Development of a rapid and validated method for investigating the metabolism of scoparone in rat using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry

Scoparone (6,7-dimethoxycoumarin) is known to have a wide range of pharmacological properties. In this study, a rapid and validated ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-QTof-MS) method was developed to investigate the metabolism of scoparone in rat for the first time. The new method reduced the sample handling and analytical time by three- to six-fold, and the detection limit by five- to 1000-fold, compared to published methods. Far more metabolites were detected and identified compared to published data, which were preliminarily identified as scopoletin, isoscopoletin, isofraxidin, and fraxidin, respectively, when subjected to tandem mass spectrometry analyses. It is found that the metabolic trajectory of scoparone in rat focused on phase I metabolism which is obviously different from published results, and revealed a wide range of pharmacological properties of scoparone partly attributed to the bioactivities of its metabolites.

Studies on Triterpenoids and Flavones in Glycyrrhiza uralensis Fisch by HPLC-ESI-MSn and FT-ICR-MSn

Seven compounds, four flavones and three triterpenoids from Glycyrrhiza uralensis Fisch. extract are identified by high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry (HPLC-ESI-MSn). The fragmentation pathways of these compounds are investigated by ESI-MSn and Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry (FT-ICR-MSn). Comparing the retention times (t(R)) and mass spectra with those of reference compounds, seven components are identified in Glycyrrhiza uralensis Fisch. and their MSn data proposed plausible schemes for their fragmentation. All the experimental results show that ESI-MSn and FT-ICR-MSn are powerful tools for the structural characterization of triterpenoids and flavones

Investigation of heptakis(2,6-di-O-methyl)-beta-cyclodextrin inclusion complexes with flavonoid glycosides by electrospray ionization mass spectrometry

The non-covalent complexes between three flavonoid glycosides (quercitrin, hyperoside and rutin) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). The 1:1 complexation of each flavonoid glycoside (guest) to the DM-beta-CD (host) was monitored in the negative ion mode by mixing each guest with an up to 30-fold molar excess of the host. The binding constants for all complexes were calculated by a linear equation in the order: DM-beta-CD:quercitrin > DM-beta-CD:rutin > DM-beta-CD:hyperoside. A binding model for the complexes has also been proposed based on the binding constants and tandem mass spectrometric data of these complexes.

Studies on electrospray mass spectrometry of some narcotic drugs

Using electrospray ionization (ESI) and tandem mass spectrometry techniques, the protonic positions in protonated molecular ions of some narcotic drugs were studied, The data of ESI/MSn experiments of morphine and deuterium-loaded morphine were first discussed. The protonic position was considered to locate on oxygen atom of cyclic ether in morphine molecular, Compared with the same and different: ions among morphine, codeine, acetylcodeine and dihydrocodeine, the protonic positions in protonated molecular ions of these compounds were further determined. The fragmentation mechanism of morphine in ESI/MSn: experiments was also repored in the paper, and there were similar dehydrolysis mechanism in gas phase or in liquid phase for morphine.

Comparison of the binding stoichiometries of positively charged DNA-binding drugs using positive and negative ion electrospray ionization mass spectrometry

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from freeDNAwere present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Pressurized electrochromatography coupled with electrospray ionization mass spectrometry for analysis of peptides and proteins

Pressurized capillary electrochromatography (pCEC) was coupled with electrospray ionization mass spectrometry (ESI-MS) using a coaxial sheath liquid interface. It was used for separation and analysis of peptides and proteins. The effects of organic modifier and applied voltage on separation were investigated, and the effects of pH value of the mobile phase and the concentration of the electrolyte on ESI-MS signal were investigated. The resolution and detection sensitivity with different separation methods (pCEC, capillary high-performance liquid chromatography) coupled on-line with mass spectrometry were compared for the separation of a peptide mixture. To evaluate the feasibility and reliability of the experimental setup of the system, tryptic digests of cytochrome c and modified protein as real samples were analyzed by using pCEC-ESI-MS.

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4'-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect.

Studies on alkaloids binding to GC-rich human survivin promoter DNA using positive and negative ion electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding of 13 alkaloids to two GC-rich DNA duplexes which are critical sequences in human survivin promoter. Negative ion ESI-MS was first applied to screen the binding of the alkaloids to the duplexes. Six alkaloids (including berberine, jatrorrhizine, palmatine, reserpine, berbamine, and tetrandrine) show complexation with the target DNA sequences. Relative binding affinities were estimated from the negative ion ESI data, and the alkaloids show a binding preference to the duplex with higher GC content. Positive ion ESI mass spectra of the complexes were also recorded and compared with those obtained in negative ion mode.

Studies of the intermolecular DNA triplexes of C+center dot GC and T center dot AT triplets by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry

Formation and stabilities of four 14-mer intermolecular DNA triplexes, consisting of third strands with repeating sequence CTCT, CCTT, CTT, or TTT, were studied by electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the gas phase. The gas-phase stabilities of the triplexes were compared with their CD spectra and melting behaviors in solution, and parallel correlation between two phases were obtained. In the presence of 20 mm NH4+ (pH 5.5), the formation of the TTT triplex was not detected in both solution and the gas phase.

