Characterization of phenolic compounds in the fruits of Forsythia suspensa by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Phenolic compounds are the major bioactive constituents of Forsythia suspensa, an important Chinese herbal medicine used for the treatment of various infectious diseases. Fragmentation behaviors of the phenolic compounds in F. suspensa were investigated by using a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS(n)) method. For common phenylethanoid glycosides, the loss of the caffeoyl moiety was the first fragmentation step, then sequential losses of rhamnose, hexose and water were observed in further fragmentations. If a substituent group presented in the beta position, the fragmentation was triggered by initial loss of a substituent group to form structures such as suspensaside A. Then it underwent the common fragmentation pathways as mentioned above, or eliminated characteristic residues of masses 134 or 152 Da, respectively. The latter pathway is reported here for the first time. The fragmentation behaviors of furofuran lignans displayed a typical cleavage of the tetrahydrofuran ring. However, the presence of a hydroxyl group at C-1 led to the successive loss of 30 Da. Neutral loss of CO(2) and benzyl cleavage were characteristic for lignans with a 2,3-dibenzylbutyrolactone skeleton. A neutral loss of 30 Da was also observed in the fragmentation pattern of flavonols. These fragmentation rules were implemented to analyze phenolic compounds in the fruits of F. suspensa. A total of 51 compounds, including 24 phenylethanoid glycosides, 21 lignans and 6 flavonols, were identified or tentatively characterized based on their retention times, UV spectra and MS fragmentation patterns.     

Characterization of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen) by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The qualitative analysis of tanshinones in the roots of Salvia miltiorrhiza (Dan-shen in Chinese) was performed using high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (ESI-MS(n)). Tanshinones are the major bioactive constituents of Dan-shen, which is used in China for the treatment of haematological abnormalities and cardiovascular diseases. The ESI-MS(n) fragmentation behavior of tanshinones was investigated. For tanshinones with the tanshinone I nucleus, the fragmentation was triggered by loss of a molecule of CO except bearing a substituent at C17 or C18, followed by sequential eliminations of CO. If C(15-16) was a saturated bond, the fragmentation was triggered by elimination of a molecule of H2O. For tanshinones with the tanshinone IIA nucleus, the fragmentation was triggered by loss of a molecule of H2O, followed by successive eliminations of CO. Ions corresponding to loss of a molecule of propylene (Delta m = 42) were also observed. Moreover, when C(15-16) was a saturated bond, ions corresponding to losses of CH3, H2O and propylene were more abundant. If no D-ring existed, the presence of isopropyl resulted in an elimination of a molecule of H2O with an adjacent CO or OH. In addition, the extension of the pi-conjugation in the A-ring (especially at C(1-2)) induced the fragmentation by loss of a molecule of CO. These fragmentation rules were applied to the identification of tanshinones in a chloroform/methanol (3:7) extract of Dan-shen, which was separated on a C18 column with gradient elution. A total of 27 tanshinones were identified, including five new constituents. The established method could be used for the sensitive and rapid identification of tanshinones in the Dan-shen drug and its pharmaceutical preparations.     

Triple quadrupole tandem mass spectrometry of sesquiterpene lactones: a study of goyazensolide and its congeners

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the fragmentation pattern of ten sesquiterpene lactones of the goyazensolide type under low-energy collision-induced dissociation (CID) using a triple quadrupole mass spectrometer. The analysis revealed that loss of CO(2)[M + H - 44](+) is the predominant process for compounds that exhibit a hydroxyl at C-8. In contrast, compounds with different acyloxy groups at C-8 fragment by means of elimination of the corresponding carboxylic acids [M + H - (R(2)CO(2)H)](+) and consecutive losses of CO and H(2)O. Our results also demonstrate the influence of both the stereochemistry of the acyloxy group at C-8 on the relative abundances of product ions and the hydroxyl at C-15, which creates an additional pathway, resulting in highly diagnostic product ions. This work clearly demonstrates the utility of tandem quadrupole low-resolution mass spectrometry for studies on the rationalization of the fragmentation of a series of compounds with a highly conserved core structure, but differing in substituent groups.     

