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Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass
Accepted Manuscript Title: Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry Author: Ji-Liang Cao Jin-Chao Wei Yuan-Jia Hu Cheng-Wei He Mei-Wan Chen Jian-Bo Wan Peng Li PII: DOI: Reference:
S0021-9673(15)01740-9 http://dx.doi.org/doi:10.1016/j.chroma.2015.11.078 CHROMA 357092
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
16-9-2015 20-11-2015 25-11-2015
Please cite this article as: J.-L. Cao, J.-C. Wei, Y.-J. Hu, C.-W. He, M.-W. Chen, J.B. Wan, P. Li, Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.11.078 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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328 peaks were detected and 7 of them were firstly discovered from Danshen. Enhanced data-dependent acquisition offers more MSn data of low abundant compounds.
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Successful quantification was achieved by using the DAD contour plot.
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Phenolic acids and diterpenoids were separated into two independent groups on the 2D contour plot.
Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry
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Ji-Liang Cao, Jin-Chao Wei, Yuan-Jia Hu, Cheng-Wei He, Mei-Wan Chen, Jian-Bo Wan, Peng Li *
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State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China
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* Corresponding author at: Institute of Chinese Medical Sciences (University of Macau), Macau, China. Dr. Peng Li Tel.: +853 8397 4874; fax: +853 2884 1358. E-mail address: [email protected]
Abstract
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Danshen is one of the most frequently used traditional Chinese herbs owing to its remarkable and reliable therapeutic effects. Phenolic acids and diterpenoids have proved to be the bioactive substance groups. In order to fully profile its chemical compositions and explore new potential bioactive compounds, a comprehensive two-dimensional liquid chromatography system coupled to DAD detector and hybrid linear ion trap (LTQ) Orbitrap mass spectrometry (LC×LC-DAD-ESI/HRMS/MSn) was set up in this study based on the
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column combination of Hypersil gold CN (150 mm × 1 mm, 3 µm) and Accucore C18 (50 mm × 4.6 mm, 2.6 µm). Using the optimal segment gradient program, phenolic acids and diterpenoids were separated into two independent groups and a total of 328 peaks were successfully detected on the contour plot of Danshen. By means of the accurate mass and reliable MSn data, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. Moreover, the LC×LC-DAD system was validated for the quantitative analysis of 14 bioactive analytes using the contour plot, exhibiting satisfactory linearity (r≥0.9976) and high precision for both peak locating (≤1.07%) and peak volume calculating (0.34%-4.11%). The established method could afford powerful separation capability, reliable identification data and accurate quantitative results, which is very suitable for analysis of complex herbal samples. Keywords
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Comprehensive two-dimensional liquid chromatography; LTQ Orbitrap mass spectrometry; Phenolic acids; Diterpenoids; Danshen. 1. Introduction
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Nowadays, with the increasing requirements for more efficient separation of complex samples, two-dimensional liquid chromatography (2D-LC) has been attracting more attentions owing to its large peak capacity and high resolving power. In 2D-LC systems, columns with different separation mechanisms, including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), size exclusion chromatography (SEC) or affinity chromatography (AC) have often been combined to achieve higher resolution and improved selectivity [1-5]. Especially when coupled with mass spectrometer (MS), 2D-LC can exhibit unparalleled capabilities for the detection and identification of minor or even trace-level components. Danshen, the dried root and rhizome of Salvia miltiorrhiza, is one of the most frequently used traditional Chinese herbs in China and, in recent years, also famous as a health product in western world thanks to its remarkable and reliable biological activities, such as therapeutic effects on cardiovascular and cerebrovascular diseases, anti-dysmenorrhea, antitumor, anti-parkinsonian, as well as neuroprotective effect [6-9]. Depending on the modern pharmacological studies and chemical investigations on Danshen, two major groups of bioactive components, phenolic acids (e.g., salvianolic acid B and rosmarinic acid) and diterpenoids (e.g., dihydrotanshinone I and tanshinone IIA) were discovered to be responsible for its pharmacological or therapeutic effects [10-14]. Therefore, chemical analysis of Danshen, mainly focused on phenolic acids and diterpenoids, have been widely reported to evaluate its quality, discover potential bioactive compounds or profile chemical compositions by using conventional chromatographic and even hyphenated techniques, such as LC, LC-MS and capillary electrophoresis [15-22]. But, restricted to limited peak capacity and resolving power of one-dimensional (1D) techniques, and also due to similar chemical properties of the preferred compounds, the chemical separation of Danshen is always insufficient, which causes the inaccurate quantification and false identification of detected components, especially the minor ones. In this study, a comprehensive 2D-LC system coupled with DAD detector and hybrid linear ion trap (LTQ) Orbitrap mass spectrometry (LC×LC-DAD-ESI/HRMS/MSn) was
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developed to separate and identify the phenolic and diterpenoid constituents in Danshen. A total of 328 peaks were successfully separated and detected. Among these peaks, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. Meanwhile, 14 bioactive compounds, including 10 phenolic acids and 4 diterpenoids (Fig. 1) were determined directly on the DAD contour plots of Danshen samples with satisfactory linearity and good precision. 2. Materials and methods 2.1. Chemicals and materials
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HPLC grade acetonitrile, formic acid and methanol were purchased from Merck (Darmstadt, Germany). Deionized water (18.2 MOhm-cm) was prepared using a Milli-Q water purification system (MA, USA). Reference standards of protocatechuic aldehyde (＞99%), protocatechuic acid (＞99%), danshensu (＞98%), caffeic acid (＞98%), rosmarinic acid (＞98%), cryptotanshinone (＞98%) and tanshinone IIA (＞98%) were purchased from Shanghai Winherb Medical Technology Co., Ltd (Shanghai, China). Salvianolic acid A (≥98%), salvianolic acid B (≥98%), salvianolic acid C (≥98%), lithospermic acid (≥98%), 9''-Methyl lithospermate B (≥96%), tanshinone I (≥99%) and dihydrotanshinone I (≥98%) were bought from Shanghai Tauto Biotech Co., Ltd (Shanghai, China). Prior to use, all the 14 reference standards were determined by HPLC for purity and confirmed with UV spectra, accurate mass and MSn data. Five batches of Danshen were collected from different provinces of China and authenticated by Prof. Shimin Guo at Yunnan Institute of Traditional Chinese Medicine and Medical Materials, Kunming, China.
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An aliquot of 2.00 g sample powder (through 50 mesh screen) was accurately weighed and transferred into a conical flask, followed with addition of 50 mL of 70% methanol (v/v). The flask along with the contents was weighed, and then the sample was extracted using an ultrasonic cleaner (Branson Ultrasonic Corp., Danbury, CT, USA) for 30 min. After ultrasonication, the flask was adjusted to its original weight with extraction solvent. Subsequently , the extract was centrifuged at 4700×g for 15 min and the supernatant was filtered through a 0.22 µm membrane (PVDF Millex-GV, 13 mm, Millipore) for the qualitative and quantitative analysis. Individual stock solutions of 14 reference standards were prepared in methanol at concentrations ranging from 1.10 to 4.44 mg mL-1. The mixed standard solution was obtained by mixing appropriate amounts of the individual stock solutions and then diluting with 70% methanol (v/v) to a desired concentration. All the solutions were stored at 4 oC before analysis. 2.3. LC×LC-DAD-ESI/HRMS/MSn analysis The LC×LC analysis was performed on Dionex UltiMate 3000×2 Dual RSLC system (Dionex, Thermo Fisher Scientific Inc., USA), equipped with a SRD-3600 degasser, a DGP3600RS dual-ternary pump (left and right pump), a WPS-3000TRS autosampler, a TCC3000RS column thermostat and a DAD-3000RS detector. An electronically controlled twoposition ten-port switching valve with two 200-µL sample loops (Rheodyne, CA, USA) was employed to connect the first and second dimension LC. A LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray
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ionization (ESI) source was connected to the LC×LC via an adjustable flow splitter (Analytical Scientific Instruments, CA, USA) at a ratio of 1:4. The LC×LC-LTQ-Orbitrap system was controlled by Xcalibur 2.1 software (Thermo Fisher Scientific, Bremen, Germany). A Hypersil gold CN column (150 mm × 1 mm, 3 µm) was used in the first dimension (1D) separation with a low flow rate of 0.08 mL min-1 and an Accucore C18 column (50 mm × 4.6 mm, 2.6 µm) was employed in the second dimension (2D) separation with a high flow rate of 2.00 mL min-1. The mobile phase, composed of 0.1% formic acid (v/v) in water (A) and acetonitrile (B), was used in both 1D and 2D separations, but different gradient programs were applied as shown in Fig. 2. Other parameters were set as follows: column temperature, 40 oC; detection wavelength, 281 nm; DAD sampling rate, 50 Hz; injection volume, 4 µL and modulation cycle, 2 min. For the ESI-HRMS-MSn analysis, samples introduced from the splitter into LTQ-Orbitrap
