Quasi-molecular Ions Research Articles

Investigation of combwax of honeybees with high-temperature gas chromatography and high-temperature gas chromatography–chemical ionization mass spectrometry: II: High-temperature gas chromatography–chemical ionization mass spectrometry

Crude combwax of six various honey bee species have been analyzed by high-temperature gas chromatography (HTGC)–chemical ionization mass spectrometry after a two-step silylation procedure. An optimized chromatographic procedure, described previously, enables the separation of high-molecular mass lipid compounds resulting in a characteristic fingerprint of the combwaxes of different honeybee species. The coupling of HTGC to mass spectrometry requires appropriate instrumentation in order to achieve sufficient sensitivity at high elution temperatures and avoid loss of chromatographic resolution. Chemical ionization was carried out using methane as reagent gas in order to determine the molecular mass of the individual compounds by means of abundant quasi molecular ions. To confirm the presence of unsaturated wax esters, ammonia was used as reagent gas. More than 80 lipid constituents were separated and characterized by their mass spectra. Representative chemical ionization mass spectra of individual compounds are presented. Both, HTGC–flame ionization detection data and the results of the HTGC–mass spectrometric investigations enabled a rapid profiling of the individual classes of compounds in crude combwaxes.

Detection of Triazolam and Its Hydroxy Metabolites in Rat Hair by Reversed-Phase Liquid Chromatography with Electrospray Ionization Mass Spectrometry

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) for simultaneous determination of triazolam (TZ) and its hydroxy metabolites in hair has been developed. After the addition of deuterium-labeled 1 -hydroxymethyltriazolam (1-HT-d4) as an internal standard, analytes in hair shaft and hair root samples were extracted with a basic medium, CH2Cl2/MeOH/28% NH4OH (20:80:2), at room temperature overnight. The chromatographic separation of the analytes was achieved using a 3-microm micro HPLC column (100 x 2.0-mm i.d.) with a gradient of acetonitrile in water containing 1% acetic acid as the mobile phase at a flow rate of 0.15 mL/min. The mass spectrometer was operated in selected-ion monitoring mode at quasi molecular ions [M+H]+ of TZ and its metabolites. Under the proposed conditions, the ranges of quantitation of TZ, 1-HT, and 4-HT were 0.1-10 ng/0.2 mL. The method has been applied to determine the hair shaft and hair root incorporation of TZ and its metabolites into Dark Agouti rats administered with 3 mg/kg or 6 mg/kg intraperitoneally twice a day for five days. Judging from the retention behavior by the chromatography and the mass spectra of the peaks detected, TZ, 1-HT, and 4-HT were incorporated in the hair shaft and the hair root. The concentration of 4-HT was the highest of all compounds detected. An unknown substance thought to be 1,4-diHT also appeared in both hair shaft and hair root samples. This substance was obtained from in vitro metabolic studies of TZ using rat liver microsome fraction and was accompanied by the other two metabolites, 1-HT and 4-HT. Structural elucidation was performed with online high-performance liquid chromatography-MS after acetylation of the substance with acetic anhydride and pyridine. This is the first report of the detection of the hydroxy metabolites of TZ in hair. The method has been found to be useful as a screening procedure of TZ intake in humans.

Fate studies of the non-ionic surfactant alkyl glucamide by liquid chromatography/electrospray mass spectrometry

Alkyl glucamides (AGs) were analyzed by reversed-phase liquid chromatography (LC) coupled to mass spectrometry with electrospray ionization (ESMS). Analytes were separated according to the chain length of two homologs, C12- and C14-glucamide. Mass spectrometric detection in the positive ion mode exhibited higher overall sensitivity where, apart from fragments, different molecular and quasi-molecular ions were obtained. However, application of the negative ion mode offered advantages in terms of reproducibility and extent of information when analyzing environmental samples. Therefore, a simple and sensitive analytical methodology was developed for the determination of AGs in municipal sewage treatment plant influent and effluent based on solid-phase enrichment, LC separation and negative ion ESMS quantification. After preconcentration of 100 ml of aqueous sample, the recoveries using polymeric LiChrolut EN cartridges exceeded 89%. A quantification limit of 0.1 microg l(-1) was achieved. Studies on the biodegradability and metabolic pathway of C10-glucamide were carried out on a laboratory-scale microbial test unit under aerobic conditions. A postulated metabolism including omega-oxidation of the alkyl chain followed by subsequent beta-oxidations was checked by LC/ESMS. Identification and formation of 'C4-glucamide acid' as a degradation intermediate was confirmed by mass spectrometric studies. Higher homolog acids such as C10-, C8- and C6-glucamide acids, which should be predicted precursors of C4-glucamide acid, and any other metabolites, were not detectable, presumably owing to rapid breakdown.

