Identification and fragmentation pathways of caffeine metabolites in urine samples via liquid chromatography with positive electrospray ionization coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry

Liquid chromatography (LC) with positive ion electrospray ionization (ESI+) coupled to a hybrid quadrupole linear ion trap (LTQ) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was employed for the simultaneous determination of caffeine and its metabolites in human urine within a single chromatographic run. LC/ESI‐FTICRMS led to the unambiguous determination of the molecular masses of the studied compounds without interference from other biomolecules. A systematic and comprehensive study of the mass spectral behaviour of caffeine and its fourteen metabolites by tandem mass spectrometry (MS/MS) was performed, through in‐source ion trap collision‐induced dissociation (CID) of the protonated molecules, [M+H]⁺. A retro‐Diels‐Alder (RDA) process along with ring‐contraction reactions were the major fragmentation pathways observed during CID. The base peak of xanthine precursors originates from the loss of methyl isocyanate (CH₃NCO, 57 Da) or isocyanic acid (HNCO, 43 Da), which in turn lose a CO unit. Also uric acid derivatives shared a RDA rearrangement as a common fragmentation process and a successive loss of CO₂ or CO. The uracil derivatives showed a loss of a ketene unit (CH₂CO, 42 Da) from the protonated molecule along with the loss of H₂O or CO. To assess the potential of the present method three established metabolite ratios to measure P450 CYP1A2, N‐acetyltransferase and xanthine oxidase activities were evaluated by a number of identified metabolites from healthy human urine samples after caffeine intake. Copyright © 2009 John Wiley & Sons, Ltd.     

Solvation of acylium fragment ions in electrospray ionization quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry

In electrospray ionization (ESI) quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry, certain fragment ions (e.g. acylium ions) generated either during the ion transportation process (in the source interface region) or in the ion trap are found to undergo ion--molecule reactions with ESI solvent molecules (water, acetonitrile and aliphatic alcohols) to form adduct species. These unexpected solvated fragment ions severely complicate the interpretation of mass spectrometic data. High-resolution accurate mass measurements are important in establishing the elemental compositions of these adduct species and preventing erroneous data interpretation.     

Enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of long-chain polysaccharides

A novel strategy was developed to extend the application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to the analysis of long-chain polysaccharides. High molecular weight polydisperse maltodextrins (poly-alpha(1-4) glucose) and dextrans (poly-alpha(1-6) glucose) were chosen as model compounds in the present study. Increased ionization efficiency of these mixtures in the positive ion mode was achieved upon modification of their reducing end with nitrogen-containing groups. The derivatization method is based on the formation of a new C--N bond between 1,6-hexamethylenediamine (HMD) and the reducing end of the polysaccharide, which exists in solution as an equilibrium between the hemiacetal and the open-ring aldehyde form. To achieve the chemical modification of the reducing end, two synthetic pathways were developed: (i) coupling of HMD by reductive amination and (ii) oxidation of the hemiacetal to lactone, followed by ring opening by HMD to yield the maltodextrin lactonamide of 1,6-hexanediamine (HMMD). Amino-functionalized polysaccharides were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR-MS) in the positive ion mode by direct flow injection. The hexamethylenediamine (HMD) and maltodextrin lactonamide of 1,6-hexanediamine (HMMD) moieties provide increased proton affinities which dramatically improve the detection of the long-chain polysaccharides by FTICR-MS. The present approach allowed for identification of single components in mixtures with prominent heterogeneity in the degree of polymerization (DP), without the need for chromatographic separation prior to MS. The high mass accuracy was essential for the unambiguous characterization of the species observed in the analyzed mixtures. Furthermore, molecular components containing up to 42 glucose residues were detected, representing the largest polysaccharide chains analyzed so far by ESI FTICR-MS.     

Characterization of oligomeric compounds in secondary organic aerosol using liquid chromatography coupled to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The components of secondary organic aerosols (SOAs) generated from the gas‐phase ozonolysis of two C₁₀H₁₆‐terpenes (α‐pinene; sabinene) and a cyclic C₆H₁₀ alkene (cyclohexene) were characterized by the use of a Fourier transform ion cyclotron mass spectrometer equipped with an electrospray ionization source operated in the negative ion mode. Reversed‐phase high‐performance liquid chromatography was used to achieve chromatographic separation of highly oxidized organic compounds. In addition to the well‐known group of low molecular weight oxidation products (monomers; e.g. dicarboxylic acids), higher molecular weight compounds (dimers) were also detected and their exact elemental compositions were determined. In order to provide additional information for the structural elucidation of these compounds, collision‐induced dissociation was applied. Based on the MS/MS spectra, two higher molecular weight products are proposed to be an ester and a peroxide. Molecular formulae calculated from the exact masses show that the SOA‐compounds are heavily oxidized and this information creates the background to a discussion of potential reaction pathways for the formation of higher molecular weight compounds. Copyright © 2009 John Wiley & Sons, Ltd.     