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(∼0.4 mL min-1) were analyzed in positive and negative ion modes, respectively. For positive
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ESI analysis, the parameters were set as follows: ion spray voltage, 5 kV; capillary temperature, 350 °C; capillary voltage, 35 V and tube lens voltage, 100 V. For negative ESI analysis, the parameters were as follows: ion spray voltage, -4 kV; capillary temperature, 350 °C; capillary voltage, -40 V and tube lens voltage, -120 V. Nitrogen (N2) was used as sheath gas and auxiliary gas with a flow rate of 60 and 20 arbitrary units, respectively. High purity helium (He) served as the collision gas in the linear ion trap. Mass spectra were recorded in six mass ranges, including m/z 100-1500, 100-350, 300-550, 500-750, 700-950, and 900-1500. The scan cycle consisted of a full scan event and six datadependent acquisition (DDA) events. In the full scan event, resolution of the Orbitrap mass analyzer was set at 30,000 (FWHM as defined at m/z 400) and, in the DDA events, two intense ions detected in the full scan event were selected to acquire MSn data (n=2-4) in the linear ion trap. The collision-induced dissociation (CID) activation was adjusted to normalized collision energy of 35% with an isolation width of m/z 2.0 for all DDA events. A series of strong background interference ions were added to the reject mass list and, with the dynamic exclusion enabled, more relative low intense ions could be targeted for MSn detection. The external mass calibration of the Orbitrap mass analyzer was performed every three days in accordance with the manufacturer’s guidelines. 2.4. Quantitative validation of LC×LC-DAD method Quantitative analysis was achieved by the DAD contour plots (281 nm) generated from LC Image LC×LC-HRMS Edition software (version 2.5, GC Image, LLC., Lincoln, NE, USA). On contour plots, the target analyte was located by the retention times (RT) of peak apex in 1 D and 2D columns and peak volume determined by LC Image was used for quantification. Thus, to evaluate the reliability of the quantitative LC×LC-DAD method, it was validated in terms of linearity, sensitivity and precision. The mixed standard solution was diluted with 70% methanol (v/v) to seven calibration levels of working solutions for LC×LC-DAD analysis in duplicates. Calibration curves were
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constructed by plotting the peak volume versus the concentration of each analyte. The sensitivity was assessed based on the limits of detection (LOD) and quantification (LOQ), which were defined at signal-to-noise ratio (S/N) of 3 and 10, respectively. The method precision was evaluated by elative standard deviation (RSD) of 2D RT and peak volume with seven replicate injections of mixed standard solution. 2.5. Data analysis The LC×LC contour plots of DAD and MS data were constructed by LC Image with 2D phase shifts of 18.0 s and 21.0 s, respectively. The LC Image was also employed for peak recognition and peak locating on the contour plot. Peak capacity and orthogonality were calculated according to the reported algorithms from References [23-25]. For the qualitative identification, Xcalibur 2.1 software was used for basic MS data reviewing and processing. Mass Frontier 7.0 software (Thermo Fisher Scientific, San Jose, CA, USA) was further employed to build the MSn spectral trees library of 14 reference standards, perform the component detection to automatically extract individual MS spectra or MSn spectral trees from the complicated DDA MSn data, and quickly screen unknown phenolic and diterpenoids using the Fragment Ion Search (FISh) feature [26]. Depending on the elemental compositions of known compounds in Danshen, the types and amounts of expected atoms for potential components were set as follows: carbons ≤60, hydrogens ≤60, oxygens ≤40 and no nitrogens. The precursor mass tolerance was defined as 5 ppm. 3. Results and discussion 3.1. Optimization of LC×LC system As described in Section 1, phenolic acids and diterpenoids are the major two groups of bioactive components in Danshen. Most of these constituents can be well separated by conventional 1D-LC techniques. However, there is still a noted cross-area of these major compounds (Fig. 3A), which is very difficult for their full group classification and structural identification. Hence, in this study, a LC×LC system was considered and optimized using a mixed standard solution (10 phenolic acids and 4 diterpenoids). The column selectivity is a key parameter when optimizing a LC×LC separation method. According to the column’s characteristics, six columns were tested for 1D separation, including Hypersil gold C18 (150 mm × 2.1 mm, 1.9 µm), Xbridge amide (150 mm × 4.6 mm, 3.5 µm), Hyperisl gold PFP (150 mm × 2.1 mm, 3 µm), Hyperisl gold C8 (150 mm × 2.1 mm, 3 µm), Syncronis C18 (150 mm × 2.1 mm, 3 µm) and Hypersil gold CN (150 mm × 1 mm, 3 µm). Four columns, which could maintain high flow speed, were studied for 2D separation, namely Accucore Phenyl-Hexyl (50 mm × 4.6 mm, 2.6 µm), Accucore PolarPremium (50 mm × 3 mm, 2.6 µm), Accucore PFP (50 mm × 4.6 mm, 2.6 µm) and Accucore C18 (50 mm × 4.6 mm, 2.6 µm). On these columns, the separation behaviors of the 14 reference standards were explored separately. Retention times of each analyte were calculated into normalized retention times (RTi(norm)) using the equation (1) [25]. RTmax and RTmin represent the retention times of the greatest and the least retained compound, respectively, and the retention times RTi were converted into normalized RTi(norm) values in the range of 0-1.
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(1) 5
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Then, the correlation coefficient (R2) of RTi(norm) value was calculated to evaluate the orthogonality between coupled columns, where 0 and 1 indicated truly orthogonal and nonorthogonal separations, respectively (Table 1). Compared with other combinations, the couplings of Hypersil gold CN to Accucore Phenyl-Hexyl, Accucore PFP and Accucore C18 showed lower R2 values as 0.9519, 0.9356 and 0.9336, respectively. Therefore, the Hypersil gold CN was selected as the optimum 1D separation column. Accucore C18 and Accucore PFP offered similar low R2 values when coupled to the Hypersil gold CN, both of which seemed to be suitable for 2D separation. As a further consideration, the chromatograms of mixed standards and Danshen samples analyzed by the two columns under the same program were shown in Fig. A.1. The Accucore PFP column exhibited high separation performance in terms of better peak resolution and shorter analysis time than Accucore C18 column. However, on the contour plot of Hypersil gold CN and Accucore PFP (Fig. 4A), peak splitting was observed for lithospermic acid (P29), salvianolic acid B (P42) and salvianolic acid C (P56), which should be one peak into two blobs. For example, Fig. 4B showed the 1D view of 2D cycle in the range of 78-80 min and salvianolic acid C (P56), identified by single standard injection, was splitting into two partially overlapped peaks as peak P56a and peak P56b, of which their UV spectra observed in Fig. 4C and Fig. 4D were matched perfectly for the confirmation of salvianolic acid C (P56). Additional optimization test, including the variations of 2D flow rate (1.5, 2.0 and 2.5 mL min-1) and gradient programs, proved to be ineffective to eliminate the peak splitting of these analytes. Therefore, Accucore C18 column was selected as the preferred 2D column in this experiment without any peak splitting. Since phenolic acids were weakly retained on the selected columns whereas diterpenoid constituents were strongly, a normal gradient program was applied for 1D separeation. For 2D separation, a segment gradient program was optimized, which consisted of seven different segments with increased acetonitrile ratios to maintain high resolution. The finally accepted gradient programs are presented in Fig. 2 and all 14 standards could be well separated without excessive bandwidths on the contour plot (Fig. 5A). Under the optimized parameters, phenolic acids and diterpenoids can be separated into two independent groups on the contour plot of Danshen (Fig. 3B), which facilitate the identification of these structural-related constituents. 3.2. Optimization of MS conditions For the two types of compounds in Danshen, positive ESI mode was found to offer better sensitivity for diterpenoids, whereas negative ESI mode was more suitable for phenolic acids. Therefore, the ESI-HRMS-MSn data was acquired in both positive and negative ion modes. For the component identification, comprehensive MS data could make it easier and more reliable. In this experiment, DDA was employed with seven scan events as a cycle. The first event was set for the mass full scan by Orbitrap with resolution of 30,000 to get the accurate mass, which was used to calculate elemental formulas. In the following six events, two most intense ions in the first event were selected to acquire MSn data (n=2-4) to get the fragment information. Due to the complex constituents in Danshen, dynamic exclusion feature was enabled in DDA mode with exclusion time set at 8 s and exclusion list up to 50. Thus, co-elution ions and relative low intensity ions have more opportunities to be selected for MS2 detection.
