Detection, identification, and occurrence of thiotetronic acids in drinking water from underground sources by electrospray ionization-high field asymmetric waveform ion mobility spectrometry-quadrupole time-of-flight-mass spectrometry.

This paper demonstrates that electrospray ionization (ESI) with differential ion mobility spectroscopy (FAIMS) and "soft" mass spectrometry (MS) provide unique analytical capabilities that led to the discovery of sulfur-containing polar congeners of thiotetronic acid (TA) in drinking water from underground sources in Canada and the United States. Polar TAs accumulate in underground aquifers and appear to be the most abundant class of organic compounds in bottled water but cannot be detected by conventional mass spectrometry methods. We show that normally stable TAs are converted into very reactive ions in ESI which have to be analyzed using special conditions in ESI-FAIMS-MS to avoid extensive dissociation and ion/molecule reactions. De novo identification of 10 TAs was accomplished by the comparative tandem mass spectrometry analysis of authentic TA derivatives from groundwater samples and synthetic TA analogues prepared for this study. We present highlights of gas phase ion chemistry of polar TAs to explain their unique properties and reactivity. TA derivatives were originally isolated from soil bacteria and are of interest in the pharmaceutical industry due to their potent activity against a broad spectrum of pathogenic bacteria and negligible toxicity to mammals. We suspect that TAs are natural disinfection agents protecting groundwater from bacterial contamination, but these compound undergo modifications or decompose during an ozonation water treatment.

Synthesis of Tumor-Associated Le(a)Le(x) Hexasaccharides: Instability of a Thiol-Containing Oligosaccharide in Mass Spectrometry and Hypermetalation Detected by ESI FAIMS.

We report the efficient synthesis of three analogues of the tumor-associated carbohydrate antigen Le(a)Le(x). This hexasaccharide was prepared as a soluble inhibitor hexyl glycoside, as a 6-aminohexyl glycoside for conjugation to proteins, and as a 6-thiohexyl glycoside for immobilization to a gold surface. These three analogues were obtained from a common hexasaccharide intermediate and isolated pure following efficient deprotection reactions that involved metal-dissolving conditions. While all other intermediates and analogues gave the expected molecular ions in ESI HRMS, the 6-thiohexyl glycoside final compound gave a complex spectrum in which no signal matched the molecular ion. Using ESI FAIMS HRMS, we were able to prevent ion dissociation reactions and obtained high quality spectral data. The ions detected could be characterized unambiguously from their accurate masses and gave insight into the behavior of the thiohexyl analogue in the gas phase. These results indicate that the 6-thiohexyl glycoside lost water and led to the formation of "hypermetalated" species which we propose are cyclic.

Commercial formaldehyde standard for mass calibration in mass spectrometry.

Common calibration standards for mass spectrometry can be a source of many problems including instrument contamination, ionization suppression and formation of unidentified ions during subsequent analysis. In this article, we present a new approach for the calibration of mass analyzers such as a quadrupole-time-of-flight mass spectrometry using a diluted solution of commercial formaldehyde. Formaldehyde is an inexpensive and commonly used solvent, and its intrinsic polymerization leads to the formation of polyoxymethylene (POM) oligomers, which are excellent multiple calibration standards for a low-mass spectral region (up to m/z 400) in the positive and negative mode of electrospray ionization. We explore the nature and origin of these polymeric species and attributed them to chemical reactions of formaldehyde and stabilizing agents in commercial formaldehyde solutions and during electrospray ionization. In contrast to other calibrants, POM oligomers do not contaminate the instrument and can easily be removed from the sample delivery system. Using tandem mass spectrometry, we elucidate the structures of the detected POM oligomers and report their reference masses, which are tightly spaced by 30 mass units. In our calibration method, mass errors of <5 ppm can be obtained from m/z 20-400 using external calibration with a simple one-point zero-order correction of spectral data and without the need for operation of a dual spray or internal calibrants. Our approach will be particularly useful for those interested in the analysis of fragile ions with low m/z values and can function at instrumental conditions required for analysis of the most labile metabolites and environmental contaminants.

Chlorine substitution promotes phenyl radical loss from C8-phenoxy-2'-deoxyguanosine adducts: implications for biomarker identification from chlorophenol exposure.

