- Email: [email protected]
Direct Molecular Analysis of Garlic Using Internal Extractive Electrospray Ionization Mass Spectrometry
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 42, Issue 11, November 2014 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2014, 42(11), 1634–1638.
RESEARCH PAPER
Direct Molecular Analysis of Garlic Using Internal Extractive Electrospray Ionization Mass Spectrometry ZHANG Hua, ZHU Liang, CHEN Huan-Wen* Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang 330013, China
Abstract: The internal extractive electrospray ionization mass spectrometry (iEESI-MS) has been applied to direct molecular analysis of garlic tissues. An extraction solvent (e.g., methanol) under a high voltage (+4.5 kV) was injected into the fused silicon capillary whose far end penetrated into the bulk tissue sample. The chemicals in the bulk tissue were selectively extracted by the working solvent and carried along the electric field toward the apex of the sample, to generate electrospray in front of an ion trap mass spectrometer. Without time-consuming sample pretreatment, active garlic substances such as organosulfur compounds (e.g., alliin, allicin), amino acids (e.g., arginine) and saccharides (glucose, polysaccharides) were successfully detected and directly identified via collision-induced dissociation (CID) in positive ion detection mode. Mass spectral fingerprints of different kinds of garlic cloves (24 samples), as well as various post-treated ones (36 samples) in the range of m/z 50–2000 Da, were classified via principal component analysis (PCA). The experimental results indicated that iEESI-MS allowed direct identification of chemical components in garlic tissue and rapid recognition of metabolic changes in the garlic tissue subjected to various external stimuli, with the analytical advantages such as simplicity, rapidity (less than 2 min per sample), good specificity, and minimal disturbance to the bioactivity of analytes. Key Words:
1
Internal extractive electrospray ionization; Garlic; Direct analysis; Mass spectrometry
Introduction
Recently, ambient mass spectrometric methods have become important for the analysis of complex raw samples, which commonly pose analytical challenges in the scientific community such as life science, chemistry, material science and forensic analysis[1,2]. Thanks to the remarkable merits of ambient ionization techniques, chemical substances spanning a wide concentration range can be directly analyzed with no or minimal sample pretreatment[3]. In the past decade, more than 50 ambient ionization techniques have been invented for practical applications[3]. Techniques such as desorption electrospray ionization (DESI)[4], low temperature plasma probe (LTP)[5], direct analysis in real time (DART)[6], microwave plasma torch (MPT)[7], air flow-assisted ionization
(AFAI)[8], surface desorption atmospheric pressure chemical ionization (SDAPCI)[9] have been applied to surface analysis, later extended to imaging studies. For liquid or gaseous substances, paper spray (PS)[10] and extractive electrospray ionization (EESI)[11] are available. Owing to laser desorption, analytes located tens of micrometres underneath the sample surface were available for MS interrogation using techniques such as ambient surface-assisted laser desorption/ionization (ambient SALDI)[12] and laser ablation electrospray ionization (LAESI)[13]. Recently emerging leaf spray ionization[14] made plant tissue analysis even easier. However, while analyzing biological samples, degradation and inactivation of bio-active chemicals may occur due to the destructive sampling of intact sample bulk, possibly affecting the measurement accuracy. Therefore, a simple and effective method is urgently needed to
Received 4 May 2014; accepted 27 August 2014 * Corresponding author. Email: [email protected] This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), China (No. IRT13054), the National Natural Science Foundation of China (No. 21105010), and the Jiangxi Provincial Department of Science and Technology, China (Nos. 2010EHA01000, 2010DD01300). Copyright © 2014, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(14)60783-0
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
meet the requirements for the rapid analysis of substances inside intact solid samples. Garlic bulb, a spicy, common vegetable in daily life, contains rich bioactive constituents such as organic sulfides, amino acids and polysaccharides. Most analytical method have limited capabilities for the rapid analysis of multicomponent garlic tissue with high molecular specificity[15,16]. In present study, garlic bulb was chosen as a representative sample. Chemical constituents such as active organosulfur compounds (e.g., alliin, allicin), amino acids (e.g., arginine), and sugars (glucose, polysaccharides) were in situ identified by internal extractive electrospray ionization mass spectrometry (iEESI-MS)[17,18] without sample pretreatment. By analyzing chemical fingerprints of garlic bulb with principal component analysis (PCA), an MS strategy for the rapid characterization of garlic tissue components at the molecular level was established.