Selection of Anionic Dopant for Quantifying Desialylation Reactions with MALDI-FTMS

Alkylsulfonates are examined as anion dopants for the matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of neutral oligosaccharides. The anion dopants allow neutral oligosaccharides to be examined in the same mixture as acidic oligosaccharides. The alkylsulfonate dopants interact strongly with the oligosaccharide to produce a quasimolecular ion composed of the oligosaccharides and the deprotonated alkylsulfonates. Compounds as small as disaccharides to large branched oligosaccharides can all be examined. The new series of alkylsulfonates works as well as HSO4- in terms of signal-to-noise ratio, sensitivity, and ease of preparation in the negative ion mode. The alkylsulfonates are used to construct a calibration curve for obtaining the relative amounts of varying neutral and acidic oligosaccharide mixtures. With the anion dopants, the MALDI-FTMS signals are shown to have a linear relationship with the molar ratios of the neutral and sialylated oligosaccharides. The method is also used to monitor the product of a desialylation reaction by acid hydrolysis.

Line emission and alignment effects in state selective electron transfer in He $\mathsf{^{2+}}$ -Li(2s, 2p ${\mathsf{\Sigma}}$ , 2p ${\mathsf{\Pi}}$ ) collisions

Classical Trajectory Monte-Carlo (CTMC) method has been used to investigate state selective electron capture by He2+ ions colliding with Li(2s) and Li*(2p) in Σ as well as Π alignments in the energy range 1–15 keV/amu. He+(4l) electron capture, line emission [He II(n = 4 → 3)] cross-sections and alignment parameters have been calculated and analyzed in the light of the available results. The undulatory structure of the capture and emission cross-sections have been explained qualitatively in terms of a quasimolecular ion formation. Projectile impact energy and spatial overlap play crucial role in determining the alignment effects.

Analysis of urinary nucleosides. I. Optimisation of high performance liquid chromatography/electrospray mass spectrometry.

In order to optimise the analysis of urinary nucleosides by high performance liquid chromatography/mass spectrometry (HPLC/MS), the HPLC separation of these compounds was performed at different 'flow rates' and 0.2mL/min was found to give both a better separation and ionisation. The ionisation conditions were optimised to give the best intensity of the molecules quasi-molecular ions. The ion distribution profile and ionisation in both positive and negative mode were examined and the detection of the protonated molecule in positive mode chosen for further analysis. The limits of detection of the method developed are reported and representative LC/MS and LC/MS/MS spectra shown. Typical urinary nucleoside chromatograms are presented.

High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood

Eleven phenothiazine derivatives with heavy side chains contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)/electrospray (ES) tandem mass spectrometry (MS). All compounds gave the base peaks due to [M + 1]+ by HPLC/ES single MS. The product ions formed from each quasi-molecular ion by HPLC/ES tandem MS showed the base peaks due to side chains liberated. The mass chromatography of HPLC/ES tandem MS showed much higher sensitivity than that of HPLC/ES single MS for phenothiazines spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and reproducibility were studied for thiethylperazine, clospirazine and flupentixol spiked to human whole blood by means of mass chromatography of HPLC/ES tandem MS. The three compounds showed good linearity in the range of 2–40 ng/mL with a detection limit of about 0.5 ng/mL. Recoveries of the three compounds spiked to whole blood (2 and 8 ng added to 1 mL whole blood) were 43.4–72.5 %; the coefficients of intraday and interday variations were 3.7–9.3 and 12.6–17.9 %, respectively. Thiethylperazine, clospirazine and flupentixol in whole blood could actually be determined with sufficient sensitivity 3 and 6 h after oral administration of 5–10 mg of each compound in a volunteer. Copyright­© 1999 John Wiley & Sons, Ltd.

High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood.

