Electrospray ionization ion-trap multiple-stage mass spectrometry of Quillaja saponins

Fifteen identified C-18 fatty acyl-containing saponin structures from Quillaja saponaria Molina have been investigated by electrospray ionization ion-trap multiple-stage mass spectrometry (ESI-IT-MS(n)) in positive ion mode. Their MS(1)-MS(3) spectra were analyzed and ions corresponding to useful fragments, important for the structural identification of Quillaja saponins, were recognized. A few key fragments could describe the structural variations in the C-3 and the C-28 oligosaccharides of the Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from the MS(1)-MS(3) spectra of these saponins, together with key fragments from these saponins and 13 previously investigated saponins, was constructed for the identification of structural elements in Quillaja saponins. Peak intensity ratios in MS(3) spectra were found to be correlated to structural features of the investigated saponins and is therefore of value for the identification of regioisomers.     

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MS(n) data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MS(n), non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools for rapid screening and structural assignment of saponins in plant extracts.     

Structural classification of acyl-substituted Quillaja saponins by electrospray ionisation ion trap multiple-stage mass spectrometry in combination with multivariate analysis

Thirty-eight saponins in two chromatographic fractions (QH-B and QH-C) from Quillaja saponaria Molina have been separated by a two-step high-performance liquid chromatography (HPLC) procedure and investigated by electrospray ionisation ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive ion mode. MS(2) and MS(3) spectra of the compounds were investigated by principal component analysis (PCA) and could be classified by partial least squares - discriminant analysis (PLS-DA) according to the structures of the oligosaccharides at C-3 and C-28 of the saponins. Four minor components with novel structures were found in a previously non-investigated fraction of QH-C. The structures of two of these components, J1 and J1a, were predicted by PLS-DA whereas the structures of the two others, J2 and J3, were only partly predicted. The structures of J1 and J1a were composed of structural elements found in the 34 known saponins whereas a new acyl substituent, not included in the training set used for calibration of the PLS-DA models, was found in J2 and J3, making these two components outliers. The complete structures of the four components were confirmed by monosaccharide analysis, MS(n) data and (1)H NMR spectroscopy.     

Characteristic fragmentation behavior of some glucuronide-type triterpenoid saponins using electrospray ionization tandem mass spectrometry

The fragmentation behavior of some glucuronide-type triterpenoid saponins from Symplocos chinensis, and their analogues escin Ia and Ib, were investigated by positive ion electrospray ionization tandem mass spectrometry using a quadrupole linear ion trap mass spectrometer. The fragmentation patterns of these saponins significantly changed in accordance with structural variations in the glucuronyl residue of the oligosaccharide chain. It was found that the carboxyl group and hydroxyl group at the C-3' position of the glucuronyl residue were the key sites for determining the fragmentation behavior of these compounds. When the carboxyl group was esterified, only the C(2alpha) ion, and no B(2alpha) ion, and cationized aglycone were observed. When the hydroxyl group at C-3' was acylated, the inherent cross-ring cleavage was hindered. However, glycosidic cleavages always occurred, regardless of the crucial structural variations. The results of the present studies can benefit the determination of trace triterpenoid saponins of this type in crude plant extracts, and also provide background information to aid the structural investigations of similar glycoconjugates.     

Fast screening of highly glycosylated plant sphingolipids by tandem mass spectrometry

The structural characterization of Glycosyl-Inositol-Phospho-Ceramides (GIPCs), which are the main sphingolipids of plant tissues, is a critical step towards the understanding of their physiological function. After optimization of their extraction, numerous plant GIPCs have been characterized by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) full scan analysis of negative ions provides a quick overview of GIPC distribution. Clear differences were observed for the two plant models studied: six GIPC series bearing from two to seven saccharide units were detected in tobacco BY-2 cell extracts, whereas GIPCs extracted from A. thaliana cell cultures and leaves were less diverse, with a dominance of species containing only two saccharide units. The number of GIPC species was around 50 in A. thaliana and 120 in tobacco BY-2 cells. MALDI-MS/MS spectra gave access to detailed structural information relative to the ceramide moiety, the polar head, as well as the number and types of saccharide units. Once released from GIPCs, fatty acid chains and long-chain bases were analyzed by GC/MS to verify that all GIPC series were taken into account by the MALDI-MS/MS approach. ESI-MS/MS provided complementary information for the identification of isobaric species and fatty acid chains. Such a methodology, mostly relying on MALDI-MS/MS, should open new avenues to determine structure-function relationships between glycosphingolipids and membrane organization.     