高速逆流色谱分离纯化防风中升麻素苷和5-O-甲基维斯阿米醇苷

建立了高速逆流色谱分离制备防风中有效成分升麻素苷和5-O-甲基维斯阿米醇苷的方法.防风根的粉末经甲醇浸泡提取和减压蒸馏,得粗提浸膏.以V(乙酸乙酯):V(正丁醇):V(水)=2:7:9为溶剂,上相为固定相,下相为流动相,流速2.0 mL/min.从316 mg防风粗提物中一步分离得到13.9 mg升麻素苷和25 mg 5-O-甲基维斯阿米醇苷,纯度分别为98.1%和99 2%.采用ESI-MS, 1H NMR 和13C NMR对目标化合物的结构进行了鉴定.     

Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids

Positive-ion mass spectral fragmentations of seven mycosporine-like amino acids (MAAs) are reported and discussed. The MAAs studied are small compounds composed of a cycloheximine ring substituted with amino acid or amino alcohol units. Techniques used include electron impact (EI) and electrospray ionization (ESI) with tandem mass spectrometry (MS/MS). ESI-MS/MS showed unusual small radical losses, generally resulting from the loss of a methyl group with the exception of shinorine and porphyra for which the initial losses were 30 and 44 Da, respectively. As expected from structural similarities, porphyra, shinorine and palythinol displayed similar fragmentation patterns, while palythenic acid and palythene fragmented in a similar manner. Overall, the ESI-MS/MS fragmentations at m/z <200 exhibited a distinctive pattern for all seven MAAs with characteristic ions at m/z 137, 168, 186, and 197 or 199. Several ions were observed for each of the MAAs analyzed, and together provide a useful and potentially diagnostic pattern for identification of MAAs and as an aid in structure elucidation of novel MAAs. For GC/EI-MS analysis, trimethylsilyl (TMS) derivatives were made. The EI-MS fragmentation patterns of TMS-MAAs showed many features typical of TMS-derivatized alpha-amines. The precursor TMS-MAA ion was not detected, but a [M-90](+ radical) ion was the highest-mass intense peak observed for palythine, palythinol and shinorine, while palythene gave a [M-116](+ radical) ion. Besides determining the number of acidic hydrogens, EI-MS of TMS-derivatized MAAs will aid in structure elucidation of novel MAAs.     

Use of diagnostic neutral losses for structural information on unknown aromatic metabolites: an experimental and theoretical study

This work presents the use of neutral losses (NL) for the identification of compounds related to the metabolism of tyrosine. The mass spectra of all the studied compounds, recorded at several collision energies, are compared. The fragmentation mechanism of protonated molecules, MH⁺, is explained by combining collision‐induced dissociation (CID) mass spectra and density functional theory (DFT) calculations. The results show that the first fragmentation is the elimination from MH⁺ of a neutral molecule including a functional group of the linear chain. Three primary neutral losses are observed: 17 u (NH₃), 18 u (H₂O) and 46 u (H₂O+CO) characterizing amino, hydroxyl and carboxylic functions on the linear chain. The presence and abundance of ions corresponding to these losses are dependent on (i) the position of the functional group on the linear chain, (ii) the initial localisation of the protonating hydrogen, and (iii) the substitution of the aromatic ring. For compounds including a functional group on the benzylic carbon atom, the investigation of the other functions requires the knowledge of secondary fragmentations. Among these secondary fragmentations we have retained the loss of NH₃ from [MH–18u]⁺ and the loss of ketene from [MH–17u]⁺. Experimentally these fragmentations are detected using losses of 35 u and 59/73 u. In other words, NL35 identifies hydroxy and amino compounds and NL 46 and/or NL59/73 identify carboxylic acids. The search for characteristic neutral losses is used for the analysis of compounds in a mixture and the analysis of biological fluid. We show that selective search of several neutral losses allows also the unambiguous differentiation of isomers and gives the opportunity to identify compounds in biological fluids. Copyright © 2008 John Wiley & Sons, Ltd.     

Structural characterization of steroidal saponins by electrospray ionization and fast-atom bombardment tandem mass spectrometry

The structural characterization of four steroidal saponin compounds involving two and three sugar groups, namely spirostanol saponins and furostanol saponins, were investigated by positive ion fast-atom bombardment (FAB), electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) techniques. Important structural information was obtained from collision-induced dissociation (CID) and FAB-MS spectra with different liquid matrices. It was found that a characteristic fragmentation involving the loss of 144 Da arising from the cleavage of the E-ring was observed when there was no sugar chain at the C-26 position. When a glucoside group was substituted at the C-26 position, this C-26 sugar moiety was preferentially eliminated. All of these compounds produced a major product ion with a stable skeleton structure at m/z 255. The results of this paper can assist structural analysis of mixtures of steroidal saponins.     

