The Applicaiton Of Liqud Chromatography Coupled With High Resolution Mass Spectrometry Technology On Chemical Study Of Two Chinese Herbs

ObjectiveThe chemical compositions and the absorbed metabolites of Chinese herbs are essential basis for exerting pharmacodynamic action. With the progress of liquid chromatography and mass spectrometry, the qualitative and quanlitative analysis of chemical compositions and absorbed components of Chinese herbs has been one of the most important means to study the material basis of bioactive compounds in traditional Chinese Medicines. Two very important Chinese herbs:Heshouwu (Polygonum multiflorum) and Fuzi (Aconitum carmichaeli) were selected as our research subjects for chemical and pharmacokinetics analyzing by ultra-high-pressure liquid chromatography coupled with linear ion trap Orbitrap high resolustion mass spectrometry methods. The fragmentation pathways of anthraquinones, stilbenes and tannins from Polygonum multiflorum and aconitine-type diterpenoid alkaloids from Aconitum carmichaeli were systematic analyzed. Besides, the quantitative methods based on UHPLC-LTQ-Orbitrap methods were developed and applied to study the quality and the pharmacokinetics properties of Polygonum multiflorum. The present study has explored new and feasible methods for material basis and the modernization research of traditional Chinese medicine.The fragmentation pathways of anthraquinones, stilbenes and tannins from Polygonum multiflorum and aconitine-type diterpenoid alkaloids from Aconitum carmichaeli were systematic analyzed. Besides, the quantitative methods based on UHPLC-LTQ-Orbitrap methods were developed and applied to study the quality and the pharmacokinetics properties of Polygonum multiflorum. The present study has explored new and feasible methods for material basis and the modernization research of traditional Chinese medicine.Methods1. The mass fragmentation regularities of THSG, anthraquinones and catechin, the main components of P. multiflorum, were systematic studied by LTQ-Orbitrap. The method was set as follows:the main components of Polygonum multiflorum stilbene glycoside, emodin, physcion, emodin-8-0-glucoside, emodin-8-0-glucoside, rhein, chrysophanol, aloe-emodin, gallic acid and catechin was selected as model components, direct infusion injected into the mass spectormetre. The sampling rate is 8 μ L/min. MS full scan range was at m/z 150-1500, the high resolution full scan resolution set at 30000 (m/z 400 full width at FWHM). The MS2-MS3 of the Orbitrap high-resolution scanning scanning resolution is set to 15000, MS4 daughter ions was used LTQ dynote detection. The highest peak of one scanning was selected for further fragmentation. The fragmentation mode was the collision induced dissociation (CID), and the collision energy was 35%.2. Based on the established the mass spectrometry database of constituents from Polygonum multiflorum Thunb and its related specis, combined with the fragmentation pattern of model components, the analysis of Polygonum multiflorum Thunb chemical composition were conducted by using UHPLC-LTQ-Orbitrap MSn technology. For liquid chromatography separations, a hypersil gold C18 column (2.1 x 100 mm,1.8 μm) was used, mobile phase consisting of acetonitrile (a) and water containing 0.1% formic acid (b), the gradient elution set as:5% a (0 min); 15% a (6 min); 15% (12 min); 38% a (25 min); 70% a (30 min) and 90% a (35 min). Flow rate was 300 μ L/min. DAD detection was ranged from 200-400 nm. Mass spectrometric conditions:electrospray ionization (ESI) was set as interface. The positive and negative ion detection mode was used, respectively. The MS scanning range was m/z 150-1500, and the ion source gas was nitrogen. High-resolution setting of full scan is 30000, 15000 resolusiton was set for MS2, and 7500 for MS3 scaninng. Dynamics scanning (data-dependent) was enabled in the data dependence scan (MSn), and the highest abundance of parent ions was selected for the dynamic exclusion.3. Reviewing the domestic and foreign literature, an innovative method was developed for determination of four representative ingredient of P. multiflorum. (emodin, emodin-8-0-glucoside, gallic acid and stilbene glycoside) by using UHPLC-LTQ-Orbitrap and applied to 20 samples collected in different areas and different processing. An UPLC BEH C18 Waters column (2.1 mm* 50 m,1.7 μm) was used for LC analysis. