Electrospray Ionization Multi-stage Tandem Mass Research Articles

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MS n ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MS n detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.

New features on the fragmentation patterns of homoisoflavonoids in Ophiopogon japonicus by high‐performance liquid chromatography/diode‐array detection/electrospray ionization with multi‐stage tandem mass spectrometry

Homoisoflavonoids, a special class of flavonoids, are mainly distributed in the Liliaceae family and have various biological activities. Previously, very little research has been reported on the gas-phase fragmentation patterns of homoisoflavonoids by electrospray ionization mass spectrometry. In this paper, we report the use of high-performance liquid chromatography with a diode-array detector (HPLC-DAD) and electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) to study the fragmentation behavior of 11 homoisoflavonoid standards and to analyze homoisoflavonoids in Ophiopogon japonicus. In total, 28 homoisoflavonoids (including seven novel constituents) were characterized. The deprotonated [M--H](-) molecules of the homoisoflavonoids containing a saturated C2--C3 bond afforded the A or B product ion (base peak) according to whether the B-ring was substituted with a hydroxyl group. For the homoisoflavonoids containing a C-2-C-3 double bond, the product ions (A or C ion) were created from the precursor [M-H](-) ion as the base peak when the B-ring was substituted with a hydroxyl group. The homoisoflavonoids carrying a formyl group in the A-ring readily eliminated one molecule of CO to form the product ion [M + H-CO](-) (base peak) irrespective whether the C-2-C-3 bond was saturated or not. This product ion afforded the [M-H-CO-B-ring--CH(2) + H](-) ion by cleavage of the C3-C9 bond. This latter product ion always appeared in tandem mass (MS/MS) spectra of type I homoisoflavonoids. The common features of flavonoids observed during the gas-phase fragmentation mechanisms were the loss of the following groups: 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)) and 46 Da (CH(2)O(2)). A retro-Diels-Alder (RDA)-like cleavage was also observed for the homoisoflavonoids. The different gas-phase fragmentation routes were characterized for the deprotonated molecules obtained from the various homoisoflavonoids and collision-induced dissociation (CID) fragmentation differences were noted for the different locations of the various substituents. In conclusion, we can say that this study allowed us to structurally elucidate and identify homoisoflavonoids distributed in related plants and their complex prescriptions.

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MS n ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MS n in negative ion mode. The MS n data of the [M−H] − ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3- O-α- l-rhamnopyranosyl(1 → 4) [α- l-rhamnopyranosyl(1 → 2)]-β- d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.

Analysis of Saponins from Leaves of Aralia elata by Liquid Chromatography and Multi-stage Tandem Mass Spectrometry

Four saponins were isolated from the leaves of Aralia elata, and established using NMR and other spectroscopic methods, as well as data reported in the literature. Three Aralia saponins from the leaves of Aralia elata sharing the same structures as those isolated from the root bark suggested that the leaves would be a good substitute for the root bark of Aralia elata. These four Aralia saponins were then extensively investigated using complementarily positive and negative electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). Two isomers of saponins with different sugar linkages were then successfully differentiated by positive ESI-MS(n) and verified with different retention times and the collision-induced dissociation (CID) spectra by LC-MS. A simple and effective LC-MS method was thus developed for the rapid identification and screening of these saponins in plant extracts from leaves of Aralia elata.

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C18 column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r2 > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL−1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.

Fragmentation Pathways of Polycyclic Polyisoprenylated Benzophenones and Degradation Profile of Nemorosone by Multiple-Stage Tandem Mass Spectrometry

Nemorosone is a polycyclic polyisoprenylated benzophenone (PPBs) with strong cytotoxic activity. It is the major constituent of Clusia rosea floral resin and brown Cuban propolis. Other PPBs found in Cuban propolis are oxidized and cyclized derivatives of nemorosone. The instability of PPBs carrying an enolizable 1,3-diketone system has been suggested, and the elucidation of this aspect is very fundamental for the evaluation of their biologic activity. Electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)) was employed to shed light on the origin of these derivatives of nemorosone and to define the stability of this natural product. For this purpose, we initially performed MS(n) experiments on seven related PPBs to obtain useful information for structural characterization of this class of compounds and to identify the degradation products of nemorosone. The proposed fragmentation pathways, supported by exact mass measurements, allowed the nature of side chains on the bicyclo core and the type and position of their modifications to be established. In a second part, the degradation profile of nemorosone under different conditions was investigated to assess the possible effects of isolation procedures, climatic, and storage conditions on its stability. Our results reveal that nemorosone undergoes rapid degradation in n-hexane and chloroform solutions. The degradation products, identified by HPLC-ESI/MS(n) and NMR, are identical to derivatives of nemorosone previously isolated from propolis and plants. Thus, these PPBs are artefacts formed predominantly during the extraction and purification procedures.