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.     

Development of a comprehensive two-dimensional hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry system and its application in separation and identification of saponins from Quillaja saponaria

Hydrophilic interaction chromatography (HILIC) was used in two dimensions in a comprehensive two-dimensional HILIC hyphenated with a quadrupole time-of-flight mass spectrometry (HILICxHILIC-Q-TOF-MS) system for the analysis of complex samples of hydrophilic compounds. A TSKgel Amide-80 column was employed as the first dimension, and a short PolyHydroxyethyl A column was as the second dimension. The column system showed moderate orthogonality at defined operational conditions. A high speed Q-TOF-MS detector as a third complementary dimension significantly improved the peak capacity. The separation capability of the developed HILICxHILIC-Q-TOF-MS system was tested by separating an extract from Quillaja saponaria. The major components, quillaja saponins, in the extract were well identified by means of [M-H](-) ions, characteristic product ions, and their two-dimensional retention behaviors. Several pairs of isomers, which were often co-eluted on conventional LC-MS methods and had similar fragmentation characteristics in MS/MS spectra, were well separated on the two-dimensional system based on their different hydrophilicity. The developed comprehensive two-dimensional HILIC system demonstrates unique selectivity for hydrophilic compounds and satisfactory peak capacity and resolution for analogues by making sufficient use of two-dimensional separation plane.     

Oligosaccharide sequences in Quillaja saponins by electrospray ionization ion trap multiple-stage mass spectrometry

Ten different samples with 13 previously identified saponin structures from Quillaja saponaria Molina were investigated by electrospray ionization ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive and negative ion modes. Both positive and negative ion mode MS(1)-MS(4) spectra were analyzed, showing that structural information on the two oligosaccharide parts in the saponin can be obtained from positive ion mode spectra whereas negative ion mode spectra mainly gave information on one of the oligosaccharide parts. Analysis of MS(1)-MS(4) spectra identified useful key fragment ions important for the structural elucidation of Quillaja saponins. A flowchart involving a stepwise procedure based on key fragments from MS(1)-MS(3) spectra was constructed for the identification of structural elements in the saponin. Peak intensity ratios in MS(3) spectra were found to be correlated with structural features of the investigated saponins and are therefore of value for the identification of terminal monosaccharide residues.     

Identification and structural characterization of steroidal glycosides in Hoodia gordonii by ion-trap tandem mass spectrometry and liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry

Electrospray ion-trap tandem mass spectrometry (ESI-MS/MS) and high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) were used to identify and characterize eight C-21 steroidal glycosides in Hoodia gordonii. A generalized fragmentation pathway was proposed by comparing the spectra acquired for eight C-21 steroidal glycosides. The steroidal glycosides in Hoodia gordonii have been classified into two major core groups: hoodigenin A and calogenin. Using the ESI-TOF method, the major core peak ions generated by hoodigenin A glycosides are m/z 313 and 295 and by calogenin glycosides are m/z 479, 461, 299 and 281, respectively. In the MS/MS spectra, fragmentation reactions of the [M+Na](+) ion were recorded to provide structural information about the glycosyl and aglycone moieties. The data illustrates the ability of positive mode ESI for the identification of hoodigenin A and calogenin glycosides, including the nature of the hoodigenin A and calogenin core, the number of sugar residues and the type of saccharide moiety.     

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.     

Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.     

