Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profiling

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.     

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 glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Determination of phenolic compounds in rose hip (Rosa canina) using liquid chromatography coupled to electrospray ionisation tandem mass spectrometry and diode-array detection

Liquid chromatography coupled with negative and positive electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) and diode-array detection (DAD) was used for determination of phenols in rose hip (Rosa canina) extract. ESI mass spectra of the chromatographically separated phenols gave the molecular weight of the compounds through prominent [M - H](-) ions for most of the compounds and M(+) ions for the anthocyanins. Collision induced dissociation (CID) of the [M - H](-) (or M(+)) precursor ions yielded product ions which determined the molecular weight of the aglycones. In-source fragmentation followed by CID of the resulting deprotonated aglycone ([A - H](-)) provided product ions for the identification of the unconjugated phenols. The identification was based on comparison with product ion spectra of commercial standards. UV-diode-array spectra were used for identity confirmation. This combined approach allowed the identification in rose hip extract of an anthocyanin, i.e. cyanidin-3-O-glucoside, several glycosides of quercetin and glycosides of taxifolin and eriodictyol. Phloridzin was identified, and several conjugates of methyl gallate were also found, one of which was tentatively identified as methyl gallate-rutinoside. Catechin and quercetin were found as the aglycones in the extract.     

Effect of phenylalanine on the fragmentation of deprotonated peptides

The fragmentation reactions of a variety of deprotonated dipeptides and tripeptides containing phenylalanine have been studied using energy-resolved collision-induced dissociation, isotopic labeling and MS/MS/MS experiments. The benzyl a-group has a substantial effect on the fragmentation reactions observed. When the phenylalanine is in the C-terminal position of dipeptides or tripeptides a major fragmentation reaction is elimination of neutral cinnamic acid to from a deprotonated amino acid amide (c1 ion) for dipeptides and a deprotonated dipeptide amide (c2 ion) for tripeptides. Fragmentation of the [M - H]- ions of tripeptides with phenylalanine in the central position also results in substantial formation of the deprotonated amide of the N-terminal amino acid residue. When the phenylalanine residue is in the N-terminal position elimination of C7H8 from the [M - H - CO2]- ion and formation of the benzyl anion become important fragmentation pathways. Sequence ions frequently observed are the y1 ions, "b2 ions and a3-Nt ions.     

Solid-phase microextraction liquid chromatography/tandem mass spectrometry for the analysis of chlorophenols in environmental samples

Liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (LC/APCI-MS), using negative ion detection in a triple quadrupole instrument, was used for the determination of chlorophenols (CPs) in environmental samples. In-source collision-induced dissociation (CID) was compared with MS/MS fragmentation. In general, less fragmentation was observed in MS/MS as compared with in-source CID, with the latter providing more intense fragment ions due to chemical ionisation. Under MS/MS conditions [M - H - HCl](-) was the main fragment ion observed for all compounds except for pentachlorophenol, which showed no fragmentation. For multiple reaction monitoring (MRM) acquisition mode, the transition from [M - H](-) to [M - H - HCl](-) was selected, leading to detection limits down to 0.3 ng injected. Direct and headspace-solid-phase microextraction (HS-SPME) were used as preconcentration procedures for the analysis of CPs in wood and in industrially contaminated soils. CPs were quantified by standard addition, which led to good reproducibility (RSD between 4 and 11%) in both SIM and MRM modes, and detection limits down to ng/g. The combination of MS/MS and in-source CID allowed confirmation of the presence of CPs in environmental samples.     

Electrospray ionisation mass spectrometric study of degradation products of quercetin, quercetin-3-glucoside and quercetin-3-rhamnoglucoside, produced by in vitro fermentation with human faecal flora

Eight phenolic compounds, obtained by in vitro fermentation of quercetin, quercetin-3-glucoside and quercetin-3-rhamnoglucoside were analysed by electrospray ionisation mass spectrometry (ESI-MS). Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) was performed on the [M - H]- precursor ions to obtain specific fragmentation. Typical fragmentation of the phenolic acids was loss of 44 (CO2) and 18 (H2O) u. Production of m/z 108 by loss of neutral radicals, e.g. HCO2, CH3 or HCO, was also favoured. Structures of the compounds, numbered 1-8, were suggested based on the fragmentation patterns.     