Silver complexation and tandem mass spectrometry for differentiation of triterpenoid saponins from the roots of Pulsatilla chinensis (Bunge) Regel

For detection and differentiation of two types of triterpenoid saponins based on different aglycons of the lupane and oleanane skeleton from the roots of Pulsatilla chinensis (Bunge) Regel, the silver ion was introduced and electrospray ionization multi-stage tandem mass spectrometry was applied to analyze eleven triterpenoid saponin silver complexes. The quasi-molecular ion [M+Ag](+) was observed in the full-scan MS spectra of all the silver complexes. The MS(2) data of the [M+Ag](+) ion provided structural information on the sugar sequence of the oligosaccharide chains and the aglycon of the saponins. There are two patterns in the cleavage pathway of oleanane-type saponins. One is elimination of the sugar chain and subsequent loss of the carboxylic group which is the same as the cleavage of lupine-type saponins. The other is loss of the distinguishing ions at m/z 72 and 28 (C(2)H(4)) followed by loss of the carboxylic group. Diagnostic fragmentation pathways of the silver complexes of the saponins allow successful identification of the two types of saponins from the roots of Pulsatilla chinensis (Bunge) Regel.

Structural characterization and identification of ecdysteroids from Sida rhombifolia L. in positive electrospray ionization by tandem mass spectrometry

Seven ecdysteroids isolated from Sida rhombifolia L. were studied by electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) in the positive ion mode using an ion trap analyzer and high-performance liquid chromatography coupled with a diode-array detector (HPLC/DAD). The HPLC experiments were performed by means of a reversed-phase C(18) column and a binary mobile phase system consisting of water (containing 0.05% formic acid) and acetonitrile (containing 0.05% formic acid) under gradient elution conditions. According to mass spectral features and the substitution at C-2, C-20, C-24 and C-25, ecdysteroids in S. rhombifolia were classified into three sub-groups. Structural identification of these three sub-groups of ecdysteroids was established by LC/multi-stage ion trap mass spectrometry on-line or off-line. The fragmentation patterns of ecdysteroids yielded ions of successive loss of 1-4 water molecules. Furthermore, ions corresponding to the complete loss of the side chain at C-17 will help to identify the sub-groups of ecdysteroids in addition to containing a hydroxyl moiety at one of the above-mentioned positions. Based on the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by ESI-MS(n) spectra, a total of nine naturally occurring ecdysteroids were identified, of these two are identified for the first time in S. rhombifolia.

Characterization of spirostanol saponins in Solanum torvum by high‐performance liquid chromatography/evaporative light scattering detector/electrospray ionization with multi‐stage tandem mass spectrometry

High-performance liquid chromatography with an evaporative light scattering detector and electrospray ionization multistage tandem mass spectrometry (HPLC/ELSD/ESI-MS(n)) was used to identify spirostanol saponins in a saponin extract of Solanum torvum. The fragmentation behavior of saponins was studied using ESI-MS(1-3) in positive ion mode. The MS(n) spectra of the [M+H](+) ions provide structural information including aglycone type and the nature and sequence of sugars. The use of ELSD allowed the profiling of the nonchromophore-containing saponins in this plant. The MS analysis established in this study with known saponins was successfully applied to tentatively identify two new siprostanol glycosides, neosolaspigenin 6-O-beta-D-quinovopyranoside and solagenin 6-O-[beta-D-xylopyranosyl-(1 --> 3)-O-beta-D-quinovopyranoside].

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.

A novel rearrangement in ESI-MS n of spirocyclic pentaerythritol di(phosphate monoamides)

Several spirocyclic pentaerythritol di(phosphate monoamides) as intumescent flame retardants were synthesized and analyzed by electrospray ionization multistage tandem mass spectrometry. A novel amino group migration from the phosphoryl group to the two methylenes was observed. This migration is believed to be a general pathway for ions with the small size and electronic donating alkyl groups of spirocyclic pentaerythritol di(phosphate monoamides), which is assisted by twice nucleophilic substitutions. Steric and electronic effects of alkyl groups might be a key factor responsible for this migration.

Rapid structural determination of modified pregnane glycosides from Cynanchum forrestii by liquid chromatography–diode-array detection/electrospray ionization multi-stage tandem mass spectrometry

The fragmentation behaviors of the two types of modified pregnane glycosides from Cynanchum forrestii were investigated by positive ion electrospray ionization multi-stage tandem mass spectrometry equipped with an ion trap analyzer. The spectral data further illuminated the predominance of ESI-MS n technique on the identification of pregnane glycosides, especially of two sorts of modified pregnane glycosides with aglycone skeletons of 13,14:14,15-disecopregnane-type and 14,15-secopregnane-type, which differed in the presence of the characteristic [M−46+Na] + ion. For sugar residues, the fragment ions were analyzed and some possible fragmentation pathways were proposed, especially for 3-demethyl-2-deoxythevetose, the glycosidic cleavage reaction was easier to occur than those of other sugar units in its moiety. The natures and differences of the pregnane cores, and the types and linked sequences of sugar residues were illustrated. According to these conclusions, eight new pregnane glycosides in the fraction of Cynanchum forrestii were structurally elucidated by HPLC/HRMS and HPLC–DAD/ESI-MS n techniques. Results of the present studies can benefit the rapid identification and structural determination of analogous constituents in crude plant extracts.

