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.     

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].     

Rapid identification of C21 steroidal saponins in Cynanchum versicolor Bunge by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with on-line electrospray ionization tandem mass spectrometry (LC/ESI-MSn) were performed to elucidate the clearage rule of nine investigated C21 steroidal saponins and identify them in the saponin fraction of 90% ethanolic extracts from the root and rhizome of Cynanchum versicolor Bunge. The fragments of C21 steroidal saponins in positive and negative ESI-MSn were used to deduce their mass spectral fragmentation mechanisms, and their structures were further confirmed by ESI-MSn in positive mode. The MSn spectra of the [M+Na]+ ions for saponins provided a wealth of structural information on glycosidic bond cleavage, which allowed a straightforward interpretation of spectra, with respect to the identifications of features such as the sequences of sugars attached to saponins and sugar type. By using LC/ESI-MSn, nine C21 steroidal saponins were detected in the saponin fraction of C. versicolor, and an isomer of atratoglaucoside A was elucidated simultaneously. All nine compounds showed an abundant ion for the loss of 46 Da (HCOOH) from [M+Na]+. The losses of monosaccharide sequences and aglycone as neutral fragmentation from [M+Na-HCOOH]+ were also acquired as the characteristic ions of these C21 steroidal saponins. It provided important information on monosaccharide sequences and in particular on sugar types and could be used to identify and elucidate other C21 steroidal saponins. These studies allowed us to rapidly identify C21 steroidal saponins from Radix cynanchi atrati. It is indicated that the described method had wide applicability to rapidly screen and provide structural confirmation on C21 steroidal saponins in crude materials.     

Rapid structural characterization of triterpenoid saponins in crude extract from Symplocos chinensis using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Triterpenoid saponins in bioactive crude extract from Symplocos chinensis were rapidly identified using electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with sequential mass spectrometry (LC-MSn). According to the characteristic fragmentation behavior of known glucuronide-type triterpenoid saponins isolated from this plant, a total of fourteen constituents in the crude extract were structurally characterized on the basis of their retention time and tandem mass spectrometric analysis, including five pairs of naturally occurring isomers. Except one known saponin formerly obtained, the other constituents were new compounds. The analytical method of LC-MSn combined with ESI-MSn in positive and negative ion modes has been developed for the direct structural elucidation of triterpenoid saponins of this kind in plant extracts.     

Characterization of steroidal saponins in crude extract from Dioscorea nipponica Makino by liquid chromatography tandem multi-stage mass spectrometry

The liquid chromatography-electrospray ionization-tandem multi-stage mass spectrometry (LC-ESI-MSⁿ) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C₁₈ column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [M − H]⁻ ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [M − H]⁻ ions at m/z 1063, 1045, 901 and 1047, respectively, were observed during the LC-ESI(−)-MS analysis, and three groups of them except the pair of isomers with the [M − H]⁻ ions at m/z 1047 could be differentiated by LC-ESI(−)-MS³. Furthermore, the ESI-MSⁿ fragmentation behaviors of the [M + Li]⁺ ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [M − H]⁻ ion at m/z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.     

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.     

Determination of Tripamide in human urine by high-performance liquid chromatography and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Tripamide is a drug widely used in clinical practice for the treatment of hypertension and edema. This work evaluated a screening method for Tripamide and its urinary metabolites in human urine, using high-performance liquid chromatography diode-array detection (HPLC/DAD). Identification of these metabolites was investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) after dosing with 15 mg Tripamide. Acid hydrolysis showed that Tripamide is conjugated in the body. Two suspected metabolites were detected by HPLC/DAD. HPLC/ESI-MS/MS analysis suggested that these metabolites were probably hydroxylated together with loss of the -NH(2) group and dehydrogenation. These results will be useful in confirmation methods for Tripamide in doping control.     

Characterization and identification of steroidal alkaloids in the Chinese herb Veratrum nigrum L. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry

Electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was performed to study the fragmentation behaviour of seventeen steroidal alkaloids (4 protoverine-type alkaloids, 10 germine-type alkaloids and 3 zygadenin-type alkaloids) from the Chinese herb Veratrum nigrum L. The MS(n) spectra of the [M+H](+) ions for steroidal alkaloids provided a wealth of structural information on the substituted groups. In positive ion mode, the three types of alkaloids showed very different characteristic ions: m/z 436 or 418 for protoverine-type alkaloids; m/z 438, 420 or 402 for germine-type alkaloids; m/z 440 or 422 for zygadenin-type alkaloids. These fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the primary compounds in methanolic extracts of the herb of Veratrum nigrum L. were investigated by using liquid chromatography (LC)/ESI-MS(n). As a result, 21 steroidal alkaloids (5 protoverine-type alkaloids, 14 germine-type alkaloids and 2 zygadenin-type alkaloids) were selectively identified from 27 determined peaks. Eleven compounds were unambiguously identified by comparing with standard compounds and ten compounds were tentatively identified or deduced according to their MS(n) data. Two of these compounds (xingangermine and deacetyl xinganveratrine) were found to be novel steroidal alkaloids. In addition, the chemical structures of two pairs of steroidal alkaloid isomers were deduced by comparing their fragment ions. Given the important structural information of known and unknown steroidal alkaloids in crude herbal extracts, this study is useful for identifying these types of steroidal alkaloids in crude materials rapidly and selectively.     

Characterization of compounds from the roots of Saposhnikovia divaricata by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

In this study, four types of compounds including coumarins, chromones, furoylmethyl amino acid derivative and benzofuran glycoside were isolated from the roots of Saposhnikovia divaricata. The electrospray ionization (ESI) mass spectral fragmentation pathways of these compounds were proposed. In particular, the ESI-MS(n) fragmentation behavior of linear dihydrofurocoumarins, dihydrofuro- and dihydropyranochromones were deduced in detail. For the linear dihydrofurocoumarins, the fragmentation was triggered by the initial loss of the C-4' substituting group. Then, the characteristic ions were observed followed by the losses of 15, 18, 28 and 46 Da. It is noteworthy that the elimination of H(2)O (18 Da) from the cleavage of the dihydrofuran ring is reported for the first time. For the linear dihydrofurochromones, characteristic eliminations of 18, 48 and 72 Da were observed. The loss of 18 Da could arise from two different fragmentation pathways, and the observed ion was composed of a mixture of two different structural ions. For the linear dihydropyranochromones, it was found that the dihydropyran ring was converted into the pyran ring by the elimination of the C-3' substituting group. This fragmentation was followed by the diagnostic losses of 18, 28, 42 and 54 Da in tandem mass spectrometry. The above fragmentation rules were successfully applied for the analysis of the chemical constituents of the roots of Saposhnikovia divaricata. A total of 32 compounds were identified or tentatively characterized by HPLC/DAD/ESI-MS(n). Among them, eight compounds were new and seven compounds were reported from that genus for the first time.     

Characterization of G-rich and T-rich oligonucleotides using ion-pair reversed-phase high-performance liquid chromatography/tandem electrospray ionization mass spectrometry

Characteristics of G-rich and T-rich oligonucleotides were investigated to compare their retention time, total ion current (TIC) intensity, charge-state distribution and product ion using ion-pair reversed-phase high- performance liquid chromatography/tandem electrospray ionization mass spectrometry (IP-RP-HPLC/ESI-MS) at room temperature. Three commonly used mobile phases for the analysis of oligonucleotides, triethylammonium acetate (TEAA), triethylammonium bicarbonate (TEAB) and triethylammonium hexafluoroisopropanol (HFIP) have been utilized. Retention time of G-rich and T-rich oligonucleotides was significantly different in TEAA and TEAB buffer systems, while in the HFIP buffer system it was affected more by the length of oligonucleotides. On the other hand, the ESI-MS ion abundance in the HFIP buffer system was higher than that in both TEAA and TEAB buffers. The TIC intensity of T-rich oligonucleotides was much higher than that of G-rich oligonucleotides in all mobile phases. In addition, much higher charge-state fragments were observed in HFIP buffer system than that in the case of TEAA and TEAB buffer systems. Product ions of both G-rich and T-rich oligonucleotides were affected by charge state of parent ions and collision energy.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

Porphomethene inhibitor of uroporphyrinogen decarboxylase: analysis by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

An uroporphomethene inhibitor of uroporphyrinogen decarboxylase, characterized by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry, was reported recently (Phillips et al., Proceedings of the National Academy of Sciences of the United States of America 2007; 104: 5079-5084). Close examination of the tandem mass spectrometric fragmentation pattern of the compound showed that it is not a tetrapyrrole or an uroporphyrinogen or uroporphyrin related molecule. The product ion spectrum showed a fragmentation pattern typical of a poly(ethylene glycol) structure. Characteristic fragmentations of the side-chain acetic acid and propionic acid substituents of a uroporphyrin or uroporphyrinogen derivative were absent.     

