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.     

Analysis of minor flavonoids in Piper hostmannianum var. berbicense using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The fragmentations of hydroxylated flavanones, chalcones and dihydrochalcones were investigated by direct loop injection using an ion trap mass spectrometry equipped with atmospheric pressure chemical ionization (APCI) probe. Some of them have been isolated from the leaves of Piper hostmannianum var. berbicense and standards were used to confirm their fragmentation behaviour. In negative ion mode, fragmentations of these three types of flavonoids revealed specific diagnostic ions which allowed us to identify aglycones in a crude plant extract. The major fragment ion obtained in MS/MS experiment for methoxylated chalcones is the neutral loss of a methyl radical whereas a H(2)O molecule is lost in the case of methoxylated dihydrochalcones. Methoxylated chalcones and flavanones isomers could be differentiated by the relative intensity ratio of [M-H-CH(3)]*(-) and [M-H-C(2)H(2)O](-) ions. Based on UV and MS data, a decision tree that includes UV lambda(max) absorptions and MS/MS diagnostic ions was built in order to obtain structural information of unknown compounds present in the extract. This tree was used to identify flavonoids in the ethyl acetate extract of P. hostmannianum var. berbicense leaves after analysis by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization ion trap multistage mass spectrometry. A total of 11 flavonoids were tentatively characterized based on the MS fragmentations pattern observed in MS(n) experiments.     

银杏叶提取物中染料木素的分离纯化及结构鉴定

对银杏叶提取物进行酸水解,然后利用正相硅胶柱色谱及重结晶法从银杏叶水解物中分离出染料木素,经紫外光谱（UV）、质谱（MSn）、核磁共振（1H NMR、13C NMR）等波谱学方法鉴定证实了其结构。高效液相色谱分析表明,所提纯的染料木素纯度达到了98%以上。     

Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry

The phenolic components from Radix Salvia miltiorrhizae Bunge, a well-known herbal medicine (Dan-Shen in Chinese), have been investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the ESI mass spectra a predominant [M-H]- ion was observed in negative mode and provided molecular mass information. ESI-MS/MS spectra of the [M-H]- ions were used for structural analysis, based on the spectra of standards. It was found that caffeic acid and its monomeric analogs containing a carboxyl group readily lost CO2, while dimers, trimers and tetramers of caffeic acid expelled successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in S. miltiorrhizae were characterized, of which eight compounds were positively identified by comparison with standards. The remaining twenty phenolics for which standards were not available were tentatively identified based on their UV spectra and MS/MS fragmentation characteristics.     

Analysis of homoisoflavonoids in <Emphasis Type="Italic">Ophiopogon japonicus</Emphasis> by HPLC-DAD-ESI-MS

The homoisoflavonoids in Ophiopogon japonicus (Thunb.) Ker-Gawler were analyzed by high-performance liquid chromatography-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MSn). Homoisoflavonoids gave prominent [M - H]- ions by electrospray ionization monitored in the negative ion mode. They could be classified into two types depending on the fragmentation behavior of their [M - H]- ions in the ion trap mass analyzer. The [M - H]- ions of homoisoflavonoids with a saturated C2-3 bond underwent C3-9 bond cleavage to lose the B-ring, which was followed by the loss of a molecule of CO. The [M - H]- ions of homoisoflavonoids with a C2-3 double bond usually eliminated a CO molecule first, and then underwent the cleavage of C3-9 or C9-1' bonds. For homoisoflavonoids with a C-6 formyl group, however, the neutral loss of CO was the first fragmentation step; the presence of a methoxyl group at C-8 could lead to the cleavage of C-ring. No retro Diels-Alder (RDA) fragmentation characteristic for normal flavonoids was observed. The above fragmentation rules were reported for the first time, and were implemented for the analysis of homoisoflavonoids in O. japonicus. The CHCl3-MeOH extract was separated on a Zorbax Extend-C18 column, eluting with a acetonitrile-0.3% acetic acid gradient. A total of 18 homoisoflavonoids, including seven new minor constituents, were identified or tentatively characterized based on the UV spectra and tandem mass spectra of the HPLC peaks.     

