Article pubs.acs.org/ac
Construction of an Ultrahigh Pressure Liquid ChromatographyTandem Mass Spectral Library of Plant Natural Products and Comparative Spectral Analyses Zhentian Lei,† Li Jing,† Feng Qiu,† Hua Zhang,‡ David Huhman,† Zhiqin Zhou,§ and Lloyd W. Sumner*,† †
Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, United States ‡ College of Life Science and Engineering, Chongqing Three Gorges University, Wanzhou, Chongqing 404100, China § College of Horticulture and Landscape Architecture, Southwest University, Beibei, Chongqing 400716, China S Supporting Information *
ABSTRACT: A plant natural product tandem mass spectral library has been constructed using authentic standards and purified compounds. Currently, the library contains 1734 tandem mass spectra for 289 compounds, with the majority (76%) of the compounds being plant phenolics such as flavonoids, isoflavonoids, and phenylpropanoids. Tandem mass spectra and chromatographic retention data were acquired on a triple quadrupole mass spectrometer coupled to an ultrahigh pressure liquid chromatograph using six different collision energies (CEs) (10−60 eV). Comparative analyses of the tandem mass spectral data revealed that the loss of ring substituents preceded the C-ring opening during the fragmentation of flavonoids and isoflavonoids. At lower CE (i.e., 10 and 20 eV), the flavonoids and isoflavonoid central ring structures typically remained intact, and fragmentation was characterized by the loss of the substituents (i.e., methyl and glycosyl groups). At higher CE, the flavonoid and isoflavonoid core ring systems underwent Cring cleavage and/or rearrangement depending on the structure, particularly hydroxylation patterns. In-source electrochemical oxidation was observed for phenolics that had ortho-diphenol moieties (i.e., vicinal hydroxyl groups on the aromatic rings). The ortho-diphenols were oxidized to ortho-quinones, yielding an intensive and, in most cases, a base ion peak corresponding to a [(M − 2H) − H]− ion in their mass spectra. The library also contains reverse-phase retention times, allowing for the construction, validation, and testing of an artificial neural network retention prediction of other flavonoids and isoflavonoids not contained within the library. The library is freely available for nonprofit, academic use and it can be downloaded at http://www.noble.org/ apps/Scientific/WebDownloadManager/DownloadArea.aspx.
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lant phenolics are a group of diverse and specialized plant natural products that include lignin, tannins, coumarins, stilbenes, flavonoids, and isoflavonoids. Phenolics are derived from the shikimate and phenylpropanoid pathways, and they play important roles in many biological processes.1 For example, flavonoids and isoflavonoids are involved in plant disease and defense responses.2 They attract pollinators and insect predators, protect against UV light, and regulate symbiotic plant−microbe interactions. They have also been reported to possess health benefits for human and animals due to their antioxidant, anti-inflammation, antimicrobial, and anticancer properties.3,4 Flavonoids and isoflavonoids comprise flavones, flavonols, flavanones, flavanonols, and isoflavones that differ in their core ring structures (Figure 1). The complexity of flavonoids and isoflavonoids is further increased by different hydroxylation, methylation, and glycosylation patterns. The diversity and complexity of these structures represent a substantial challenge to the large-scale, quantitative, and qualitative profiling of flavonoids and isoflavonoids in plant and animal metabolomics. © XXXX American Chemical Society
Figure 1. Flavonoid structures can be generally classified as isoflavonoids, flavones, flavonols, flavanones, and flavanonols.