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In addition, because the major components in Danshen have been identified and reported in many literatures [16-18,22,27], our study paid more attention to the characterization of minor compounds. Except for using dynamic exclusion feature, we have divided the scan range m/z 100-1500 into five narrower and partially overlapped scan ranges, as described in Section 2.3. Under this strategy, more components with low abundance could be detected for MSn analysis as illustrated in Fig. A.2. Besides, because a single compound can generate many fragments with different m/z values in full scan mode, it may result to time-wasting and increased risk of false identification. Therefore, the scan range of m/z 100-1500 was also employed to decrease the false selection of precursor ions during data process. 3.3. Peak capacity and orthogonality of the system To evaluate the performance of the optimized LC×LC system, peak capacity and system orthogonality were calculated as measurements. As a result, a total of 328 blobs in Danshen can be recognized by the LC Image on the DAD contour plot (281 nm). The effective gradient time for 1D and 2D analysis were 115 min and 1 min, respectively. The average 4σ peak widths were 2.497 min and 0.032 min in 1D and 2D chromatograms. So, the theoretical peak capacities for 1D and 2D analysis were 46.06 and 31.25, respectively. The lowest USP plate counts were 451 (calculated by Caffeic acid) and 108335 (calculated by Danshensu) for 1D and 2D analysis, respectively. Furthermore, according to the reported equations [23,24], the theoretical peak capacity and effective peak capacity of the developed system were calculated as 1439 and 830, respectively. The constructed LC×LC system provided remarkably effective peak capacity with 17-fold higher than that of 1D LC analysis, exhibiting its powerful separation capability. By referring the literature [23-25], the orthogonality and practical peak capacity were calculated. Specifically, the separation space was divided into 18×18 rectangular bins close to Pmax (328) and was superimposed with the data points (Fig. A.3). The result showed that about 41% of the separation space was covered by bins containing data points. Thus, the orthogonality was calculated as 53.9% and the practical peak capacity was 575. 3.4. Quantitative validation and sample analysis For conventional 1D-LC analysis of complex herbs, such as Danshen, the accurate quantification of interested analytes often encountered the problems of co-elution and matrix effect. LC×LC system, as a powerful separation technique, was then considered for the quantification. But, since peaks for each analyte were distributed into several modulation cycles in 2D systems, they have to be summed for calculation with the help of extracted ion chromatograms obtained from specific MS detectors [28-30], which seemed to be more complicated than 1D-LC. Therefore, in this study, we employed the contour plots for the quantification analysis. With LC Image [31], target analytes on the DAD contour plot (281nm) can be easily recognized and located, and meanwhile the peak volumes were automatically integrated for quantification instead of peak areas. At the same time, due to the complex matrix of Danshen, quantitative results were further confirmed by the coupled LTQOrbitrap to avoid false-positive identifications. The proposed LC×LC-DAD method was then validated to test its quantitative reliability by the contour plots. The standard curves, correlation coefficient (r), linear range, LOD, LOQ and RSDs (2D RT and peak volume) for each analyte are summarized in Table 2. All 14 seven-point standard curves offered the r values ranging from 0.9976 to 1.0000 for all
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analytes using the plotting of peak volumes and concentrations, highlighting its feasibility and reliability for the quantification. LODs and LOQs were in the range of 0.16-1.18 µg mL-1 and 0.52-3.89 µg mL-1 for investigated compounds, respectively, demonstrating the good sensitivity of the proposed method. The precision for the analyte locating and peak volume calculating was also tested using seven injection of a mixed standard solution. The RSD values were lower than 1.07% for 2D RT, which showed the highly locating accuracy; and peak volumes also gave a low RSD range from 0.34% to 4.11%. In conclusion, all these validation results showed satisfactory linearity, sensitivity and precision of the established LC×LC-DAD method, which was then applied to sample quantitative analysis. Typical 2D contour plots of the 14 mixed standards and a Danshen sample are shown in Fig. 5. As can be seen in Fig. 5A, all 14 standards were well separated as single blobs with the varied color representing different peak intensities. Also, the contour plot of a Danshen sample (no.1) is shown in Fig. 5B and the detected blobs were highlighted with bubbles, which indicated both retention time (by image location) and peak volume (by disk size). The recognition of the investigated analytes was carried out by comparing their retention times with those of corresponding genuine standards and was further confirmed by the coupled LTQ-Orbitrap. According to these procedures, all samples were analyzed and the calculated contents of target compounds are presented in Table 3. Among the 14 compounds, it can be observed that salvianolic acid B (P42) (13.44-36.44 mg g-1) was the most dominant component in the 10 phenolic acids, as well as tanshinone IIA (D41) (0.26-0.86 mg g-1) in the 4 diterpenoids, which were both used as the chemical markers for the quality control of Danshen in Chinese Pharmacopoeia (edition 2010) [32] . 3.5. Characterization of chemical constituents in Danshen Chemical constituents in Danshen were analyzed by the constructed LC×LC system and, thanks to its remarkable separation power, the interested phenolic acids and diterpenoids were detected as blobs into two individual groups on the 2D contour plot (Fig. 3B), since each of them shared common structural motifs. Meanwhile, 14 available standards were analyzed using the LTQ Orbitrap by direct infusion to explore their fragmental behavior and to build an MSn spectral library for structural characterization of those unknown constituents. According to the number of phenyls in the structure, phenolic acids in Danshen could be classified into monomers (e.g. danshensu and caffeic acid) and polymers, including dimers, trimers and tetramers, which consist of several monomers [27]. Therefore, phenolic acids always exhibited a unique fragmentation behavior. Based on our fragmental study of the reference standards and literature reports [16,27,33], in the MSn spectra of phenolic acids, the loss of small molecules such as CO2, CO and H2O were produced by the monomers, due to the existence of either carboxyl, carbonyl or hydroxyl groups. Moreover, the fragment ions [M-H-198]- and [M-H-180]- were derived from the neutral loss of danshensu and caffeic acid in polymers. For the diterpenoid constituents, their protonated molecules [M+H]+ or [M+Na]+ could be observed in full MS spectra under positive ion mode. The fragments of [M+H-15]+, [M+H-18]+, [M+H-28]+ and [M+H-32]+ can be found in the MS and MSn spectra, indicating the loss of CH3, H2O, CO and CH3OH, respectively [17,33]. Therefore, based on the fragmental pattern and comparing with reference standards and literature data [17,22,27,33-35], a total of 95 compounds including 52 phenolic acids and 43 diterpenoids were identified or tentatively identified and detailed information were listed in
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Table A.1 and A.2. In addition, although there have been lots of reports for the discovery and identification of phenolic acids in Danshen [17,27,33-35], 7 new compounds were detected for the first time and tentatively characterized in the current study, which were summarized in Table 4. Also, their MS2 spectra together with proposed chemical structures were interpreted in Fig. 6. Peak P17 and P27 showed similar [M−H]− ions at m/z 735.1555 and 735.1556, respectively, and gave the same element composition of C36H31O17 with errors lower than 0.90 ppm. Their molecular weights were 18 Da higher than that of salvianolic acid B, while their MS2 spectra were similar and yielded a series of same fragments such as m/z 717, 537, 519, 493, 339, 321 and 295. These evidences allowed to deduce that peak P17 and P27 were the addition product of H2O toward the double bond of salvianolic acid B. Therefore peak P17 and P27 were tentatively characterized as 8-hydroxy-salvianolic acid B and its isomer. Peak P28 gave the [M−H]− ion at m/z 699.1555 (C33H32O17). The MS2, MS3 and MS4 spectra of peak P28 produced prominent ions at m/z 519, 321 and 279, resulting from the loss of 180 Da, danshensu and 42 Da, which were similar with those of salvianolic acid B. In addition, the high resolution MS2 spectra of peak P28 showed the [M-H-180]− ion at 519.0917 and the calculated element composition is C27H19O11 (Fig. 6). Therefore, the neutral loss of 180 Da in MS2 spectra was C6H12O6, which may be attributed to glucose. Thus, peak P28 was tentatively identified as lithospermate-9'-O-glucoside. Peak P39 displayed a [M−H]− ion at m/z 523.1236 (C27H23O11). Its MS2 spectra yielded ions at m/z 505, 491, 325 and 293 (Fig. 6), representing the ions of [M-H-H2O]-, [M-HCH3OH]-, [M-H-danshensu]- and [M-H-danshensu-CH3OH]-, which suggested the existence of methoxyl. Further compared MS3 and MS4 spectra of peak P39 with that of salvianolic acid C, it could be further deduced that a H2O was added toward the double bond of salvianolic acid C besides the group of methoxyl. Thus, peak P39 was proposed to be 8hydroxy-4-methoxyl-salvianolic acid C. Peak P45 showed a [M−H]− ion at m/z 701.1503 and gave the element composition of C36H29O15, which was only one oxygen atom less than that of salvianolic acid B ([M-H]-, C36H29O16). In the MS2 spectra, the ions at m/z 519 (100%, relative intensity) and 503 (24%) were observed, indicating the presence of deoxy-danshensu and danshensu, and also meaning that deoxy-danshensu was easier to be lost. Moreover the fragment ions at 519 and 503 could both produced prominent ion at m/z 321 in the MS3 spectra, resulting from the loss of danshensu and deoxy-danshensu. According to literature [27], fragmentation behaviors of salvianolic acid B showed that the cleavage of a bond was easier than b bond (Fig. 1). Therefore, compared with salvianolic acid B, Peak P45 was tentatively identified as 3'''deoxy-salvianolic acid B. Peak P48 gave a [M−H]− ion at m/z 747.1544 (C37H31O17). The MS2 spectra of peak P48 generated ions at m/z 715, 549 and 517, representing the ions of [M-H-CH3OH]-, [M-Hdanshensu]- and [M-H-danshensu-CH3OH]- , respectively, which suggested the presence of methoxyl (Fig. 6). The fragment ions in MS3 and MS4 were similar with that of salvianolic acid B. Thus, peak P48 was tentatively identified as 5''-hydroxyl-9''-methyl-salvianolic acid B. Peak P50 provided a [M−H]− ion at m/z 685.1550 and gave the element composition of C36H29O14, which were two oxygen atoms less than that of salvianolic acid B ([M-H]-,
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347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
9
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391 392 393 394 395
C36H29O16). The MS2 and MS3 spectra of peak P50 yielded prominent ions at m/z 503 and 321, resulting from the loss of deoxy-danshensu and the second deoxy-danshensu. Compared with peak 45 and salvianolic acid B, peak P50 could be tentatively characterized as 3''-deoxy3'''-deoxy-salvianolic acid B.
396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412
In this work, a LC×LC-DAD-ESI/HRMS/MSn system was constructed and evaluated for the qualitative and quantitative characterization of phenolic acids and diterpenoid components in Danshen. Columns with different selectivity were employed to improve orthogonality of the first and second dimensions. By using the optimal segment gradient program, phenolic acids and diterpenoids were separated into two independent groups, which facilitated the identification of unknown compounds. Besides, the combination of DDA and partially overlapped scan ranges make it possible to detect and fragment more components with low abundance in Danshen. Under this strategy, a total of 328 peaks were separated and detected successfully. Among these peaks, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. The proposed LC×LC system showed good orthogonality (53.9%) and high effective peak capacity (830). Furthermore, the contents of 14 bioactive compounds in different Danshen samples were simultaneously quantified directly on the DAD contour plots. Above all, the established LC×LC-DAD-ESI/HRMS/MSn system has proved to be powerful to discover minor compounds, as well as be reliable for the accurate quantification of interested analytes in complex herbs.
413 414 415 416 417 418 419 420 421 422
This research was supported by the National Natural Science Foundation of China (31160065), the Macao Science and Technology Development Fund (No. 052/2012/A2) and the Research Committee of the University of Macau (No. MYRG109-ICMS13-LP and MYRG2014-00089-ICMS-QRCM). We are grateful to Prof. Qingping Tao (GC Image, LLC., Lincoln, NE, USA) and Yanhai Zhang (Thermo Fisher Scientific) for their professional technical support.