Characterization of acylated flavonoid-O-glycosides and methoxylated flavonoids from Tagetes maxima by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Liquid chromatography coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) employing a triple quadrupole mass spectrometer was used in the structural determination of acylated flavonoid-O-glycosides and methoxylated flavonoids occurring in Tagetes maxima. The compounds were identified by experiments in full scan mode (MS), and tandem mass experiments (MS/MS) of precursor ion scan, product ion scan, and neutral loss scan modes. In order to characterize the aglycones of the flavonoid glycosides, in-source fragmentation of the deprotonated molecule [M-H]- followed by product ion scan of the resulting aglycone [A-H]- were performed. This combined approach allowed the identification of 51 phenolic compounds, including flavonoid-O-glycosides acylated with galloyl, protocatechuoyl, coumaroyl or caffeoyl groups, methoxylated flavonoids, and hydroxycinnamic acid and phenolic acid derivatives, none of them previously reported in Tagetes maxima.     

Analysis of fungal cyclopentenone derivatives from Hygrophorus spp. by liquid chromatography/electrospray-tandem mass spectrometry

Fruitbodies of the genus Hygrophorus (Basidiomycetes) contain a series of anti-biologically active compounds. These substances named hygrophorones possess a cyclopentenone skeleton. LC/ESI-MS/MS presents a valuable tool for the identification of such compounds. The mass spectral behaviour of typical selected members of this group under positive and negative ion electrospray conditions is discussed. Using the ESI collision-induced dissociation (CID) mass spectra of the [M + H]+ and [M - H]- ions, respectively, the compounds can be classified with respect to the substitution pattern at the cyclopentenone ring and the type of oxygenation at C-6 (hydroxy/acetoxy or oxo function) of the side chain. The elemental composition of the fragment ions was determined by ESI-QqTOF measurements. Thus, in case of the negative ion CID mass spectra an unusual loss of CO2 from the deprotonated molecular ions could be observed.     

Furofuranic glycosylated lignans: a gas-phase ion chemistry investigation by tandem mass spectrometry

Alkali metal cation adducts, [M+Alk](+), and [M-H](-) ions of four known glycosylated furofuran lignans, (+)-pinoresinol 4-O-beta-D-glucopyranoside, (+)-phylliroside, (+)-8-hydroxypinoresinol 4-O-beta-D-glucopyranoside, and (+)-8-hydroxypinoresinol 8-O-beta-D-glucopyranoside, recently isolated from Carex distachya, were generated by electrospray ionization and allowed to undergo collisionally activated dissociation (CAD) in a quadrupole ion trap (QIT) and in a triple quadrupole (TQ) mass spectrometer. CAD mass spectra of [M+Na](+) and [M+Li](+) adducts revealed the presence of structurally diagnostic product ions. CAD mass spectra of deprotonated glycosylated furofuran lignans showed the typical neutral loss of 162 Da when the glucose residue was bound to a phenolic oxygen atom. When glycosylation occurred at an alcoholic oxygen, as for (+)-8-hydroxypinoresinol 8-O-beta-D-glucopyranoside, a neutral loss of 180 Da represented the main fragmentation pathway. Selective hydrogen/deuterium (H/D) exchange of all the acidic hydrogen atoms of furofuran glycosides, performed by introducing lignan glycosides in D(2)O/CH(3)OD solutions, were employed to obtain information on the nature of the product ions generated during TQ/CAD processes. Energy-resolved TQ/CAD mass spectra of deprotonated lignan glycosides and their deprotonated aglycones were used in a qualitative way to infer information on the integrated energetic picture of CAD fragmentations and to investigate the mechanism of the predominant dissociation/isomerization processes. On the basis of the hypothesized fragmentation mechanisms, gas-phase features of the furofuran ring were derived. The presence of an OH substituent in the C8 position decreased the electron density in the adjacent C8' position, modifying the fragmentation pathway.     

Direct determination of resin and fatty acids in process waters of paper industries by liquid chromatography/mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS)-based methods were developed for the analysis of 10 resin acids and five fatty acids in process waters of paper industries. No fragmentation of target compounds was observed using atmospheric pressure chemical ionization (APCI) with negative ionization. The [M - H](-) ion permitted the individual quantification of fatty and aromatic resin acids, whereas the non-aromatic resin acids presented a single and common ion at m/z 301. Separation with two columns of different polarity permitted peak confirmation. The method that used a C(8) column with 2-propanol in the mobile phase allowed a certain separation and identification of the non-aromatic resin acids, whereas the method using a C(18) column provided detection limits 10-fold lower for fatty acids. Limits of detection were 0.10 ng for all compounds. Direct sample introduction was compared with liquid-liquid extraction, with similar recoveries (70-101%). Whereas slightly lower detection limits were obtained with liquid-liquid extraction, better reproducibility was observed for direct sample introduction. Resin and fatty acids were determined in process waters of several paper industries. Palmitic, dehydroabietic and non-aromatic resin acids were encountered in most water samples, at levels between 22 and 403 micro g l(-1). LC/MS with direct sample introduction was found to be a good alternative to traditional liquid-liquid extraction and gas chromatography for the analysis of such compounds since no derivatization was required and sample manipulation was minimal.     