Characterization of fifty-one flavonoids in a Chinese herbal prescription Longdan Xiegan Decoction by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection

High-performance liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry and photodiode array detection (HPLC-DAD-ESI-MS(n)) was developed to identify and characterize the flavonoids in a Chinese formulated preparation, Longdan Xiegan Decoction (LXD). In total, fifty-one flavonoids (27 flavones, 10 flavanones, 7 chalcones, 5 flavonols and 2 isoflavones) were characterized. Eighteen compounds among them including a newly detected flavonoid, naringin, from the ingredient herbs, were unambiguously determined by comparing the retention times (t(R)), UV spectral data and mass fragmentation behaviors with those of the reference compounds. Another thirty-three compounds were tentatively identified by referencing to the reported data of their UV and MS spectra. The ESI-MS/MS fragmentation behavior of flavones (OMe-substituted, O-glycosides, C-glycosides), chalcones, flavonols and their appropriate characteristic pathways were proposed. In negative ion ESI-MS all the flavonoids yielded prominent [M--H](-) ions in the first order mass spectra. Fragmentation with a loss of mass of 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)), 56 Da (2CO) and the residues of glucose and glucuronic acid observed in the MS/MS spectra were useful for aiding the structural identification of the flavonoids investigated.     

Preparative separation of chromones in plant extract of Saposhnikovia divaricata by high-performance counter-current chromatography

Four chromones, prim-O-glucosylcimifugin, 4'-O-β-D-glucosyl-5-O-methylvisamminol, cimifugin and sec-O-glucosylhamaudol, were isolated and purified from Saposhnikovia divaricata for the first time by high-performance counter-current chromatography (HPCCC) using a system consisting of ethyl acetate/n-butanol/ethanol/water (1:1:0.1:2, v/v/v/v). The separation parameters were first performed on the analytical HPCCC and the optimized conditions were then scaled up to preparative HPCCC. A total of 72.1 mg of prim-O-glucosylcimifugin, 27 mg of 4'-O-β-D-glucosyl-5-O-methylvisamminol, 14.1 mg of cimifugin and 1.1 mg of sec-O-glucosylhamaudol were purified from 960 mg of the n-butanol extract of S. divaricata, each at over 90% purity as determined by high-performance liquid chromatography (HPLC). The structures of four compounds were identified by their retention time, the liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) in the positive ion mode, and confirmed by NMR. The characteristic LC-ESI-MS fragmentation patterns of the four compounds were discussed, and found to be a very specific and useful tool for the structural identification of chromones from S. divaricata.     

Fragmentation patterns of novel dithiocarbamate derivatives with pharmaceutical activity under electrospray ionization tandem mass spectrometry conditions

The fragmentation patterns of a novel series of dithiocarbamate derivatives with pharmaceutical activity were investigated by positive ion electrospray ionization mass spectrometry in conjunction with tandem mass spectrometry (ESI-MS(n)). In the gas phase, the dithiocarbamate protonated molecules containing the piperazinium moiety undergo losses of bromide to form the piperazinium cation by ionization, followed by subsequent losses of methyl bromide, ring opening and rearrangement of piperazinium. Furthermore, the dithiocarbamate derivatives and their intermediates both undergo cleavage of the C-S bond to produce two common fragment ions. The different fragmentation observed for these compounds facilitated their identification and could be valuable in the further study of their metabolic pathways as prodrugs.     

Gas Phase Chemistry of Li+ with Amides: the Observation of LiOH Loss in Mass Spectrometry

Collision-induced dissociation (CID) of Li(+) adducts of three sets of compounds that contains an amide bond, including 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N-phenylbenzamide, its derivatives and simpler structures was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Observed fragment ions include those that reflect loss of LiOH. Other product ions result from the Smiles rearrangement and direct C-S bond cleavage. MS/MS of H/D exchange products demonstrated occurrence of a 1,3-H shift from the amide nitrogen atom to the phenyl ring of these compounds. The LiOH loss from Li(+) adducts of amides was further examined by CID of [M + Li](+) ions of N-phenylbenzamide and N-phenylcinnamide. Loss of LiOH was essentially the sole fragmentation reaction observed for the former. For the latter, both losses of LiOH and H(2)O were discovered. The presence of electron-donating substituents of the phenyl ring of these compounds was found to facilitate elimination of LiOH, while that loss was retarded by electron-withdrawing substituents. Proposed fragment ion structures were supported by elemental compositions deduced from ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR-MS/MS) m/z value determinations. Density functional theory-based (DFT) calculations were performed to evaluate potential mechanisms for these reactions.     

