Structural Characterization and Identification of Major Constituents in Jitai Tablets by High-Performance Liquid Chromatography/Diode-Array Detection Coupled with Electrospray Ionization Tandem Mass Spectrometry

In the present study a universally applicable HPLC-DAD/ESI-MS/MS method was developed for carrying out the comprehensive characterization of Jitai tablets (JTT). Based on the ESI-MSn fragmentation patterns of the reference standards, a total of 101 components were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of reference standards or through the matching of empirical information with those of published components in the in-house library. The characteristic fragmentation pattern of alkaloids, phenolic acids, tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides, 2-(2-phenylethyl) chromones, phthalides and gingerol-related compounds were tentatively elucidated using structurally-relevant product ions. It was observed that neutral losses of C9H10O3 and C9H8O2 were the characteristic product ions of scopola alkaloids. Neutral fragment mandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge, tropylium ion and C4H2O unit were the characteristic ions of 2-(2-phenylethyl) chromone, which resulted from the Retro-Diels-Alder (RDA) cleavage of the C ring. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in TCM. This work is expected to provide comprehensive information for the quality evaluation and pharmacokinetic studies of JTT.


Results and Discussion
All the constituents were identified by the interpretation of their mass spectral behavior obtained from HPLC-DAD/ESI-MS/MS spectra and also taking into account the data provided by the 23 reference standards and literature. Figure 1 shows the chromatogram of JTT registered at 280 nm (A), the HPLC-ESI-MS/MS base peak chromatogram (BPC) of JTT in positive (B) and negative (C) ion mode.    were used to determine molecular weight. The identification of these compounds was carried out referring to their MS and MS n spectra, and the characteristic fragmentation pattern of alkaloids, phenolic acids, tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides, 2-(2-phenylethyl) chromones, phthalides and gingerol-related compounds are summarized as follows.

Identification of Alkaloids in JTT
Maximum absorptions in the range from 260 to 300 nm are exhibited by alkaloids in their UV spectra, and the preliminary identification could be facilitated by this characteristic absorption [14]. Consequently, 18 peaks with suitable maximum absorptions were detected as alkaloids in JTT. Furthermore, three major types including isoquinoline alkoloids, scopola alkaloids and aconitine-type alkaloids were summarized mainly based on their mass fragmentation behavior.
Using the proposed method, 10 isoquinoline alkoloids including four tertiary alkaloids, and six quaternary alkaloids were identified. In the positive ionization mode, the protonated molecule [M+H] + ions of tertiary alkaloids in ESI-MS/MS spectra were observed to undergo the Retro-Diels-Alder (RDA) cleavage. The fragmentation pathway is shown in Figure 3, and the same fragmentation behavior could also be applied for identifying tetra-proberberine alkaloids.  3 ] + were observed in the MS/MS spectra, which indicated unique fragments produced by the losses of CO and CH 3 . By using structurally-relevant product ions in literature data [15], six quaternary alkaloids were tentatively identified.
It is reported that the specific fragmentation pathways of the aconitine-type alkaloids were the neutral losses of H 2 O, CO and CH 3 OH in MS n spectra [16]. In the present study, highly abundant protonated molecule [M+H] + ions of peak 1, 39, 48 and 55 were observed in the MS spectra and fragment ions [M+H−H 2 O−CH 3 OH] + and [M+H−H 2 O−2CH 3 OH] + were exhibited in their MS/MS spectra, which indicated a further fragmentation by the neutral losses of CH 3 OH eliminated from the methoxyl on C 16 position and the proton on the C 15 position. By using structurally-relevant product ions in literature data [17], they were tentatively identified as mesaconitine, benzoylmesaconitine, benzoylaconine and benzoylhypoaconine, respectively.
For scopola alkaloids, protonated molecular ions [M+H] + were observed in the MS spectra. In the MS/MS spectra, m/z 138 [M+H−C 9 H 10 O 3 ] + was formed by the loss of tropic acid, while m/z 156 [M+H−C 9 H 8 O 2 ] + was produced by the loss of C 9 H 8 O 2 unit resulted from the fragmentation reaction of the ester bond. It could be concluded that the ions at m/z 138 and 156 were the characteristic product ions of scopola alkaloids. By comparison with reference standards and literature data [18], the [M+H] + ions of scopolamine and atropine eluting at 16.8 and 22.6 min were tentatively identified, respectively.

Identification of Phenolic Acids in JTT
Phenolic acids exhibited a unique fragmentation pattern in the negative ionization mode. They could be classified into monomers and polymers including dimers, trimers and tetramers, which were calculated by the number of phenyls in the structure accordingly [19]. which also showed the same general fragmentation pattern for their quasi-molecular ions [20,21]. This strategy was successfully applied for identifying or tentatively characterizing 16 phenolic acids with the structurally-relevant product ions.