2 2.1
Experimental Instruments and reagents
MS experiments were carried out using an LTQ-XL MS instrument equipped with Xcalibur data processing system (Thermo Scientific, CA). A homemade iEESI apparatus was coupled in the front of MS inlet. The LTQ-XL MS instrument was set at positive ionization detection mode with a mass range of 50–2000 Da. The ion introduction capillary temperature was 150 °C. In the collision-induced dissociation (CID) experiments, the precursor ions of interest were isolated with a window width of 1.5 Da and the collision energy was set at 10%–30% in an arbitrary unit. Other parameters were set as default values and no further optimization was performed. The fused silicon capillary (inner diameter 0.25 mm, outer diameter 0.30 mm) was purchased from Agilent (USA). HPLC grade methanol and acetic acid were from ROE Scientific® Inc. Newark, USA. The double-distilled water was used throughout the experiment. Two kinds of garlic bulbs (common garlic bulb and single-clove garlic bulb) were purchased from a local supermarket. 2.2
Experimental method
The conceptual illustration of iEESI-MS was shown in Fig.1. A fused silicon capillary was inserted into the bulk sample in parallel with the sample surface, allowing a gap of ca. 2 mm between the capillary orifice and the edge of sample apex. The apex of the tissue was intentionally pointed toward the ion entrance of the mass spectrometer, keeping a distance of ca. 5 mm in between. An extraction solvent (e.g., methanol) biased with high voltage (+4.5 kV) was injected into the fused silicon capillary at a flow rate of 2 µL min–1. The chemicals in the bulk tissue were selectively extracted by the working solvent
Fig.1 Schematic diagram of the internal extractive electrospray ionization process
and carried along the electric field toward the apex of tissue sample, where an electro-spraying was induced. The charged micro-scale droplets were subsequently desolvated, producing gaseous ions ready for MS analysis. Garlic cloves with similar size were chosen, and triangularshaped tissues were sampled from the similar part of each garlic clove, in order to assure the repeatability. In the case of characterization of different kinds of garlic, two sets of garlic cloves (12 common garlic cloves and 12 single-clove garlic cloves) were analyzed in triplicate. In addition, three sets of garlic cloves (untreated garlic cloves, frozen garlic cloves and greened garlic cloves) were measured with 12 garlic cloves in each set, respectively. The frozen garlic cloves were stored in the refrigerator (–18 °C) for 6 h, and the greened ones were soaked in 10% acetic acid aqueous solution for 6 days. Fresh ones were taken as the control group. All of the three groups of garlic cloves were used directly without further treatments, and each garlic clove was analyzed once by iEESI-MS. All the mass spectral fingerprint data were further exported to Excel software for principal component analysis (PCA) performed using the Matlab (version 7.8.0, Mathworks, Inc., Natick, MA, USA).
3 3.1
Results and discussion Analysis of different kinds of garlic cloves
Two sets of garlic cloves (common garlic and single-clove garlic) were subjected to iEESI-MS analysis and the chemical fingerprints were shown in Fig.2. Due to the inherent nature of iEESI extraction, rich inorganic ions in garlic tissue (e.g., Na+, K+) contributed in the ionization reaction, [M + H]+, [M + Na]+, [M + K]+ and [M + NH4]+. The precursor ions of interest were isolated in the following collision-induced dissociation (CID) experiments, and characteristic fragment ions were obtained for the identification purpose. Based on characteristic fragments and the results of relevant literatures[15,19], active organosulfur compounds in the garlic tissue were identified. Peaks such as m/z 178 [alliin + H]+, m/z 216 [alliin + K]+, m/z 163 [allicin + H]+, m/z 180 [allicin + NH4]+, m/z 185 [allicin + Na]+, and m/z 201 [allicin + K]+ were assigned to ionization products of alliin (Mw 177) and allicin (Mw 162), respectively (Fig.2a). Moreover, MS/MS spectra of m/z 201 [allicin + K]+ and m/z 216 [alliin + K]+ were exhibited in Fig.3a and Fig.3b,
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
respectively. Major fragments at m/z 111 and m/z 139 were produced after the loss of CH2=CH-CH2-S(O)-H and CH2=CH-CH=S from the precursor of [allicin + K]+. Similarly, major fragment at m/z 126 was present after the loss of CH2=CH-CH2-S(O)-H from m/z 216 [alliin + K]+ (Fig.3b). These CID experiments results were in good agreement with previous literature[20]. Alliin is a natural sulfoxide constituent in the cytoplasm of normal fresh garlic. When the damage to garlic tissue occurs, the stable alliin turns to allicin under the catalysis of alliinase. This is a very quick enzymatic reaction which can be completed with 10 s at room temperature[21]. In this perspective, the direct detection of alliin and fragile allicin from bulk garlic tissue by iEESI-MS indicated the rapidity and soft ionization nature of iEESI analysis. Therefore, alliinase
denaturation, derivatization or addition of external standard required in conventional chromatographic methods could be obviated. The ability of iEESI to simultaneously and continuously profile the metabolic precursors and corresponding products in situ in a rapidly changing environment allows it to probe the activity of enzymes of interest either in the native environment or under external stimuli. Moreover, dominant peaks at m/z 381, 543, 705, 867, 1029, 1191, 1353, 1515, were present with a fixed mass shift of 162 Da, corresponding to the characteristic residue of fructosans ((–C6H10O5–), 162 Da). This observation was most likely attributed to the ionization of oligosaccharides and polysaccharides in garlic tissue. For example, assuming the degree of polymerization (DP) of a polysaccharide is 8 or 9, the respective [M + K]+ peak is estimated to be 1353 Da or 1515
Fig.2 iEESI-MS analysis of two garlic species (a) Common garlic bulb; (b) Single-clove garlic bulb; the insets: the zoomed-in mass spectra