Liquid chromatography (LC) coupled to mass spectrometry (MS) is an important analytical platform for large-scale, nontargeted metabolomics owing to its excellent combination of sensitivity and selectivity.5 It has been successfully utilized in the analysis of flavonoids and isoflavonoids in complex plant Received: April 24, 2015 Accepted: June 24, 2015
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DOI: 10.1021/acs.analchem.5b01559 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry extracts.6−8 Metabolites observed in LC-MS based metabolic profiling are typically annotated by comparing the analyte’s retention time and mass-to-charge ratio (m/z) to those of authentic standards acquired in the same manner.9 This approach is effective when the sample mixture is simple. However, in large-scale metabolomics, this approach can be complicated by a large number of stereoisomers that have similar chromatographic behavior and UV absorption properties. This is particularly true with plant natural products such as flavonoids and isoflavonoids that have many possible substitution positions available on the central ring structures, which often result in a large number of isobaric compounds that cannot be differentiated simply based upon their m/z values. Identification of these isomers typically requires tandem mass spectrometry (MS/MS).10−12 To increase our peak annotation confidence in large-scale metabolomics, a custom plant natural product MS/MS spectral library was constructed using authentic standards and purified compounds. The MS/ MS spectra were acquired using fixed collision energy (CE) from 10 to 60 eV (i.e., 10, 20, 30, 40, 50, and 60 eV). The varying fixed energies used in the MS/MS acquisitions provided fundamental insights into the fragmentation patterns of flavonoids and isoflavonoids. Loss of functional substituents (methyl and glycosyl groups) was observed at lower CE for flavonoids and isoflavonoids, while fragmentation of the core ring systems was observed at higher CE. The fragmentation patterns of the core ring systems were greatly influenced by the hydroxylation patterns. In-source, electrochemical oxidation was also observed for phenolics, flavonoids, and isoflavonoids that possessed ortho-diphenol moieties (i.e., vicinal hydroxyl groups on the A-ring and/or B-ring). The ortho-diphenol moieties were oxidized to ortho-quinone species, resulting in a shift of their m/z values. Understanding this in-source oxidation phenomenon facilitates the correct identification of these compounds. Currently, the library contains 289 compounds with the majority (76%) being phenolics such as flavonoids, isoflavonoids, and phenylpropanoids. Other compounds include fatty acids, carbohydrates, and triterpenes. The library is an extremely valuable source for metabolite identification in both large-scale targeted and nontargeted metabolomics. It also served as a valuable training set for retention prediction of other phenolics not present in the library and as orthogonal data for confirming metabolite identification. The library is free for academic use and can be downloaded from http://www. n o b l e . o r g / a p p s / S c ie n t i fi c / W e b D o w n l o a d M a n a g e r / DownloadArea.aspx.
mobile phases were 0.1% aqueous acetic acid (A) and acetonitrile (B). UHPLC separations were performed with a Waters BEH C18 column (2.1 × 150 mm, 1.7 μm particles) and the following gradient: the initial mobile phase ratio (A:B) 95%:5% was changed linearly to 30%:70% over 30 min, then to 5%:95% over 3 min, held at 5%:95% for 3 min and returned to 95%:5% for equilibration over 3 min. The flow rate was 0.56 mL/min and the column temperature was 60 °C. MS analyses were performed in negative electrospray ionization (ESI) mode (or positive ionization mode if analytes did not ionize sufficiently in negative ion mode) with a scan range of 100 to 1000 m/z. The following ESI source parameters were used: drying gas flow (N2): 6 L/m at 300 °C; nebulizer gas pressure (N2): 15 p.s.i.; capillary voltage: 4000 V. The default fragmentor voltage of 135 V was used, and the voltage was applied at the exit end of the ion transmission capillary, similar to cone voltage in other instruments. Retention Prediction. The library also contains reversephase retention times for the 289 compounds. The retention data were used to construct, validate, and test an artificial neural network (ANN) for the retention prediction of other flavonoids and isoflavonoids not contained within the library. Structural descriptors were generated using binary codes (1 or 0) to denote the substitution patterns of flavonoids. Log P values were calculated using JChem for Excel 6.1.0.688 (ChemAxon, Hungary) and simplified molecular-input lineentry system (SMILES) strings. The number of each functional group including hydroxyl, methoxyl, prenyl, glucosyl, glucuronyl, and rhamnosyl was also used for the ANN modeling. Training of the network was performed in MATLAB R2012b (Mathworks, MA) using the Neural Network Toolbox. Initially, the input (molecular descriptors) and output (retention time) variables were nominalized to the range of −1 to 1 using the “mapminmax” function in MATLAB. Then, a total number of 126 compounds were divided into training (80%), validation (10%), and testing (10%) sets. The number of nodes in the hidden layer was determined by trial and error. Training of the network was performed using the training function “trainlm” and transfer function “tansig”. The training process was stopped when the satisfactory correlation coefficients (R2 > 0.97) were achieved for all three data sets. The contribution of each descriptor to the retention prediction was evaluated using the approach developed by Chastrette et al.13
