The characterisation of selected antidepressant drugs using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

The electrospray ionisation ion trap tandem mass spectrometry (ESI-MS(n)) of selected antidepressant drugs, i.e., citalopram, fluoxetine, mirtazapine, paroxetine, sertraline, and venlafaxine, has been investigated. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M+H](+) ions and their predominant product ions. These MS(n) experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as molecules such as H(2)O, amines and phenols. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with mirtazapine, fragmentation initially occurs at the latter ring with the former being predictably resistant to fragmentation. Also, when an amine-containing drug molecule such as fluoxetine also contains a functional group, which liberates a phenol with a significantly lower DeltaH(f) (0) value than that of the corresponding amine, the phenol is preferentially liberated. The structures of product ions proposed for ESI-MS(n) can be supported by electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (ESI-QToF-MS/MS). These molecules can be identified and determined in mixtures at low ng/mL concentrations by the application of high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS(2)), which can also be used for their analysis in hair samples.     

Effect of thermal processing on the flavonols rutin and quercetin

The current research involves the study of the thermal treatment of quercetin and rutin in an aqueous model system (cooking). These substances were heated and their degradation was followed by high-performance liquid chromatography/diode-array detection (HPLC/DAD). The influence of pH and the involvement of oxygen in the degradation were studied. HPLC/electrospray ionization multi-stage mass spectrometry (ESI-MS(n)) was used for the structural characterization of the compounds produced. The influence of the degradation of the phenolic compounds on their antioxidant properties was elucidated by a electron spin resonance (ESR) spectrometry study of the reaction samples mixed with the stabilized radical, Fremy's salt. Strong degradation of the model substances took place under weak basic and oxidative conditions. Quercetin showed the most intense degradation. Protocatechuic acid could be identified as a cleavage reaction product by analyzing its retention time and molar mass during the degradation of quercetin. The structure of a second cleavage product could be identified on the basis of ESI-MS(n) fragmentation data. Also, several structures for reaction products of oxidized quercetin are suggested. The ESR analysis showed a decrease in the antioxidant activity of the reaction samples after heat treatment in aqueous solution.     

Structural analysis of monoterpene glycosides extracted from Paeonia lactiflora Pall. using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Paeoniflorin standard was first investigated by electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) using a sustained off-resonance irradiation (SORI) collision-induced dissociation (CID) method at high mass resolution. The experimental results demonstrated that the unambiguous elemental composition of product ions can be obtained at high mass resolution. Comparing MS/MS spectra and the experimental methods of hydrogen and deuterium exchange, the logical fragmentation pathways of paeoniflorin have been proposed. Then, the extracts of the traditional Chinese medicine Paeonia lactiflora Pall. were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). By comparison with the ESI-FTICR-MS/MS data of paeoniflorin, the isomers paeoniflorin and albiflorin in Paeonia lactiflora Pall. have been identified using HPLC/MS with CID in an ion trap and in-source CID. Furthermore, using the characteristic fragmentation pathways, the retention times (t(R)) in HPLC and MS/MS spectra, the structures of three other kinds of monoterpene glycoside compounds have been identified on-line without time-consuming isolation. Thus an HPLC/ESI-MS method for the analysis of constituents in Paeonia lactiflora Pall. has been established.     

A new solution for an old problem: the regiochemical distribution of the acyl chains in galactolipids can be established by electrospray ionization tandem mass spectrometry

Electrospray ionization-quadrupole ion trap mass spectrometry (ESI-QITMS), either in positive- or in negative-ion mode, has been used to establish the chemical structures (chain length, degree of unsaturation, positional distribution) of the fatty acids attached to the primary (sn-1) and secondary (sn-2) hydroxyl groups of the glycerol moiety of natural monogalactosyl- (MGDG) and digalactosyldiacylglycerols (DGDG), isolated from the freshwater dinoflagellate Glenodinium sanguineum and from a marine diatom belonging to the genus Chaetoceros. Fragmentation by collision-induced dissociation of a single component in MGDG and DGDG mixtures, separated by high-performance liquid chromatography (HPLC) and detected on-line by tandem positive-ion ESI-MS, leads to a clear-cut determination of the positional distribution of the sn-glycerol-bound fatty acyl chains. Reversed-phase liquid chromatography allowed a partial resolution of the component mixture before ESI-MS/MS analysis. These results were validated by comparison with ESI-MS data obtained for the sn-2 lysoglyceroglycolipids synthesized via regiospecific enzymatic hydrolysis of the corresponding diacylglycerols by Rhizopus arrhizus lipase.     