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid water system. System program was: 13% a (0 min),35% a (3.5 min),90% a (7.5 min),95% a (8.5 min) and 95% a (10 min). Flow rate was 400 μ L/min.LTQ-Orbitrap high resolution multistage mass spectrometry was adopted and the ESI interface was connected with LC. The full scan mode of targeted ions were qualitatively detected by comparing retention time, fragment ion abundance, fragments ion with high resolution data for qualitative detection. The method of SRM was used (sellected reaction monitoring) and the highest response sub ion was monitored. The ion pairs were as follows:gallic acid: m/z 169→125; emodim m/z 269→225; THSG:m/z 405→243; Emodin-8-glu:m/z 431-269.4. The identification of the absorbed components and metabolites after oral administration of P. multiflorum in rats were conducted by UHPLC-LTQ-Orbitrap. A systematic strategy, including mass database hitting, diagnostic fragment ion searching and high accurate MS/MS information was used. Methods:SD rats were divided into urine, blood and control groups. Each group at a dose of 2 mL/kg and control group given corresponding dose of saline. The blood samples were kept in the lower velocity centrifugation (5000 rpm, 10 min), and the upper plasma was obtained with-80 C preservation. Plasma and urine samples were treated by organic solvent precipitation method. Accela UHPLC Thermo-Fisher system was used for LC separations. The chromatographic column was Phemomenex Kinetex C18 (2.1* 100 mm,2.6 μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid water system with gradient elution. System program:5% a (0 min),35% a (13.5 min), 60% a (23.5 min) and 90% a (30 min). Flow rate of 200 L/min. The sample amount is 5 μL. Online DAD detection record 200-400 nm spectral data. LTQ Orbitrap high-resolution multistage mass spectrometry was usedand an ESI interface connected to LC.5. A method was developed for blood drug concentration determination of tetrahydroxystilbene glucoside and emodin by LC-MS and pharmacokinetic parameters were studied. Given the dose of 1 ml/lOOg P. multiflorum, blood samples were obtained at 5,10,15,20,30,45,60,240,120,480, and 750 min.A Waters UPLC BEH C18 (2.1* 100 m,1.7 μmm) column was used in the LC separations. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid water system with gradient elution. The system program is:8% A (0 min), 30% A (3.5 min),85% A (7.5 min),90% A (8.5 min) and 90% A (10 min). Flow rate was 400 μL/min. The sample volume was 5 μL. The mass spectrometry qualitative detection was conducted in high resolution full scan mode, by comparing the retention time, abundance of fragment ion and fragment ions high resolution MS data for the qualitative detection. Orbitrap high resolution selective ion monitoring mode (HR-SIM) was used for quantitative detection.6. MSn fragmentation behaviors of representative alkaloid components were sysmaticly studied in UHPLC-LTQ-Orbitrap. Methods:the sample rate was 3 u L/min. MS full scan range was m/z 150-1500, high resolution full scan resolution set to 30000 (m/z 400 full width at half maximum), MS2-MS3 of the Orbitrap high-resolution scanning resolution is set to 15000, MS4 ions was detected by LTQ dynote. Choose the most abundant ions of parent scanning for further collision induced dissociation (CID) fragmentations. The collision energy was 40%, the peak wide range was m/z±1, and other parameters set as default. Based on the ion characteristic signals and multistage mass spectrometry data, supplemented with high resolution mass spectrometry data combined with literature confirmed, the possible fragmentation patterns of representative aconite alkaloids were comprehensively studied.7. The qualatively analysis of the chemical constituents of radix aconiti carmichaeli was conducted by UHPLC-LTQ-Orbitrap MSn method. Several strategies, including self-established diterpenoid alkaloids mass spectrometry database hitting and literature searching were used for systematic identification of unknown aconite alkaloids by LC-MS techniques.Results1. Based on the analysis and simulation of the key fragmentation site of THSG, the fragment ions at m/z 225、215、137 were proved to correspond to the cleavage of the ring A, and m/z 149 was originated from the cleavage of the alkene bond after rearrangement. The key fragmentation patterns of several anthraquinones with different substituents were conprehensively studied by LTQ-Orbitrap MS and their 0E fragment ions were identified. Also, the fragmentation pathways of two important phenolic compounds from P. multif lorum, catechin and epicatechin in LTQ-Orbitrap were systematic studied.2. A simple and effective method was developed for characterization of phenolic constituents in the roots of P. multiflorum by UHPLC-LTQ-Orbitrap. Stilbenes, anthraquinones, tannins