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RESULTS AND DISCUSSION A tandem mass spectral library of plant natural products was generated from 289 compounds, most of which were flavonoids and isoflavonoids. Both the retention time and the tandem spectra contained in the library are characteristic of each individual compound and are valuable in large-scale metabolomics peak annotation. For example, retention time has been concluded as a reliable diagnostic tool in peak annotation,14 and retention time prediction algorithms were also developed to aid in compound identification.15,16 As flavonoids and isoflavonoids constitute a majority of the compounds in the library, their chromatographic behavior, fragmentation patterns, and insource electrochemical oxidation are discussed below in more detail. Chromatographic Behavior of Flavonoids. Polarity is an important factor that influences the retention of organic compounds in HPLC. Generally, polarity is negatively correlated to the carbon content of a molecule. Moderate correlations (Figure S1 in the Supporting Information) were
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EXPERIMENTAL SECTION Materials and Reagents. Authentic standards were purchased from various commercial sources (Sigma-Aldrich, A-Apin, Fluka, Quality Phytochemicals LLC, Chromadex, Extrasynthese, ICN Biomedicals, Supelco, Cerilliant, and Indofine) and used without further purification. Additional phenolics that were not commercially available were gifts from Drs. Tom J. Mabry at the University of Texas and Paul Paré at the Texas Tech University. All solvents were HPLC grade. Water and acetonitrile were purchased from Honeywell Burdick and Jackson, Muskegon, MI, and acetic acid was from Fisher Chemical, Fairlawn, NJ. UHPLC-MS. An Agilent Infinity 1290 ultrahigh pressure liquid chromatography (UHPLC) system coupled to an Agilent 6430 triple quadrupole mass spectrometer was used. UHPLC B
DOI: 10.1021/acs.analchem.5b01559 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
hydroxyflavone to form the hydrogen bond. This conclusion was further supported by the longer retention of 5,7,3′,4′,5′pentahydroxyflavone (tricetin, 7.01 min) relative to 3,7,3′,4′,5′pentrahydroxyflavone (robinetin, 5.2 min) and the longer retention time of 5,7,4′-trihydroxyflavone (apigenin, 10.29 min) relative to 7,4′-dihydroxyflavone (7.71 min) even though the former, apigenin, has one more hydroxyl group. 4′-Hydroxyflavone had the shortest retention time, which indicated that it was the most polar mono-hydroxyflavone in Table 1. We propose that this is due to the resonance stabilization offered by the 4′-hydroxyflavone phenoxides. In solution, 4′-hydroxyflavones readily dissociate into 4′-hydroxyflavone phenoxides that are resonance stabilized and more polar due to the increase in nonbonded electrons. A similar effect was also observed for other 4′-multihydroxyisoflavones. For example, 3,6,2′,4′tetrahydroxyflavone (7.21 min) eluted earlier than 3,6,2′,3′tetrahydroxyflavone (8.63 min) and 5,7,4′-trihydroxyflavone (apigenin, 10.29 min) eluted earlier than 5,6,7-trihydroxyflavone (baicalein, 12.02 min); both due to the presence of the C4′-hydroxyl group. Similarly, 7,4′-dihydroxyflavone (7.71 min) and 7,4′-dihydroxyisoflavone (daidzein, 8.07 min) eluted much earlier than 5,7-dihydroxyflavone (chrysin, 14.66 min) due to the presence of the C4′-hydroxy group and the presence of a C5-hydroxy group that reduces polarity of the C4-keto group in 5,7-dihydroxyflavone. Methylation and prenylation decreased the polarity of flavonoids and isoflavonoids, whereas glycosylation increased the polarity. For example, methylated luteolin (6-methoxyluteolin) had a longer retention time (9.33 min) compared to luteolin (8.77 min) while glycosylated luteolin (luteolin-7-O-glucoside) had a significantly shorter retention time (5.82 min). The effects of structural modifications on retention time were also additive. For example, the retention time of kaempferol (10.39 min) was shorter than that of monomethylated kaempferol (4′-methoxykaempferol, 15.34 min) which was shorter than the retention time of 3,7,4′-trimethoxykaempferol (21.37 min). Similarly, 6,8-diprenylnaringenin (24.88 min) was less polar than 8-prenylnaringenin (16.61 min) which was less polar than naringenin (9.91 min). The effect of these modifications on the retention of flavonoids and isoflavonoids was dependent on the type of the modifications and the position of the modifications. For example, luteolin-4′-Oglucoside (6.73 min) was less polar than luteolin-7-O-glucoside (5.82 min). This was because glycosylation at the C4′-hydroxy group in luteolin-4′-O-glucoside prevented the formation of the more polar luteolin-4′-phenoxide. In contrast, in luteolin-7-Oglucoside, the free C4′-hydroxy group favored the formation of the resonance stabilized and more polar luteolin-4′-phenoxide, resulting in shorter retention time. Similarly, kaempferol-3-Oglucoside (6.66 min) was more polar than kaempferol-7-Oglucoside (7.12 min) due to the presence of the C3-hydroxy group in the latter that formed the intramolecular hydrogen bond with the C4-keto group to decrease its polarity. The combination of different modifications (i.e., number of hydroxyl, methyl, glycosyl, and prenyl groups and their position) and different core ring systems resulted in a wide range of retention times for flavonoids and isoflavonoids, ranging from 2.23 min (catechin) to 27.57 min (artocarpin, 5,2′,4′-trihydroxy-3,6-diprenyl-7-methoxyflavone) in this study. The overall elution order of the different classes of compounds analyzed and reported here was: benzoic acids < cinnamic acids < flavonoid C-glycosides < flavonoid O-glycosides < flavonoid and isoflavonoid aglycones < prenylated flavonoids