Structural analysis of naphthoquinone protein adducts with liquid chromatography/tandem mass spectrometry and the scoring algorithm for spectral analysis (SALSA)

The relative reactivities of various naphthoquinone isomers (1,4-, 1,2- and 2-methyl-1,4-naphthoquinone) to two test proteins, apomyoglobin and human hemoglobin, were evaluated via liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The structural characterization of the resulting adducts was also obtained by LC/ESI-MS analysis of the intact proteins. The reactive sites of apomyoglobin and human hemoglobin with 1,4-naphthoquinone and 1,2-naphthoquinone were also identified through characterization of adducted tryptic peptides by use of high-pressure liquid chromatography/electrospray ionization with tandem mass spectrometry (HPLC/ESI-MS/MS), TurboSEQUEST, and the scoring algorithm for spectral analysis (SALSA). Four adducted peptides, which were formed by nucleophilic addition of a lysine amino acid residue to 1,4-naphthoquinone, were also identified, as was an adducted peptide from incubation of 1,2-naphthoquinone with apomyoglobin. In the case of incubation of human hemoglobin with the two naphthoquinones, two adducted peptides were identified from the N-terminal valine modification of the alpha and beta chains of human hemoglobin. The adducted protein formation may imply that naphthalene produces its in vivo toxicity through 1,2- and 1,4-naphthoquinone metabolites reacting with biomolecular proteins.     

On-line identification of phenanthroindolizidine alkaloids in a crude extract from Tylophora atrofolliculata by liquid chromatography combined with tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)) and liquid chromatography coupled with sequential mass spectrometry (LC/MS(n)) were applied to identify trace-level phenanthroindolizidine alkaloids in crude extracts from Tylophora atrofolliculata. Based on the relationship between the characteristic fragmentation reactions and the structural features of related compounds of known structure from this plant, the bioactive crude extract was analyzed in detail by positive and negative ion ESI-MS(n), LC/UV-MS and LC/MS(n) techniques. A total of nine constituents in the crude extract were identified rapidly, including several isomers; seven of these constituents are new and two are known compounds. The structures of four of these constituents were subsequently confirmed by nuclear magnetic resonance (NMR) and accurate mass measurements using high-resolution fast-atom bombardment mass spectrometry (FAB-HRMS).     

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

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

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.     

Identification of C-21 steroidal glycosides from the roots of Cynanchum chekiangense by high-performance liquid chromatography/tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) was used to identify C-21 steroidal glycosides with immunological activities in roots of Cynanchum chekiangense. In the MS/MS spectra, fragmentation reactions of the [M + Na]⁺ were recorded to provide structural information about the glycosyl and aglycone moieties. To further confirm the fragments structures, off-line Fourier transform ion cyclotron resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. In the study, four known steroidal glycosides cynascyroside C, chekiangensosides A and B, glaucoside H, and four novel steroidal glycosides chekiangensosides C, D, E and chekiangensoside A isomer were identified based on mass spectral data, NMR spectral data and standards. This is the first report on identifying steroidal glycosides in roots of C. chekiangense by HPLC/ESI-MS/MS directly, which could save time and material consuming efforts in traditional phytochemistry analysis.     

Rapid protein identification using a disposable on-line clean-up/concentrating device and electrospray ionization mass spectrometry

A simple, low-cost, expedient method has been developed for identification of proteins isolated from two-dimensional (2D) gels. The method described uses a disposable on-line clean-up device, a syringe infusion pump and electrospray ionization mass spectrometry (ESI-MS). The on-line clean-up and concentrating device is a tapered capillary column filled with 1.5 cm of 5 microm C18 particles. The short column was easily prepared and was connected directly to the ESI source through a low-flow ESI sprayer. Peptides resulting from enzymatic digestion of proteins were eluted from the short column isocratically using a syringe infusion pump and analyzed by ESI-MS. This simple set-up was found useful in the analysis of proteins isolated from 2D gels. Compared to the more conventional micro-liquid chromatography/tandem mass spectrometry (microLC/MS/MS), this method can identify proteins rapidly without the need for an HPLC pump and removes the problem of cross-contamination caused by system carryover. These advantages make the method described competitive with conventional LC/MS even though the latter method gives slightly expanded sequence coverage.     

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.     

Identification of phenolic constituents in Radix Salvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry

The phenolic components from Radix Salvia miltiorrhizae Bunge, a well-known herbal medicine (Dan-Shen in Chinese), have been investigated by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). HPLC analyses were performed on a reversed-phase C18 column using gradient elution. In the ESI mass spectra a predominant [M-H]- ion was observed in negative mode and provided molecular mass information. ESI-MS/MS spectra of the [M-H]- ions were used for structural analysis, based on the spectra of standards. It was found that caffeic acid and its monomeric analogs containing a carboxyl group readily lost CO2, while dimers, trimers and tetramers of caffeic acid expelled successively danshensu or caffeic acid or their esters. Twenty-eight phenolic compounds in S. miltiorrhizae were characterized, of which eight compounds were positively identified by comparison with standards. The remaining twenty phenolics for which standards were not available were tentatively identified based on their UV spectra and MS/MS fragmentation characteristics.     

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

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

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

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].     