Fig.3 MS2 spectrum of allicin (m/z 201), alliin (m/z 216), polysaccharide (m/z 1515, DP = 9), and arginine (m/z 175), respectively. The inset shows MS3 spectra of polysaccharide (DP = 9)
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
Da based on molecular weight calculation. Subsequent CID measurement on m/z 1515 was performed to test this hypothesis (Fig.3c). The generated fragments, such as m/z 1353, 1191, 1029, 867, 705, corresponded well with [M + K]+ of the polysaccharide with DP of 8, 7, 6, 5 and 4, respectively, indicating that peak at m/z 1515 lost several fructosan residues (162 Da) in the CID process. The generated fragment at m/z 1353 was further subjected to MS3 experiment. Major fragments such as m/z 1191, 1173, 1029, 867, 849 and 705 were observed, indicating the loss of several fructosan residues or a combination of fructosan residue and H2O (inset of Fig.3c). These results of CID experiments were consistent with previous literature[22]. All these results proved that iEESI-MS could be applied to the direct MS analysis of organic sulfides and polysaccharides in the complex medium of garlic tissue without pretreatment, which provided an applicable strategy for the direct, rapid analysis of unstable, macromolecular active components in biological tissues. Striking differences were revealed from the mass spectra of common garlic cloves and single-clove ones (Fig.2a and Fig.2b). The dominant peak in the single-clove garlic spectra, m/z 175, which produced MS2 fragments such as m/z 158, 157, 130, 116 and 60, could be ascribed to protonated arginine according to the analysis of characteristic fragment ions and the result of previous literature[23]. As shown in Fig.3d, major fragment ions at m/z 158, 157, 130 and 116 Da were produced after the loss of NH3 (17), H2O (18), (NH3 + CO) (45) and (NH=C(NH2)2) (59) group from protonated arginine, respectively. Moreover, peaks of allicin and alliin (m/z 201, 216) were also found in the mass spectra fingerprint of signalclove garlic; however, the abundances were much lower. Moreover, a very low abundance was found for the garlic polysaccharide in the signal-clove ones. The difference between the two sets garlic cloves samples was visualized after PCA analysis (Fig.4A). Corresponding PCA loading results revealed that the peaks at m/z 163, 175, 201, 381, 543, 705, 1029, 1191, 1353 and 1515 contributed notably to the differentiation of these two kinds of garlic cloves (Fig.4B), suggesting that organosulfur and polysaccharide contents were significantly different between
these two kinds of garlics. Previous studies showed that garlic’s healthy benefits are mostly attributed to the characteristic organosulfur compounds[21]. Moreover, garlic polysaccrcharides have shown to be beneficial in lowering blood sugar levels as well as facilitating growth of probiotics[24,25]. Therefore, the rapid detection of various healthy nutrients at molecular level in the bulk garlic tissue using iEESI-MS provides a practical way of nutrition evaluation of fruits and vegetables. 3.2
iEESI-MS analysis of differently processed garlic cloves
Many nutrients in garlic can be destroyed during preservation or processing, which may greatly affect the nutritional value of garlic cloves. Three sets of garlic clove (untreated garlic cloves, frozen garlic cloves and greened garlic cloves) were chosen for iEESI-MS proof-of-principle test. Compared with the untreated garlic clove (Fig.5A), the abundance of low DP polysaccharides (m/z 867, 1029, 1191 and 1353) in greened garlic (Fig.5B) was much higher than that of the untreated garlic clove. The acetic acid might facilitate the hydrolysis of glucosidic bond in polysaccharide, thus the low DP polysaccharide content increased. Moreover, the abundance of peak at m/z 216 (alliin) was higher in the greened garlic clove than that in the untreated garlic clove, most likely attributed to the low enzymatic activity of alliinase under low pH condition. The effect of freezing treatment on the alliinase activity was more remarkable (Fig.5C). Low temperature has been shown to possess a negative effect on the activity of alliinase, thus showing a higher abundance of alliin (m/z 216) in the frozen garlic clove. The molecular difference between these three sets of garlic cloves was discriminated well by PCA analysis of the iEESI-MS fingerprints (Fig.5D). All these results indicated that iEESI- MS could be used in analyzing changes of the components in garlic samples after different treatments, thus further validated the applicability of iEESI-MS in direct analysis of biological tissue samples. 3.3
Speed and stability of iEESI-MS analysis
Fig.4 (A) 3D-PCA score plots of iEESI-MS fingerprints of two garlic species (common garlic and single-clove one) and (B) PCA loading plots
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
Fig.5 Mass spectra of three kinds of garlic samples: (A) untreated garlic; (B) greened garlic; (C) frozen garlic; (D) 3D-PCA score plots of iEESI- MS fingerprints of three sets of garlic: a. untreated garlic; b. greened garlic; c. frozen garlic
iEESI-MS allows recognition of chemical changes in the garlic cloves with simple operation and high analytical speed. The analysis of a single sample took less than 2 min. Satisfactory RSD (n = 6) of the polysaccharide (m/z 705, DP = 4) in untreated garlic clove, greened garlic clove and frozen garlic clove (7.35%, 11.24% and 9.73%, respectively) was achieved. The relatively large RSD values might be due to the manual injection. During the iEESI-MS development toward quantitative analysis, parameters including the sample shape and size, and geometrical alignment in front of MS inlet are under strict control to reduce the manual operation errors. Apart from that, external standards may be added in the iEESI extraction solution to avoid the artificial errors and improve the quantitative capability of iEESI measurements in future studies.
biological tissue samples.