Characterization of alpha- and gamma-glutamyl dipeptides by negative ion collision-induced dissociation

The low-energy CID mass spectra of the [M-H](-) ions of a variety of dipeptides containing glutamic acid have been obtained using cone-voltage collisional activation. Dipeptides with the gamma-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the alpha-linkage, H-Glu-Xxx-OH, by the much more prominent elimination of H-Xxx-OH from the [M-H](-) ions of the former isomers, resulting in formation of m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M-H](-) ions including enhanced elimination of CO(2) and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuterium has shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded) hydrogens prior to most fragmentation reactions. All dipeptides show loss of H(2)O from [M-H](-). MS(3) studies show that the [M-H-H(2)O](-) ion derived from H-Glu-Gly-OH has the structure of deprotonated pyroglutamylglycine while the [M-H-H(2)O](-) ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.     

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.     

Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.     

Structural characterization of isoprenylated flavonoids from Kushen by electrospray ionization multistage tandem mass spectrometry

Eighteen isoprenylated flavonoids (8 flavanones, 3 flavanols, and 7 chalcones) isolated from Kushen or synthesized were studied by positive and negative ion electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Plausible fragmentation patterns were obtained by comparing their MS(n) spectra with each other, which were further supported by high-resolution MS data and two model compounds. It was shown that the 2'-OH group would make the C-ring of flavonoids studied more labile through a six-membered mechanism, resulting in base peaks of (1,3)A+ (positive mode) and (1,4)A(-) (negative mode). In addition, the 2'-OH is also responsible for the neutral loss of water in (+)ESI/MS(2) of flavanones. The neutral loss of water (or methanol) in (-)ESI/MS(2) of flavanols was elucidated by a E2 elimination mechanism. Different relative abundances (RA) of (1,3)A(+) and S(+) in (+)ESI/MS(2) spectra were used to discriminate flavanones with their open-ring products, chalcones, since the equilibrium for flavanone<-->chalcone isomerization in ESI ion source could not be obtained in positive mode.     

Identification and determination of major flavonoids in rat urine by HPLC-UV and HPLC-MS methods following oral administration of Dalbergia odorifera extract

Flavonoids are the main active constituents of Dalbergia odorifera. The excretion of the major flavonoids in rat urine after oral administration of D. odorifera extract was investigated by HPLC-UV and HPLC-MS methods. Utilizing the HPLC-MS technique, 18 flavonoids, including five isoflavones, four isoflavanones, four neoflavones, two flavanones, two chalcones and one isoflavanonol were identified in free form in a urine sample based on the direct comparison of the corresponding tR, UV maximum absorbance (lambda(max)) values and MS data with the authentic standards. The amounts of the prominent flavonoids, (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone and vestitone, were determined by HPLC-UV with the internal standard method, and the validation procedure confirmed that it afforded reliable analysis of these two analytes in urine after oral administration of D. odorifera extract.     

Fragmentation study of protonated chalcones by atmospheric pressure chemical ionization and tandem mass spectrometry

Fragmentation mechanisms of protonated chalcone and its derivatives with different functional groups were investigated by atmospheric pressure chemical ionization with tandem mass spectrometry (MS/MS). The major fragmentation pathways were loss of the phenyl group from the A or B ring, combined with loss of CO. Losses of H(2)O and CO from the precursor ions of [M+H](+) are proposed to occur via rearrangements. Elimination of water from protonated chalcones was observed in all the title compounds to yield a stable ion but it was difficult to obtain skeletal fragmentation of a precursor ion. Loss of CO was found in the MS/MS spectra of all the compounds except the nitro-substituted chalcones. When the [M+H--CO](+) ion was fragmented in the MS/MS experiments, there were distinctive losses of 15 and 28 Da, as the methyl radical and ethylene, respectively. The ion at m/z 130, found only in the nitro-substituted chalcones, was assigned as C(9)H(6)O by Fourier transform ion cyclotron resonance (FTICR)-MS/MS; m/z 130 is a common fragment ion in the electron ionization (EI) spectra of chalcones. In order to more easily distinguish the constitutional isomers of these chalcones, breakdown curves were produced and these provided strong support in this study.     

