Fourier Transform Ion Cyclotron Research Articles

Production and Characterization of Hydrothermal Extracts of the Needles from Four Conifer Tree Species: Scots Pine, Norway Spruce, Common Juniper, and European Larch.

Coniferous trees are the most dominant trees in Finland with a great economic value for pulp, paper, and timber making. Thus, their utilization also results in large quantities of residues, especially bark and needles. Tree needles are a rich source of bioactive compounds, which have a considerable utilization potential in different pharmaceutical or techno-chemical applications. In this study, hydrothermal extraction (HTE) of the needles from four conifer tree species, namely, Scots pine, Norway spruce, common juniper, and European larch, was performed. Besides water, ethanol was also used as a solvent to enhance extraction efficiency and selectivity. All of the HTE experiments were conducted with a customized high-pressure reactor operated at 120 °C and 5 bar. The obtained needle extracts were then analyzed using a direct-infusion ultrahigh-resolution Fourier transform ion cyclotron (FT-ICR) mass spectrometry. The FT-ICR analysis of water and ethanol extracts allowed identification of over 200 secondary plant metabolites, including monosaccharides, organic acids, terpenoids, a variety of phenolic compounds, and nitrogen alkaloids. The use of ethanol as the extraction solvent considerably enhanced the recovery of lipids, especially terpenoids, some polyphenols, and other unsaturated hydrocarbon species.

Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging.

(1) Imaging of pharmaceutical compounds in tissue is an increasingly important subsection of Mass Spectrometry Imaging (MSI). Identifying proper target engagement requires MS platforms with high sensitivity and spatial resolution. Three prominent categories of drugs are small molecule drugs, antibody-drug conjugate payloads, and protein degraders. (2) We tested six common MSI platforms for their limit of detection (LoD) on a representative compound for each category: a Matrix-Assisted Laser Desorption/Ionization (MALDI) Fourier Transform Ion Cyclotron, a MALDI-2 Time-of-Flight (ToF), a MALDI-2 Trapped Ion Mobility Spectrometry ToF, a Desorption Electrospray Ionization Orbitrap, and 2 Atmospheric Pressure-MALDI Triple Quadrupoles. Samples were homogenized tissue mimetic models of rat liver spiked with known concentrations of analytes. (3) We found that the AP-MALDI-QQQ platform outperformed all 4 competing platforms by a minimum of 2- to 52-fold increase in LoD for representative compounds from each category of pharmaceutical. (4) AP-MALDI-QQQ platforms are effective, cost-efficient mass spectrometers for the identification of targeted analytes of interest.

Towards Higher Sensitivity of Mass Spectrometry: A Perspective From the Mass Analyzers.

Mass spectrometry (MS) is one of the most widely used analytical techniques in many fields. Recent developments in chemical and biological researches have drawn much attention to the measurement of substances with low abundances in samples. Continuous efforts have been made consequently to further improve the sensitivity of MS. Modifications on the mass analyzers of mass spectrometers offer a direct, universal and practical way to obtain higher sensitivity. This review provides a comprehensive overview of the latest developments in mass analyzers for the improvement of mass spectrometers’ sensitivity, including quadrupole, ion trap, time-of-flight (TOF) and Fourier transform ion cyclotron (FT-ICR), as well as different combinations of these mass analyzers. The advantages and limitations of different mass analyzers and their combinations are compared and discussed. This review provides guidance to the selection of suitable mass spectrometers in chemical and biological analytical applications. It is also beneficial to the development of novel mass spectrometers.

Compositional analysis of essential oil and solvent extracts of Norway spruce sprouts by ultrahigh-resolution mass spectrometry.