References [1]
Spectrom. Rev., 2013, 32(3): 218–243 [2] [3]
Chen H W, Hu B, Zhang X. Chinese J. Anal. Chem., 2010, 38(8): 1069–1088
[4]
Badu-Tawiah A K, Eberlin L S, Ouyang Z, Cooks R G. Annu. Rev. Phy. Chem., 2013, 64: 481–505
[5]
Liu Y Y, Ma X X, Lin Z Q, He M J, Han G J, Yang C D, Xing Z, Zhang S C, Zhang X R. Angew. Chem. Int. Edit., 2010, 49(26): 4435–4437
Conclusions
Chang C L, Xu G G, Bai Y, Zhang C S, Li X J, Li M, Liu Y, Liu H W. Anal. Chem., 2013, 85(1): 170–176
[7]
iEESI-MS allowed the direct analysis of endogenous chemicals inside garlic clove tissues without tedious sample pretreatment. It was demonstrated that chemical constituents such as active organosulfur compounds (e.g., alliin, allicin), amino acid (e.g., arginine), and saccharide (glucose, polysaccharides) in different kinds of garlic cloves or garlic under different treatment could be readily analyzed and identified based on chemical fingerprints. With the advantages such as simplicity, high throughput, high analytical speed and no requirement of pretreatment, iEESI-MS is potentially applicable for the direct analysis of unstable active constituents and biomacromolecules in solid and semi-solid
Wang X, Wang S J, Cai Z W. Trac-trend. Anal. Chem, 2013, 52: 170–185
[6]
4
Wu C P, Dill A L, Eberlin L S, Cooks R G, Ifa D R. Mass.
Zhang T Q, Zhou W, Jin W, Zhou J G, Handberg E, Zhu Z Q, Chen H W, Jin Q. J. Mass. Spectrom., 2013, 48(6): 669–676
[8]
Luo Z G, He J M, Chen Y, He J J, Gong T, Tang F, Wang X H, Zhang R P, Huang L, Zhang L F, Lü H N, Ma S G, Fu Z D, Chen X G, Yu S S, Abliz Z. Anal. Chem., 2013, 85(5): 2977–2982
[9]
Zhu Z Q, Yan J P, Wang Y, Chen S Z, Chen H W. Chinese J. Anal. Chem., 2013, 41(6): 905–910
[10] Zhang Y, Ju Y, Huang C, Wysocki V H. Anal. Chem., 2014,
86(3): 1342–1346 [11] Zhang Y, Pan S S, Zhu Z Q, Zhang X L, Xu G S, Wei Y P, Chen
H W, Ding J H. Chinese J. Anal. Chem., 2013, 41(8): 1220–1225
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
[12] Zhang J L, Li Z, Zhang C S, Feng B S, Zhou Z G, Bai Y, Liu H
W. Anal. Chem., 2012, 84(7): 3296–3301 [13] Nemes P, Barton A A, Vertes A. Anal. Chem., 2009, 81(16):
6668–6675 [14] Liu J J, Wang H, Cooks R G, Ouyang Z. Anal. Chem., 2011,
83(20): 7608–7613 [15] Huang X S, Lü M S. Chinese J. Anal. Chem., 2012, 40(2):
309–312 [16] Huang X S, Yan F Co, Wu J Z. Food Sci. (in Chinese), 2011,
(02): 146–149 [17] Zhang H, Zhu L, Luo L P, Wang N N, Chingin K, Guo X L,
Chen H W. J. Agric. Food Chem., 2013, 61(45): 10691–10698 [18] Zhang H, Gu H W, Yan F Y, Wang N A, Wei Y P, Xu J J, Chen
H W. Sci. Rep., 2013, 3: 2495 [19] Lee J, Harnly J M. J. Agric. Food Chem., 2005, 53(23):
9100-9104 [20] Chang J M, Xiang Y, Wang Y, Chen J. Food Sci. (in Chinese),
2008, 29(9): 485–486 [21] Lanzotti V. J. Chromatogr. A, 2006, 1112(1-2): 3–22 [22] Losso J N, Nakai S. J. Agric. Food Chem., 1997, 45(11):
4342–4346 [23] Xu N, Zhu Z Q, Yang S P, Wang J, Gu H W, Zhou Z, Chen H W.
Chinese J. Anal. Chem., 2013, 41(4): 523–528 [24] Yan C K, Zeng F D. Chinese J. New Drugs, 2004, (08):
688–691 [25] Agarwal K C. Med. Res. Rev., 1996, 16(1): 111–124
Cite this article as: Chin J Anal Chem, 2014, 42(11), 1634–1638.