Chlorophenols are persistent organic pollutants, which undergo peroxidase-mediated oxidation to afford phenolic radical intermediates that react at the C8-site of 2'-deoxyguanosine (dG) to generate oxygen-linked C8-dG adducts. Such adducts are expected to contribute to chlorophenol toxicity and serve as effective dose biomarkers for chlorophenol exposure. Electrospray ionization mass spectrometry (ESI-MS) was employed to study collision induced dissociation (CID) for a family of such phenolic O-linked C8-dG adducts. Fragmentation of the deprotonated nucleosides demonstrates that an unexpected homolytic cleavage of the ether linkage to release phenyl radicals and a nucleoside distonic ion with m/z 281 competes effectively with commonly observed breakage of the glycosidic bond to release the deprotonated nucleobase. Increased chlorination of the phenyl ring enhances phenyl radical loss. Density functional theory calculations demonstrate that Cl-substitution decreases phenyl radical stability but promotes homolytic breakage of the C8-phenyl bond in the C8-dG adduct. The calculations suggest that phenyl radical loss is driven by destabilizing steric (electrostatic repulsion) interactions between the ether oxygen atom and ortho-chlorines on the phenyl ring. The distonic ion at m/z 281 represents a unique dissociation product for deprotonated O-linked C8-dG adducts and may prove useful for selective detection of relevant biomarkers for chlorophenol exposure by tandem mass spectrometry using selective reaction monitoring.

Linear and nonlinear regimes of electrospray signal response in analysis of urine by electrospray ionization-high field asymmetric waveform ion mobility spectrometry-MS and implications for nontarget quantification.

Quantitative nontarget analysis is intended to provide a measurement of concentration of newly identified components in complex biological or environmental samples for which authentic or labeled standard do not exist. Electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) has unique advantages that allowed us to develop a novel approach for quantification of nontarget analytes. In the nontarget analysis of urinary metabolites by ESI-FAIMS-MS, we find it practical and beneficial to analyze highly diluted urine samples. We show that urine extracts can be analyzed directly at very high dilutions (up to 20,000 times) by extending MS analysis times during slow FAIMS scanning. We explore the effects of sample dilution on ionization efficiency and ionization suppression in direct electrospray of complex sample matrixes. We consistently observe two distinct regimes in ESI operation related to the limited ionization capacity of this method. In the linear dynamic concentration range below the limiting ionization capacity, the analytical sensitivity of an analyte is constant and does not depend on matrix composition and concentration. Once the capacity of ESI is exceeded, all species exhibit log-log linearity in signal response. We show how quantification can be carried out using two different approaches, one for analytes which can be detected in the linear regime and another for those only detected in the suppression regime that overcomes the effects of ionization suppression. Our new insight into ionization suppression effects in ESI is of broad interest to anyone using ESI as an ionization technique for the MS analysis of complex samples.

Revisiting the reactivity of uracil during collision induced dissociation: tautomerism and charge-directed processes.

In our recent work towards the nontarget identification of products of nucleic acid (NA) damage in urine, we have found previous work describing the dissociation of NA bases not adequate to fully explain their observed reactivity. Here we revisit the gas-phase chemistry of protonated uracil (U) during collision induced dissociation (CID) using two modern tandem mass spectrometry techniques; quadrupole ion trap (QIT) and quadrupole time of flight (Q-TOF). We present detailed mechanistic proposals that account for all observed products of our experiments and from previous isotope labeling data, and that are supported by previous ion spectroscopy results and theoretical work. The diverse product-ions of U cannot be explained adequately by only considering the lowest energy form of protonated U as a precursor. The tautomers adopted by U during collisional excitation make it possible to relate the complex reactivity observed to reasonable mechanistic proposals and feasible product-ion structures for this small highly conjugated heterocycle. These reactions proceed from four different stable tautomers, which are excited to a specific activated precursor from which dissociation can occur via a charge-directed process through a favorable transition state to give a stabilized product. Understanding the chemistry of uracil at this level will facilitate the identification of new modified uracil derivatives in biological samples based solely on their reactivity during CID. Our integrated approach to describing ion dissociation is widely applicable to other NA bases and similar classes of biomolecules.

Hydroxyl radical-induced oxidation of a phenolic C-linked 2'-deoxyguanosine adduct yields a reactive catechol.