423 424 425 426 427 428 429 430 431 432 433 434
[1] J.L. Cao, J.C. Wei, M.W. Chen, H.X. Su, J.B. Wan, Y.T. Wang, P. Li, Application of twodimensional chromatography in the analysis of Chinese herbal medicines, J. Chromatogr. A 1371 (2014) 1-14. [2] L. Hu, X. Chen, L. Kong, X. Su, M. Ye, H. Zou, Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights, J. Chromatogr. A 1092 (2005) 191-198. [3] J. Zhang, D. Tao, J. Duan, Z. Liang, W. Zhang, L. Zhang, Y. Huo, Y. Zhang, Separation and identification of compounds in Adinandra nitida by comprehensive two-dimensional liquid chromatography coupled to atmospheric pressure chemical ionization source ion trap tandem mass spectrometry, Anal. Bioanal. Chem. 386 (2006) 586-593. [4] D.X. Li, O.J. Schmitz, Use of shift gradient in the second dimension to improve the
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Acknowledgement
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an
us
cr
ip t
Fig. 1 The chemical structures of 14 reference standards. Fig. 2 Gradient elution programs for the first and second dimension separations. Fig. 3 Comparison of 1D-LC chromatogram (A) with 2D contour plot (B) for the separation of Danshen (281 nm). The conventional 1D-LC conditions for (A) are provided in Supporting Information. Fig. 4 Peak splitting of the primary LC×LC system for the analysis of 14 reference standards. (A) 2D contour plot with DAD detection at 281nm; (B) 1D-LC chromatogram view for the time range of 78-80 min; (C) The UV spectra of peak P56a; (D) The UV spectra of peak P56b. Hypersil gold CN (150 mm × 1 mm, 3 µm) and Accucore PFP (50 mm × 4.6 mm, 2.6 µm) were used for 1D and 2D separation, respectively. Fig. 5 2D contour plots for separation of 14 reference standards (A) and no. 1 Danshen sample (B) at 281 nm.
M
Fig. 6 Proposed chemical structures and LTQ-Orbitrap ddMS2 spectra for the interested compounds.
d
547 548
[35] G.F. Zeng, H.B. Xiao, J.X. Liu, X.M. Liang, Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun. Mass Sp. 20 (2006) 499-506.
te
523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546
1
1
549 550 551
D column Hypersil gold C18
Xbridge amide
Hyperisl gold PFP
Hyperisl gold C8
Syncronis C18
Hypersil gold CN
0.9928
0.9775
0.9981
0.9969
0.9948
0.9519
0.9612
0.9576
0.9830
0.9722
0.9636
0.9806
0.9947 0.9991
0.9836 0.9853
0.9951 0.9956
0.9964 0.9989
0.9980 0.9997
0.9356 0.9336
Ac ce p
2
D column
R2
552 553
2
Table 1 Orthogonal test between the D and D columns
Accucore PhenylHexyl Accucore PolarPremium Accucore PFP Accucore C18
Table 2 Quantitative performance for 14 analytes by using the constructed LC×LC-DAD
system. 1
Peak no.
Analyte
P3
Danshensu
P4
Protocatechuic acid
D RT (min)
2
Linearity
2
8.40
4
12.78
Standard curve y = 15x – 16 y = 49x + 321
D RT (sec)
r 0.9998 0.9990
Range (µg mL-1) 6.30126.00 2.56102.55
LOD (µg mL-1)
LOQ (µg mL1 )
Precision (RSD, %) 2 D Peak RT volume
1.18
3.89
0.85
1.35
0.58
1.93
0.83
1.06
13
Page 13 of 21
P7
Caffeic acid
4
71.84
P26
Rosmarinic acid
30
84.48
P29
Lithospermic acid
40
77.90
P42
Salvianolic acid B
52
79.60
P44
Salvianolic acid A
58
82.20
P52
9''-Methyl lithospermate B
66
79.04
P56
Salvianolic acid C
76
81.10
D19
Dihydrotanshinone I
76
91.00
D32
Cryptotanshinone
86
93.38
D33
Tanshinone I
92
97.14
D41
Tanshinone IIA
104
107.28
y = 147x + 116 y = 112x - 194 y = 61x 239 y = 39x + 222 y = 35x 273 y = 78x 453 y = 43x +6 y = 81x + 48 y = 119x - 29 y = 77x 17 y = 63x 44 y = 101x - 303
-1
Retention time 1st dim.
2nd dim. (sec) 79
a
P3
1
Hebei
2.09
2
Hebei
-
3
Hebei
1.59
P4 -
a
d
Origin
P5 +
0.9999 0.9985 1.0000 0.9999 0.9999 0.9999 0.9999 0.9997
1.07
1.20
0.29
0.95
0.35
4.11
0.22
0.73
0.06
0.49
0.55
1.81
0.10
1.31
0.59
1.95
0.06
0.90
0.92
0.05
0.53
1.02
0.07
1.05
0.28 0.31 0.16
0.52
0.06
1.09
0.22
0.74
0.19
0.90
0.36
1.17
0.16
0.51
0.42
1.40
0.19
0.77
0.24
0.79
0.13
0.34
b
P7
P26
P29
P42
P44
P52
P56
D19
D32
D33
0.09
1.15
1.16
23.44
0.22
+
0.02
0.13
0.36
0.36
0.15
-
+
0.77
1.19
16.23
0.04
0.11
0.04
0.11
0.27
0.38
-
-
0.05
1.01
1.42
20.04
0.11
0.09
0.02
0.05
0.10
0.19
Ac ce p
564 565
0.9976
1.28
detected by the LC×LC-DAD system. No.
559 560 561 562 563
0.9995
0.39
Table 3 Contents (mg g ) of 14 standards in different Danshen (Salvia miltiorrhiza) samples
te
556 557 558
0.9993
1.5562.00 1.6766.60 7.16286.50 9.18367.00 20.00800.00 6.01240.50 2.1987.50 2.59103.50 1.3855.00 2.3092.00 2.0180.50 5.25210.50
M
554 555
0.9996
ip t
39.78
cr
4
us
Protocatechuic aldehyde
an
P5
4
Shandong
2.08
-
+
-
0.55
1.25
13.44
0.05
0.09
0.03
0.05
0.11
0.23
5
Sichuan
1.60
-
-
0.05
1.36
1.62
36.44
0.21
0.04
0.02
0.01
0.10
0.18
not detected; b under the limit of quantification.
Table 4 Charaterization of unknown compounds by UHPLC-LTQ-Orbitrap from Danshen
(Salvia miltiorrhiza)a Measured [M-H](m/z)
Theoretical [M-H]- (m/z)
Error (ppm)
Molecular formula
LC/ESI-MSn m/z (% base peak)
735.1555
735.1561
-0.90
C36H32O17
MS2[735]: 295(8),297(6),321(5),339(3), 357(4),493(4),519(20),537(100),555(3),717(2) MS3[537]:179(2),185(2),253(8),269(22),277(2),279(2),2 95(75),297(100),321(64),339(24),357(70),449(11), 475(9),493(13),519(52) MS4[297]:161(5),175(7),225(16),241(3),253(100),269(4 7),279(6)
Identification
8-Hydroxy-salvianolic acid B
14
Page 14 of 21
82
699.1555
699.1561
-0.86
C33H32O17
85
523.1236
523.1240
-0.82
C27H24O11
84
701.1503
701.1506
-0.44
C36H30O15
75
747.1544
747.1561
-2.36
C37H32O17
80
685.1550
685.1557
-1.02
C36H30O14
a
Lithospermate-9'-O-
8-Hydroxy-4-methoxyl acid C
3'''-Deoxy-salvianolic acid B
5''-Hydroxyl- 9'' -methyl acid B
3''-Deoxy-3'''-deoxyB
d
Identified from Salvia miltiorrhiza for the first time.
te
569 570
a
8- Hydroxy-salvianolic isomer
Ac ce p
566 567 568
MS2[735]: 295(8),297(5),321(10),339(2), 357(3),493(6),519(56),537(100),555(3),717(7) MS3[537]: 253(5),269(29),277(1),279(2), 295(78),297(100),321(76),339(24),357(54),449(10), 475(7),493(24),519(63) MS4[297]:161(5),175(7),225(16),241(2),253(100),269(4 3),279(8) MS2[699]:321(11),339(3),493(9),519(100),537(7),655(2 0) MS3[519]: 279(3),321(100),339(35) MS4[321]:249(7),265(2),277(47),279(100),293(16) MS2[523]:179(6),197(2),293(60),311(19),325(100),343( 35),491(68),505(2) MS3[325]:175(12),251(15),265(10),283(25),293(100),30 7(2),311(29) MS4[293]:221(10),237(19),247(16),249(33),251(51),265 (100),275(20) MS2[701]:321(18),339(4),503(24),519(100) MS3[519]:277(3),279(3),295(4),321(100),339(25),475(2) MS3[503]:279(5),305(5),321(100),339(20),441(4) MS4[503→321]:249(8),275(3),277(40),279(100), 293(20),303(11) MS2[747]:295(1),321(8),339(4),517(3),519(2),549(100), 567(2), 715(1) MS3[549]:277(3),279(12),295(7),321(100),339(39),517( 40) MS4[321]:249(3),251(1),265(2),275(1),277(33),279(100) ,293(10),303(2) MS2[685]:321(11),339(2),503(100) MS3[503]:279(6),295(2),321(100),339(20),441(3) MS4[321]:249(8),265(3),275(3),277(45),279(100),293(1 7),303(8)
ip t
C36H32O17
cr
-0.65
us
735.1561
an
735.1556
M
81
15
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Li et al., 2015 Page 16 of 21
Fig. 1
i cr us M an ed ce pt Ac
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Fig. 2
i cr us M an ed ce pt Ac
Li et al., 2015 Page 18 of 21
Fig. 3
i cr us M an ed ce pt Ac
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Fig. 4
i cr us M an ed ce pt Ac
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Fig. 5
i cr us M an ed ce pt Ac
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Fig. 6
S0021-9673(15)01740-9 http://dx.doi.org/doi:10.1016/j.chroma.2015.11.078 CHROMA 357092
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
16-9-2015 20-11-2015 25-11-2015
Please cite this article as: J.-L. Cao, J.-C. Wei, Y.-J. Hu, C.-W. He, M.-W. Chen, J.B. Wan, P. Li, Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.11.078 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2
328 peaks were detected and 7 of them were firstly discovered from Danshen. Enhanced data-dependent acquisition offers more MSn data of low abundant compounds.
us
cr
Successful quantification was achieved by using the DAD contour plot.
ip t
Phenolic acids and diterpenoids were separated into two independent groups on the 2D contour plot.