A new technique for unbiased external ion accumulation in a quadrupole two-dimensional ion trap for electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

External ion accumulation in a two-dimensional (2D) multipole trap has been shown to increase the sensitivity, dynamic range and duty cycle of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. However, it is important that trapped ions be detected without significant bias at longer accumulation times in the external 2D multipole trap. With increasing ion accumulation time pronounced m/z discrimination was observed when trapping ions in an accumulation quadrupole. In this work we show that superimposing lower rf-amplitude dipolar excitation over the main rf-field in the accumulation quadrupole results in disruption of the m/z discrimination and can potentially be used to achieve unbiased external ion accumulation with FTICR.     

Manipulating internal energy of protonated biomolecules in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The internal energy of protonated leucine enkephalin has been manipulated in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with two newly designed pump-probe experiments. Blackbody infrared radiation was applied to pump an ion population into a well-defined internal energy distribution below the dissociation threshold. Following this pumping stage, the internal energy distribution was probed using on-resonance collisional activation to dissociate the ions. These pump-probe experiments were carried out in two different ways: (a) using on-resonance collisional activation with variable kinetic energies to dissociate the ions at a constant initial ion temperature (determining the precursor ion survival percentage as a function of kinetic energy) and (b) using on-resonance collisional activation with a constant kinetic energy to dissociate the ions at variable initial ion temperatures (to investigate the ion survival yield-initial ion temperature dependence). Using this approach, a detailed study of the effects of the initial ion temperature, the probing kinetic energy and the internal energy loss rate on the effective conversion efficiency of (laboratory-frame) kinetic energy to internal energy was conducted. This conversion efficiency was found to be dependent on the initial ion temperature. Depending on the experimental conditions the conversion efficiency (for collisions with argon) was estimated to be about 4.0 +/- 1.7%, which agrees with that obtained from a theoretical modeling. Finally, the reconstructed curves of the ion survival yield versus the mode of the (final) total internal energy distribution of the activated ion population (after pump and probe events) at different pump-probe conditions reveal the internal energy content of the activated ions.     

Analysis of strychnos alkaloids using electrospray ionization Fourier transform ion cyclotron resonance multi-stage tandem mass spectrometry

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.     

Mass spectrometric evidence for collisionally induced removal of H2 from monoanions of 10B nido‐carborane derivatives investigated by electrospray ionization quadrupole linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry

Some newly synthesized ¹⁰B nido‐carborane derivatives, i.e., 7,8‐dicarba‐nido‐undecaborane monoanions ([7‐Me‐8‐R‐C₂B₉H₁₀]^‐K⁺, R = H, butyl, hexyl, octyl and decyl), have been fully characterised and examined by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry with liquid chromatographic separation (LC/ESI‐FTICR‐MS). These boron‐containing compounds exhibit abundant molecular ions ([M]^?) at m/z 140.22631 [CB₉H₁₄]^?, m/z 196.28883 [CB₉H₂₂]^?, m/z 224.32032 [CB₉H₂₆]^?, m/z 252.35133 [CB₉H₃₀]^? and m/z 280.38354 [CB₉H₃₄]^? at the normal tube lens voltage setting of ?90 V, which was an instrumental parameter value selected in the tuning operation. Additional [M–nH₂]^? (n = 1?4) ions were observed in the mass spectra when higher tube lens voltages were applied, i.e., ?140 V. High‐resolution FTICR‐MS data revealed the accurate masses of fragment ions, bearing either an even or an odd number of electrons. Collision‐induced dissociation of the [M–nH₂]^? ions (n = 0–4) in the quadrupole linear ion trap (LTQ) analyzer confirmed the loss of hydrogen molecules from the molecular ions. It is suggested that the loss of H₂ molecules from the alkyl chain is a consequence of the stabilization effect of the nido‐carborane charged polyhedral skeleton. Copyright © 2009 John Wiley & Sons, Ltd.     

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.     

Direct characterization of intact polypeptides by matrix-assisted laser desorption electrospray ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometry

We report the characterization of a recently introduced hybrid ionization source, matrix-assisted laser desorption electrospray ionization (MALDESI), coupled to a quadrupole Fourier transform ion cyclotron resonance mass spectrometry (QFT-ICR-MS) system. We first demonstrate the ability of MALDESI-QFT-ICR MS to directly analyze and provide high mass measurement accuracy (approximately 1 part-per-million) of a polypeptide using internal calibration. Second, we show the potential of MALDESI-QFT-ICR MS for the top-down characterization of multiply charged polypeptide cations. Finally, we demonstrate sub-femtomole detection limits in MALDESI-QFT-ICR MS using a combination of naturally occurring peptides and their respective stable isotope labeled forms. The results presented herein demonstrate the feasibility of several potential applications for MALDESI-QFT-ICR MS for the direct analysis of intact biological molecules.     