and naphthalenes were differentiated by diagnostic fragment ions with accurate mass measurements and characteristic fragmentation pathways. Based on the proposed strategy, eighty-seven constituents were characterized or tentatively identified from P. multiflorum, including fifteen stilbene glycosides, nineteen anthraquinones, and thirteen tannins. Furthermore, twenty-eight new dianthrone glycosides were characterized and their fragmentation behaviors were firstly exploited by our LC-HR-MSn method. It’s improved that the new dianthrone glycosides were originated from emodin (10→10’) emodin or emodin (10→10’) physcion basic parent nucleus by the high accurate tandem MS. It’s first time that dianthrone glycosides were reported from genus of Fallopia.3. We innovatively developed liquid chromatography coupled with linear ion trap MS quantitative methods for determination of four representative components emodin, emodin-8-0-glucoside, gallic acid and THSG in P. mult if lorum. It was applied to evaluate the quality of 20 batches P. multiflorum of different origin and different processing methods. The results demonstrated that the linear ion trap MS shows effective and high sensitive advantage, which could used for quality control of P. multiflorum. The quantitative analysis result also showed that the contents of four target compounds vary greatly among 20 batches of P. multiflorum.4. The identification of the absorbed components and metabolites after oral administration of P. multiflorum in rats were conducted by UHPLC-LTQ-Orbitrap for the first time. Using the systematic strategy, including mass database hitting, diagnostic fragment ion searching and high accurate MS/MS information, five absorbed components and forty metabolites were identified. The results showed that the metabolic mechanisms were dominated by the Phase II metabolic products of emodin, THSG and physcion with glucuronidation and sulfation reaction. Phase I metabolites such as the oxidation and methylation products of emodin, as well as the hydrolysis and reduction products of THSG were also observed.5. Based on the UHPLC-LTQ-Orbitrap LC-MS methods, the pharmacokinetics study of the two main components emodin and THSG after oral administration of P. multiflorum in rats was conducted. By using the high resolution SIM mode, the in-vivo quantitative determination of THSG and emodin were developed, which showed high interference rejection ability, high sensitivity and advantage at convenience.6. The mass fragmentation behaviors of the representative diterpenoid alkaloids from Fuzi (the roots of Aconitum carmichaeli) were systematic studied by LTQ-Orbitrap MSn. Based on the analysis of the characteristic fragments and tandem mass spectrometry, the key fragmentation patterns of several representative diterpenoid alkaloids were plausibly speculated and proved by high accurate MS detection. The results showed that the C1, C3 and C6 site of diterpenoid alkaloids were the preferred active sites for fragmentation. The fragmentation referring to loss of H20 were originated from C3-OH cleavage, and loss of CO were from C16-OH rearrangement. For ADA type alkaloids, the base peaks of the MS2 spectra could be used for distinguishing the substituents mode of C1 and C3. These findings are conducive to the identification of unknown components from Fuzi.7. A rapid and effective method was developed for separation and identification of diterpenoid alkaloids in the roots of Aconitum carmichaeli by ultra-high-pressure liquid chromatography coupled with high resolution LTQ-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MSn). According to accurate mass measurement and the characteristic neutral loss filtering strategy, more than 120 diterpenoid alkaloids were rapidly detected and characterized or tentatively identified, including potential new and minor DDA^ MDA, LDA and ADA. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources. ConclusionIn this paper, we selected two popular TCM herbs:Aconitum carmichaeli and Polygonum multiflorum as the objects to lauch the Traditional Chinese medicine material basis, the metabolism and absorption material basis and the pharmacokinetics researches by using ultra high pressure liquid chromatography coupled with high resolustion mass spectrometry. The present study reveals the chemical foundation of Aconitum carmichaeli and Polygonum multiflorum, provides methodological reference for the on-line rapid detection of the Chinese traditional medicine in vivo and in vitro composition, and further explore feasible technology and method for the modernization of Chinese medicine.