RESEARCH PAPER
Direct Molecular Analysis of Garlic Using Internal Extractive Electrospray Ionization Mass Spectrometry ZHANG Hua, ZHU Liang, CHEN Huan-Wen* Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology, Nanchang 330013, China
Abstract: The internal extractive electrospray ionization mass spectrometry (iEESI-MS) has been applied to direct molecular analysis of garlic tissues. An extraction solvent (e.g., methanol) under a high voltage (+4.5 kV) was injected into the fused silicon capillary whose far end penetrated into the bulk tissue sample. The chemicals in the bulk tissue were selectively extracted by the working solvent and carried along the electric field toward the apex of the sample, to generate electrospray in front of an ion trap mass spectrometer. Without time-consuming sample pretreatment, active garlic substances such as organosulfur compounds (e.g., alliin, allicin), amino acids (e.g., arginine) and saccharides (glucose, polysaccharides) were successfully detected and directly identified via collision-induced dissociation (CID) in positive ion detection mode. Mass spectral fingerprints of different kinds of garlic cloves (24 samples), as well as various post-treated ones (36 samples) in the range of m/z 50–2000 Da, were classified via principal component analysis (PCA). The experimental results indicated that iEESI-MS allowed direct identification of chemical components in garlic tissue and rapid recognition of metabolic changes in the garlic tissue subjected to various external stimuli, with the analytical advantages such as simplicity, rapidity (less than 2 min per sample), good specificity, and minimal disturbance to the bioactivity of analytes. Key Words:
1
Internal extractive electrospray ionization; Garlic; Direct analysis; Mass spectrometry
Introduction
Recently, ambient mass spectrometric methods have become important for the analysis of complex raw samples, which commonly pose analytical challenges in the scientific community such as life science, chemistry, material science and forensic analysis[1,2]. Thanks to the remarkable merits of ambient ionization techniques, chemical substances spanning a wide concentration range can be directly analyzed with no or minimal sample pretreatment[3]. In the past decade, more than 50 ambient ionization techniques have been invented for practical applications[3]. Techniques such as desorption electrospray ionization (DESI)[4], low temperature plasma probe (LTP)[5], direct analysis in real time (DART)[6], microwave plasma torch (MPT)[7], air flow-assisted ionization
(AFAI)[8], surface desorption atmospheric pressure chemical ionization (SDAPCI)[9] have been applied to surface analysis, later extended to imaging studies. For liquid or gaseous substances, paper spray (PS)[10] and extractive electrospray ionization (EESI)[11] are available. Owing to laser desorption, analytes located tens of micrometres underneath the sample surface were available for MS interrogation using techniques such as ambient surface-assisted laser desorption/ionization (ambient SALDI)[12] and laser ablation electrospray ionization (LAESI)[13]. Recently emerging leaf spray ionization[14] made plant tissue analysis even easier. However, while analyzing biological samples, degradation and inactivation of bio-active chemicals may occur due to the destructive sampling of intact sample bulk, possibly affecting the measurement accuracy. Therefore, a simple and effective method is urgently needed to
Received 4 May 2014; accepted 27 August 2014 * Corresponding author. Email: [email protected] This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), China (No. IRT13054), the National Natural Science Foundation of China (No. 21105010), and the Jiangxi Provincial Department of Science and Technology, China (Nos. 2010EHA01000, 2010DD01300). Copyright © 2014, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(14)60783-0
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
meet the requirements for the rapid analysis of substances inside intact solid samples. Garlic bulb, a spicy, common vegetable in daily life, contains rich bioactive constituents such as organic sulfides, amino acids and polysaccharides. Most analytical method have limited capabilities for the rapid analysis of multicomponent garlic tissue with high molecular specificity[15,16]. In present study, garlic bulb was chosen as a representative sample. Chemical constituents such as active organosulfur compounds (e.g., alliin, allicin), amino acids (e.g., arginine), and sugars (glucose, polysaccharides) were in situ identified by internal extractive electrospray ionization mass spectrometry (iEESI-MS)[17,18] without sample pretreatment. By analyzing chemical fingerprints of garlic bulb with principal component analysis (PCA), an MS strategy for the rapid characterization of garlic tissue components at the molecular level was established.