Electrospray ionization mass spectrometry of ginsenosides

Ginsenosides R(b1), R(b2), R(c), R(d), R(e), R(f), R(g1), R(g2) and F(11) were studied systematically by electrospray ionization mass spectrometry in positive- and negative-ion modes with a mobile-phase additive, ammonium acetate. In general, ion sensitivities for the ginsenosides were greater in the negative-ion mode, but more structural information on the ginsenosides was obtained in the positive-ion mode. [M + H](+), [M + NH(4)](+), [M + Na](+) and [M + K](+) ions were observed for all of the ginsenosides studied, with the exception of R(f) and F(11), for which [M + NH(4)](+) ions were not observed. The signal intensities of [M + H](+), [M + NH(4)](+), [M + Na](+) and [M + K](+) ions varied with the cone voltage. The highest signal intensities for [M + H](+) and [M + NH(4)](+) ions were obtained at low cone voltage (15-30 V), whereas those for [M + Na](+) and [M + K](+) ions were obtained at relatively high cone voltage (70-90 V). Collision-induced dissociation yielded characteristic positively charged fragment ions at m/z 407, 425 and 443 for (20S)-protopanaxadiol, m/z 405, 423 and 441 for (20S)-protopanaxatriol and m/z 421, 439, 457 and 475 for (24R)-pseudoginsenoside F(11). Ginsenoside types were identified by these characteristic ions and the charged saccharide groups. Glycosidic bond cleavage and elimination of H(2)O were the two major fragmentation pathways observed in the product ion mass spectra of [M + H](+) and [M + NH(4)](+). In the product ion mass spectra of [M - H](-), the major fragmentation route observed was glycosidic bond cleavage. Adduct ions [M + 2AcO + Na](-), [M + AcO](-), [M - CH(2)O + AcO](-), [M + 2AcO](2-), [M - H + AcO](2-) and [M - 2H](2-) were observed at low cone voltage (15-30 V) only.     

Different behavior of dextrans in positive-ion and negative-ion mass spectrometry

A mass spectrometric (MS) comparative study of dextran samples using two different ionization techniques (matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI)) in both positive- and negative-ion modes is reported. The experiments were carried out with two polydisperse dextrans (1000 and 8800 Da) and isomaltotriose. In the positive-ion mode, the expected alkali metal ion adducts of dextrans were observed in both techniques. In contrast, the expected preferential formation of deprotonated molecules [M - H](-) was not confirmed in negative mode MALDI time-of-flight (TOF) MS, where the series of ions [M(x)- H +42](-) or [M(x+1)- H - 120](-), coming either from some addition or fragmentation, were observed. In both ionization techniques, these ions formed the main distributions of dextrans in the negative-ion mode. It seems that the negative molecular ions formed from the alpha1 --> 6 linkage of polyglucose oligomers easily decompose, and the product ions [M - H - 120](-) markedly dominate. The fragmentation experiments and especially the investigation of the fundamental role of the nozzle-skimmer potential in ESI-MS supported our explanation of the observed behavior because its higher values caused higher fragmentation. The experiments with isomaltotriose excluded any addition of 42 Da during the MS procedures, which is not distinguishable from the loss of 120 Da in the case of polydisperse dextrans. MALDI-TOFMS was found to be more sensitive for the detection of higher oligosaccharides and ESI-MS more useful for structural studies.     

Mass spectrometric and tandem mass spectrometric behavior of nitrocatechol glucuronides: A comparison of atmospheric pressure chemical ionization and electrospray ionization

The mass spectrometric (MS) and tandem mass spectrometric (MS/MS) behavior of six nitrocatechol-type glucuronides using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) was systematically studied, and the effect of operation parameters on the fragmentations are presented. The positive ion APCI- and ESI-MS spectra showed an intense protonated molecule and the respective negative ion spectra a deprotonated molecule with minimal fragmentation. The main fragment ions in the MS/MS spectra of the protonated and deprotonated molecules were [M + H - Glu]+ and [M - H - Glu]-, respectively, formed by the loss of the glucuronide moiety. The measured limits of detection indicated that ESI is a significantly more efficient ionization method than APCI in the negative and positive ion modes for the compounds studied. MS/MS was found to be less sensitive, but more reliable and simple than MS due to the absence of chemical noise.     

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.     

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]-.     