Qualitative and quantitative characterization of phenolic and diterpenoid constituents in Danshen (Salvia miltiorrhiza) by comprehensive two-dimensional liquid chromatography coupled with hybrid linear ion trap Orbitrap mass spectrometry
an
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Ji-Liang Cao, Jin-Chao Wei, Yuan-Jia Hu, Cheng-Wei He, Mei-Wan Chen, Jian-Bo Wan, Peng Li *
23 24 25 26 27 28 29 30
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China
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* Corresponding author at: Institute of Chinese Medical Sciences (University of Macau), Macau, China. Dr. Peng Li Tel.: +853 8397 4874; fax: +853 2884 1358. E-mail address: [email protected]
Abstract
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Danshen is one of the most frequently used traditional Chinese herbs owing to its remarkable and reliable therapeutic effects. Phenolic acids and diterpenoids have proved to be the bioactive substance groups. In order to fully profile its chemical compositions and explore new potential bioactive compounds, a comprehensive two-dimensional liquid chromatography system coupled to DAD detector and hybrid linear ion trap (LTQ) Orbitrap mass spectrometry (LC×LC-DAD-ESI/HRMS/MSn) was set up in this study based on the
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column combination of Hypersil gold CN (150 mm × 1 mm, 3 µm) and Accucore C18 (50 mm × 4.6 mm, 2.6 µm). Using the optimal segment gradient program, phenolic acids and diterpenoids were separated into two independent groups and a total of 328 peaks were successfully detected on the contour plot of Danshen. By means of the accurate mass and reliable MSn data, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. Moreover, the LC×LC-DAD system was validated for the quantitative analysis of 14 bioactive analytes using the contour plot, exhibiting satisfactory linearity (r≥0.9976) and high precision for both peak locating (≤1.07%) and peak volume calculating (0.34%-4.11%). The established method could afford powerful separation capability, reliable identification data and accurate quantitative results, which is very suitable for analysis of complex herbal samples. Keywords
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Comprehensive two-dimensional liquid chromatography; LTQ Orbitrap mass spectrometry; Phenolic acids; Diterpenoids; Danshen. 1. Introduction
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Nowadays, with the increasing requirements for more efficient separation of complex samples, two-dimensional liquid chromatography (2D-LC) has been attracting more attentions owing to its large peak capacity and high resolving power. In 2D-LC systems, columns with different separation mechanisms, including reversed-phase (RP), normal-phase (NP), ion exchange (IEX), size exclusion chromatography (SEC) or affinity chromatography (AC) have often been combined to achieve higher resolution and improved selectivity [1-5]. Especially when coupled with mass spectrometer (MS), 2D-LC can exhibit unparalleled capabilities for the detection and identification of minor or even trace-level components. Danshen, the dried root and rhizome of Salvia miltiorrhiza, is one of the most frequently used traditional Chinese herbs in China and, in recent years, also famous as a health product in western world thanks to its remarkable and reliable biological activities, such as therapeutic effects on cardiovascular and cerebrovascular diseases, anti-dysmenorrhea, antitumor, anti-parkinsonian, as well as neuroprotective effect [6-9]. Depending on the modern pharmacological studies and chemical investigations on Danshen, two major groups of bioactive components, phenolic acids (e.g., salvianolic acid B and rosmarinic acid) and diterpenoids (e.g., dihydrotanshinone I and tanshinone IIA) were discovered to be responsible for its pharmacological or therapeutic effects [10-14]. Therefore, chemical analysis of Danshen, mainly focused on phenolic acids and diterpenoids, have been widely reported to evaluate its quality, discover potential bioactive compounds or profile chemical compositions by using conventional chromatographic and even hyphenated techniques, such as LC, LC-MS and capillary electrophoresis [15-22]. But, restricted to limited peak capacity and resolving power of one-dimensional (1D) techniques, and also due to similar chemical properties of the preferred compounds, the chemical separation of Danshen is always insufficient, which causes the inaccurate quantification and false identification of detected components, especially the minor ones. In this study, a comprehensive 2D-LC system coupled with DAD detector and hybrid linear ion trap (LTQ) Orbitrap mass spectrometry (LC×LC-DAD-ESI/HRMS/MSn) was
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developed to separate and identify the phenolic and diterpenoid constituents in Danshen. A total of 328 peaks were successfully separated and detected. Among these peaks, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. Meanwhile, 14 bioactive compounds, including 10 phenolic acids and 4 diterpenoids (Fig. 1) were determined directly on the DAD contour plots of Danshen samples with satisfactory linearity and good precision. 2. Materials and methods 2.1. Chemicals and materials
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HPLC grade acetonitrile, formic acid and methanol were purchased from Merck (Darmstadt, Germany). Deionized water (18.2 MOhm-cm) was prepared using a Milli-Q water purification system (MA, USA). Reference standards of protocatechuic aldehyde (＞99%), protocatechuic acid (＞99%), danshensu (＞98%), caffeic acid (＞98%), rosmarinic acid (＞98%), cryptotanshinone (＞98%) and tanshinone IIA (＞98%) were purchased from Shanghai Winherb Medical Technology Co., Ltd (Shanghai, China). Salvianolic acid A (≥98%), salvianolic acid B (≥98%), salvianolic acid C (≥98%), lithospermic acid (≥98%), 9''-Methyl lithospermate B (≥96%), tanshinone I (≥99%) and dihydrotanshinone I (≥98%) were bought from Shanghai Tauto Biotech Co., Ltd (Shanghai, China). Prior to use, all the 14 reference standards were determined by HPLC for purity and confirmed with UV spectra, accurate mass and MSn data. Five batches of Danshen were collected from different provinces of China and authenticated by Prof. Shimin Guo at Yunnan Institute of Traditional Chinese Medicine and Medical Materials, Kunming, China.
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An aliquot of 2.00 g sample powder (through 50 mesh screen) was accurately weighed and transferred into a conical flask, followed with addition of 50 mL of 70% methanol (v/v). The flask along with the contents was weighed, and then the sample was extracted using an ultrasonic cleaner (Branson Ultrasonic Corp., Danbury, CT, USA) for 30 min. After ultrasonication, the flask was adjusted to its original weight with extraction solvent. Subsequently , the extract was centrifuged at 4700×g for 15 min and the supernatant was filtered through a 0.22 µm membrane (PVDF Millex-GV, 13 mm, Millipore) for the qualitative and quantitative analysis. Individual stock solutions of 14 reference standards were prepared in methanol at concentrations ranging from 1.10 to 4.44 mg mL-1. The mixed standard solution was obtained by mixing appropriate amounts of the individual stock solutions and then diluting with 70% methanol (v/v) to a desired concentration. All the solutions were stored at 4 oC before analysis. 2.3. LC×LC-DAD-ESI/HRMS/MSn analysis The LC×LC analysis was performed on Dionex UltiMate 3000×2 Dual RSLC system (Dionex, Thermo Fisher Scientific Inc., USA), equipped with a SRD-3600 degasser, a DGP3600RS dual-ternary pump (left and right pump), a WPS-3000TRS autosampler, a TCC3000RS column thermostat and a DAD-3000RS detector. An electronically controlled twoposition ten-port switching valve with two 200-µL sample loops (Rheodyne, CA, USA) was employed to connect the first and second dimension LC. A LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray
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2.2. Sample preparation and standard solutions
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ionization (ESI) source was connected to the LC×LC via an adjustable flow splitter (Analytical Scientific Instruments, CA, USA) at a ratio of 1:4. The LC×LC-LTQ-Orbitrap system was controlled by Xcalibur 2.1 software (Thermo Fisher Scientific, Bremen, Germany). A Hypersil gold CN column (150 mm × 1 mm, 3 µm) was used in the first dimension (1D) separation with a low flow rate of 0.08 mL min-1 and an Accucore C18 column (50 mm × 4.6 mm, 2.6 µm) was employed in the second dimension (2D) separation with a high flow rate of 2.00 mL min-1. The mobile phase, composed of 0.1% formic acid (v/v) in water (A) and acetonitrile (B), was used in both 1D and 2D separations, but different gradient programs were applied as shown in Fig. 2. Other parameters were set as follows: column temperature, 40 oC; detection wavelength, 281 nm; DAD sampling rate, 50 Hz; injection volume, 4 µL and modulation cycle, 2 min. For the ESI-HRMS-MSn analysis, samples introduced from the splitter into LTQ-Orbitrap
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(∼0.4 mL min-1) were analyzed in positive and negative ion modes, respectively. For positive