Coniferous trees, especially their needles and bark, are a rich source of bioactive compounds. The developing needles of Norway spruce (Picea abies), also known as spruce sprouts, are enriched with vitamin C and other antioxidants, and thus they are used as a dietary supplement and have been traditionally used to treat various inflammatory disorders such as rheumatism and gout. Their chemical composition is only limitedly known, however. The main objective of this work was to have a deeper understanding on the chemical composition of spruce sprouts to assess their full potential in different pharmaceutical, nutraceutical, or technochemical applications. Ultrahigh-resolution Fourier-transform ion cyclotron (FT-ICR) mass spectrometry, coupled to direct-infusion electrospray ionisation (ESI) or atmospheric pressure photoionisation (APPI) techniques, was used for in-depth compositional analysis of solvent extracts and essential oil of spruce sprouts. A combined use of ESI and APPI techniques offered a great complementary insight into the rich chemistry of different spruce sprout extracts, allowing detection of thousands of chemical constituents with over 200 secondary metabolites tentatively identified. These compounds belonged to different classes such as organic acids, terpenes, flavonoids, stilbenes, sterols, and nitrogen alkaloids. Spruce sprouts have a complex metabolite profile that differs considerably from that of the old, developed needles.

ADVANCES IN HIGH-RESOLUTION MALDI MASS SPECTROMETRY FOR NEUROBIOLOGY.

Research in the field of neurobiology and neurochemistry has seen a rapid expansion in the last several years due to advances in technologies and instrumentation, facilitating the detection of biomolecules critical to the complex signaling of neurons. Part of this growth has been due to the development and implementation of high-resolution Fourier transform (FT) mass spectrometry (MS), as is offered by FT ion cyclotron resonance (FTICR) and Orbitrap mass analyzers, which improves the accuracy of measurements and helps resolve the complex biological mixtures often analyzed in the nervous system. The coupling of matrix-assisted laser desorption/ionization (MALDI) with high-resolution MS has drastically expanded the information that can be obtained with these complex samples. This review discusses notable technical developments in MALDI-FTICR and MALDI-Orbitrap platforms and their applications toward molecules in the nervous system, including sequence elucidation and profiling with de novo sequencing, analysis of post-translational modifications, in situ analysis, key advances in sample preparation and handling, quantitation, and imaging. Notable novel applications are also discussed to highlight key developments critical to advancing our understanding of neurobiology and providing insight into the exciting future of this field. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Carbon-carbon bond formation in the reaction of hydrated carbon dioxide radical anions with 3-butyn-1-ol

Electrochemical activation of carbon dioxide in aqueous solution is a promising way to use carbon dioxide as a C1 building block. Mechanistic studies in the gas phase play an important role to understand the inherent chemical reactivity of the carbon dioxide radical anion. Here, the reactivity of CO2−(H2O)n with 3-butyn-1-ol is investigated by Fourier transform ion cyclotron (FT-ICR) mass spectrometry and quantum chemical calculations. Carbon-carbon bond formation takes places, but is associated with a barrier. Therefore, bond formation may require uptake of several butynol molecules. The water molecules slowly evaporate from the cluster due to the absorption of room temperature black-body radiation. When all water molecules are lost, butynol evaporation sets in. In this late stage of the reaction, side reactions occur including H atom transfer and elimination of HOCO.

Generation of (M = K, Rb, Cs, 2n = 80–220) by laser ablation of graphene

RationaleThe yield of endohedral metallofullerene (EMF) ions is greatly related to the encaged metal atoms. For alkali metals, the generation of corresponding large‐sized monometallofullerene ions by traditional methods is still difficult. The aim of this work is to study the generation and the distribution of alkali‐metallofullerene ions by the method of laser ablation with graphene as the precursor.MethodsThe experiment was performed with a Fourier transform ion cyclotron (FTICR) mass spectrometer. Mass spectra were obtained by laser ablation of the mixtures of graphene and KCl, RbCl or CsCl in the positive ion mode. Collision‐induced dissociation (CID) experiments were also performed for some selected ions.ResultsEMF ions of (M = K, Rb, Cs, 2n = 80–220) can be generated by using graphene as the precursor. Other EMF ions with smaller sizes, including , were also observed under the optimized experimental conditions. CID experiments of some selected ions confirmed their endohedral structures.ConclusionsMetallofullerene ions of (M = K, Rb, Cs, 2n = 80–220) were generated by laser ablation of graphene and corresponding metal salts. Compared with previous results of alkaline earth metals, the yields of EMF ions are generally lower. However, for large‐sized EMF ions, the relative intensities of EMF ions to their corresponding fullerene ions / generally increase with the increase in cluster sizes, which is opposite to the trend observed for alkaline earth metals. Copyright © 2015 John Wiley & Sons, Ltd.