A non-covalent dimer formed in electrospray ionisation mass spectrometry behaving as a precursor for fragmentations

A non-covalent-bonded dimer was detected in the positive ion electrospray ionisation (ESI) mass spectra of a synthetic impurity. In tandem mass spectrometry (MS/MS) experiments using collision-induced dissociation (CID), the ion was found to behave as a [M+H]+-type precursor ion for fragmentation until MS5. The dimer was probably formed through multi-hydrogen bonds over a proton bridge. When the fragmentation occurred at the center of the bridge, the dimer was broken apart to give monomer fragments at MS6. However, no corresponding deprotonated dimer [2M-H]- was found in the negative ion ESI spectra. The dimer was extremely stable, and it could still be observed when a fragmentation voltage of up to 50 V was applied in the ionisation source. The formation of the non-covalent dimer was also found to be instrument-dependent, but independent of sample concentration. Accurate mass measurements of the [2M+H]+ and [M+H]+ ions, and their MSn product ions, provided the basis for assessing the fragmentation mechanism proposed for [2M+H]+. The fragmentation pathway was also illustrated for the deprotonated molecule [M-H]-.     

苦参中黄酮类化合物的电喷雾质谱研究

采用电喷雾多级串联质谱对苦参中黄酮类化合物二氢黄酮及二氢黄酮醇类化合物的特征质谱行为进行了研究.实验结果表明,两类化合物在电喷雾多级串联质谱条件下均可以在C环发生开环断裂,但断裂的位点不同;两类化合物生成的碎片离子也有很大差异,提出了由二氢黄酮醇类化合物C环上3位连接的-OH所诱发的不同反应过程的质谱碎裂机理.     

Practical guidelines for characterization of O‐diglycosyl flavonoid isomers by triple quadrupole MS and their applications for identification of some fruit juices flavonoids

Fifteen flavonoid O‐diglycosides with different interglycosidic linkage isomery and glycosylation position have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high performance liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Some useful differences in their MS spectra have been found and discussed. As it has already been reported, [Y*]⁺/[Y₀]⁺ ratio for flavanones and [Y₁]⁺/[Y₀]⁺ ratio for other flavonoids is specific for each isomeric interglycosidic linkage. In this work it has also been observed that the abundance of these ions is dependent on the position of glycosylation. On the basis of these differences, systematic guidelines for our experimental conditions have been proposed for the differentiation of not only isomeric interglycosidic linkage but also glycosylation position using collision‐induced dissociation MS/MS (CID‐MS/MS) spectra in positive mode. These results have been successfully applied for the characterization of three diglycosyl flavonoids found in Citrus fruit juices and these conclusions have also been extrapolated for characterizing two triglycosides in the same fruits. Copyright © 2009 John Wiley & Sons, Ltd.     

Generation of alkoxide anions from a series of aliphatic diols and alcohols and their ion-molecule reactions with carbon dioxide in the gas phase

Alkoxide anions, [M-H](-) from a series of aliphatic diols and alcohols are generated in the source under negative ion electrospray ionisation conditions by cone-voltage fragmentation of the corresponding [M + F](-) ions. The collision-induced dissociation (CID) spectra of [M-H](-) ions consist of [M-H-2H](-) ions, in addition to the other characteristic fragment ions, and the relative abundance of [M-H-2H(-) ions among the series of diols varies as a function of chain length that could be explained based on their stabilities through intramolecular hydrogen bonding. The reactivity of alkoxide anions is studied through ion-molecule reactions with CO(2) in the collision cell of a triple quadrupole mass spectrometer. All the alkoxide anions reacted with CO(2) and formed corresponding carbonate anions, [M-H + CO(2)](-) ions. The reactivity of alkoxide anions within the series of diols also reflected the stability of their [M-H](-) ions.     

Studies on Fragmentation Pathways of N-Ethoxy （phenyl） phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

IntroductionPhosphorylation often acts as a molecular switchcontrolling the protein activity in different pathways asin metabolism,signal transduction,cell division,andso on.Therefore,N-phosphoryl amino acids play aspecial and important role in biological…     

Identification of phenolics and nucleoside derivatives in Gastrodia elata by HPLC-UV-MS

An HPLC-UV-MS method for simultaneous identification of predominant phenolics and minor nucleoside derivatives in Gastrodia elata was developed, which was based on their UV and MS characteristics summarized through a series of homemade reference standard experiments. Phenolics showed characteristic UV lambda(max) at 267 nm, [M + NH(4)](+) base peak in positive mode and [M-H](-) base peak in negative mode while nucleosides exhibited UV lambda(max) at 255 nm, [M + H](+), [M-H + 2H(2)O](-) or [M-H + CH(3)COOH](-). Phenolics conjugates mainly underwent the consecutive loss of gastrodin residue (-268 U) and the combined loss of H(2)O and CO(2 )from the citric acid unit under negative MS/MS conditions whereas nucleosides simply lost the ribose (-132 U) under positive MS/MS conditions. According to these characteristics, a special pattern under MS/MS conditions and reported compound data for G. elata in the literature, not only 15 phenolics were identified but also 6 nucleoside derivatives were identified. Among these compounds, seven phenolics and three nucleoside derivatives have not been reported yet from G. elata.     