High-resolution liquid chromatography/electrospray ionization time-of-flight mass spectrometry combined with liquid chromatography/electrospray ionization tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber

Grape antioxidant dietary fiber (GADF) is a dietary supplement that combines the benefits of both fiber and antioxidants that help prevent cancer and cardiovascular diseases. The antioxidant polyphenolic components in GADF probably help prevent cancer in the digestive tract, where they are bioavailable. Mass spectrometry coupled to liquid chromatography is a powerful tool for the analysis of complex plant derivatives such as GADF. We use a combination of MS techniques, namely liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) on a triple quadrupole, for the identification of the polyphenolic constituents of the soluble fraction of GADF. First, we separated the mixture into four fractions which were tested for phenolic constituents using the TOF system in the full scan mode. The high sensitivity and resolution of the TOF detector over the triple quadrupole facilitate the preliminary characterization of the fractions. Then we used LC/ESI-MS/MS to identify the individual phenols through MS/MS experiments (product ion scan, neutral loss scan, precursor ion scan). Finally, most of the identities were unequivocally confirmed by accurate mass measurements on the TOF spectrometer. LC/ESI-TOF-MS combined with MS/MS correctly identifies the bioactive polyphenolic components from the soluble fraction of GADF. High-resolution TOF-MS is particularly useful for identifying the structure of compounds with the same LC/ESI-MS/MS fragmentation patterns.     

Characterization of ageing products of ester-based synthetic lubricants by liquid chromatography with electrospray ionization mass spectrometry and by electrospray ionization (tandem) mass spectrometry

Ageing products of a commercial jet engine oil based on pentaerythritol tetraesters which were formed upon operation in an aviation turbine were detected by electrospray ionization mass spectrometry (ESI-MS) and characterized by LC-ESI-MS. The fatty acid composition of these ageing products was investigated by ESI-MS-MS analysis. The ammonium adducts of the newly formed pentaerythritol tetraester degradation products were found to be suitable parent ions for further structure elucidation work. ESI-MS, LC-ESI-MS and ESI-MS-MS proved to be versatile tools to study the chemical composition (distribution of homologues) as well as the mechanism of ageing of ester based lubricants on a molecular level. Due to its high sensitivity, ESI-MS can also be used to characterize and identify trace levels of ester-based lubricants.     

Quantitation of glutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. It is present in practically all cells and has several important roles, such as preventing the oxidation of the sulfhydryl groups of proteins within a cell. Evidence for GSH deficiency or depletion has been found in a variety of diseases and toxicity-related studies, including diabetes and induction of oxidative stress to form reactive oxygen species which cause DNA, lipid, and protein oxidations. A simple, selective, and sensitive analytical method for measuring low levels of GSH in biological fluids would therefore be desirable to conduct GSH deficiency or depletion-related mechanistic toxicity studies. Here a method for both low- and high-level quantitation of GSH from cultured cells and rat liver tissues via liquid chromatography/positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed. The lower limit of quantitation (LOQ) of the method was 5 ng/mL. The method is linear over a wide dynamic concentration range of 5.0 to 5000.0 ng/mL, with a correlation coefficient R2 > 0.99. The intra-day assay precision relative standard deviation (RSD) values for all quality control (QC) samples were < or =16.31%, with accuracy values ranging from 94.13 to 97.80%. The inter-day assay precision RSD values for all QC samples were < or =15.94%, with accuracy values ranging from 94.51 to 100.29%. With this method, low levels of GSH from diethyl maleate (DEM)-treated mouse lymphoma cells, and GSH in rat liver tissues, were quantified.     

Congener-specific analysis of hexabromocyclododecane by high-performance liquid chromatography/electrospray tandem mass spectrometry

A congener-specific method based on high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ES-MS/MS) in the negative ion mode was developed for the analysis of hexabromocyclododecane (HBCDD). On a C(18) analytical column, with a methanol/water mobile phase, the alpha-isomer was completely resolved from the beta- and gamma-isomers while the beta- and gamma-isomers were sufficiently resolved at half their peak heights. The ES spray voltage strongly influenced the intensity of the ion signal. For MS, a source temperature of 500 degrees C and a collision energy of 50 eV were found to be optimum for the [M-H](-) to Br(-) transition. Run-to-run and day-to-day (n = 3) variability was minimal, with relative standard deviations of 2.6-4.1 and 2.4-4.4%, respectively. The limit of detection was 4-6 pg on-column. When applied to tissue samples from Lake Winnipeg fish both alpha- and gamma-isomers of HBCDD were found in low-ng/g (lipid corrected) concentrations.