Nontarget analysis of Murchison soluble organic matter by high-field NMR spectroscopy and FTICR mass spectrometry.

High-field NMR spectra of Murchison meteorite methanolic extracts revealed primarily aliphatic extraterrestrial organic matter (EOM) with near statistical branching of commonly C(3-5) units separated by heteroatoms and aromatic units. The ratios of CCH, OCH and C(sp2)H units were 89 : 8 : 3, whereas carbon-based aliphatic chain termination was in the order methyl > -COOH > -CH(CH3)COOH. Aliphatic methine carbon was abundant, but its weak NMR signatures were primarily deduced from JRES (J-resolved) NMR spectra. Carbon NMR spectra were dominated by methylene and methyl carbon; strong apodization revealed methine carbon, of which about 20% was aromatic. Extrapolation provided 5-7% aromatic carbon present in Murchison soluble EOM. Compositional heterogeneity in Murchison methanolic extracts was visible in NMR and Fourier transform ion cyclotron (FTICR) mass spectra obtained from a few cubic millimeters of solid Murchison meteorite; increasing sample size enhanced uniformity of NMR spectra. Intrinsic chemical diversity and pH-dependent chemical shift variance contributed to the disparity of NMR spectra. FTICR mass spectra provided distinct clustering of CHO/CHOS and CHNO/CHNOS molecular series and confirmed the prevalence of aliphatic/alicyclic (73%) over single aromatic (21%) and polyaromatic (6%) molecular compositions, suggesting extensive aliphatic substitution of aromatic units as proposed by NMR. Murchison soluble EOM molecules feature a center with enhanced aromatic and heteroatom content, which provides rather diffuse and weak NMR signatures resulting from a huge overall chemical diversity. The periphery of Murchison EOM molecules comprises flexible branched aliphatic chains and aliphatic carboxylic acids. These project on narrow ranges of chemical shift, facilitating observation in one-dimensional and two-dimensional NMR spectra. The conformational entropy provided by these flexible surface moieties facilitates the solubility of EOM.

Solutions for low and high accuracy mass spectrometric data matching: a data-driven annotation strategy in nontargeted metabolomics.

Ultra high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) has become a widespread analytical technique in metabolomics investigations, however the benefit of high-performance chromatographic separation is often blunted due to insufficient mass spectrometric accuracy. A strategy that allows for the matching of UHPLC-MS data to highly accurate direct infusion electrospray ionization (DI-ESI) Fourier transform ion cyclotron resonance/mass spectrometry (FTICR/MS) data is developed in this manuscript. Mass difference network (MDiN) based annotation of FTICR/MS data and matching to unique UHPLC-MS peaks enables the consecutive annotation of the chromatographic data set. A direct comparison of experimental m/z values provided no basis for the matching of both platforms. The matching of annotation-based exact neutral masses finally enabled the integration of platform specific multivariate statistical evaluations, minimizing the danger to compare artifacts generated on either platform. The approach was developed on a non-alcoholic fatty liver disease (NAFLD) data set.

Mapping the protein‐binding sites for novel iridium(III) anticancer complexes using electron capture dissociation

Application of Fourier transform ion cyclotron (FT-ICR) tandem mass spectrometry reveals the binding sites for novel cyclopentadienyl Ir(III) anticancer complexes on calmodulin. The conventional fragmentation methods, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), failed to define the Ir modification, but these binding sites were located via electron capture dissociation (ECD). A combination of top-down and bottom-up methods was used to generate detailed information about the reaction of these compounds with a common signalling protein, calmodulin. The research shows that such Ir-based complexes preferentially bind to methionine sites in the protein, and interestingly, the very low efficiency of the Ir modification is different compared to reactions of Pt(II) complexes, which can lead to protein crosslinking. This is the first report on reactions of novel Ir-based anticancer complexes with proteins, which provides helpful information for studying the protein targets of this category of metallodrug and the transportation mechanisms which allow them to inhibit cancer cell growth.