Cyclodextrin-based liquid chromatographic enantiomeric separation of chiral dihydrofurocoumarins, an emerging class of medicinal compounds

A set of 28 racemic dihydrofurocoumarins in which the stereogenic center is located in the furan ring have been synthesized. Currently no effective asymmetric synthesis of this class of compounds exists, although their enantiomers are produced biologically by certain plants. Their diverse medicinal properties are being investigated in several laboratories. The enantioselective separation of these dihydrofurocoumarins by three native and six derivatized cyclodextrins has been evaluated in the reversed-phase mode, the polar organic mode, and normal-phase mode. The hydroxypropyl-beta-cyclodextrin is the most effective chiral stationary phase (CSP) at separating the dihydrofurocoumarins into enantiomers, showing some enantioselectivity for 22 dihydrofurocoumarins, and baseline resolving 16 of the 28 compounds in the reversed-phase mode. The acetyl-beta-cyclodextrin and 2,3-dimethyl-beta-cyclodextrin also showed enantioselectivity for a large number (18 and 17, respectively) of dihydrofurocoumarins in the reversed-phase mode. The native cyclodextrins are ineffective and the aromatic derivatized beta-cyclodextrins are only marginally effective at separating the furocoumarin enantiomers in the reversed-phase mode. The polar organic mode and the normal-phase mode have also been evaluated with these CSPs, but no enantioseparations were observed.     

Negative ion electrospray ionization mass spectrometry of nucleoside phosphoramidate monoesters: elucidation of novel rearrangement mechanisms by multistage mass spectrometry incorporating in-source deuterium labelling

Several O-2',3'-isopropylideneuridine-O-5'-phosphoramidate monoesters were synthesized and analyzed by negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)). Two kinds of novel rearrangement reactions were observed due to the difference in the amino acid in the nucleoside phosphoramidate monoesters, and possible mechanisms were proposed. One involves a five-membered cyclic transition state. The other is formation of a stable five-membered ring intermediate by Michael addition. Results were confirmed by tandem mass spectrometry and isotopically labeled hydrogen atoms. Furthermore, the internal hydrogen exchange between active hydrogen and methyl acrylate in the heated capillary of the mass spectrometer was found. The characteristic fragmentation behavior in ESI-MS may be used to monitor this kind of compounds in the biological metabolism.     

Characterization of cationic glycoporphyrins by electrospray tandem mass spectrometry

Novel cationic porphyrin derivatives having a galactose or a bis(isopropylidene)galactose unit linked directly to a pyridine or to an aminophenyl group were characterized by electrospray tandem mass spectrometry (ESI-MS/MS). The electrospray mass spectra (ESI-MS) show the M(+) ions, since these porphyrins are already monocharged in solution. The fragmentation of these ions under ESI-MS/MS conditions was studied and it was found that elimination of the sugar residue as a radical (-163 or -243 Da) is a common fragmentation pathway. Loss of the sugar unit as a neutral fragment (-162 or -242 Da) and cross-ring fragmentations typical of glyco-derivatives are also observed for the pyridinium glycoporphyrins, but they are absent in the case of ammonium glycoporphyrins. The cationic beta-pyridiniumvinyl porphyrins show an atypical fragmentation due to the cleavage of the C(5)-C(6) bond of the sugar unit. Overall, the different patterns of fragmentation observed in the ESI-MS/MS spectra of the sugar pyridinium porphyrins and of the sugar ammonium phenyl porphyrins can give important information about the type of spacer between the porphyrin and the sugar unit.     

Tracing glycoprotein structures: electrospray ionization tandem mass spectrometric analysis of sugar-peptide adducts

Owing to the diversity of carbohydrate structures and their significance for the function of many biopolymers, structural analysis of various carbohydrate-related compounds is of great importance. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to establish the fragmentation behaviour of a range of sugar-peptide adducts as model compounds of widespread glycoprotein structures. The compounds used in this study were chosen to provide correlation of distinct fragment ions with specific structural differences, namely position and type of carbohydrate-peptide bond and structure of the sugar moiety. All compounds show N- and C-terminal sequence ions along with losses of up to three water molecules. Fructose-related Amadori compounds exhibit M + 78 modified N-terminal peptide fragment ions. Fragmentation of glucose-peptide esters is characterized by the sugar ring fragmentation. Additionally, under the ESI-MS conditions applied, the esters studied undergo intramolecular reaction giving cyclic sugar-peptide structures that can be traced by the presence of N-terminal peptide M + 42 adducts. Detailed analysis of cyclic fructose-related compound comprising structural features of both studied groups revealed a rich fragmentation pattern derived from amino acid residues and water molecules losses from [M - 2H(2)O + H](+) ion. Also, some interesting differences were found with respect to the nature of carbohydrate moieties.     