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ESI analysis, the parameters were set as follows: ion spray voltage, 5 kV; capillary temperature, 350 °C; capillary voltage, 35 V and tube lens voltage, 100 V. For negative ESI analysis, the parameters were as follows: ion spray voltage, -4 kV; capillary temperature, 350 °C; capillary voltage, -40 V and tube lens voltage, -120 V. Nitrogen (N2) was used as sheath gas and auxiliary gas with a flow rate of 60 and 20 arbitrary units, respectively. High purity helium (He) served as the collision gas in the linear ion trap. Mass spectra were recorded in six mass ranges, including m/z 100-1500, 100-350, 300-550, 500-750, 700-950, and 900-1500. The scan cycle consisted of a full scan event and six datadependent acquisition (DDA) events. In the full scan event, resolution of the Orbitrap mass analyzer was set at 30,000 (FWHM as defined at m/z 400) and, in the DDA events, two intense ions detected in the full scan event were selected to acquire MSn data (n=2-4) in the linear ion trap. The collision-induced dissociation (CID) activation was adjusted to normalized collision energy of 35% with an isolation width of m/z 2.0 for all DDA events. A series of strong background interference ions were added to the reject mass list and, with the dynamic exclusion enabled, more relative low intense ions could be targeted for MSn detection. The external mass calibration of the Orbitrap mass analyzer was performed every three days in accordance with the manufacturer’s guidelines. 2.4. Quantitative validation of LC×LC-DAD method Quantitative analysis was achieved by the DAD contour plots (281 nm) generated from LC Image LC×LC-HRMS Edition software (version 2.5, GC Image, LLC., Lincoln, NE, USA). On contour plots, the target analyte was located by the retention times (RT) of peak apex in 1 D and 2D columns and peak volume determined by LC Image was used for quantification. Thus, to evaluate the reliability of the quantitative LC×LC-DAD method, it was validated in terms of linearity, sensitivity and precision. The mixed standard solution was diluted with 70% methanol (v/v) to seven calibration levels of working solutions for LC×LC-DAD analysis in duplicates. Calibration curves were
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constructed by plotting the peak volume versus the concentration of each analyte. The sensitivity was assessed based on the limits of detection (LOD) and quantification (LOQ), which were defined at signal-to-noise ratio (S/N) of 3 and 10, respectively. The method precision was evaluated by elative standard deviation (RSD) of 2D RT and peak volume with seven replicate injections of mixed standard solution. 2.5. Data analysis The LC×LC contour plots of DAD and MS data were constructed by LC Image with 2D phase shifts of 18.0 s and 21.0 s, respectively. The LC Image was also employed for peak recognition and peak locating on the contour plot. Peak capacity and orthogonality were calculated according to the reported algorithms from References [23-25]. For the qualitative identification, Xcalibur 2.1 software was used for basic MS data reviewing and processing. Mass Frontier 7.0 software (Thermo Fisher Scientific, San Jose, CA, USA) was further employed to build the MSn spectral trees library of 14 reference standards, perform the component detection to automatically extract individual MS spectra or MSn spectral trees from the complicated DDA MSn data, and quickly screen unknown phenolic and diterpenoids using the Fragment Ion Search (FISh) feature [26]. Depending on the elemental compositions of known compounds in Danshen, the types and amounts of expected atoms for potential components were set as follows: carbons ≤60, hydrogens ≤60, oxygens ≤40 and no nitrogens. The precursor mass tolerance was defined as 5 ppm. 3. Results and discussion 3.1. Optimization of LC×LC system As described in Section 1, phenolic acids and diterpenoids are the major two groups of bioactive components in Danshen. Most of these constituents can be well separated by conventional 1D-LC techniques. However, there is still a noted cross-area of these major compounds (Fig. 3A), which is very difficult for their full group classification and structural identification. Hence, in this study, a LC×LC system was considered and optimized using a mixed standard solution (10 phenolic acids and 4 diterpenoids). The column selectivity is a key parameter when optimizing a LC×LC separation method. According to the column’s characteristics, six columns were tested for 1D separation, including Hypersil gold C18 (150 mm × 2.1 mm, 1.9 µm), Xbridge amide (150 mm × 4.6 mm, 3.5 µm), Hyperisl gold PFP (150 mm × 2.1 mm, 3 µm), Hyperisl gold C8 (150 mm × 2.1 mm, 3 µm), Syncronis C18 (150 mm × 2.1 mm, 3 µm) and Hypersil gold CN (150 mm × 1 mm, 3 µm). Four columns, which could maintain high flow speed, were studied for 2D separation, namely Accucore Phenyl-Hexyl (50 mm × 4.6 mm, 2.6 µm), Accucore PolarPremium (50 mm × 3 mm, 2.6 µm), Accucore PFP (50 mm × 4.6 mm, 2.6 µm) and Accucore C18 (50 mm × 4.6 mm, 2.6 µm). On these columns, the separation behaviors of the 14 reference standards were explored separately. Retention times of each analyte were calculated into normalized retention times (RTi(norm)) using the equation (1) [25]. RTmax and RTmin represent the retention times of the greatest and the least retained compound, respectively, and the retention times RTi were converted into normalized RTi(norm) values in the range of 0-1.
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(1) 5
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Then, the correlation coefficient (R2) of RTi(norm) value was calculated to evaluate the orthogonality between coupled columns, where 0 and 1 indicated truly orthogonal and nonorthogonal separations, respectively (Table 1). Compared with other combinations, the couplings of Hypersil gold CN to Accucore Phenyl-Hexyl, Accucore PFP and Accucore C18 showed lower R2 values as 0.9519, 0.9356 and 0.9336, respectively. Therefore, the Hypersil gold CN was selected as the optimum 1D separation column. Accucore C18 and Accucore PFP offered similar low R2 values when coupled to the Hypersil gold CN, both of which seemed to be suitable for 2D separation. As a further consideration, the chromatograms of mixed standards and Danshen samples analyzed by the two columns under the same program were shown in Fig. A.1. The Accucore PFP column exhibited high separation performance in terms of better peak resolution and shorter analysis time than Accucore C18 column. However, on the contour plot of Hypersil gold CN and Accucore PFP (Fig. 4A), peak splitting was observed for lithospermic acid (P29), salvianolic acid B (P42) and salvianolic acid C (P56), which should be one peak into two blobs. For example, Fig. 4B showed the 1D view of 2D cycle in the range of 78-80 min and salvianolic acid C (P56), identified by single standard injection, was splitting into two partially overlapped peaks as peak P56a and peak P56b, of which their UV spectra observed in Fig. 4C and Fig. 4D were matched perfectly for the confirmation of salvianolic acid C (P56). Additional optimization test, including the variations of 2D flow rate (1.5, 2.0 and 2.5 mL min-1) and gradient programs, proved to be ineffective to eliminate the peak splitting of these analytes. Therefore, Accucore C18 column was selected as the preferred 2D column in this experiment without any peak splitting. Since phenolic acids were weakly retained on the selected columns whereas diterpenoid constituents were strongly, a normal gradient program was applied for 1D separeation. For 2D separation, a segment gradient program was optimized, which consisted of seven different segments with increased acetonitrile ratios to maintain high resolution. The finally accepted gradient programs are presented in Fig. 2 and all 14 standards could be well separated without excessive bandwidths on the contour plot (Fig. 5A). Under the optimized parameters, phenolic acids and diterpenoids can be separated into two independent groups on the contour plot of Danshen (Fig. 3B), which facilitate the identification of these structural-related constituents. 3.2. Optimization of MS conditions For the two types of compounds in Danshen, positive ESI mode was found to offer better sensitivity for diterpenoids, whereas negative ESI mode was more suitable for phenolic acids. Therefore, the ESI-HRMS-MSn data was acquired in both positive and negative ion modes. For the component identification, comprehensive MS data could make it easier and more reliable. In this experiment, DDA was employed with seven scan events as a cycle. The first event was set for the mass full scan by Orbitrap with resolution of 30,000 to get the accurate mass, which was used to calculate elemental formulas. In the following six events, two most intense ions in the first event were selected to acquire MSn data (n=2-4) to get the fragment information. Due to the complex constituents in Danshen, dynamic exclusion feature was enabled in DDA mode with exclusion time set at 8 s and exclusion list up to 50. Thus, co-elution ions and relative low intensity ions have more opportunities to be selected for MS2 detection.