Identification of Tanshinones in JTT
In the positive ionization mode, the protonated molecule [M+H] + ions of tanshinones were observed in the MS spectra. It is reported that the hydrogen at C-1 and oxygen at C-11 of tanshinones were the source of the dissociated H 2 O, the loss of CO, O, CH 3 and H 2 O were also observed in the MS/MS spectra [22].

Identification of Flavonoid Glycosides in JTT
Flavonoid glycosides are the main constituents originating from Flos Carthami. In the full scan mass spectra, most ions were selected to be dissociated with CID in the MS/MS spectra. Although flavonoid glycosides can be ionized under both positive and negative ionization conditions, more relative intense signals were obtained in negative ionization mode. Usually, high abundant quasi-molecular [M−H] − ion was observed in the MS spectra. In the MS/MS spectra, aglycone ions m/z 285, m/z 301 and m/z 287 were finally formed by losing several glycosidic units, which were identified as the characteristic ions of kaempferol, quercetin/hydroxy kaempferol and chalcone, respectively [23,24]. In the present study, a total of 21 flavonoid glycosides were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of reference standards or through the matching of empirical information with those of published components in the in-house library.

Identification of Cyanogenic Glycosides in JTT
The UV spectra of peaks 10 and 16 showed maximum absorption bands at 210-230 nm, consistent with the characteristic absorption of cyanogenic glycosides reported in Semen Persicae [25].

Identification of Ginsenosides in JTT
Due to the lack of a conjugated system, no ginsenoside peaks have been found in the UV chromatograms. Therefore, the constituents were characterized mainly based on their mass fragmentation behavior. In the negative ion mode, high abundance quasi-molecular ions [M−H] − and [M−H+HCOOH] − were formed first. In the MS/MS spectra, aglycone ions at m/z 475 and 459 were finally formed by losing several glycosidic units, which were also the characteristic ions of panaxatriols and panaxadiols, respectively. Usually, the ginsenosides could be quickly identified by their big molecular weight (>600 Da). However, the comparison with reference standards should also be needed due to the similarity of the structures of some ginsenosides [26]. The ion at m/z 455 was produced by successive losing of all linked glucosidic bonds, which was a characterized fragmentation of oleanolic acid type ginsenoside. By comparison with the literature data, the compound was tentatively identified as ginsenoside Ro [28]. With the same method, a total of 15 ginsenosides were tentatively identified with the structurally-relevant product ions.
In the full scan mass spectra,  Figure 5 for the first time.

Identification of Phthalides in JTT
It is reported that two main fragmentation patterns exist in the fragmentation pathway of phthalides: side chain fragmentation reaction corresponding to loss of alkenyl group or ring cleavage corresponding to the loss of H 2 O and carbonyl group [31]. The most abundant product ion at m/z 105 was a characteristic ion to identify the structures of these constituents. According to the law that the trans-production peaks eluted early than cis peaks in reversed-phase column and comparing with the literature data [32], they were identified as senkyunolide I and senkyunolide H, respectively. By using structurally-relevant product ions [33], peaks 79, 83, 96, 98 and 99 were identified as senkyunolide A, E-butenylphthalide, tokinolide B, riligustilide, and levistolide A, respectively.

Identification of Gingerol-Related Compounds in JTT
The gingerols showed a characteristic UV absorption maximum at 280 and 425 nm, suggesting the presence of an extended conjugation system. It was also found to be useful in the structure confirmation and especially for compounds belonging to homologous series.  [35], it was tentatively identified as 6-gingerol. Therefore, peaks 85 and 95 were identified as 8-gingerol and 10-gingerol, respectively, by the same method.
For shogaols, high abundant product ions at m/z 137[M+H−C 13 H 22 O] + of peaks 89 and 101 were formed in the MS/MS spectra, which indicated the characteristic fragmentation of the keto group on the alkyl chain. By comparison with the literature data [36], peaks 89 and 101 were tentatively identified as 6-shogaol and 10-shogaol, respectively.

Chemicals and Reagents
Reference standards such as danshensu, scopolamine, caffeic acid, amygdalin, hydroxysafflor yellow A, ferulic acid, tetrahydropulmatine, salvianolic acid B, pseudoginsenoside-F11, protopine, costunolide, dihydrocostunolide, cryptotanshinone, tanshinone IIA, ginsenoside Rg1, Re, Rb1, Rb2, Rb3, F1, Rd and Rg3 were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 6-Hydroxy-2-[(2-phenyl)ethyl] chromone was isolated and purified from Lignum Aquilariae Resinatum, the structure was identified by spectroscopic methods (UV, IR, MS, 1 H-NMR and 13 C-NMR), and the purity was determined to be over 98% by HPLC. Acetonitrile and formic acid were of HPLC grade (Merck, Darmstadt, Germany). Ultrapure water from a Milli-Q50 SP Reagent Water System (Millipore Corporation, MA, USA) was used for the preparation of samples and mobile phase. Other reagents were of analytical grade. JTT samples (batch number: 050602) and 15 comprised drugs were kindly offered by National Engineering Research Center for TCM (Shanghai, China).

Preparation of Analytical Sample of JTT
JTT and each comprised drug were ground into a fine powder and accurately weighed (400 mg) in a vial. 50% Methanol solvent (4.0 mL) was added and ultrasonic extracted for 30 min to prepare a uniform suspension, then centrifuged at 13,680 g for 10 min (Universal 320R, Hettich, Germany). At last, the supernatant was filtered using a syringe filter (0.22 µm).

Preparation of Standard Solutions
All reference standards were accurately weighed, and dissolved in methanol to obtain stock solutions with proper concentrations (50-1,000 ng/mL). All the stock solutions were stored in the refrigerator at 4 °C until analysis.

HPLC-DAD/ESI-MS/MS
HPLC/DAD analysis was carried out on an Agilent 1100 series HPLC system (Agilent Series 1100, Palo Alto, CA, USA) equipped with a quaternary pump with on-line degasser, auto-sampler, column oven and diode array detector (DAD), scanning from 200-400 nm and the wavelength was then selected and fixed at 280 nm since many peaks could only be found under this condition. Chromatographic separation was performed on a XTerra MS C18 column (5 µm, 4.6 × 250 mm, Waters, Milford, MA, USA) equipped with an XTerra MS C18 guard column (5 µm, 3.9 × 20 mm) (Waters), with the column temperature was set at 25 °C. The mobile phase consisted of 0.1% aqueous formic acid (A) and (B) acetonitrile using a gradient elution of 5-30% B at 0-50 min, 30-100% B at 50-90 min, 100% B at 90-100 min. The flow rate was kept at 1.0 mL/min and 10 µL of sample solution was injected in each run. By solvent splitting, 0.2 mL/min portions of the column effluent were delivered into the ion source of an Agilent LC-MSD Trap XCT mass spectrometer. The MS conditions were fixed as follows: drying gas (nitrogen) flow rate 10 L/min, gas temperature 350 °C, pressure of nebulizer gas 30 psi, HV voltage 3.5 kV, compound stability 100%, threshold 50,000 (ESI + ) and 10,000 (ESI − ) and scan range 100-1,500 amu. The amplitude voltage used for fragmentation was 1.0 V. The capillary exit voltage was set at 121 V for both positive and negative ion modes. Positive and negative mode data were acquired using Agilent chemstation software (Agilent Technologies, Palo Alto, MA, USA).

Data Analysis
Data analysis was performed on Microsoft Excel 2003 (Microsoft Corporation). An in-house library was established by searching from online websites such as Google Scholar, PubMed of the US National Library Medicine and Chinese National Knowledge Infrastructure (CNKI) of Tsinghua University, all components of comprised drugs were summarized in a Microsoft Office Excel sheet, which included the name, molecular weight, deprotonated molecular ions, protonated molecular ions, fragment ions and literatures of each published known compound. The "Find" function of Microsoft Office Excel was applied to match the detected mass value with that of published known compounds in the library. The characteristic fragmentation pattern of all components was tentatively elucidated using structurally-relevant product ions.

Conclusions
In the present study, a reliable and combinative analytical method using HPLC-DAD/ESI-MS/MS was established for rapid identification of multiple constituents in JTT. As a result, a total of 101 constituents were successfully separated and identified, and the fragmentation patterns of nine types of natural products in the complex system were successfully elucidated by the proposed method.
The present study, compared with the previous studies, showed differences or improvements as follows. First of all, it is the first time a combinative LC/MS (MS n ) method for screening the chemical constituents in such complex formula comprised of fifteen drugs has been developed. Furthermore, according to the interpretation of their mass behavior obtained from HPLC-DAD/ESI-MS/MS spectra and also taking into account the data provided by the 23 reference standards and the established in-house library, a total of 101 constituents were systematically characterized and identified in a single run. Neutral losses of C 9 H 10 O 3 and C 9 H 8 O 2 were the characteristic product ions of scopola alkaloids, and neutral fragment mandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge, losses of tropylium ions and C 4 H 2 O unit resulted from the RDA cleavage of C ring existed in fragmentation of 2-(2-phenylethyl) chromones. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in TCMs. This work is expected to provide comprehensive information for the quality evaluation and pharmacokinetic studies of JTT.