Phenolic toxins stimulate oxidative stress and generate C-linked adducts at the C8-site of 2'-deoxyguanosine (dG). We previously reported that the C-linked adduct 8-(4″-hydroxyphenyl)-dG (p-PhOH-dG) undergoes oxidation in the presence of Na(2)IrCl(6) or horseradish peroxidase (HRP)/H(2)O(2) to generate polymeric adducts through phenoxyl radical production [ Weishar ( 2008 ) Org. Lett. 10 , 1839 - 1842 ]. We now report on reaction of p-PhOH-dG with two radical-generating systems, Cu(II)/H(2)O(2) or Fe(II)-EDTA/H(2)O(2), which were utilized to study the fate of the C-linked adduct in the presence of hydroxyl radical (HO(•)). The radical-generating systems facilitate (i) hydroxylation of the phenolic ring to afford the catechol adduct 8-(3″,4″-dihydroxyphenyl)-dG (3″,4″-DHPh-dG) and (ii) H-atom abstraction from the sugar moiety to generate the deglycosylated base p-PhOH-G. The ratios of 3″,4″-DHPh-dG to p-PhOH-G were ∼1 for Cu(II)/H(2)O(2) and ∼0.13 for Fe(II)-EDTA/H(2)O(2). The formation of 3″,4″-DHPh-dG was found to have important consequences in terms of reactivity. The catechol adduct has a lower oxidation potential than p-PhOH-dG and is sensitive to aqueous basic media, undergoing decomposition to generate a dicarboxylic acid derivative. In the presence of excess N-acetylcysteine (NAC), oxidation of 3″,4″-DHPh-dG produced mono-NAC and di-NAC conjugates. Our results imply that secondary oxidative pathways of phenolic-dG lesions are likely to contribute to toxicity.

Nontarget analysis of urine by electrospray ionization-high field asymmetric waveform ion mobility-tandem mass spectrometry.

Nearly a decade after first commercialization, high field asymmetric waveform ion mobility spectrometry (FAIMS) has yet to find its place in routine chemical analysis. Prototypes have been used to demonstrate the utility of this separation technique combined with mass spectrometry (MS). Unfortunately, first generation commercial FAIMS instruments have gone practically unused by early adopters. Here, we show this to be due to poor ion transmission in the FAIMS-MS source interface. We present simple instrumental modifications and optimization of experimental conditions to achieve good performance from the first generation commercial FAIMS device (the Ionalytics Selectra) coupled to a high resolution Q-TOF-MS. In combination with nanospray ionization, we demonstrate for the first time the nontarget analysis of urine by FAIMS with minimal sample preparation. We show the unique suitability of electrospray ionization (ESI)-FAIMS-MS for identification of low abundance species such as urinary biomarkers of damage of nucleic acids in a complex biological matrix. The elimination of electrospray noise and matrix components by FAIMS and the continuous flow of analytes through FAIMS for accurate and tandem mass analysis produce high quality spectral data suitable for structural identification of unknowns. These characteristics make ESI-FAIMS-MS ideal for nontarget identification, even when compared to high efficiency LC-ESI-MS.

Tautomerization in gas-phase ion chemistry of isomeric C-8 deoxyguanosine adducts from phenol-induced DNA damage.

Collision-induced dissociation (CID) of 8-(4''-hydroxyphenyl)-2'-deoxyguanosine and 8-(2''-hydroxyphenyl)-2'-deoxyguanosine was investigated using sequential tandem mass spectrometry. These adducts represent biomarkers of DNA damage linked to phenolic radicals and were investigated to gain insight into the effects of chemical structure of a C-8 modification on fragmentation pathways of modified 2'-deoxyguanosine (dG). CID in MS(2) of the deprotonated molecules of both the isomers generated the same product ion having the same m/z values. CID in MS(3) of the product ion at m/z 242 and CID in MS(4) experiments carried out on the selected product ions at m/z 225 and m/z 218 afford distinct fragmentation patterns. The conformational properties of isomeric product ions from CID showed that the ortho-isomers possess the unique ability to tautomerize through an intramolecular proton transfer between the phenolic OH group and the imine nitrogen (N7). Tautomerization of ortho-isomers to their keto-tautomers led to differences in their system of conjugated double bonds compared with either their enol-tautomer or the para-isomer. The charge redistribution through the N-7 site on the imidazole ring is a critical step in guanosine adduct fragmentation which is disrupted by the formation of the keto-tautomer. For this reason, different reaction pathways are observed for 8-(4''-hydroxyphenyl)-2'-deoxyguanosine and 8-(2''-hydroxyphenyl)-2'-deoxyguanosine. We present herein the dissociation and the gas-phase ion-molecule reactions for highly conjugated ions involved in the CID ion chemistry of the investigated adducts. These will be useful for those using tandem mass spectrometry for structural elucidation of C-8 modified dG adducts. This study demonstrates that the modification at the C-8 site of dG has the potential to significantly alter the reactivity of adducts. We also show the ability of tandem mass spectrometry to completely differentiate between the isomeric dG adducts investigated.

Postsynthetic guanine arylation of DNA by Suzuki-Miyaura cross-coupling.