Molecular characterization of dissolved organic matter (DOM): a critical review

Advances in water chemistry in the last decade have improved our knowledge about the genesis, composition, and structure of dissolved organic matter, and its effect on the environment. Improvements in analytical technology, for example Fourier-transform ion cyclotron (FT-ICR) mass spectrometry (MS), homo and hetero-correlated multidimensional nuclear magnetic resonance (NMR) spectroscopy, and excitation emission matrix fluorimetry (EEMF) with parallel factor (PARAFAC) analysis for UV-fluorescence spectroscopy have resulted in these advances. Improved purification methods, for example ultrafiltration and reverse osmosis, have enabled facile desalting and concentration of freshly collected DOM samples, thereby complementing the analytical process. Although its molecular weight (MW) remains undefined, DOM is described as a complex mixture of low-MW substances and larger-MW biomolecules, for example proteins, polysaccharides, and exocellular macromolecules. There is a general consensus that marine DOM originates from terrestrial and marine sources. A combination of diagenetic and microbial processes contributes to its origin, resulting in refractory organic matter which acts as carbon sink in the ocean. Ocean DOM is derived partially from humified products of plants decay dissolved in fresh water and transported to the ocean, and partially from proteinaceous and polysaccharide material from phytoplankton metabolism, which undergoes in-situ microbial processes, becoming refractory. Some of the DOM interacts with radiation and is, therefore, defined as chromophoric DOM (CDOM). CDOM is classified as terrestrial, marine, anthropogenic, or mixed, depending on its origin. Terrestrial CDOM reaches the oceans via estuaries, whereas autochthonous CDOM is formed in sea water by microbial activity; anthropogenic CDOM is a result of human activity. CDOM also affects the quality of water, by shielding it from solar radiation, and constitutes a carbon sink pool. Evidence in support of the hypothesis that part of marine DOM is of terrestrial origin, being the result of a long-term carbon sedimentation, has been obtained from several studies discussed herein.

FT-ICR/MS and GC-EI/MS metabolomics networking unravels global potato sprout's responses to Rhizoctonia solani infection.

The complexity of plant-pathogen interactions makes their dissection a challenging task for metabolomics studies. Here we are reporting on an integrated metabolomics networking approach combining gas chromatography/mass spectrometry (GC/MS) with Fourier transform ion cyclotron resonance/mass spectrometry (FT-ICR/MS) and bioinformatics analyses for the study of interactions in the potato sprout-Rhizoctonia solani pathosystem and the fluctuations in the global metabolome of sprouts. The developed bioanalytical and bioinformatics protocols provided a snapshot of the sprout's global metabolic network and its perturbations as a result of pathogen invasion. Mevalonic acid and deoxy-xylulose pathways were substantially up-regulated leading to the biosynthesis of sesquiterpene alkaloids such as the phytoalexins phytuberin, rishitin, and solavetivone, and steroidal alkaloids having solasodine and solanidine as their common aglycons. Additionally, the perturbation of the sprout's metabolism was depicted in fluctuations of the content of their amino acids pool and that of carboxylic and fatty acids. Components of the systemic acquired resistance (SAR) and hypersensitive reaction (HR) such as azelaic and oxalic acids were detected in increased levels in infected sprouts and strategies of the pathogen to overcome plant defense were proposed. Our metabolic approach has not only greatly expanded the multitude of metabolites previously reported in potato in response to pathogen invasion, but also enabled the identification of bioactive plant-derived metabolites providing valuable information that could be exploited in biotechnology, biomarker-assisted plant breeding, and crop protection for the development of new crop protection agents.