Mass spectral fragmentation of the intravenous anesthetic propofol and structurally related phenols

Propofol (2,6-diisopropyl phenol) is a widely used intravenous anesthetic. To define its pharmacokinetics and pharmacodynamics, methods for its quantitation in biological matrixes have been developed, but its pattern of mass spectral fragmentation is unknown. We found that fragmentation of the [M - H](-) ion (m/z 177) of propofol in both APCI MS/MS and ESI MS/MS involves the stepwise loss of a methyl radical and a hydrogen radical from one isopropyl side chain to give the most intense product ion, [M -H - CH(4)](-), at m/z 161. This two-step process is also the preferred mode of fragmentation for similar branched alkyl substituted phenols. This mode of fragmentation of the [M - H](-) ion is supported by three independent lines of evidence: (1) the presence of the intermediary [M - H - CH(3)](-) radical ion under conditions of reduced collision energy, (2) the determination of the mass of the predominant [M - H - CH(4)](-) product ion by high resolution mass spectrometry, and (3) the pattern of product ions resulting from further fragmentation of the [M - H - CH(4)](-) product ion. Phenols with a single straight chain alkyl substituent, in contrast, undergo beta elimination of the alkyl radical irrespective of the length of the alkyl chain, yielding the most intense product ion at m/z 106. This product ion represents a special case of a stable intermediary radical for the two-step process described for branched side chains, because further elimination of a hydrogen radical from the beta carbon is not possible.     

Qualitative analysis of phenolic compounds in apple pomace using liquid chromatography coupled to mass spectrometry in tandem mode

The occurrence of phenolic compounds in apple residues resulting from the juice industry was investigated to provide an alternative use for this raw material. For the identification of these compounds, liquid chromatography coupled to ionspray mass spectrometry in tandem mode (LC/MS/MS) with negative ion detection was used. The residues were first extracted and then chromatographed on Sephadex LH-20 to yield 13 fractions. Positive identification of the compounds was based on their retention times and mass spectra in full scan mode (MS), and in different MS/MS modes (product ion scan, precursor ion scan and neutral loss scan). In this way, 60 compounds, including cinnamic and benzoic acid derivatives and flavonoids, were identified, some of them not previously reported in apple waste.     

Dereplication of known pregnane glycosides and structural characterization of novel pregnanes in Marsdenia tenacissima by high‐performance liquid chromatography and electrospray ionization‐tandem mass spectrometry

In the search for novel natural products in plants, particularly those with potential bioactivity, it is important to efficiently distinguish novel compounds from previously isolated, known compounds, a process known as dereplication. In this study, electrospray ionization‐multiple stage tandem mass spectrometry (ESI‐MSⁿ) was used to study the behaviour of 12 pregnane glycosides and genins previously isolated from Marsdenia tenacissima, a traditional Chinese medicinal plant, as a basis for dereplication of compounds in a plant extract. In addition to [M + Na]⁺ and [M + NH₄]⁺ ions, a characteristic [M‐glycosyl + H]⁺ ion was observed in full‐scan mode with in‐source fragmentation. Sequential in‐trap collision‐induced dissociation of [M + Na]⁺ ions from 11,12‐diesters revealed consistent preferred losses of substituents first from C‐12, then from C‐11, followed by losses of monosaccharide fragments from the C‐3 tri‐ and tetrasaccharide substituents. A crude methanol extract of M. tenacissima stems was analysed using high‐performance liquid chromatography coupled to ESI‐MS. Several previously isolated pregnane glycosides were dereplicated, and the presence of an additional nine novel pregnane glycosides is predicted on the basis of the primary and fragment ions observed, including two with a previously unreported C₄H₇O C‐11/C‐12 substituent of pregnane glycosides. This study is the first report of prediction of the structures of novel pregnane glycosides in a crude plant extract by a combination of in‐source fragmentation and in‐trap collision‐induced dissociation and supports the usefulness of LC‐ESI‐MSⁿ not only for dereplication of active compounds in extracts of medicinal plants but also for detecting the presence of novel related compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of glucuronide conjugates of ketobemidone and its phase I metabolites in human urine utilizing accurate mass and tandem time-of-flight mass spectrometry

The glucuronide conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone were identified in human urine post-intravenous administration of Ketogan Novum. The human urine was extracted on a mixed-mode solid-phase micro-column before analysis with liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) and tandem MS (MS/MS). Accurate mass and collision-induced dissociation product ion spectra were used for identification of the glucuronide conjugates. Two different TOF mass spectrometers were used and the accurate mass measurements were performed on three separate days with each instrument. The accuracy of the mass measurements was better than 2.1 ppm for two out of three conjugates and the inter-day relative standard deviation was within +/-0.00049%. The MS/MS fragmentation patterns of the conjugates were in accordance with those of the synthetic aglycones and included peaks originating from the [M + H](+) ion of the respective aglycone.