Eleven phenothiazine derivatives with heavy side chains contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)/electrospray (ES) tandem mass spectrometry (MS). All compounds gave the base peaks due to [M + 1](+) by HPLC/ES single MS. The product ions formed from each quasi-molecular ion by HPLC/ES tandem MS showed the base peaks due to side chains liberated. The mass chromatography of HPLC/ES tandem MS showed much higher sensitivity than that of HPLC/ES single MS for phenothiazines spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and reproducibility were studied for thiethylperazine, clospirazine and flupentixol spiked to human whole blood by means of mass chromatography of HPLC/ES tandem MS. The three compounds showed good linearity in the range of 2-40 ng/mL with a detection limit of about 0.5 ng/mL. Recoveries of the three compounds spiked to whole blood (2 and 8 ng added to 1 mL whole blood) were 43.4-72.5 %; the coefficients of intraday and interday variations were 3.7-9.3 and 12.6-17.9 %, respectively. Thiethylperazine, clospirazine and flupentixol in whole blood could actually be determined with sufficient sensitivity 3 and 6 h after oral administration of 5-10 mg of each compound in a volunteer.

Simultaneous determination of the enantiomers of leucine and [ 2H 7]leucine in plasma by capillary gas chromatography–mass spectrometry

A method for the stereoselective assay of d- and l-enantiomers of both leucine and [ 2H 7]leucine in rat plasma was developed using gas chromatography–mass spectrometry–selected-ion monitoring. dl-[ 2H 3]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.

Stereochemical effects enhanced by using selective “self-ionization” under electron ionization conditions in a quadrupole ion trap mass spectrometer

From a previous study on the reactivity of the cis and trans (4-hydroxy-4-methyl-cyclohexyl)-benzoates (Mw = 234) under ammonia chemical ionization conditions in high pressure source, it was demonstrated that competitive decompositions, have specific orientations. In particular, highly stereospecific benzoic acid loss from the trans isomer takes place, suggesting that protonation by NH 4 + regioselectively occurred at the benzoate site rather than at the hydroxyl group. From the cis epimer elimination of water was only observed. This behavior could be explained by (1) possible proton transfer to both the functional groups and (2) decomposition occurring via water loss due to a higher rate constant compared with that of the benzoic acid loss (PA C 6H 5 COOH > PA H 2O ), which is not observed. These epimers are now studied under low pressure chemical ionization (CI) and electron impact (EI) conditions by using an ion trap mass spectrometer. Epimer differentiation from the stereospecific benzoic acid loss can be achieved independently of the gas-phase reagent used for chemical ionization, even if quasimolecular ions are entirely decomposed (i.e. in low pressure isobutane CI and methane CI). More spectacular differentiation is displayed in the ammonia CI mass spectra of epimers. Among the various quasimolecular species, the product [MNH 4–H 2O] + ions, termed substituted ions, usually containing a covalent C–NH 3 + bond are herein in fact a noncovalent form similar to the ammonia solvating protonated(4-hydroxy-4-methyl-cyclohexyl)-benzoate structure, as shown by deuterium labeling. Other product ions such as C 7H 11NH 3 + and {NH 3, C 6H 5COOH 2 +} are detected in contrast to those observed under high pressure source conditions. Collision induced dissociation spectra of the adduct MNH 4 + ions in addition to those of protonated molecules are investigated to obtain information about the location of ammonium (or proton) attachment. It appears for the trans isomer, a regioselective approach to the benzoate group, yielding [MH–C 6H 5COOH] +, takes place rather than attack at the OH site resulting into [MH–H 2O] +. This difference is at the origin of the observed stereospecific decomposition, which occurs via anchimeric assistance. Alternatively, these epimers give similar EI mass spectra, masking all the stereochemical effects, which reappear when using a residence time of 80 ms prior to apply the analytical scan. Under such conditions, ion–molecule reactions are enhanced, yielding formation of the epimeric MH + ions. These “ self-ionization” processes are induced through exothermical proton transfers from many EI fragment ions. Competitive decompositions of the product even-electron MH + species provide a direct cis/ trans differentiation. The diagnostic cleavages involved are comparable to those observed under low pressure CI conditions except that the loss of water occurs from both the precursors. The latter loss indicates that protonation is in competition and takes place at either of the basic sites, in contrast with that observed in chemical ionization. Furthermore, the exothermicity of proton transfer is preserved as internal energy of MH + which allows consecutive decompositions. At higher m/z ratio ranges, several adduct species are stereoselectively observed in the self-ionization mass spectrum of the trans epimer. These specific processes provide enhancement of the stereochemical cis/ trans differentiation.