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin, Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn(167). Highly sialylated O-glycans were found to be present in the Thr(110)-Thr(124) region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

Cover Picture: ZAAC ‐ Journal of Inorganic and General Chemistry 7/2010

AbstractThe cover picture shows in the centre the metalloid cluster anion {Ge9[Si(SiMe3)3]3}– (1) that is transferred intact from the solution into the gas phase through the mild electro spray method. Subsequently the fragmentation of single molecules of 1 after resonant excitation and collision with argon atoms (lower side), as well as the oxidation reaction with O2 and Cl2 (upper side) are investigated in the ICR cell of an FT‐ICR mass spectrometer (Fourier transform ion cyclotron). The reductive elimination of the ligands finally leads to Zintl‐like cluster species (lower side: Ge9– (left); Ge9Si– (right)) exhibiting a negative average oxidation state of the group 14 element. Thus these single molecule experiments show for the first time the transition from metalloid clusters to Zintl‐ions. DFT calculations additionally show that the delocalized bonding situation within the Ge9 core of 1 is strongly distorted upon oxidation. This behavior is in contrast to that of similar Zintl‐Ions. More details are discussed in the article by A. Schnepf et al. on page 1173ff.

Natural Organic Matter and the Event Horizon of Mass Spectrometry

Soils, sediments, freshwaters, and marine waters contain natural organic matter (NOM), an exceedingly complex mixture of organic compounds that collectively exhibit a nearly continuous range of properties (size-reactivity continuum). NOM is composed mainly of carbon, hydrogen, and oxygen, with minor contributions from heteroatoms such as nitrogen, sulfur, and phosphorus. Suwannee River fulvic acid (SuwFA) is a fraction of NOM that is relatively depleted in heteroatoms. Ultrahigh resolution Fourier transform ion cyclotron (FTICR) mass spectra of SuwFA reveal several thousand molecular formulas, corresponding in turn to several hundred thousand distinct chemical environments of carbon even without accountancy of isomers. The mass difference deltam among adjoining C,H,O-molecules between and within clusters of nominal mass is inversely related to molecular dissimilarity: any decrease of deltam imposes an ever growing mandatory difference in molecular composition. Molecular formulas that are expected for likely biochemical precursor molecules are notably absent from these spectra, indicating that SuwFA is the product of diagenetic reactions that have altered the major components of biomass beyond the point of recognition. The degree of complexity of SuwFA can be brought into sharp focus through comparison with the theoretical limits of chemical complexity, as constrained and quantized by the fundamentals of chemical binding. The theoretical C,H,O-compositional space denotes the isomer-filtered complement of the entire, very vast space of molecular structures composed solely of carbon, hydrogen, and oxygen. The molecular formulas within SuwFA occupy a sizable proportion of the theoretical C,H,O-compositional space. A 100 percent coverage of the theoretically feasible C,H,O-compositional space by SuwFA molecules is attained throughout a sizable range of mass and H/C and O/C elemental ratios. The substantial differences between (and complementarity of) the SuwFA molecular formulas that are observed using six different modes of ionization (APCI, APPI, and ESI in positive and negative modus) imply considerable selectivity of the ionization process and suggest that the observed mass spectra represent simplified projections of still more complex mixtures.

Screening potential inhibitors against Alzheimer's amyloidosis using electrospray ionization mass spectrometry

Alzheimer's disease (AD) is associated with neurodegeneration, premature death and has a major impact on health along with economical ramifications in Western world. The prevailing hypotheses regarding the cause of AD have centered on the amyloid beta protein (Aβ)-containing senile plaques. In light of the suggested link between oxidative stress and Aβ's aggregation, the use of antioxidants as a therapeutic regime for protection against the risk of this disease has to be explored. The binding of Aβ with endogenous or plant-derived bioactive antioxidants via the formation of noncovalent complexes has been studied by electrospray ionization (ESI) mass spectrometry (MS). The stability and specificity of the complexes has been examined under several experimental parameters, while the topology of the interacting species has been revealed by proteolytic mapping, in conjunction with ESI Fourier Transform Ion Cyclotron (FTICR) MS analysis. The ligands that bind to Aβ could be used as potential aggregation inhibitors and serve as lead compounds for the synthesis of anti-amyloidogenic agents against AD.

