affect the reproducibility of ion signals.34,35 This creates challenges to the determination of chemical distribution profiles in herbal and mammalian tissues. A number of studies on sample preparation of mammalian tissues for MALDI-MS analysis have been reported.36,37 The experimental conditions might not be applicable to plant tissues, as the chemical nature of plant tissue coated with cellulose cell wall is largely different from that of animal tissue. Caulis Sinomenii, the dried stem of Sinomenium acutum (Thunb) Rehd. et Wils, has long been used in traditional Chinese medicine for treatment of inflammatory and rheumatic diseases.38,39 Abundant alkaloids isolated from the herbal materials, as shown in Figure 1, were found to be bioactive.38-45 In this report, the stem of Sinomenium acutum is chosen as the model system to establish the MALDI-MS method for in vivo analysis of phytochemicals/secondary metabolites within herbal tissues. Our objectives are to apply MALDI-MS to analyze original chemical compositions directly from integrated herbal tissue without solvent extraction and to determine their spatial distributions in different tissue regions. Differentiation of the herbal samples collected from different growing areas based on specific distributions of the metabolites is explored. Formation of MALDI matrix crystals on the tissue surface and effects of different matrixes on the desorption/ionization efficiency of the metabolites are also investigated. EXPERIMENTAL SECTION Chemicals and Reagents. Reference standards of the alkaloids, including sinomenine and magnoflorine iodide, were purchased from ChromaDex (Santa Ana, CA). The purity of these chemicals is >95%. (+)-Menisperine and stepharanine standards in the forms of perchlorate are generously provided by Professor Yumi Nishiyama of Kobe Pharmaceutical University in Japan. R-Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA) (32) Fukai, T.; Kuroda, J.; Nomura, T. J. Am. Soc. Mass Spectrom. 2000, 11, 458-463. (33) Domin, M. A.; Welham, K. J.; Ashton, D. S. Rapid Commun. Mass Spectrom. 1999, 13, 222-226. (34) Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30-36. (35) Luxembourg, S. L.; McDonnell, L. A.; Duursma, M. C.; Guo, X.; Heeren, R. M. A. Anal. Chem. 2003, 75, 2333-2341. (36) Schwartz, S. A.; Reyzer, M. L.; Caprioli, R. M. J. Mass Spectrom. 2003, 38, 699-708. (37) Todd, P. J.; Schaaff, G.; Chaurand, P.; Caprioli, R. M. J. Mass Spectrom. 2001, 36, 355-369. (38) Wang, Y.; Zhou, L.; Li, R. Zhong Yao Cai. 2002, 25 (3), 209 - 211. (39) Lou, Z. C.; Qin, B. In Species Systematization and Quality Evaluation of Commonly Used Chinese Traditional Drugs, North-China ed.; Peking University Medical Press: Beijing, 2003; Vol. III, pp 95-143. (40) Liu, L.; Riese, J.; Resch, K.; Kaever, V. Arzneimittelforschung 1994, 44, 12231226. (41) Liu, L.; Resch, K.; Kaever, V. Int. J. Immunopharmacol. 1994, 16, 685691. (42) Liu, L.; Buchner, E.; Beitze, D.; Schmidt-Weber, C. B.; Kaever, V.; Emmrich, F.; Kinne, R. W. Int. J. Immunopharmacol. 1996, 18, 529-543. (43) Li, X. J.; Yue, P. Y. K.; Ha, W. Y.; Wong, D. Y. L.; Tin, M. M. Y.; Wang, P. X.; Wong, R. N. S.; Liu, L. Life Sci. 2006, 79, 665-673. (44) Bao, G. H.; Qin, G. W.; Wang, R.; Tang, X. C. J. Nat. Prod. 2005, 68, 11281130. (45) Min, Y. D.; Choi, S. U.; Lee, K. R. Arch. Pharm. Res. 2006, 29, 627-632.