2 2.1
Experimental Instruments and reagents
MS experiments were carried out using an LTQ-XL MS instrument equipped with Xcalibur data processing system (Thermo Scientific, CA). A homemade iEESI apparatus was coupled in the front of MS inlet. The LTQ-XL MS instrument was set at positive ionization detection mode with a mass range of 50–2000 Da. The ion introduction capillary temperature was 150 °C. In the collision-induced dissociation (CID) experiments, the precursor ions of interest were isolated with a window width of 1.5 Da and the collision energy was set at 10%–30% in an arbitrary unit. Other parameters were set as default values and no further optimization was performed. The fused silicon capillary (inner diameter 0.25 mm, outer diameter 0.30 mm) was purchased from Agilent (USA). HPLC grade methanol and acetic acid were from ROE Scientific® Inc. Newark, USA. The double-distilled water was used throughout the experiment. Two kinds of garlic bulbs (common garlic bulb and single-clove garlic bulb) were purchased from a local supermarket. 2.2
Experimental method
The conceptual illustration of iEESI-MS was shown in Fig.1. A fused silicon capillary was inserted into the bulk sample in parallel with the sample surface, allowing a gap of ca. 2 mm between the capillary orifice and the edge of sample apex. The apex of the tissue was intentionally pointed toward the ion entrance of the mass spectrometer, keeping a distance of ca. 5 mm in between. An extraction solvent (e.g., methanol) biased with high voltage (+4.5 kV) was injected into the fused silicon capillary at a flow rate of 2 µL min–1. The chemicals in the bulk tissue were selectively extracted by the working solvent
Fig.1 Schematic diagram of the internal extractive electrospray ionization process
and carried along the electric field toward the apex of tissue sample, where an electro-spraying was induced. The charged micro-scale droplets were subsequently desolvated, producing gaseous ions ready for MS analysis. Garlic cloves with similar size were chosen, and triangularshaped tissues were sampled from the similar part of each garlic clove, in order to assure the repeatability. In the case of characterization of different kinds of garlic, two sets of garlic cloves (12 common garlic cloves and 12 single-clove garlic cloves) were analyzed in triplicate. In addition, three sets of garlic cloves (untreated garlic cloves, frozen garlic cloves and greened garlic cloves) were measured with 12 garlic cloves in each set, respectively. The frozen garlic cloves were stored in the refrigerator (–18 °C) for 6 h, and the greened ones were soaked in 10% acetic acid aqueous solution for 6 days. Fresh ones were taken as the control group. All of the three groups of garlic cloves were used directly without further treatments, and each garlic clove was analyzed once by iEESI-MS. All the mass spectral fingerprint data were further exported to Excel software for principal component analysis (PCA) performed using the Matlab (version 7.8.0, Mathworks, Inc., Natick, MA, USA).
3 3.1
Results and discussion Analysis of different kinds of garlic cloves
Two sets of garlic cloves (common garlic and single-clove garlic) were subjected to iEESI-MS analysis and the chemical fingerprints were shown in Fig.2. Due to the inherent nature of iEESI extraction, rich inorganic ions in garlic tissue (e.g., Na+, K+) contributed in the ionization reaction, [M + H]+, [M + Na]+, [M + K]+ and [M + NH4]+. The precursor ions of interest were isolated in the following collision-induced dissociation (CID) experiments, and characteristic fragment ions were obtained for the identification purpose. Based on characteristic fragments and the results of relevant literatures[15,19], active organosulfur compounds in the garlic tissue were identified. Peaks such as m/z 178 [alliin + H]+, m/z 216 [alliin + K]+, m/z 163 [allicin + H]+, m/z 180 [allicin + NH4]+, m/z 185 [allicin + Na]+, and m/z 201 [allicin + K]+ were assigned to ionization products of alliin (Mw 177) and allicin (Mw 162), respectively (Fig.2a). Moreover, MS/MS spectra of m/z 201 [allicin + K]+ and m/z 216 [alliin + K]+ were exhibited in Fig.3a and Fig.3b,
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
respectively. Major fragments at m/z 111 and m/z 139 were produced after the loss of CH2=CH-CH2-S(O)-H and CH2=CH-CH=S from the precursor of [allicin + K]+. Similarly, major fragment at m/z 126 was present after the loss of CH2=CH-CH2-S(O)-H from m/z 216 [alliin + K]+ (Fig.3b). These CID experiments results were in good agreement with previous literature[20]. Alliin is a natural sulfoxide constituent in the cytoplasm of normal fresh garlic. When the damage to garlic tissue occurs, the stable alliin turns to allicin under the catalysis of alliinase. This is a very quick enzymatic reaction which can be completed with 10 s at room temperature[21]. In this perspective, the direct detection of alliin and fragile allicin from bulk garlic tissue by iEESI-MS indicated the rapidity and soft ionization nature of iEESI analysis. Therefore, alliinase
denaturation, derivatization or addition of external standard required in conventional chromatographic methods could be obviated. The ability of iEESI to simultaneously and continuously profile the metabolic precursors and corresponding products in situ in a rapidly changing environment allows it to probe the activity of enzymes of interest either in the native environment or under external stimuli. Moreover, dominant peaks at m/z 381, 543, 705, 867, 1029, 1191, 1353, 1515, were present with a fixed mass shift of 162 Da, corresponding to the characteristic residue of fructosans ((–C6H10O5–), 162 Da). This observation was most likely attributed to the ionization of oligosaccharides and polysaccharides in garlic tissue. For example, assuming the degree of polymerization (DP) of a polysaccharide is 8 or 9, the respective [M + K]+ peak is estimated to be 1353 Da or 1515
Fig.2 iEESI-MS analysis of two garlic species (a) Common garlic bulb; (b) Single-clove garlic bulb; the insets: the zoomed-in mass spectra