Direct radical addition reactions at the C(8)-site of 2'-deoxyguanosine (dG) can afford C(8)-Ar-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons, and certain phenolic toxins. Such modified nucleobases are also highly fluorescent for sensing applications and possess useful electron transfer properties. The site-specific synthesis of oligonucleotides containing the C(8)-Ar-G adduct can be problematic. These lesions are sensitive to acids and oxidants that are commonly used in solid-phase DNA synthesis and are too bulky to be accepted as substrates for enzymatic synthesis by DNA polymerases. Using the Suzuki-Miyaura cross-coupling reaction, we have synthesized a number of C(8)-Ar-G-modified oligonucleotides (dimers, trimers, decamers, and a 15-mer) using a range of arylboronic acids. Good to excellent yields were obtained, and the reaction is insensitive to the nature of the bases flanking the convertible 8-Br-G nucleobase, as both pyrimidines and purines are tolerated. The impact of the C(8)-Ar-G lesion was also characterized by electrospray ionization tandem mass spectrometry, UV melting temperature analysis, circular dichroism, and fluorescence spectroscopy. The C(8)-Ar-G-modified oligonucleotides are expected to be useful substrates for diagnostic applications and understanding the biological impact of the C(8)-Ar-G lesion.

Oxidation of a biomarker for phenol carcinogen exposure: expanding the redox chemistry of 2'-deoxyguanosine.

A biomarker for phenolic carcinogen exposure, 8-(4''-hydroxyphenyl)-2'-deoxyguanosine, has been found to undergo oxidative coupling in the presence of Na2IrCl6 to afford ortho-ortho C-C-coupled polyphenols through the intermediacy of a phenoxyl radical. One can envision using such unique chemistry to oxidatively couple strands of DNA for the generation of new biomaterials. Our results also demonstrate the potential for phenolic adducts of DNA to undergo further oxidation reactions that may contribute to phenol-mediated cytotoxicity and genotoxicity.

Structural identification of highly polar nontarget contaminants in drinking water by ESI-FAIMS-Q-TOF-MS.

Drinking water is a complex mixture that contains thousands of naturally occurring and anthropogenic contaminants. Liquid chromatography-mass spectrometry (LC-MS) methods have gained a tremendous popularity in monitoring nonvolatile, highly polar, and thermally labile components in drinking water. It is well recognized, however, that there are difficulties or limitations of LC-MS methods associated with (1) significant resources (time and effort) involved in sample preparation (preconcentration, fractionation, separation), (2) low screening capacity for target contaminants, and (3) insufficient capabilities for structural identification (elucidation) of nontarget contaminants. Consequently, LC-MS methods are mainly used for the detection of target contaminants (compounds identified in drinking water before), seldom for the structural identification of abundant nontarget pollutants (unidentified pollutants in drinking water), and almost never for the structural identification of nontarget components at a trace level. The paper presents a new method of electrospray ionization high field asymmetric waveform ion mobility spectrometry mass spectrometry (ESI-FAIMS-MS), which can detect a large number of water pollutants in a quick and convenient fashion without preconcentration, fractionation, derivatization, or column separation. Most importantly, the method provides structural identification of nontarget contaminants including species present in drinking water at a sub-parts-per-billion concentration level. The identification of previously unknown contaminants was based on mass measurements of investigated ions and their fragments in mass and tandem mass spectrometry. Elemental compositions of these ions, determined by mass measurements, were used to link dissociation patterns of investigated species with their chemical structures. Characterization of nontarget contaminants of chlorine-treated drinking water by ESI-FAIMS-MS has revealed many previously unknown disinfection byproducts. The most intriguing compound, from a group of highly polar hydroxycarboxylic acids discovered in the study, was the most abundant component of drinking water, glycolic acid. Glycolic acid (toxic to kidneys and associated with a moderate maternal toxicity) has never been considered as a drinking water contaminant, despite the fact that it is present in drinking water at a higher concentration (high ppm) than concentrations of highly polar water pollutants that had attracted most attention in the past. The process of structural elucidation of discovered pollutants, including ultratrace contaminants representing a variety of carboxylic acids, will be presented in detail. The structural identification of highly polar contaminants in drinking water presented in the paper is rarely reported in the literature. The key experimental feature of the ESI-FAIMS-MS method is FAIMS separation, which significantly improves the identification capabilities of mass spectrometry.

Characterization of naphthenic acids by electrospray ionization high-field asymmetric waveform ion mobility spectrometry mass spectrometry.