LC-MS Method for Structural Characterization of Ecdysteroids in Sida rhombifolia L.

The genus Sida is used extensively in the herbal medicine industry [1]. Roots and aerial parts of S. rhombifolia are used in the treatment of gonorrhoea, anti-soud, diuretic, aphrodisiac and rheumatism [1]. Ecdysteroids are insect moulting hormones [2], and are generally considered to have a significance and specific role in the plant-insect chemical interactions [2]. Ecdysteroids isolated from Sida rhombifolia L. were studied by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) in the positive ion mode using ion trap analyzers and high performance liquid chromatography coupled with photodiode array detector (HPLC-PDA). The HPLC experiments were performed by means of a reversed-phase C18 column and a binary mobile phase system consisting of water (containing 0.05% formic acid) and acetonitrile (containing 0.05% formic acid) under gradient elution conditions. According to the substitution at C-2, C-5, C-20 and C-24, ecdysteroids in S. rhombifolia were classified into 4 sub-groups. Structural identification of these four sub-groups of ecdysteroids was established by multi-stage ion trap mass spectrometry. The fragmentation patterns of ecdysteroids yielded ions of successive loss of 1–3 water molecules. Furthermore, ions corresponding to the complete loss of the side chain at C-17 will help to identify the sub-groups of ecdysteroids in addition to containing a hydroxy moiety at one of the above mentioned positions. Based on the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by electrospray multi-stage mass spectrometry spectra, a total of eleven naturally occurring ecdysteroids were identified, of these four are identified for the first time in S. rhombifolia. Acknowledgements: This research is funded in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 2 U01 FD 002071-07.

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.

Structural analysis of platycosides in Platycodi Radix by liquid chromatography/electrospray ionization-tandem mass spectrometry

Platycosides extracted from Platycodi Radix were analyzed by HPLC coupled with electrospray ionization multistage tandem mass spectrometry (HPLC/ESI-MS n ). Predominant [ M + Na] + ions in positive mode and [ M − H] − ions in negative mode in the direct ESI-MS spectra of extract provided information on molecular weights, but minor components and isomers could not be discriminated. However, combining HPLC and ESI-MS n , allowed eleven platycosides, including four acetylated platycodin isomers and two prosapogenines to be analyzed. During MS 2 analysis conducted to elucidate the structures of platycosides, fragment ions provided information on sugar moieties attached at C-28 of triterpene structure of the platycosides. Glycosidic bond cleavages at C-3 were revealed by fragment ions in MS 3 spectra. Some characteristic fragment ions not related to sugar bond cleavage revealed that an esterified triterpene is linked to sugars at C-28. The only sugar ring-cross cleavage corresponding to 90 Da in the negative MS 2 spectrum took place at an arabinosyl sugar moiety. By using HPLC/ESI-MS n , three acetylated platycosides in Platycodi Radix extract were newly identified.

Electrospray ionization ion‐trap time‐of‐flight tandem mass spectrometry of two furofurans: sesamin and gmelinol

High-resolution electrospray ionization multistage tandem mass spectrometry (MS(1-7)) in positive ion mode was used to determine the accurate masses and the fragmentation pathways of two furofurans, sesamin and gmelinol. The protonated molecules [M+H]+ were absent in the conventional mass spectra of both compounds, but two characteristic ions, [M+H-H(2)O]+ and [M+H-H(2)]+, were always observed. Successive losses of CH(2)O and CO are the common characteristic fragmentations. Based on the exact masses acquired from 21 different tandem mass spectra, two or three fragmentation pathways for each compound are proposed. The consecutive losses of two H(2)O molecules and one H(2) molecule readily take place from the furan rings for both sesamin and gmelinol, resulting in the absence of the protonated molecules in the single-stage experiments. HCHO loss is observed at least three times in the tandem mass spectra, mainly from methylenedioxy groups (OCH(2)O) for sesamin but only from tetrahydrofuran rings for gmelinol. Moreover, CO loss is found at least three times in the tandem mass spectra of both sesamin and gmelinol from the 3,4-methylenedioxyphenyl (ArOCH(2)O) moieties for sesamin and from both the dimethoxyphenyl and the tetrahydrofuran ring moieties for gmelinol. In addition, the disubstituted benzyl cation ArCH(2)+ at m/z 135 for sesamin and at m/z 151 for gmelinol was found in the MS(3) spectra of both sesamin and gmelinol, which is very helpful in the identification of the compositions of 3,4-disubstituted groups on the benzene rings of the furofurans.