Discovery of Quasi-Molecular Ions in Electrospray Spectra by Automated Searching for Simultaneous Adduct Mass Differences

Our high-throughput flow injection analysis mass spectrometry (FIA-MS) system using electrospray ionization steers the majority of ion current into quasi molecular ion and adduct m/z positions. One advantage of this dawned on us, i.e., that adducted electrospray spectra are amenable to automated quasi molecular ion discovery by searching for the simultaneous occurrence of adduct mass differences. Therefore, an adduct scanning algorithm was developed, given a graphical user interface, and then incorporated into a Visual Basic application originally developed for reporting of high-throughput FIA-MS results.

Fast, sensitive and selective liquid chromatographic–tandem mass spectrometric determination of tumor-promoting diterpene esters

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to detect tumor-promoting diterpene esters of the tigliane and ingenane types within plant extracts. Fractionation on a C 18 high-performance liquid chromatography (HPLC) column was followed by MS–MS–multiple reaction monitoring (MRM) using the precursor→product ion pairs of m/z 311→293 and 293→265 for phorbol esters. The ion pairs m/z 313→295 and 295→267 were used for ingenol and deoxyphorbol esters. In a second run, the characteristic ions at m/z 311 and 313 were followed in precursor ion scan mode. These quasi-molecular ions were utilized to obtain full scan spectra of the compounds in product ion scan mode. Due to its selectivity, the present on-line method can be applied for plant cultivar selection and plant product control without time-consuming extraction procedures and complex bioassays.

Liquid chromatography–fast atom bombardment mass spectrometry for detection and determination of pentazocine in human tissues

A reliable and sensitive method was developed for the detection and determination of pentazocine in human solid tissues using liquid chromatography–dynamic fast atom bombardment (FAB) mass spectrometry, combined with a three-step liquid–liquid extraction procedure. Levallorphan tartrate served as an internal standard. The extract was evaporated to dryness and dissolved in the mobile phase, acetonitrile–10 mM ammonium acetate solution (20:80, pH 4.0) containing 0.5% glycerol as FAB matrix. The eluent was pumped at a flow rate of 25 μl/min and split before introduction to FAB mass spectrometer. Quantitative analysis was carried out by means of monitoring quasi-molecular ions with m/z 286 for pentazocine and m/z 284 for levallorphan. The lower limit of detection of pentazocine in each tissue tested was 1 ng/g with scan mode and 0.1 ng/g with SIM mode. Using this method, the concentrations of pentazocine were determined in the tissues of an autopsied individual to perform toxicological evaluation.

Organic substances in cometary grains: comparison of secondary ion mass spectral data and californium-252 plasma desorption data from reference compounds

Secondary ion mass spectra (SIMS) and 252Cf plasma desorption ionization mass spectra (PDMS) of two carboxylic acids and two purine nucleobases have been compared. Results demonstrate that the positive ion mass spectra as well as the negative ion mass spectra are very similar in SIMS and PDMS. The principal characteristics are formation of quasimolecular ions (M + H) + or (M−H) − and loss of small molecules producing nonradical fragment ions; fumaric acid exhibits a remarkable formation of radical cations and anions. A set of simplified rules has been formulated for typical ion production processes involved in these techniques. This work contributes to the development of methods for data interpretation applicable to in situ analyses of cometary grains during the ROSETTA space mission of European Space Agency to the comet 46p/Wirtanen.

Determination of polar terpene oxidation products in aerosols by liquid chromatography–ion trap mass spectrometry