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In addition, because the major components in Danshen have been identified and reported in many literatures [16-18,22,27], our study paid more attention to the characterization of minor compounds. Except for using dynamic exclusion feature, we have divided the scan range m/z 100-1500 into five narrower and partially overlapped scan ranges, as described in Section 2.3. Under this strategy, more components with low abundance could be detected for MSn analysis as illustrated in Fig. A.2. Besides, because a single compound can generate many fragments with different m/z values in full scan mode, it may result to time-wasting and increased risk of false identification. Therefore, the scan range of m/z 100-1500 was also employed to decrease the false selection of precursor ions during data process. 3.3. Peak capacity and orthogonality of the system To evaluate the performance of the optimized LC×LC system, peak capacity and system orthogonality were calculated as measurements. As a result, a total of 328 blobs in Danshen can be recognized by the LC Image on the DAD contour plot (281 nm). The effective gradient time for 1D and 2D analysis were 115 min and 1 min, respectively. The average 4σ peak widths were 2.497 min and 0.032 min in 1D and 2D chromatograms. So, the theoretical peak capacities for 1D and 2D analysis were 46.06 and 31.25, respectively. The lowest USP plate counts were 451 (calculated by Caffeic acid) and 108335 (calculated by Danshensu) for 1D and 2D analysis, respectively. Furthermore, according to the reported equations [23,24], the theoretical peak capacity and effective peak capacity of the developed system were calculated as 1439 and 830, respectively. The constructed LC×LC system provided remarkably effective peak capacity with 17-fold higher than that of 1D LC analysis, exhibiting its powerful separation capability. By referring the literature [23-25], the orthogonality and practical peak capacity were calculated. Specifically, the separation space was divided into 18×18 rectangular bins close to Pmax (328) and was superimposed with the data points (Fig. A.3). The result showed that about 41% of the separation space was covered by bins containing data points. Thus, the orthogonality was calculated as 53.9% and the practical peak capacity was 575. 3.4. Quantitative validation and sample analysis For conventional 1D-LC analysis of complex herbs, such as Danshen, the accurate quantification of interested analytes often encountered the problems of co-elution and matrix effect. LC×LC system, as a powerful separation technique, was then considered for the quantification. But, since peaks for each analyte were distributed into several modulation cycles in 2D systems, they have to be summed for calculation with the help of extracted ion chromatograms obtained from specific MS detectors [28-30], which seemed to be more complicated than 1D-LC. Therefore, in this study, we employed the contour plots for the quantification analysis. With LC Image [31], target analytes on the DAD contour plot (281nm) can be easily recognized and located, and meanwhile the peak volumes were automatically integrated for quantification instead of peak areas. At the same time, due to the complex matrix of Danshen, quantitative results were further confirmed by the coupled LTQOrbitrap to avoid false-positive identifications. The proposed LC×LC-DAD method was then validated to test its quantitative reliability by the contour plots. The standard curves, correlation coefficient (r), linear range, LOD, LOQ and RSDs (2D RT and peak volume) for each analyte are summarized in Table 2. All 14 seven-point standard curves offered the r values ranging from 0.9976 to 1.0000 for all
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analytes using the plotting of peak volumes and concentrations, highlighting its feasibility and reliability for the quantification. LODs and LOQs were in the range of 0.16-1.18 µg mL-1 and 0.52-3.89 µg mL-1 for investigated compounds, respectively, demonstrating the good sensitivity of the proposed method. The precision for the analyte locating and peak volume calculating was also tested using seven injection of a mixed standard solution. The RSD values were lower than 1.07% for 2D RT, which showed the highly locating accuracy; and peak volumes also gave a low RSD range from 0.34% to 4.11%. In conclusion, all these validation results showed satisfactory linearity, sensitivity and precision of the established LC×LC-DAD method, which was then applied to sample quantitative analysis. Typical 2D contour plots of the 14 mixed standards and a Danshen sample are shown in Fig. 5. As can be seen in Fig. 5A, all 14 standards were well separated as single blobs with the varied color representing different peak intensities. Also, the contour plot of a Danshen sample (no.1) is shown in Fig. 5B and the detected blobs were highlighted with bubbles, which indicated both retention time (by image location) and peak volume (by disk size). The recognition of the investigated analytes was carried out by comparing their retention times with those of corresponding genuine standards and was further confirmed by the coupled LTQ-Orbitrap. According to these procedures, all samples were analyzed and the calculated contents of target compounds are presented in Table 3. Among the 14 compounds, it can be observed that salvianolic acid B (P42) (13.44-36.44 mg g-1) was the most dominant component in the 10 phenolic acids, as well as tanshinone IIA (D41) (0.26-0.86 mg g-1) in the 4 diterpenoids, which were both used as the chemical markers for the quality control of Danshen in Chinese Pharmacopoeia (edition 2010) [32] . 3.5. Characterization of chemical constituents in Danshen Chemical constituents in Danshen were analyzed by the constructed LC×LC system and, thanks to its remarkable separation power, the interested phenolic acids and diterpenoids were detected as blobs into two individual groups on the 2D contour plot (Fig. 3B), since each of them shared common structural motifs. Meanwhile, 14 available standards were analyzed using the LTQ Orbitrap by direct infusion to explore their fragmental behavior and to build an MSn spectral library for structural characterization of those unknown constituents. According to the number of phenyls in the structure, phenolic acids in Danshen could be classified into monomers (e.g. danshensu and caffeic acid) and polymers, including dimers, trimers and tetramers, which consist of several monomers [27]. Therefore, phenolic acids always exhibited a unique fragmentation behavior. Based on our fragmental study of the reference standards and literature reports [16,27,33], in the MSn spectra of phenolic acids, the loss of small molecules such as CO2, CO and H2O were produced by the monomers, due to the existence of either carboxyl, carbonyl or hydroxyl groups. Moreover, the fragment ions [M-H-198]- and [M-H-180]- were derived from the neutral loss of danshensu and caffeic acid in polymers. For the diterpenoid constituents, their protonated molecules [M+H]+ or [M+Na]+ could be observed in full MS spectra under positive ion mode. The fragments of [M+H-15]+, [M+H-18]+, [M+H-28]+ and [M+H-32]+ can be found in the MS and MSn spectra, indicating the loss of CH3, H2O, CO and CH3OH, respectively [17,33]. Therefore, based on the fragmental pattern and comparing with reference standards and literature data [17,22,27,33-35], a total of 95 compounds including 52 phenolic acids and 43 diterpenoids were identified or tentatively identified and detailed information were listed in
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Table A.1 and A.2. In addition, although there have been lots of reports for the discovery and identification of phenolic acids in Danshen [17,27,33-35], 7 new compounds were detected for the first time and tentatively characterized in the current study, which were summarized in Table 4. Also, their MS2 spectra together with proposed chemical structures were interpreted in Fig. 6. Peak P17 and P27 showed similar [M−H]− ions at m/z 735.1555 and 735.1556, respectively, and gave the same element composition of C36H31O17 with errors lower than 0.90 ppm. Their molecular weights were 18 Da higher than that of salvianolic acid B, while their MS2 spectra were similar and yielded a series of same fragments such as m/z 717, 537, 519, 493, 339, 321 and 295. These evidences allowed to deduce that peak P17 and P27 were the addition product of H2O toward the double bond of salvianolic acid B. Therefore peak P17 and P27 were tentatively characterized as 8-hydroxy-salvianolic acid B and its isomer. Peak P28 gave the [M−H]− ion at m/z 699.1555 (C33H32O17). The MS2, MS3 and MS4 spectra of peak P28 produced prominent ions at m/z 519, 321 and 279, resulting from the loss of 180 Da, danshensu and 42 Da, which were similar with those of salvianolic acid B. In addition, the high resolution MS2 spectra of peak P28 showed the [M-H-180]− ion at 519.0917 and the calculated element composition is C27H19O11 (Fig. 6). Therefore, the neutral loss of 180 Da in MS2 spectra was C6H12O6, which may be attributed to glucose. Thus, peak P28 was tentatively identified as lithospermate-9'-O-glucoside. Peak P39 displayed a [M−H]− ion at m/z 523.1236 (C27H23O11). Its MS2 spectra yielded ions at m/z 505, 491, 325 and 293 (Fig. 6), representing the ions of [M-H-H2O]-, [M-HCH3OH]-, [M-H-danshensu]- and [M-H-danshensu-CH3OH]-, which suggested the existence of methoxyl. Further compared MS3 and MS4 spectra of peak P39 with that of salvianolic acid C, it could be further deduced that a H2O was added toward the double bond of salvianolic acid C besides the group of methoxyl. Thus, peak P39 was proposed to be 8hydroxy-4-methoxyl-salvianolic acid C. Peak P45 showed a [M−H]− ion at m/z 701.1503 and gave the element composition of C36H29O15, which was only one oxygen atom less than that of salvianolic acid B ([M-H]-, C36H29O16). In the MS2 spectra, the ions at m/z 519 (100%, relative intensity) and 503 (24%) were observed, indicating the presence of deoxy-danshensu and danshensu, and also meaning that deoxy-danshensu was easier to be lost. Moreover the fragment ions at 519 and 503 could both produced prominent ion at m/z 321 in the MS3 spectra, resulting from the loss of danshensu and deoxy-danshensu. According to literature [27], fragmentation behaviors of salvianolic acid B showed that the cleavage of a bond was easier than b bond (Fig. 1). Therefore, compared with salvianolic acid B, Peak P45 was tentatively identified as 3'''deoxy-salvianolic acid B. Peak P48 gave a [M−H]− ion at m/z 747.1544 (C37H31O17). The MS2 spectra of peak P48 generated ions at m/z 715, 549 and 517, representing the ions of [M-H-CH3OH]-, [M-Hdanshensu]- and [M-H-danshensu-CH3OH]- , respectively, which suggested the presence of methoxyl (Fig. 6). The fragment ions in MS3 and MS4 were similar with that of salvianolic acid B. Thus, peak P48 was tentatively identified as 5''-hydroxyl-9''-methyl-salvianolic acid B. Peak P50 provided a [M−H]− ion at m/z 685.1550 and gave the element composition of C36H29O14, which were two oxygen atoms less than that of salvianolic acid B ([M-H]-,
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C36H29O16). The MS2 and MS3 spectra of peak P50 yielded prominent ions at m/z 503 and 321, resulting from the loss of deoxy-danshensu and the second deoxy-danshensu. Compared with peak 45 and salvianolic acid B, peak P50 could be tentatively characterized as 3''-deoxy3'''-deoxy-salvianolic acid B.
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In this work, a LC×LC-DAD-ESI/HRMS/MSn system was constructed and evaluated for the qualitative and quantitative characterization of phenolic acids and diterpenoid components in Danshen. Columns with different selectivity were employed to improve orthogonality of the first and second dimensions. By using the optimal segment gradient program, phenolic acids and diterpenoids were separated into two independent groups, which facilitated the identification of unknown compounds. Besides, the combination of DDA and partially overlapped scan ranges make it possible to detect and fragment more components with low abundance in Danshen. Under this strategy, a total of 328 peaks were separated and detected successfully. Among these peaks, 102 compounds were identified or tentatively identified and 7 of them were discovered from Danshen for the first time. The proposed LC×LC system showed good orthogonality (53.9%) and high effective peak capacity (830). Furthermore, the contents of 14 bioactive compounds in different Danshen samples were simultaneously quantified directly on the DAD contour plots. Above all, the established LC×LC-DAD-ESI/HRMS/MSn system has proved to be powerful to discover minor compounds, as well as be reliable for the accurate quantification of interested analytes in complex herbs.
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This research was supported by the National Natural Science Foundation of China (31160065), the Macao Science and Technology Development Fund (No. 052/2012/A2) and the Research Committee of the University of Macau (No. MYRG109-ICMS13-LP and MYRG2014-00089-ICMS-QRCM). We are grateful to Prof. Qingping Tao (GC Image, LLC., Lincoln, NE, USA) and Yanhai Zhang (Thermo Fisher Scientific) for their professional technical support.