Characterisation of membrane phospholipids and glycolipids from a halophilic archaebacterium by high-performance liquid chromatography/electrospray mass spectrometry

Combined high-performance liquid chromatography and electrospray mass spectrometry (LC/ES-MS) has been used for direct characterisation of the polar membrane lipids in total lipid extracts from Halobacterium salinarium, a species of halophilic archaebacterium. The principle phospholipids found were the diphytanyl archaeol phosphatidylglycerol and diphytanyl archaeol phosphatidylglycerolphosphate methyl ester. The application of LC/ES-MS revealed the additional presence of diphytanyl archaeol phosphatidylglycerol sulphate The extracts also contained an archaeol glycolipid, initially detected in preliminary offline ES-MS studies, which was further characterised by LC/ES-MS and by product ion tandem mass spectrometry (MS/MS) as a sulphate ester of diglycosyl-2,3-di-O-phytanyl-sn-glycerol. Whilst archaeol phospho- and glycolipids containing a (C(20)-C(20))-isopranyl glycerol ether core predominated, LC/ES-MS of the extracts from Halobacterium salinarium indicated the presence of an analogue containing one double bond in its isoprenyl ether core as a minor component of the phosphatidylglycerolphosphate methyl ester fraction, providing a further example of the previously recognised existence of isoprenologues of diphytanyl archaeols which occur as minor components of archaebacterial membrane lipids. The value of these techniques in compositional analysis of archaebacterial lipid extracts is discussed.     

Tandem mass spectrometric analysis of a complex triterpene saponin mixture of Chenopodium quinoa

A nano-HPLC electrospray ionization multi-stage tandem mass spectrometry (nLC-ESI-MS/MS) approach was applied to a complex crude triterpene saponin extract of Chenopodium quinoa seed coats. In ESI-MS/MS spectra of triterpene saponins, characteristic fragmentation reactions are observed and allow the determination of aglycones, saccharide sequences, compositions, and branching. Fragmentation of aglycones provided further structural information. The chemical complexity of the mixture was resolved by a complete profiling. Eighty-seven triterpene saponins comprising 19 reported and 68 novel components were identified and studied by MS. In addition to four reported, five novel triterpene aglycones were detected and characterized according to their fragmentation reactions in ESI-MS/MS and electron ionization mass spectrometry (EI-MS). As a novelty fragmentation pathways were proposed and analyzed based upon quantum chemical calculations using a hybrid Hartree-Fock density functional method. Accuracy of the assignment procedure was proven by isolation and structure determination of a novel compound. As the relative distribution and composition of saponins varies between different cultivars and soils, the presented strategy allows a rapid and complete analysis of Chenopodium quinoa saponin distribution and composition, and is particularly suitable for quality control and screening of extracts designated for pharmaceutical, agricultural, and industrial applications.     

Rapid characterization of triterpene saponins from Conyza blinii by liquid chromatography coupled with mass spectrometry

Conyza blinii Le'vl is a medicinal herb used for the treatment of inflammation in Chinese folk medicine. Its major bioactive constituents are triterpene saponins, most of which contain 6–8 sugar residues. In this report, electrospray ionization tandem mass spectrometry fragmentation behaviors of bisdesmosidic triterpene saponins (conyzasaponin A, B, and C) were studied in both positive and negative ion modes with an ion‐trap mass spectrometer. In full scan mass spectrometry, these saponins gave predominant [M–H]^? and [M+Na]⁺ ions, which determined the molecular weights. In tandem mass spectrometry (MSⁿ, n = 2–4), the [M–H]^? and [M+Na]⁺ ions yielded fragments [Y_0α–H]^? and [B_α+Na]⁺, which were diagnostic for the structures of the triterpene skeleton and sugar chains. The structural elucidation was approved by accurate mass data using IT‐TOF‐MS. An interpretation guideline based on MSⁿ (n = 2–4) diagnostic ions was proposed in order to elucidate the chemical structures of unknown triterpene saponins in C. blinii extract. The saponins in C. blinii were separated by liquid chromatography with a methanol/acetonitrile/water solvent system, and then analyzed by ion‐trap and IT‐TOF mass spectrometers. Based on the interpretation guideline, a total of 35 triterpenoid saponins were tentatively identified. Among them, 15 saponins had been previously reported, and the other 20 saponins were reported from Conyza species for the first time. This study indicates that LC/MS is a powerful technology for the rapid characterization of complicated saponins in herbal extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

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).     