Figure 1. Chemical structures of alkaloids previously found in Sinomenium acutum.

with purity >98% were purchased from Sigma (St. Louis, MO). 2,5-Dihydroxylbenzoic acid (DHB) (AR grade) was purchased from ICN Biomedicals (Costa Mesa, CA). Methanol (AR grade) was purchased from Mallinckrodt, and acetone (ACS grade) was from Tedia. Water was purified through a Milli-Q water purification system. All chemicals were used as received. Herbal Materials. Two different herbal samples (0311007 and 0311008) of Caulis Sinomenii were collected from the same growing area in Shaanxi province in China. The other two, including 0311011 and 0312032, were collected from Anhui province and Chongqing city, respectively. The diameter of the herbal samples (stems) chosen in the present study was in the range of 12-13 mm. All the raw herbs adopted in the present study were authenticated by Professor Z. Z. Zhao and are kept in the Bank of China (HK) Chinese Medicines Centre of Hong Kong Baptist University. Sample Preparation. Stock solutions (200 µM) of sinomenine, magnoflorine iodide, (+)-menisperine perchlorate, and stepharanine perchlorate were prepared individually in methanol. Working solutions were prepared by serial dilution with methanol or acetone to reach desired final concentrations. The MALDI matrix solutions of CHCA, SA, and DHB at 10 mg/mL were prepared separately in acetone containing 0-40% (v/v) water content. CHCA solution at 1 mg/mL was used for recording the MALDI mass spectra of reference standards. To prepare mixture solutions of reference standards and CHCA matrix, 2 µL of individual standard (10 µM in methanol or acetone) was mixed with 2 µL of CHCA [1 mg/mL in acetone containing 20% (v/v) water content] in a centrifuge tube, and 1.5 µL of the mixture solution was applied on a sample spot (a circle spot with 5.08 mm diameter) of a stainless steel MALDI plate, or else specified where appropriate. The samples were left to air-dry prior to MALDI-MS analysis.

The herbal stems were sectioned (with thickness of ∼20 µm) by a cryostat (Shandon As620 cryotome, U.K.) operated at -20 °C. Cryomatrix (Shandon, Pittsburgh, PA) was applied to hold the stem on a cutting platform. Each sectioned tissue slice was then placed on an indium-tin oxide- (ITO-) coated polyester microscope slide (with thickness of 0.2 mm, and resistivities of 8-12 Ω; from SPI Supplies). In order to maintain the integrity of the tissue slices during sample preparation, the samples were softened by wrapping with fully wet tissue papers (Kimwipes, Ontario, Canada) overnight before sectioning. To apply MALDI matrix on the surface of the tissue samples, the matrix solution was aerosprayed on the surface with an airbrush (Badger model 175-7) at a distance of ∼15-18 cm. The nozzle of the airbrush was opened at ∼300 µm in diameter. The airbrush was connected to a mini-air compressor operated at an outlet pressure of 20 psi. The matrix solution (at 10 mg/mL) was applied onto the tissue surface in 10 spraying cycles, and each cycle lasted for 1 min. The total consumption of the matrix solution was about 30 mL. The matrix-coated tissues were left to air-dry and then kept in a desiccator overnight prior to MALDI-MS analysis. Scanning Electron Microscopic Examination. The formation of MALDI matrix crystals coated on the surface of the herbal tissue was investigated by using field emission scanning electron microscopy (LEO 1530 FEG, Zeiss/LEO, Oberkochen) operating at 5 kV. All the tissue samples for SEM observations were sputtered with a thin layer of gold with thickness e10 nm. Mass Spectrometric Measurements. All mass spectrometric measurements were performed with a QSTAR-XL hybrid quadrupole time-of-flight (Q-TOF) tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Ontario, Canada) equipped with a matrix-assisted laser desorption/ionization (MALDI) source and a N2 laser (337 nm with pulse width 0.99) of its ion intensity, accumulated from 1.5 µL of the magnoflorine solution at 1.25, 2.5, and 5 µM containing 0.5 mg/mL CHCA, was 5079 counts/pmol. In addition, the ion intensity of magnoflorine ion (for 5 µM magnoflorine solution containing 0.5 mg/mL CHCA) was about 12 times higher than that of protonated CHCA. Our results suggested that CHCA matrix is essential for the desorption/ionization of the alkaloids under our MALDI conditions. Abundant alkaloids could possibly suppress the desorption/ ionization of CHCA. Formation of MALDI Matrix Crystals on Stem Tissue and Effects of Solvent Composition on Ion Signal Intensities. Previous studies demonstrated that an aerospraying method37,46 for applying matrix could allow the formation of homogeneous crystal layers, which could improve spot-to-spot variations of MALDI mass spectra and prevent proteins from lateral diffusion within biological tissues. Here, by using the aerospraying method, the formation of CHCA (light yellow) and SA (white-yellow) matrixes on the surface of the herbal tissue had already been noted by visual inspection. When examined under a scanning electron microscope, the relatively even layers of CHCA crystals (∼0.2-2 µm) and SA crystals (∼1-5 µm) on the tissue surface were clearly observed, as represented by the phloem tissue shown (46) Miliotis, T.; Kjellstro¨m, S.; Nilsson, J.; Laurell, T.; Edholm, L.-E.; MarkoVarga, G. Rapid Commun. Mass Spectrom. 2002, 16, 117-126.