Characterization and identification of triterpenoid saponins in crude extracts from Clematis spp. by high‐performance liquid chromatography/electrospray ionization with multi‐stage tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC/ESI-MS(n)) was applied to characterize and identify triterpenoid saponins in crude extracts from nine species of Clematis L. After separation on a Zorbax SB-C(18) column, negative ESI-MS experiments were performed. The quasi-molecular ions [M-H]-, [M+Cl]- and [M+HCOO]- were observed in the full-scan MS spectra of all compounds. The MS(n) (n = 2-4) data of the [M-H]- ions provided structural information on the sugar sequence of the oligosaccharide chains and on the aglycone of the saponins. In addition, the fragmentation mechanisms could be deduced from the fragment ions. As a result, eight saponins were unambiguously identified in C. ganpiniana by comparison with reference compounds. In addition, a new compound was tentatively identified as 3-O-ribopyranosyl --> rhamnopyranosyl --> (glucopyranosyl) --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 1), and another one was tentatively deduced to be 3-O-glucopyranosyl --> ribopyranosyl --> rhamnopyranosyl --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 5) from the genus Clematis L. for the first time. By ESI-MS(n), non-isomeric saponins could be discriminated rapidly. It is of interest that cleavage preferentially occurrs at the ester bond at C-28 and the charge is easy to transfer onto the oligosaccharide chain when the ester bond of a monodesmosidic saponin like HNH cleaves.

The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography–diode array detector coupled with electrospray ionization/mass spectrometry

An high performance liquid chromatography–diode array detector coupled with electrospray ionization/mass spectrometry (HPLC–DAD/MS) based method has been developed for the simultaneous determination of nine coumarin compounds, nodakenin ( 1), peucedanone ( 2), marmesin ( 3), decursinol ( 4), 7-hydroxy-6-(2 R-hydroxy-3-methylbut-3-enyl)coumarin ( 5), demethylsuberosin ( 6), decursin ( 7), decursinol angelate ( 8) and isoimperatorin ( 9) in the Korean medicinal herb, Cham-Dang-Gui, the dried root of Angelica gigas (Umbelliferae). The methanol extracts were analyzed by HPLC using a reversed-phase C18 column (5 μm, 4.5 mm × 250 mm) using a gradient acetonitrile–water solvent system at a flow rate of 1.0 ml/min. The analysis of six coumarins ( 1, 3, 4 and 6– 8) with DAD was done at 330 nm and showed excellent linearity ( r 2 = 0.998–0.999) in a range of 0.2–250 μg/ml for all the compounds. The average recoveries ( n = 3) were between 96.5% and 110.8%. Identification of each peak was also discussed with the electrospray ionization multi-stage tandem mass spectroscopy (ESI-MS n ). The amount of these coumarin compounds was evaluated in A. gigas samples. Meanwhile, three coumarins ( 2, 5 and 9) could not been quantified by DAD because these peaks were overlapped with others. Determination of these compounds could be successfully accomplished with the HPLC–ESI/MS in selected ion monitoring/selected reaction monitoring mode.

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.

A fragmentation study of podophyllotoxin and its 4′‐demethyl‐4β‐substituted derivatives by electrospray ionization ion‐trap time‐of‐flight tandem mass spectrometry

High-resolution electrospray ionization multistage tandem mass spectrometry (MS(1-9)) was used to determine the accurate masses and the fragmentation pathways of protonated podophyllotoxin (1) and its corresponding 4'-demethyl-4beta-substituted derivatives (2-4). The protonated molecules, [M + H](+), of all the four compounds were observed in the conventional single-stage mass spectra. Two fragmentation pathways, that appear to be characteristic of the four compounds, are proposed on the basis of their multistage tandem mass spectrometric data. The characteristic elimination, from the precursor protonated ions, of the neutral groups 4-R(1)H, 1-ArH, CO, CH(2)O and C(4)H(4)O(2), in which R is located on C-4, is the common elimination, and the product ions at m/z 267, 239, 229, 181, 173, 153, 143 and 115 are the common diagnostic masses. The elimination of the R(1) group substituent located on the C-4 position of compounds 1-4 has a significant influence on the fragmentation pathway obtained in the conventional single-stage mass spectra. A large R(1) group would be unfavorable for this elimination, unless the collision energy is raised. Apart from the common fragmentations obtained for the protonated molecules 1-4, significant additional product ions were detected in the various multistage tandem mass spectrometric analyses, particularly in the case of the product ions derived initially from the phenolic hydroxyl group of 2-4, which are different from those of 1. Based on these additional formed product ions, several additional fragmentation pathways for 1 or 2-4 are also presented.