A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry

Liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) has been employed to identify carotenoid esters present in raw organic extracts of pigmented freshwater microalgae and to gain structural information on these compounds. In particular, acyl carotenoid derivatives of Haematococcus pluvialis and Euglena sanguinea have been characterised by tandem mass spectrometry (MS/MS) in a quadrupole ion trap. ESI-MS/MS allows recognition of the presence of carotenoid esters in complicated mixtures without any initial chromatographic work-up and without the need to use UV-Vis photo-diode array (PDA) detectors. Product ion scans of the [M + Na]+ ion lead to known neutral losses of the C7H8 and C8H10 residues from the conjugated polyene moiety of the carotenoid unit, that permit the unambiguous identification of the carotenoid itself. These structurally relevant ions are not observed in positive or negative ion APCI (atmospheric pressure chemical ionisation) mass spectra. Moreover, the several product ions observed in positive and/or negative ion ESI-MS/MS not only are a diagnostic signature of the main structural features of the acyl chains such as length, position and unsaturation, but also display the nominal mass of the parent xanthophyll. Our methodology has been validated (i) by using esters of astaxanthin obtained from off-line purification of the H. pluvialis extracts and structurally elucidated through proton nuclear magnetic resonance (1H-NMR) spectroscopy and (ii) by product analysis of esters by alkaline hydrolysis. The characterisation of the unknown carotenoid esters of E. sanguinea is a demonstration of the capabilities of this methodology.     

Determination of free fatty acids in chocolate by liquid chromatography with tandem mass spectrometry

This paper describes a rapid extraction method, based on a matrix solid-phase dispersion technique using diatomaceous earth as solid support and 50:50 (v/v) chloroform/methanol as extracting solvent, that can determine 11 free fatty acids in chocolate. The extraction procedure is followed by reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a normal-bore (4.6 mm i.d.) C-18 column and an electrospray interface operating in the negative ion mode. The tandem mass spectra of selected compounds show that charge-remote fragmentation (CRF) mechanisms are occurring; the intensities of the CRF reactions increase with the carbon number and degree of unsaturation of the fatty acids. Average recoveries, evaluated by the standard addition method, vary between 79-103%, and the estimated quantification limits are less than 153 ng/g. The proposed method has been used to analyse nine chocolate samples from various price ranges, bought from supermarkets.     

The detection of nicotine in a Late Mayan period flask by gas chromatography and liquid chromatography mass spectrometry methods

Several ancient Mayan vessels from the Kislak Collection of the US Library of Congress were examined for the presence of alkaloids. One of them, a codex‐style flask, bears a text that appears to read yo‐'OTOT‐ti 'u‐MAY, spelling y‐otoot 'u‐may 'the home of its/his/her tobacco'. Samples extracted from this Late Classic period (600 to 900 AD) container were analyzed by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) methods. Nicotine was identified as the major component of the extracts. LC/MS analyses also yielded signals due to nicotine mono‐oxides. The identities of the compounds were determined by comparison of the chromatographic and/or mass spectral characteristics with those from standards and literature data. High‐resolution high mass accuracy tandem mass spectrometry (MS/MS) spectra of protonated nicotine and nicotine mono‐oxides were measured to verify and to correct previous product ion assignments. These analyses provided positive evidence for nicotine from a Mayan vessel, indicating it as a likely holder of tobacco leafs. The result of this investigation is the first physical evidence of tobacco from a Mayan container, and only the second example where the vessel content recorded in a Mayan hieroglyphic text has been confirmed directly by chromatography/mass spectrometry trace analysis. Copyright © 2012 John Wiley & Sons, Ltd.     

On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.