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Figure 4. Competitive desorption/ionization of magnoflorine (A) versus CHCA (B) under MALDI conditions. Magnoflorine solution (50 µL) at the respective concentrations of 50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.08 µM was mixed with 50 µL of CHCA solution at 1.0 mg/mL in 80% (v/v) acetone and 20% (v/v) H2O. Aliquots (1.5 µL) of the individual mixture solutions were applied on sample spots of the MALDI plate. The ion current profiles were obtained by N2 laser ablation at 4-5 positions in each sample spot. The ion current of magnoflorine ion was extracted by integrating the ion intensity over the mass profile from m/z 342.1 to 342.3, and that of protonated CHCA was extracted by integrating the ion intensity from m/z 190.0 to 190.1.

in Figure 5. In fact, the formation of CHCA matrix on the whole surface was also implied from the ion current profile of protonated CHCA (extracted by integrating the ion intensity from m/z 190.0 to 190.1) recorded along the tissue surface, as shown in Figure 6. However, the formation of DHB crystals on the tissue surface was not obvious (Figure S-2, Supporting Information). The favorable formation of CHCA and SA crystals on the tissue surface under our aerospraying conditions could possibly be due to their stronger interaction with the plant tissue rich in cellulose, although the underlying mechanisms are not well understood. On the microscopic scale (Figure 5B), although the CHCA crystals formed on the tissue surface were relatively scattered and even not observed in some positions (< 1 × 1 µm), the layer of CHCA crystals was still relatively uniform when compared with the spot size of the N2 laser beam. The much larger spot area (∼ 100 × 300 µm) of the laser beam than the size of the microcrystals (∼0.2-2 µm) allowed recording of the MALDI mass spectra resulting from the average of a large number of microcrystals coated on specific regions of the tissue. This could minimize the variation of ion signals resulting from the relatively

Figure 5. Scanning electron microscopic images of MALDI matrix crystals formed on the stem tissue (phloem region) of Sinomenium acutum: with CHCA (panels A and B, recorded at different magnifications) and SA (panels C and D, recorded at different magnifications). The individual CHCA and SA (at 10 mg/mL), dissolved in 80% (v/v) acetone and 20% (v/v) H2O, was sprayed on the tissue surface for 10 cycles and each cycle lasted for 1 min.

scattered matrix crystals, as demonstrated in our subsequent study, and thus allow the spatial profiling of phytochemicals. The point-to-point variations of MALDI signal intensities of magnoflorine and sinomenine standards sprayed with different matrixes on blank microscope slides were also investigated (Figure 7). The results of using CHCA as the matrix were acceptable with coefficient of variance (CV) smaller than 22%, while those of SA and DHB were larger than 55%. Our findings suggested that the variation effect, resulting from the scattered or heterogeneous CHCA crystals if any, was not significant. In addition, it was noted that the ion intensity of the reference standards desorbed from CHCA was 10 times (on average) higher than that from SA and DHB. Although MALDI has been widely applied to the analysis of biopolymers since the 1980s, its underlying mechanisms of chemical and physical events involved are still not well understood.47,48 Here our findings on the stronger ion intensities of the metaboltes desorbed from CHCA-coated stem tissue suggested that CHCA was the more effective matrix reagent for the MALDI-MS analysis of the alkaloids. We also investigated the effect of water content in the CHCA matrix solution on the ion signal intensity. As demonstrated from