Interactions of mitoxantrone with duplex and triplex DNA studied by electrospray ionization mass spectrometry

We have examined interactions between mitoxantrone (MXT) and DNA duplexes or triplexes with different base compositions by using electrospray ionization mass spectrometry (ESI-MS), respectively. MXT interacts preferentially with DNA duplexes compared to the triplexes. In the mass spectrum of the duplex-MXT mixture, the complex peaks dominated in the ratios of duplex/MXT of 1:1, 1:2 and 1:3, and the 1:2 duplex/MXT peak was the most abundant. In contrast, only 1:1 triplex-MXT complexes were observed in the mass spectrum of the triplex-MXT mixture, and the most intensive peak was a free triplex ion without MXT.

Screening and Structural Characterization of alpha-Glucosidase Inhibitors from Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS

In vitro a-glucosidase inhibition assays and ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC-DAD-ESI-MSn) were combined to screen a-glucosidase inhibitors from hawthorn leaf flavonoids extract (HLFE). As a result, four compounds were identified as alpha-glucosidase inhibitors in the HLFE, and their structures were confirmed to be quercetin-3-O-rha-(1-4)-glc-rha and C-glycosylflavones (vitexin-2 ''-O-glucoside, vitexin-2 ''-O-rhamnoside and vitexin) by high-resolution sustained off resonance irradiation collision-induced dissociation (SORI-CID) data obtained by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS).

A study of the non-covalent interaction between flavonoids and DNA triplexes by electrospray ionization mass spectrometry

The binding interactions of 22 flavonoids (9 aglycones and 13 glycosides) with DNA triplexes were investigated using electrospray ionization mass spectrometry (ESI-MS). The results revealed that the hydroxyl positions of aglycones. the locations and numbers of saccharide, as well as the aglycone skeletons play roles in the triplex-binding properties of flavonoids. The presence of 3-OH, or 3'-OH, or replacement of 4'-OH with methoxy group in aglycones decreased the fraction of bound DNA sharply. Flavonoid glycosides exhibit higher binding affinities towards the DNA triplexes than their aglycone counterparts. Glycosylations of flavones at the 8-C position and isoflavones at the 7-O position show higher binding affinities than those on the other positions of ring A of aglycones. Glycosylation with a disaccharide on 0 position of flavonol results in higher binding affinity than that with monosaccharide. Flexibility of the ring B is favorable for its interaction with DNA triplex. According to sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments, glycosylation and non-planarity of flavonoid aglycones lead to different dissociation pathways of the flavonoid/triplex complexes.

A study of isomeric diglycosyl flavonoids by SORI CID of Fourier transform ion cyclotron mass Spectrometry in negative ion mode

High-resolution Sustained off resonance irradiation (SORI) CID was employed to distinguish four pairs of isomeric diglycosyl flavonoids in the negative mode using the electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). All of these isomers can be distinguished via MS/MS data. For these diglycosyl flavones and flavanones, the deprotonated alpha 1-->6 linkage diglycosyl flavonoids produce fewer fragments than the alpha 1-->2 linkage type compounds and the Retro-Diels-Alder (RDA) reaction in MS/MS only takes place when the aglycone is a flavanone and glycosylated with an alpha 1-->2 intersaccharide linkage disaccharide. The deprotonation sites after collisional activation are discussed according to the high mass accuracy and high-resolution data of tandem spectrometry. Some of these high-resolution SORI CID product ions from alpha 1-->2 linkage diglycosyl flavonoids involve multibond cleavages; the possible mechanism is discussed based on the computer modeling using Gaussian 03 program package at the B3LYP/6-31G level of theory. Unambiguous elementary composition data provides fragmentation information that has not been reported previously.

Characterization of triterpenoidic saponin mixture in crude extracts from leaves of Acanthopanax senticosus Harms by saponin structural correlation and mass spectrometry

Eighteen triterpenoidic saponins in crude extracts from leaves of Acanthopanax senticous Harms have been investigated by electrospray ionization multi-stage tandem mass spectrometry and high-resolution mass spectrometry. In ESI-MS spectra, predominant [M + Na](+) ions in the positive ion mode have been observed for molecular mass information. Meanwhile, specific structural correlations between these ions are firstly found. The 18 peaks (ions) can be classified into three groups (group D, E, and F with mass increase) with each group including six peaks. There is a mass difference of 132 Da between group D and E for each corresponding peak in turn (for example peak 1 to peak 7), indicating one more pentose residue was attached to saponins in group E than those corresponding in group D. The mass difference of 146 Da between group E and F implies one more deoxy-hexose attached to saponins in group F than those corresponding in group E. The structural correlations of the corresponding ions are confirmed by tandem mass spectrometry and high-re solution mass spectrometry. These structural features can not only facilitate the rapid characterization of the native known saponins in crude plant extracts, thus avoiding tedious derivation and separation of saponins, but also help find novel compounds of the same type in a specific medicinal plant.