Study of the structure of poly(methyl methacrylate) obtained in the presence of potassium hydride

Electrospray ionization mass spectrometry (ESI-MS) in positive- and negative-ion mode and multi-step tandem mass spectrometry (ESI-MS ( n) ) were selected for the determination of the structure of poly(methyl methacrylate) obtained using potassium hydride as initiator. Macromolecules with methoxy and methyl starting groups, i. e. those situated at the beginning of the polymer backbone, were observed in this case. No other differences in the chain structure were found in the polymer.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Thermo-oxidative processes in biodegradable poly(butylene succinate)

Aliphatic polyesters have acquired significant interest as environmentally friendly thermoplastics for a wide range of applications, and understanding their degradation behaviour has relevance both for processing and end uses. We have investigated the thermal and thermo-oxidative degradation processes occurring in synthetic and commercial poly(butylene succinate) (PBSu). Thermal oxidation was performed in atmospheric air using extremely thin polymer films at 170 °C for up to 6 h. The oxidized compounds were analyzed by size exclusion chromatography (SEC), NMR spectroscopy, and Mass Spectrometry (MALDI-TOF MS). A measurable reduction of the molar mass of the polyesters was soon apparent, promoting the formation of PBSu oligomers with different end groups. MALDI mass spectrometry combined with the use of extremely thin polyester films provided a virtual magnifying glass to obtain exhaustive information on the structure of the oxidation products. An α-H abstraction mechanism has been unambiguously ascertained to be the primary step in PBSu oxidation. The oxidized polymer chains originating from the decomposition of the hydroperoxide intermediate by radical rearrangement reactions had not been revealed before. The latter products subsequently undergo chain scission processes, which can be accurately traced from the chemical species identified in our work. Thermal degradation experiments were also performed under nitrogen at 240-260 °C. The new species identified in the MALDI spectra support a decomposition pathway taking place through a β-hydrogen-transfer mechanism, followed by the production of succinic anhydride from succinic acid end molecules via a back-biting process.     

Partial characterization of glycosphingolipids of Agelas sponges in their peracetylated form by liquid secondary ionization mass spectrometry and high-performance liquid chromatography combined with direct electrospray ionization mass spectrometric detection

Electrospray ionization (ESI) and liquid secondary ionization (LSI) mass spectrometry were applied for characterization of glycosphingolipids (GSLs) isolated in their peracetylated form from four Agelas marine sponge species. Since peracetylated glycosphingolipids are not soluble in solvents traditionally used for ESI, lithium chloride was added to the samples in order to obtain lithium cationized molecules. Although the preferred fragmentation seems to be the sequential loss of acetic acid molecules, it was found that tandem mass spectra obtained from peracetylated diglycosyl ceramides might provide direct information about the structure of the long-chain base (formation of W'/Z0 fragments). The utility of ESI and LSI in the analysis of these compounds has also been compared. It was found that the tandem mass spectra obtained by LSI-MS/MS experiments could provide information about the chain-length (carbon atom number) variations within a certain ceramide mass. Thus, from one of our samples, 25 different ceramide compositions have been identified from 8 precursor (Z0) ions. Comparison of the two ionization modes (LSI and ESI) highlights the fact that molecular mass distributions obtained by LSI-MS, especially the presence of unsaturated species, have to be interpreted carefully. For the first time a direct high-performance liquid chromatography (HPLC)/ESI-MS method was used for characterization of complex mixtures of peracetylated GSLs. The results demonstrate that HPLC/ESI-MS is able to analyze mono- and diglycosylated GSLs, and other kinds of glycolipids that are actually present in the sample.     

Complex analytical approach to characterization of the influence of carbon dioxide concentration on carbohydrate composition in Norway spruce needles

Water-soluble non-structural carbohydrates (NSC) in the needles of Norway spruce Picea abies [L.] Karst have been studied by using a combination of several separation techniques, having various detectors, with mass spectrometry. The intent was to find a suitable methodology that enables the characterization and determination of NSC, covering a wide range of molar masses, and being suitable to assess how NCS are influenced by both external conditions, e.g. different carbon dioxide (CO(2)) concentrations, light intensity, and by internal conditions such as the needle age. The techniques were liquid-liquid extraction, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), asymmetrical flow field-flow fractionation (AsFlFFF), electrospray ionization mass spectrometry (ESI-MS), and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). NSC were extracted by a methanol/chloroform/water mixture into the water-rich phase. Application of AsFlFFF and SEC, using refractive index (RI) and multi-angle light scattering (MALS) detectors to the water-rich extracts resulted in three or four main fractions covering molar masses from 10(3) to 10(6)g/mol. Individual fractions collected from SEC were directly subjected to both MALDI and ESI-MS analysis in order to identify NSC. MALDI mass spectra confirmed the presence of hexose oligomers in individual fractions while ESI-MS was used for evaluation of low mass NSC. HPLC-RI was used for quantification of NSC and predominant carbohydrates were found to be fructose, glucose, and sucrose. The changes in their content during seasonal course were studied in detail. HPLC coupled to ESI-MS enabled the identification of low concentration NSC like raffinose that occurred in the needles of autumn samplings. An influence of the increased CO(2) concentration on sucrose and glucose accumulation was observed and it was found that the light intensity as well as the needle age has significant influence on the sucrose content.