Catalyst poisoning in the conversion of CO and N 2O to CO 2 and N 2 on Pt 4 - in the gas phase

Pt4 - catalyses the conversion of CO and N2O to CO2 and N2 in the gas phase, as observed by Fourier transform ion cyclotron (FT-ICR) mass spectrometry. The partial pressures of CO and N2O determine the extent of poisoning and the turnover numbers that can be achieved. The catalytic conversion terminates as soon as two CO are adsorbed on the cluster. With N2O, the reactivity of Pt4O2 - and Pt4O3 - is reduced to 41% and 34% compared to Pt4O-, respectively, and with Pt4O4 - this value is reduced to 1%. In contrast, Pt4 + shows no apparent catalytic activity. Density functional theory calculations of Pt4 +/- with CO and N2O adsorbates reveal significantly different stabilities of the reaction intermediates for the different charge states.

Direct mass spectrometric identification of ABCB1 (P-glycoprotein/MDR1) from the apical membrane fraction of human placenta using fourier transform ion cyclotron mass spectrometry

Identification and quantification of proteins from human tissue by electrophoresis and subsequent mass spectrometry (MS) has been mainly restricted to high abundance, non-membrane-bound molecules. Pharmacologically relevant structures such as cytochrome P450 enzyme, drug transporters and receptors were not accessible by this technology. We developed a method to identify an integral membrane-bound protein (ABCB1) from human placenta using Fourier transform ion cyclotron (FTICR)-MS. Apical and basal membrane fractions were enriched from term human placenta using differential centrifugation. Following SDS-page these fractions were cleaved with trypsin, separated by nano-HPLC and subjected to FTICR-MS. We identified a total of 70 and 89 proteins in the apical and basal membranes, respectively. Among these proteins, and restricted to the apical membrane, was the transport protein ABCB1, with 10 peptides identified by MS, covering a total of 10% of the entire protein. The study describes a method suitable for direct monitoring of membrane-bound proteins from human tissue using FTICR-MS.

Gaseous H5P2O8- ions: a theoretical and experimental study on the hydrolysis and synthesis of diphosphate ion.

The structure and reactivity of gaseous H5P2O8- ions obtained from the chemical ionization (CI) of an H4P2O7/H2O mixture and from electrospray ionization (ESI) of CH3CN/H2O/H4P2O7 solutions were investigated by Fourier transform ion cyclotron (FTICR) and triple quadrupole mass spectrometry. Theoretical calculations performed at the B3LYP/6-31+G* level of theory and collisionally activated dissociation (CAD) mass spectrometric results allowed the ionic population obtained in the CI conditions to be structurally characterized as a mixture of gaseous [H3P2O7...H2O]-, [H3PO4...H2PO4]-, and [PO3...H3PO4...H2O]- clusters. The energy profile emerging from theoretical calculations affords insight into the mechanism of diphosphate ion hydrolysis and synthesis.

Experimental validation of theoretical potassium and sodium cation affinities of amides by mass spectrometric kinetic method measurements.

In this study the theoretical Gaussian-2 K(+)/Na(+) binding affinities (enthalpies) at 0 K (in kJ mol(-1)) for six amides in the order: formamide (109.2/138.5) < N-methylformamide (117.7/148.6) < acetamide (118.7/149.5) < N,N-dimethylformamide (123.9/156.4) < N-methylacetamide (125.6/157.7) < N,N-dimethylacetamide (129.2/162.6), reported previously (Siu et al., J. Chem. Phys. 2001; 114: 7045-7051), were validated experimentally by mass spectrometric kinetic method measurements. By monitoring the collision-induced dissociation (CID) of K(+)/Na(+)-bound heterodimers of the amides, the relative affinities were shown to be accurate to within +/-2 kJ mol(-1). With these six theoretical K(+)/Na(+) binding affinities as reference values, the absolute K(+)/Na(+) affinities of imidazole, 1-methylimidazole, pyridazine and 1,2-dimethoxyethane were determined by the extended kinetic method, and found to be consistent (to within +/-9 kJ mol(-1)) with literature experimental values obtained by threshold-CID, equilibrium high-pressure mass spectrometry, and Fourier transform ion cyclotron resonance/ligand-exchange equilibrium methods. A self-consistent resolution is proposed for the inconsistencies in the relative order of K(+)/Na(+) affinities of amides reported in the literature. These two sets of validated K(+) and Na(+) affinity values are useful as reference values in kinetic method measurements of K(+)/Na(+) affinity of model biological ligands, such as the K(+) affinities of aliphatic amino acids.