Fig.3 MS2 spectrum of allicin (m/z 201), alliin (m/z 216), polysaccharide (m/z 1515, DP = 9), and arginine (m/z 175), respectively. The inset shows MS3 spectra of polysaccharide (DP = 9)
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
Da based on molecular weight calculation. Subsequent CID measurement on m/z 1515 was performed to test this hypothesis (Fig.3c). The generated fragments, such as m/z 1353, 1191, 1029, 867, 705, corresponded well with [M + K]+ of the polysaccharide with DP of 8, 7, 6, 5 and 4, respectively, indicating that peak at m/z 1515 lost several fructosan residues (162 Da) in the CID process. The generated fragment at m/z 1353 was further subjected to MS3 experiment. Major fragments such as m/z 1191, 1173, 1029, 867, 849 and 705 were observed, indicating the loss of several fructosan residues or a combination of fructosan residue and H2O (inset of Fig.3c). These results of CID experiments were consistent with previous literature[22]. All these results proved that iEESI-MS could be applied to the direct MS analysis of organic sulfides and polysaccharides in the complex medium of garlic tissue without pretreatment, which provided an applicable strategy for the direct, rapid analysis of unstable, macromolecular active components in biological tissues. Striking differences were revealed from the mass spectra of common garlic cloves and single-clove ones (Fig.2a and Fig.2b). The dominant peak in the single-clove garlic spectra, m/z 175, which produced MS2 fragments such as m/z 158, 157, 130, 116 and 60, could be ascribed to protonated arginine according to the analysis of characteristic fragment ions and the result of previous literature[23]. As shown in Fig.3d, major fragment ions at m/z 158, 157, 130 and 116 Da were produced after the loss of NH3 (17), H2O (18), (NH3 + CO) (45) and (NH=C(NH2)2) (59) group from protonated arginine, respectively. Moreover, peaks of allicin and alliin (m/z 201, 216) were also found in the mass spectra fingerprint of signalclove garlic; however, the abundances were much lower. Moreover, a very low abundance was found for the garlic polysaccharide in the signal-clove ones. The difference between the two sets garlic cloves samples was visualized after PCA analysis (Fig.4A). Corresponding PCA loading results revealed that the peaks at m/z 163, 175, 201, 381, 543, 705, 1029, 1191, 1353 and 1515 contributed notably to the differentiation of these two kinds of garlic cloves (Fig.4B), suggesting that organosulfur and polysaccharide contents were significantly different between
these two kinds of garlics. Previous studies showed that garlic’s healthy benefits are mostly attributed to the characteristic organosulfur compounds[21]. Moreover, garlic polysaccrcharides have shown to be beneficial in lowering blood sugar levels as well as facilitating growth of probiotics[24,25]. Therefore, the rapid detection of various healthy nutrients at molecular level in the bulk garlic tissue using iEESI-MS provides a practical way of nutrition evaluation of fruits and vegetables. 3.2
iEESI-MS analysis of differently processed garlic cloves
Many nutrients in garlic can be destroyed during preservation or processing, which may greatly affect the nutritional value of garlic cloves. Three sets of garlic clove (untreated garlic cloves, frozen garlic cloves and greened garlic cloves) were chosen for iEESI-MS proof-of-principle test. Compared with the untreated garlic clove (Fig.5A), the abundance of low DP polysaccharides (m/z 867, 1029, 1191 and 1353) in greened garlic (Fig.5B) was much higher than that of the untreated garlic clove. The acetic acid might facilitate the hydrolysis of glucosidic bond in polysaccharide, thus the low DP polysaccharide content increased. Moreover, the abundance of peak at m/z 216 (alliin) was higher in the greened garlic clove than that in the untreated garlic clove, most likely attributed to the low enzymatic activity of alliinase under low pH condition. The effect of freezing treatment on the alliinase activity was more remarkable (Fig.5C). Low temperature has been shown to possess a negative effect on the activity of alliinase, thus showing a higher abundance of alliin (m/z 216) in the frozen garlic clove. The molecular difference between these three sets of garlic cloves was discriminated well by PCA analysis of the iEESI-MS fingerprints (Fig.5D). All these results indicated that iEESI- MS could be used in analyzing changes of the components in garlic samples after different treatments, thus further validated the applicability of iEESI-MS in direct analysis of biological tissue samples. 3.3
Speed and stability of iEESI-MS analysis
Fig.4 (A) 3D-PCA score plots of iEESI-MS fingerprints of two garlic species (common garlic and single-clove one) and (B) PCA loading plots
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
Fig.5 Mass spectra of three kinds of garlic samples: (A) untreated garlic; (B) greened garlic; (C) frozen garlic; (D) 3D-PCA score plots of iEESI- MS fingerprints of three sets of garlic: a. untreated garlic; b. greened garlic; c. frozen garlic
iEESI-MS allows recognition of chemical changes in the garlic cloves with simple operation and high analytical speed. The analysis of a single sample took less than 2 min. Satisfactory RSD (n = 6) of the polysaccharide (m/z 705, DP = 4) in untreated garlic clove, greened garlic clove and frozen garlic clove (7.35%, 11.24% and 9.73%, respectively) was achieved. The relatively large RSD values might be due to the manual injection. During the iEESI-MS development toward quantitative analysis, parameters including the sample shape and size, and geometrical alignment in front of MS inlet are under strict control to reduce the manual operation errors. Apart from that, external standards may be added in the iEESI extraction solution to avoid the artificial errors and improve the quantitative capability of iEESI measurements in future studies.
biological tissue samples.