Speciation of M+3-hydroxypyrone chelation complexes by electrospray ionization ion trap and Fourier transform ion cyclotron resonance mass spectrometry

Kojic acid (5-hydroxy-2-hydroxymethyl-4-pyrone) is known to have a high affinity for transition metals, and it and its derivatized cogeners are used both analytically and clinically. The interactions between kojic acid (KA) and eleven +3 metals (Al(+3), As(+3), Cr(+3), Ga(+3), Fe(+3), In(+3), Yb(+3), Y(+3), Gd(+3), Nd(+3), La(+3)) were examined by electrospray ionization mass spectrometry (ESI-MS) using an ion trap in an aqueous medium. For a subset of five ions, Fourier transform ion cyclotron resonance (FTICR)-MS was conducted to provide accurate mass confirmation of peak assignments for metals having clustering of abundant isotopes. KA readily formed complexes with all the metal ions tested. The most common complexes observed were ML(3)H(+) and M(2)L(5). Different behavior was seen for small and large ionic radius ions, with a relative cut-off between In(+3) ( approximately 80 pm) and Yb(+3) ( approximately 87 pm); a striking trend in % collision energy vs. cluster complexity was revealed. The KA-Cr(+3)complex shows a high affinity for H(2)O molecules in the gas phase, whilst In(+3) shows a preference for dimetal complexes and Y(+3) a deviant behavior when complexed to two neutrals.     

Identification of unsaturated N-acylhomoserine lactones in bacterial isolates of Rhodobacter sphaeroides by liquid chromatography coupled to electrospray ionization-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry

The identification of two unsaturated N‐acylhomoserine lactones (AHLs) produced by Rhodobacter sphaeroides bacteria, based on liquid chromatography (LC) coupled to a hybrid quadrupole linear ion trap (LTQ)‐Fourier transform ion cyclotron resonance (FTICR) mass spectrometer upon electrospray ionization (ESI), is presented. Besides the confirmation of the signaling molecule already described in the literature, i.e. (Z)‐N‐tetradec‐7‐enoyl‐homoserine lactone (C_14:1‐HSL), we have discovered the occurrence, at low, yet significant levels, of another monounsaturated compound, C_12:1‐HSL, which may extend the number of small diffusible chemical signals known for R. sphaeroides. Both unsaturated AHLs were identified by high‐resolution FTICR mass spectrometry in extracts of bacterial culture media and the occurrence of a C=C bond was assessed upon their conversion into bromohydrins. Collision‐induced dissociation (CID) spectra were then collected on the LTQ mass analyzer. A careful comparison of tandem MS spectra of monounsaturated (i.e., C_12:1‐HSL and C_14:1‐HSL) and saturated AHLs (i.e. C₁₂‐HSL and C₁₄‐HSL) led to the emphasis of two series of product ions, exhibiting 14 Da spaced m/z ratios. Both series were referred to progressive fragmentations at the aliphatic end of the AHL acyl chains, followed by neutral losses of terminal alkenes (i.e. CH₂=CH(CH₂)_nH). In particular, the series located at the higher end of the explored m/z range (>200 Da), observed only for monounsaturated species, enabled the location of the C=C bond between carbons 7 and 8 of the acyl chain. Copyright © 2011 John Wiley & Sons, Ltd.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Endgroup determination of synthetic polymers by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) was performed on a Fourier transform ion cyclotron resonance mass spectrometer for the endgroup and monomer mass determination of three poly(oxyalkylene)s in the mass range of 400–8000 Da. A combined use of the multiple charge states observed with ESI, leads to a threefold increase in accuracy of the endgroup and monomer determination. The improvement is attributed to the increased number of datapoints used for the regression procedure, yielding more accurate results. Endgroup masses are determined with a mass error better than 5 and 75 millimass units for the molecular weight range of 400–4200 and 6200–8000 Da, respectively. A mass error of better than 1 millimass unit was observed for all monomer mass determinations. With ESI, endgroup and monomer masses have been determined for poly(ethylene glycol) oligomers with a mass higher than 8000 Da. This is almost two times higher than observed with matrix-assisted laser desorption/ ionization on the same instrument.     

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.     

Electron beam potential depression as an ion trap in Fourier transform ion cyclotron resonance mass spectrometry

Trapping of ions in the electron beam of a FTICR mass spectrometer is investigated and a simple model describing the confinement process is presented. Detection of resistive-wall destabilization of the magnetron motion of ions in the trapped-ion cell is used to determine conditions for ion trapping within and escape from the electron beam. The model predicts a potential well that is dependent on electron beam current, energy, and dimension in defining its capacity for low energy ions. Plots of ion retention time versus ion number are consistent with a model in which ions are initially trapped in the electron beam but with increasing ion formation will eventually overcome the potential depression in the electron beam and escape into magnetron orbits. Based upon this model, expressions are derived for ion retention time which are then fit to the experimental data. The model is used to estimate ion number, initial magnetron radius and ion cloud shape and density. One example in which electron trapping is important in the FTICR experiment is in the efficient transfer of ions between dual trapped-ion cells. Ion transfer within the potential depression of the electron beam environment is shown to be virtually 100% efficient over a 10 ms interval whereas all ions are lost to collisions with the conductance limit after 2 ms when transferring without the confining aid of the electron beam. Several analytical applications of electron traps in the ICR cell are now being investigated.