Investigations of taxoids by electrospray and atmospheric pressure chemical ionization with tandem mass spectrometry

The fragmentation behavior of taxoids was studied using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources with multi-stage tandem mass spectrometry. In the positive ion mode taxoids gave prominent [M+Na]+ and [M+K]+ ions with the ESI source, and [M+NH4]+ or [M+H]+ ions with the APCI source. The MS/MS fragmentations of ions produced by APCI and ESI sources were very similar. For both sources, the presence of cinnamoyl or benzoyl groups could be characterized by initial losses of 148 or 122 u, respectively, from molecular adduct ions. However, the elimination of cinnamic acid was relatively difficult for the molecular adduct ions formed by APCI, and was comparable in importance to the loss of acetic acid. The other fragments involved losses of CH2CO, CO, and H2O. The 5/7/6 type taxoids underwent characteristic losses of 58 or 118 u from ions produced by both APCI and ESI sources. The fragmentation behavior was remarkably influenced by substitution locations. The elimination of the C-10 benzoyl group was usually the first fragmentation step, while that of the C-2 benzoyl group was relatively difficult. The acetoxyl group at C-7 was more active than those at C-2, C-9, and C-10, which in turn were more active than that at C-4. These fragmentation rules could facilitate the rapid screening and structural characterization of taxoids in plant extracts by high-performance liquid chromatography/mass spectrometry (HPLC/MS).     

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H₂O, CO, and CH₂O₂ losses, together with retro‐Diels–Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C‐ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C₄H₈) and 68 Da (C₅H₈) were yielded from a prenyl chain; neutral losses of 42 (C₃H₆), 54 (C₄H₆), and 70 Da (C₄H₆O) were generated by a pyran ring. Particularly, neutral losses of 18 (H₂O), 16 (CH₄), 112 (C₈H₁₆), and 98 Da (C₇H₁₄) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.     

Characterization of phenolic compounds in the crude extract of Hedysarum multijugum by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry

The fragmentation behavior of four types of phenolic compounds: coumestans, pterocarpenes, benzofurans and isoflavones, were studied using electrospray ionization multistage mass spectrometry (ESI-MS(n)) (n = 2-5) in negative ion mode. Losses of methyl (15 Da), CO (28 Da), CO(2) (44 Da), isopropyl (43 Da) and isobutenyl (55 Da) were dominating fragmentation patterns. Different positions and numbers of the substituents also led to the different fragmentation behavior. These fragmentation rules were applied for the identification of constituents in methanolic extracts of Hedysarum multijugum, in which 29 compounds were characterized, including nine new compounds. The method established in this study could be applied to the comprehensive quality control of the herb and its formulations. It could also be applied to the biological and pharmacological research of traditional Chinese medicines.     

Positive-ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: in-source fragmentation of sodium adducts

Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na](+). In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1' and C-2' of the alpha-D-Glcp-(1-2)-beta-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry.     

Analysis of bufadienolides in the Chinese drug ChanSu by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

The qualitative analysis of bufadienolides in the Chinese drug ChanSu was performed using high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS). Bufadienolides are the major bioactive constituents of ChanSu, which is used to treat heart failure and cancer in traditional Chinese medicine. The APCI-MS fragmentation behavior of bufadienolides was studied. For bufadienolides with only hydroxyl substituents, the fragmentation was characterized by successive eliminations of H(2)O and CO molecules, and the profile of MS/MS product ions was correlated with the number of hydroxyl groups. If a C-16 acetoxyl group was present, the fragmentation of [M+H](+) ions was triggered by initial loss of 60 Da (HOAc). The elimination of CO was significant for bufadienolides with a 19-formyl group, and the 19-hydroxyl group could be characterized by the loss of 30 Da (HCHO). These fragmentation rules were applied to the identification of bufadienolides in a methanolic extract of ChanSu, which was separated on a C(18) column with gradient elution. A total of 35 bufadienolides were identified, including four new constituents. The method established here facilitated the convenient and rapid quality control of ChanSu crude drug and its pharmaceutical preparations.