References [1]
Spectrom. Rev., 2013, 32(3): 218–243 [2] [3]
Chen H W, Hu B, Zhang X. Chinese J. Anal. Chem., 2010, 38(8): 1069–1088
[4]
Badu-Tawiah A K, Eberlin L S, Ouyang Z, Cooks R G. Annu. Rev. Phy. Chem., 2013, 64: 481–505
[5]
Liu Y Y, Ma X X, Lin Z Q, He M J, Han G J, Yang C D, Xing Z, Zhang S C, Zhang X R. Angew. Chem. Int. Edit., 2010, 49(26): 4435–4437
Conclusions
Chang C L, Xu G G, Bai Y, Zhang C S, Li X J, Li M, Liu Y, Liu H W. Anal. Chem., 2013, 85(1): 170–176
[7]
iEESI-MS allowed the direct analysis of endogenous chemicals inside garlic clove tissues without tedious sample pretreatment. It was demonstrated that chemical constituents such as active organosulfur compounds (e.g., alliin, allicin), amino acid (e.g., arginine), and saccharide (glucose, polysaccharides) in different kinds of garlic cloves or garlic under different treatment could be readily analyzed and identified based on chemical fingerprints. With the advantages such as simplicity, high throughput, high analytical speed and no requirement of pretreatment, iEESI-MS is potentially applicable for the direct analysis of unstable active constituents and biomacromolecules in solid and semi-solid
Wang X, Wang S J, Cai Z W. Trac-trend. Anal. Chem, 2013, 52: 170–185
[6]
4
Wu C P, Dill A L, Eberlin L S, Cooks R G, Ifa D R. Mass.
Zhang T Q, Zhou W, Jin W, Zhou J G, Handberg E, Zhu Z Q, Chen H W, Jin Q. J. Mass. Spectrom., 2013, 48(6): 669–676
[8]
Luo Z G, He J M, Chen Y, He J J, Gong T, Tang F, Wang X H, Zhang R P, Huang L, Zhang L F, Lü H N, Ma S G, Fu Z D, Chen X G, Yu S S, Abliz Z. Anal. Chem., 2013, 85(5): 2977–2982
[9]
Zhu Z Q, Yan J P, Wang Y, Chen S Z, Chen H W. Chinese J. Anal. Chem., 2013, 41(6): 905–910
[10] Zhang Y, Ju Y, Huang C, Wysocki V H. Anal. Chem., 2014,
86(3): 1342–1346 [11] Zhang Y, Pan S S, Zhu Z Q, Zhang X L, Xu G S, Wei Y P, Chen
H W, Ding J H. Chinese J. Anal. Chem., 2013, 41(8): 1220–1225
ZHANG Hua et al. / Chinese Journal of Analytical Chemistry, 2014, 42(11): 1634–1638
[12] Zhang J L, Li Z, Zhang C S, Feng B S, Zhou Z G, Bai Y, Liu H
W. Anal. Chem., 2012, 84(7): 3296–3301 [13] Nemes P, Barton A A, Vertes A. Anal. Chem., 2009, 81(16):
6668–6675 [14] Liu J J, Wang H, Cooks R G, Ouyang Z. Anal. Chem., 2011,
83(20): 7608–7613 [15] Huang X S, Lü M S. Chinese J. Anal. Chem., 2012, 40(2):
309–312 [16] Huang X S, Yan F Co, Wu J Z. Food Sci. (in Chinese), 2011,
(02): 146–149 [17] Zhang H, Zhu L, Luo L P, Wang N N, Chingin K, Guo X L,
Chen H W. J. Agric. Food Chem., 2013, 61(45): 10691–10698 [18] Zhang H, Gu H W, Yan F Y, Wang N A, Wei Y P, Xu J J, Chen
H W. Sci. Rep., 2013, 3: 2495 [19] Lee J, Harnly J M. J. Agric. Food Chem., 2005, 53(23):
9100-9104 [20] Chang J M, Xiang Y, Wang Y, Chen J. Food Sci. (in Chinese),
2008, 29(9): 485–486 [21] Lanzotti V. J. Chromatogr. A, 2006, 1112(1-2): 3–22 [22] Losso J N, Nakai S. J. Agric. Food Chem., 1997, 45(11):
4342–4346 [23] Xu N, Zhu Z Q, Yang S P, Wang J, Gu H W, Zhou Z, Chen H W.
Chinese J. Anal. Chem., 2013, 41(4): 523–528 [24] Yan C K, Zeng F D. Chinese J. New Drugs, 2004, (08):
688–691 [25] Agarwal K C. Med. Res. Rev., 1996, 16(1): 111–124