Electrospray ionization (ESI) high-field asymmetric waveform ion mobility spectrometry (FAIMS) was combined with quadrupole, time-of-flight, and tandem mass spectrometry to characterize commercial and naturally occurring naphthenic acids (NA) mixtures. This new method provides quantitatively reliable mass and isomer distributions of NA components in approximately 3 min without extensive sample preparation. ESI-FAIMS-MS seems to be especially useful for characterization of fragile ions that cannot be detected by other methods. A unique part of this technique is separation of structural isomers that proved to be critical in determination of elemental composition and in structure elucidation. Tandem mass spectrometry of NA ions separated by FAIMS provides more information about the structure of NA than other methods in the field of NA analysis.

Comparison of high-field asymmetric waveform ion mobility spectrometry with GC methods in analysis of haloacetic acids in drinking water.

Haloacetic acids (HAAs) are major byproducts of chlorination of drinking water. Electrospray ionization high-field asymmetric waveform ion mobility spectrometry mass spectrometry (ESI-FAIMS-MS) provides a tool for direct monitoring of these compounds. However, treated drinking water samples can be challenging to analyze due to the large number of chemicals present and due to matrix effects that can hinder quantitation of analytes. We developed a standard addition ESI-FAIMS-MS method that permits submicrogram per liter detection of haloacetic acids and overcomes matrix effects. An advantage of FAIMS is increased selectivity through a significant reduction in the chemical background from ESI. Moreover, detection limits with this method are much lower than with previously existing GC and GC/MS methods, and quantitation results compare favorably with other existing methods. This new method does not require sample preparation or chromatographic separation and provides a fast, simple, sensitive, and selective method for monitoring HAAs.

Rapid and sensitive differentiation of anomers, linkage, and position isomers of disaccharides using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS).

A challenging aspect of structural elucidation of carbohydrates is gaining unambiguous information for anomers, linkage, and position isomers. Such isomers with identical mass can't be easily distinguished in mass spectrometry and a separation step is required prior to mass spectrometry identification. In our laboratory, gas-phase separation and differentiation of anomers, linkage, and position isomers of disaccharides was achieved using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). The FAIMS method responds to changes in ion mobility at high field rather than absolute values of ion mobility, and was shown to provide efficient separation and identification of disaccharide isomers at high sensitivity. Separation of analyzed disaccharide isomers can be accomplished at low nM level in a matter of seconds without sample purification or fractionation. Capability for examining a large population of ionic species of disaccharides by this method allowed for correlating structural details of disaccharide isomers with their separation properties in FAIMS. Results for disaccharide isomers indicate that this method could be applied to a larger group of carbohydrates.

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices.

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.

Photoinduced dissociation of electrospray-generated ions in an ion trap/time-of-flight mass spectrometer using a pulsed CO2 laser.

An ion trap/time-of-flight (IT/TOF) mass spectrometer was developed and applied to infrared multiphoton dissociation (IRMPD) studies of ions generated by electrospray ionization. A pulsed 10.6- micro m laser beam from a CO(2) laser was used for excitation of trapped ions. Results from IRMPD of peptide ions show that this method provides useful information related to the amino acid sequence of analyzed peptides. Comparative studies show that IRMPD spectra are similar to those obtained using a 266-nm UV laser beam for excitation. However, in contrast to multiple-pulse excitation required at 266 nm, the energy of a single laser pulse in IRMPD is sufficient to induce dissociation of peptide ions. The laser power is practically an exclusive parameter that must be controlled in order to obtain IRMPD spectra that will provide the optimal structural information. It is further demonstrated that the IRMPD IT/TOF technique has the potential to probe the structural features of larger ions that cannot be readily fragmented by collision-induced dissociation (CID). A multiply charged ion of equine cytochrome c is successfully fragmented in a single laser pulse experiment. The IRMPD IT/TOF technique is also shown to be a promising tool for studying dissociation kinetics of peptide and protein ions. Unlike other methods that usually monitor the dissociation ion kinetics in a dissociation time frame of greater than milliseconds, the IT/TOF can promptly detect all product ions generated by the dissociation process, and thus monitor the dissociation process of peptides and proteins in a sub-millisecond time frame. This instrument allows us to determine the dissociation rates of cytochrome c ions using high-energy photoexcitation. It is found that the charge state of the protein ion has a significant effect on dissociation kinetics, which is consistent with that found under low-energy excitation experiments. It is shown that the increase in energy of a laser pulse from 130 to 180 mJ changes the dissociation rate constant for the +12 ion from k = 2.4 x 10(3) x s(-1) to k = 7.3 x 10(4) x s(-1). The +8 ion following excitation at 130 mJ dissociates slower with a rate constant of k = 2.6 x 10(2) x s(-1). The rate difference observed is attributed to conformational differences among the ions with different charge states.