the metabolite ion at m/z 356 depicted in Figure 8, increasing water content (from 0% to 20% v/v) could significantly enhance the ion intensity by more than 20 times (on average). Further increasing the water content (to 40% v/v) did not yield further improvement. Similar results were also observed for the other ions. This could be due to the better formation of CHCA crystals on the tissue surface when the water content increased, as implied in Figure 6. Another reason could be attributed to increasing dissolution of the metabolites from the stem tissue, thus facilitating their incorporation into the matrix crystals. However, the effect was not observed for SA or DHB. Moreover, the similar distribution profiles of the metabolite, not affected by the different water content, suggested that delocalization of the metabolite during the matrix deposition was not significant. We also noted that increasing the water content (to 40% v/v) in CHCA solution did not affect the homogeneity of the crystals coated on the tissue surface, but the tissue became poorly coated with the crystals when pure acetone was used (Figure S-3, Supporting Information, and Figure 6). In our subsequent analysis, the solvent composition (47) Dreisewerd, K. Chem. Rev. 2003, 103, 395-425. (48) Karas, M.; Kruger, R. Chem. Rev. 2003, 103, 427-439.

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Figure 6. Desorption/ionization of CHCA matrix, detected as [CHCA + H]+ at m/z 190, along the stem tissue surface coated with CHCA crystal layer. CHCA (10 mg/mL) used for the matrix deposition was dissolved in 80% (v/v) acetone containing 20% (v/v) H2O (2) or in 100% acetone (]). The ion current of the protonated CHCA was integrated from the ion intensity over the mass profile from m/z 190.0 to 190.1.

Figure 7. Spot-to-spot variations of MALDI ion signals of sinomenine and magnoflorine: (0) sinomenine and (9) magnoflorine mixed with CHCA; (4) sinomenine and (2) magnoflorine mixed with SA; (O) sinomenine and (b) magnoflorine mixed with DHB. The mixture solution of sinomenine (10 µM), magnoflorine iodide (10 µM), and the matrix (10 mg/mL) in 80% (v/v) acetone and 20% (v/v) H2O was sprayed onto blank microscope slides continuously for 5 min.

in matrix solution was optimized at 80% (v/v) acetone containing 20% (v/v) water. Spatial Distributions of the Metabolites and Its Application for Differentiation of Herbal Samples Collected from Different Sources. Under our optimized experimental conditions, MALDI mass spectra of the CHCA-coated stem tissue of Sinomenium acutum collected from Shaanxi, Anhui, and Chongqing in China were recorded. To semiquantitatively determine the spatial distributions of the individual metabolites in different tissue regions, percentage total ion intensities of the metabolites at m/z 356, 342, 338, 330, 324, and 314 against radial distance of the stem tissue were determined and are depicted in Figure 9. Our results showed that MALDI-MS was a sensitive method to measure the variations of metabolite distributions. It could determine their 2752 Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

Figure 8. Effect of solvent composition of CHCA solution on signal intensity of metabolite ion (at m/z 356) desorbed from CHCA-coated stem tissue: (]) 0% (v/v) H2O, (0) 10% (v/v) H2O, (2) 20% (v/v) H2O, and (b) 40% (v/v) H2O, in acetone.

spatial distributions and measure their relative abundances localized in different tissue regions, including cortex, phloem, xylem, rim, and pith. For the herbal sample (0312032) collected from Chongqing city, the metabolite at m/z 356 was mainly localized (with ∼30-60% of the total ion intensity) in the cortex and phloem tissue (Figure 9A), while the relative abundance of the metabolite localized in the xylem was less intense (∼10-30%), and its relative abundance in the rim and pith regions was minimal (