Collision-induced fragmentation of deprotonated methoxylated flavonoids, obtained by electrospray ionization mass spectrometry

Electrospray operated in the negative mode was used to analyse methoxylated flavonoids. They were found to produce radical anions by collision-induced fragmentation of the aglycones. Loss of a methyl group from the deprotonated molecule corresponding to [M - H - 15]-* ions, as well as [M - H - 15-28]-* and [M - H - 15-29]- fragment ions, were found to constitute the characteristic fragmentation for the monomethoxylated species, whereas [M - H - 15]-*, [M - H - 30]- and [M - H - 30-28]- were predominant for the polymethoxylated species. Obtained under similar conditions, the product-ion spectra of isomeric compounds were characteristically different. It is therefore possible to distinguish between methoxylated flavonoids with identical molecular mass, e.g. when screening plant extracts for flavonoid composition. However, comparison with standard compounds is necessary for the identification of unknown flavonoid aglycones.     

Use of solid-phase extraction, reverse osmosis and vacuum distillation for recovery of aromatic sulfonic acids from aquatic environment followed by their determination using liquid chromatography-electrospray ionization tandem mass spectrometry

Three different sample preparation techniques (i) solid-phase extraction, (ii) reverse osmosis and (iii) vacuum distillation have been investigated and the recoveries were compared for determination of highly water-soluble benzene and stilbene sulfonic acids in aqueous environment by liquid chromatography with photodiode array (PDA) and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The recoveries were quite high using vacuum distillation (>90%) compared to solid phase extraction and reverse osmosis. The negative ion ESI mass spectra containing the peaks of quasimolecular ion [M-H]- allow the molecular mass determination of unknown compounds whereas the structures were proposed using fragments obtained from MS/MS analysis of [M-H]- ions. At lower fragmentation voltages only the quasimolecular ion [M-H]- was observed and as fragmentation voltages increased, it led to the formation of fragment ions corresponding to [M-H-SO3]-, [M-H-SO2]-, and SO3-. The detection limits were 1-28 microg/L with LC-ESI-MS. The sample collected from wastewater treatment plant was found to contain 21.1, 13.3, 12.1, 41.8 and 9.9 microg/L of cis-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (cis-DASDA), trans-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (trans-DASDA), 3-amino acetanilide-4-sulfonic acid (3-AASA), 4-chloroaniline-2-sulfonic acid (4-CASA), 2-chloroaniline-5-sulfonic acid (2-CASA), respectively.     

A fragmentation study of two compounds related to 4'-demethylepipodophyllotoxin in negative ion electrospray ionization by MSn ion-trap time-of-flight mass spectrometry

High-resolution electrospray ionization multistage tandem mass spectrometry (MS 1-7) in negative ion mode was used to determine the accurate masses and fragmentation pathways of two compounds, 4'-demethylepipodophyllotoxin and 4'-demethyl-4-azido-4-deoxyepipodophyllotoxin, which are key intermediate compounds for the preparation of podophyllotoxin-type anti-cancer drugs. The deprotonated molecules [M-H]* of both compounds were readily observed in the conventional single-stage mass spectra due to the presence of the phenolic hydroxyl group in the molecules. Abundant information on the product ions was obtained from tandem mass spectra (MS 2-7) in negative ion mode. Based on the exact masses acquired from 14 different tandem mass spectra, a similar MSn fragmentation pathway was proposed for both compounds. A characteristic product ion produced in the MS 2-4 product ion scan experiments is the cyclohexylenetrione anion [M-H-2Me-RH]* or [M-H-RH-2Me]* at m/z 351 (C19H11O7) formed by the consecutive losses of two CH3 radicals at the 3'- and 5'-positions and the neutral loss of RH, where R = a 4-substituted group (-OH or -N3), from the [M-H]* ion. This anion may be considered as diagnostic for the presence of this type of compound. The other common cleavages are the neutral losses of CO at least two times in the MS 6,7 product ion spectra. The results of this work could serve as an effective tool for the detection or determination of other derivatives of 4'-demethyl-4beta-substituted podophyllotoxin, which are widely used as intermediates for the preparation of anti-tumor drugs.