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[1] J.L. Cao, J.C. Wei, M.W. Chen, H.X. Su, J.B. Wan, Y.T. Wang, P. Li, Application of twodimensional chromatography in the analysis of Chinese herbal medicines, J. Chromatogr. A 1371 (2014) 1-14. [2] L. Hu, X. Chen, L. Kong, X. Su, M. Ye, H. Zou, Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights, J. Chromatogr. A 1092 (2005) 191-198. [3] J. Zhang, D. Tao, J. Duan, Z. Liang, W. Zhang, L. Zhang, Y. Huo, Y. Zhang, Separation and identification of compounds in Adinandra nitida by comprehensive two-dimensional liquid chromatography coupled to atmospheric pressure chemical ionization source ion trap tandem mass spectrometry, Anal. Bioanal. Chem. 386 (2006) 586-593. [4] D.X. Li, O.J. Schmitz, Use of shift gradient in the second dimension to improve the
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4. Conclusions
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Acknowledgement
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Ac ce p
479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522
12
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an
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ip t
Fig. 1 The chemical structures of 14 reference standards. Fig. 2 Gradient elution programs for the first and second dimension separations. Fig. 3 Comparison of 1D-LC chromatogram (A) with 2D contour plot (B) for the separation of Danshen (281 nm). The conventional 1D-LC conditions for (A) are provided in Supporting Information. Fig. 4 Peak splitting of the primary LC×LC system for the analysis of 14 reference standards. (A) 2D contour plot with DAD detection at 281nm; (B) 1D-LC chromatogram view for the time range of 78-80 min; (C) The UV spectra of peak P56a; (D) The UV spectra of peak P56b. Hypersil gold CN (150 mm × 1 mm, 3 µm) and Accucore PFP (50 mm × 4.6 mm, 2.6 µm) were used for 1D and 2D separation, respectively. Fig. 5 2D contour plots for separation of 14 reference standards (A) and no. 1 Danshen sample (B) at 281 nm.
M
Fig. 6 Proposed chemical structures and LTQ-Orbitrap ddMS2 spectra for the interested compounds.
d
547 548
[35] G.F. Zeng, H.B. Xiao, J.X. Liu, X.M. Liang, Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun. Mass Sp. 20 (2006) 499-506.
te
523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546
1
1
549 550 551
D column Hypersil gold C18
Xbridge amide
Hyperisl gold PFP
Hyperisl gold C8
Syncronis C18
Hypersil gold CN
0.9928
0.9775
0.9981
0.9969
0.9948
0.9519
0.9612
0.9576
0.9830
0.9722
0.9636
0.9806
0.9947 0.9991
0.9836 0.9853
0.9951 0.9956
0.9964 0.9989
0.9980 0.9997
0.9356 0.9336
Ac ce p
2
D column
R2
552 553
2
Table 1 Orthogonal test between the D and D columns
Accucore PhenylHexyl Accucore PolarPremium Accucore PFP Accucore C18
Table 2 Quantitative performance for 14 analytes by using the constructed LC×LC-DAD
system. 1
Peak no.
Analyte
P3
Danshensu
P4
Protocatechuic acid
D RT (min)
2
Linearity
2
8.40
4
12.78
Standard curve y = 15x – 16 y = 49x + 321
D RT (sec)
r 0.9998 0.9990
Range (µg mL-1) 6.30126.00 2.56102.55
LOD (µg mL-1)
LOQ (µg mL1 )
Precision (RSD, %) 2 D Peak RT volume
1.18
3.89
0.85
1.35
0.58
1.93
0.83
1.06
13
Page 13 of 21
P7
Caffeic acid
4
71.84
P26
Rosmarinic acid
30
84.48
P29
Lithospermic acid
40
77.90
P42
Salvianolic acid B
52
79.60
P44
Salvianolic acid A
58
82.20
P52
9''-Methyl lithospermate B
66
79.04
P56
Salvianolic acid C
76
81.10
D19
Dihydrotanshinone I
76
91.00
D32
Cryptotanshinone
86
93.38
D33
Tanshinone I
92
97.14
D41
Tanshinone IIA
104
107.28
y = 147x + 116 y = 112x - 194 y = 61x 239 y = 39x + 222 y = 35x 273 y = 78x 453 y = 43x +6 y = 81x + 48 y = 119x - 29 y = 77x 17 y = 63x 44 y = 101x - 303
-1
Retention time 1st dim.
2nd dim. (sec) 79
a
P3
1
Hebei
2.09
2
Hebei
-
3
Hebei
1.59
P4 -
a
d
Origin
P5 +
0.9999 0.9985 1.0000 0.9999 0.9999 0.9999 0.9999 0.9997
1.07
1.20
0.29
0.95
0.35
4.11
0.22
0.73
0.06
0.49
0.55
1.81
0.10
1.31
0.59
1.95
0.06
0.90
0.92
0.05
0.53
1.02
0.07
1.05
0.28 0.31 0.16
0.52
0.06
1.09
0.22
0.74
0.19
0.90
0.36
1.17
0.16
0.51
0.42
1.40
0.19
0.77
0.24
0.79
0.13
0.34
b
P7
P26
P29
P42
P44
P52
P56
D19
D32
D33
0.09
1.15
1.16
23.44
0.22
+
0.02
0.13
0.36
0.36
0.15
-
+
0.77
1.19
16.23
0.04
0.11
0.04
0.11
0.27
0.38
-
-
0.05
1.01
1.42
20.04
0.11
0.09
0.02
0.05
0.10
0.19
Ac ce p
564 565
0.9976
1.28
detected by the LC×LC-DAD system. No.
559 560 561 562 563
0.9995
0.39
Table 3 Contents (mg g ) of 14 standards in different Danshen (Salvia miltiorrhiza) samples
te
556 557 558
0.9993
1.5562.00 1.6766.60 7.16286.50 9.18367.00 20.00800.00 6.01240.50 2.1987.50 2.59103.50 1.3855.00 2.3092.00 2.0180.50 5.25210.50
M
554 555
0.9996
ip t
39.78
cr
4
us
Protocatechuic aldehyde
an
P5
4
Shandong
2.08
-
+
-
0.55
1.25
13.44
0.05
0.09
0.03
0.05
0.11
0.23
5
Sichuan
1.60
-
-
0.05
1.36
1.62
36.44
0.21
0.04
0.02
0.01
0.10
0.18
not detected; b under the limit of quantification.
Table 4 Charaterization of unknown compounds by UHPLC-LTQ-Orbitrap from Danshen
(Salvia miltiorrhiza)a Measured [M-H](m/z)
Theoretical [M-H]- (m/z)
Error (ppm)
Molecular formula
LC/ESI-MSn m/z (% base peak)
735.1555
735.1561
-0.90
C36H32O17
MS2[735]: 295(8),297(6),321(5),339(3), 357(4),493(4),519(20),537(100),555(3),717(2) MS3[537]:179(2),185(2),253(8),269(22),277(2),279(2),2 95(75),297(100),321(64),339(24),357(70),449(11), 475(9),493(13),519(52) MS4[297]:161(5),175(7),225(16),241(3),253(100),269(4 7),279(6)
Identification
8-Hydroxy-salvianolic acid B
14
Page 14 of 21
82
699.1555
699.1561
-0.86
C33H32O17
85
523.1236
523.1240
-0.82
C27H24O11
84
701.1503
701.1506
-0.44
C36H30O15
75
747.1544
747.1561
-2.36
C37H32O17
80
685.1550
685.1557
-1.02
C36H30O14
a
Lithospermate-9'-O-
8-Hydroxy-4-methoxyl acid C
3'''-Deoxy-salvianolic acid B
5''-Hydroxyl- 9'' -methyl acid B
3''-Deoxy-3'''-deoxyB
d
Identified from Salvia miltiorrhiza for the first time.
te
569 570
a
8- Hydroxy-salvianolic isomer
Ac ce p
566 567 568
MS2[735]: 295(8),297(5),321(10),339(2), 357(3),493(6),519(56),537(100),555(3),717(7) MS3[537]: 253(5),269(29),277(1),279(2), 295(78),297(100),321(76),339(24),357(54),449(10), 475(7),493(24),519(63) MS4[297]:161(5),175(7),225(16),241(2),253(100),269(4 3),279(8) MS2[699]:321(11),339(3),493(9),519(100),537(7),655(2 0) MS3[519]: 279(3),321(100),339(35) MS4[321]:249(7),265(2),277(47),279(100),293(16) MS2[523]:179(6),197(2),293(60),311(19),325(100),343( 35),491(68),505(2) MS3[325]:175(12),251(15),265(10),283(25),293(100),30 7(2),311(29) MS4[293]:221(10),237(19),247(16),249(33),251(51),265 (100),275(20) MS2[701]:321(18),339(4),503(24),519(100) MS3[519]:277(3),279(3),295(4),321(100),339(25),475(2) MS3[503]:279(5),305(5),321(100),339(20),441(4) MS4[503→321]:249(8),275(3),277(40),279(100), 293(20),303(11) MS2[747]:295(1),321(8),339(4),517(3),519(2),549(100), 567(2), 715(1) MS3[549]:277(3),279(12),295(7),321(100),339(39),517( 40) MS4[321]:249(3),251(1),265(2),275(1),277(33),279(100) ,293(10),303(2) MS2[685]:321(11),339(2),503(100) MS3[503]:279(6),295(2),321(100),339(20),441(3) MS4[321]:249(8),265(3),275(3),277(45),279(100),293(1 7),303(8)
ip t
C36H32O17
cr
-0.65
us
735.1561
an
735.1556
M
81
15
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i cr us M an ed ce pt Ac
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Fig. 2
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Fig. 3
i cr us M an ed ce pt Ac
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Fig. 4
i cr us M an ed ce pt Ac
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Fig. 5
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Fig. 6