A new method for the determination of polar terpene oxidation products in secondary aerosols is described. It is based on collection of particles on PTFE filters and extraction with dichloromethane followed by analysis with liquid chromatography–ion-trap mass spectrometry (MS n ) using pneumatically assisted electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) with the ion-trap operated in the product scan mode. Separations were achieved on a C 18 reversed-phase column with methanol–water (0.1% acetic acid) as eluent. The method has a high sensitivity (instrument detection limit 0.7–7 pg/μl at S/N=3) and precision (5–10%) and a good linearity. Acidic oxidation products produce strong signals with ESI. They appear as negative quasimolecular ions [M−H] −, acetate adducts [M+CH 3COO] − and molecular clusters [2M−H] −. In the MS 2 mode these acids show strong signals from neutral loss of CO 2: [M−H−CO 2] −, and/or weaker signals from loss of H 2O: [M−H−H 2O] −, [M−H−H 2O−CO 2] −. Mass spectra were recorded by APCI for a number of oxygenated terpenoid standards containing keto groups, hydroxy groups, aldehyde groups, or epoxy groups. These compounds give intense signals as their positive quasimolecular ions [M+H] +, methanol adducts [M+H+CH 3OH] + and fragments from loss of water, such as [M+H−H 2O] +, [M+H+CH 3OH−H 2O] + and [M+H−2H 2O] +. By MS 2 and MS 3 neutral loss of H 2O and 2H 2O is observed. The method has been tested in analysis of aerosol from O 3–α-pinene and O 3–myrtenol and has been proved to compare well with classical methods based on derivatisation and gas chromatography mass spectrometry. Three new compounds, tentatively identified in aerosol are reported here for the first time: 10-hydroxypinonic acid, 9-hydroxynorpinonic acid and pinalic 4-acid.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF VITAMIN D3 3-FATTY ACID ESTERS AND THEIR LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

The separation of authentic vitamin D3 3-stearate, -palmitate, -oleate, and -linoleate, possible metabolites of vitamin D3, was carried out using reversed-phase high performance liquid chromatography. Liquid chromatography/atmospheric pressure chemical ionization - mass spectrometry (LC/APCI–MS) of these esters was also examined, and a deesterified peak was detected as a base peak. On the contrary, the fatty acid ester derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione, showed a quasi-molecular ion as an intense peak. The LC/APCI–MS data on the adducts of another Cookson-type reagent having an electron-capture substituent were also reported.

Anion dopant for oligosaccharides in matrix-assisted laser desorption/ionization mass spectrometry.

A new anion dopant for oligosaccharides is developed for use in matrix-assisted laser desorption/ionization mass spectrometry. Two types of sulfate-attached quasimolecular ions are formed in the negative ion mode when neutral oligosaccharides are doped with dilute H2SO4 solutions. Under mild conditions, i.e., low H2SO4 concentration (approximately 10(-3) M) and threshold laser fluence, a sulfate adduct [M + HSO4]- is formed. With more concentrated H2SO4 solutions (approximately 10(-2) M) and higher laser fluence, in situ derivatization of the oligosaccharides occurs to produce an ion whose m/z corresponds to a sulfate derivative [M + HSO4 - H2O]-. Hydrogen sulfate appears to be a general anion dopant because it forms complexes with a wide variety of neutral oligosaccharides. Conversely, anionic oligosaccharides form neither the adduct nor the derivative. The combination of complex formation (with neutral oligosaccharides) and the deprotonation of acidic oligosaccharides allows simultaneous detection of the respective mixture.

Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry

Several fractions of oligomeric and polymeric procyanidins have been obtained from grape seeds by gel chromatography on Fractogel TSK. Their molecular weights (Mw) were directly determined by Liquid Secondary Ion Mass Spectrometry (LSIMS). We observed that tannins were molecules with a wide range of Mw: from 290 to 3100 (decamers). Catechins (Mw 290), procyanidin dimers (Mw 578), (−)-epicatechin gallate (Mw 442), procyanidin dimer gallates (Mw 730), procyanidin dimer digallates (Mw 882), procyanidin trimers (Mw 866) and procyanidin trimer gallates (Mw 1018), were identified as (M−H) − quasimolecular ions by negative LSIMS. Some structures were elucidated by partial thiolysis and enzymatic hydrolysis.

Separatory determination of diastereomeric ibuprofen glucuronides in human urine by liquid chromatography/electrospray ionization-mass spectrometry.

A method for the separatory determination of diastereomeric isomers of glucuronic acid conjugates of ibuprofen having a carboxyl group at the chiral center by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. The authentic specimens of acyl glucuronides of R(-)- and S(+)-ibuprofen were chemically synthesized by the Mitsunobu reaction. In the ESI mode, the glucuronides were characterized by an abundant quasi-molecular ion [M-H]-, and the formation of the negative ion was markedly influenced by a drift voltage. The resolution of diastereomeric isomers was achieved on a Develosil ODS-HG-5 column with 20 mM ammonium acetate (pH 5.0):acetonitrile (5:2, v/v) as a mobile phase where diastereomers were monitored with a corresponding quasi-molecular ion. After oral administration of racemic ibuprofen, a preferential excretion of (S)-ibuprofen glucuronide into the urine was observed.

Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry.

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans.