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Choline is a quaternary ammonium bioactive compound that is used as a dietary supplement. Direct determination of choline in pharmaceutical formulations is a challenging analytical task. The strong UV absorbance of p-sulfonatocalix[4]arene (SC4) and its outstanding complexation properties towards quaternary ammonium compounds have been utilized to develop a method to detect and quantify choline in aqueous solution. This method has been applied to a commercial pharmaceutical choline formulation showing results with no statistically significant difference from those obtained by the official U.S. Pharmacopeial method. The method was validated according to ICH guidelines and the limit of detection for choline was calculated to be 7.2 × 10−6 mol L−1 (1.30 μg mL−1). The host-guest complexation between SC4 and choline were investigated using UV and 1H NMR spectroscopy and DFT calculations. The peak amplitude of the host-guest complexes formed were found to be linearly proportional to the concentration of the choline and l -carnitine analytes in the ranges of 1.0 × 10−5 to 1.6 × 10−4 mol L−1 and 1.0 × 10−5 to 1.8 × 10−4 mol L−1 respectively. The reaction stoichiometry between either choline or l -carnitine with SC4 in the formed complexes were determined to be 1:1 while the stability constants for the complexes formed were estimated to be 8.3 × 104 M−1 and 2.8 × 104 M−1 in the case of choline and l -carnitine, respectively. Both 1H NMR spectroscopy and DFT calculations suggest the complexation between choline and SC4 involves the insertion of the trimethylammonium group of choline into the cavity of SC4 in agreement with previously published X-ray data. The spectrophotometric method presented here should not be limited to the determination of choline. The linear relationship between the peak amplitude of the host-guest complex formed between SC4 and l -carnitine presented here suggests the applicability of the method for the detection of other small molecular species possessing a quaternary ammonium group.

Macromolecules including macrocyclic species have been reported to have the potential to encapsulate biologically active compounds such as drugs through host-guest complexation to increase their solubility, stability and bioavailability. In this paper the first experimental and theoretical investigation of the complexation between nedaplatin, a second generation antineoplastic drug, and p-4-sulfocalix[4]arene, a macromolecule possessing a bipolar amphiphilic structure with good biocompatibility and relatively low haemolytic toxicity for potential use as a drug delivery system is presented. Data from 1H NMR, UV, Job's plot analysis, HPLC and DFT calculations are detailed and suggest the formation of a 1:1 complex. The stability constant of the complex was experimentally estimated to be 3.6 × 104 M− 1 and 2.1 × 104 M− 1 which correspond to values of − 6.2 and − 5.9 kcal mol− 1, respectively for the free energy of complexation while the interaction free energy is calculated to be − 4.9 kcal mol− 1. The formed species is shown to be stabilised in solution through hydrogen bonding between the host and the guest which may allow for this strategy to be effective for potential use in drug delivery.

Protein phosphorylation and de-phosphorylation, provide one of the most common signalling pathways within cells, being involved in regulating cellular processes, mediating enzyme inhibition, protein–protein recognition and protein degradation. Compared with normal proteomics, phosphoproteomics poses some additional challenges requiring more initial separation and additional sensitivity to detect and quantify potentially ultra-low abundance species. In this work, the selective detection of phosphopeptides is described based on the incorporation of a metal tag, gallium–N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC–ICP-MS). Experimental and theoretical characterisation of the resulting Ga–phosphopeptide complex is presented based on linear ion trap electrospray ionisation mass spectrometry (ESI-MS), Fourier transform mass spectrometry (FT-MS) and molecular modelling data. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (s-PDGF), a small phosphopeptide. In addition high resolution Fourier transform mass spectrometry (FT-MS) was used for accurate mass determination and multistage tandem mass spectrometry of the gallium–s-PDGF complex identified the fragmentation pathway. Finally, molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the s-PDGF–Ga-LysNTA complex.

The fragmentations of [AA + M](+) complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag(•)).

This paper describes methodologies, based on sector field inductively-coupled plasma mass spectrometry (SF-ICP-MS), and their application in the holistic study of the fate of Pt in human cell populations following treatment with cis- or oxaliplatin and combination treatments. Pt–DNA adduct formation data at several time points has been determined in the leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 10 6 nucleotides using 10 μg of patient DNA. Further, the first ever reported in vivo sub-cellular Pt fractionation data on a patient sample is presented indicating the feasibility of applying the methods presented here in a clinical environment. For in vitro studies, three cell models were used: A549 human lung adenocarcinoma epithelial cells were exposed to 50 μM cisplatin for 1 h; HCA7 human colorectal cancer cells were treated with either FOLFOX (200 μM 5-fluorouracil, 200 μM folinic acid and 50 μM oxaliplatin) or 50 μM oxaliplatin; and HT29 human colorectal cancer cells were treated with 50 μM oxaliplatin in combination with 20 μM methaneseleninic acid, CH 3 SeO 2 H (MSA). The cells were harvested and either the DNA extracted and/or a commercially available kit used to fractionate the treated cells into four sub-cellular compartments. Each of the sub-cellular fractions and extracted DNA were digested separately, evaporated to dryness and reconstituted in 2% nitric acid for analysis by SF-ICP-MS. The sub-cellular Pt distribution for cisplatin treated A549 cells was shown to be as follows: ∼70% localized in the cytosol, ∼17% in the membrane and membrane localized fraction, ∼9% in the nuclear fraction and ∼4% in the cytoskeletal fraction. Both FOLFOX and oxaliplatin treated HCA7 cells showed comparable sub-cellular Pt distributions, and Pt–DNA adduct formation was similar for the oxaliplatin and FOLFOX treatments with adduct yields of 5.6 and 5.5 adducts per 10 6 nucleotides respectively. It was found that the combination of oxaliplatin with 20 μM MSA did not change the distribution of Pt or significantly alter its accumulation in the cytosol of the HT29 cells. Mass balance experiments showed a >99% recovery of the total Pt in the sub-cellular fractions. These experiments are the first to provide such a detailed quantitation of Pt-drug partitioning and they show that the Pt broadly follows the total protein content of the individual compartments with the majority being scavenged in the cytosol compartment.

This paper presents size exclusion chromatography data with on-line coupling to UV and inductively coupled plasma mass spectrometry (ICP-MS) of water soluble metal-binding compounds present in zinc, copper, chromium and iodine enriched yeast nutritional supplements. Molecular weight estimates of the extracted metal-containing compounds are given and are shown to vary substantially from 1.2 kDa to larger than 668 kDa. Seven proteins suspected of containing chromium were identified from one of the chromium-containing fractions. Four of these identified proteins are known to form complexes with other metal ions. The metal chromatographic profiles of zinc, copper and chromium-enriched yeasts were compared to their respective native metal profiles in non-enriched yeast samples. The chromium profiles are shown to be markedly different while those of zinc and copper are qualitatively similar. Only iodide ions or weakly bound, non-aromatic, low molecular weight (∼ 1.2 kDa) iodine species were observed in the iodine-enriched yeast samples.

The gas-phase dissociation chemistries of protonated 3-iodo- l -tyrosine, 3,5-diiodo- l -tyrosine and 3,3′,5,5′-tetraiodo-thyronine (thyroxine) have been examined using a combination of tandem mass spectrometry and density functional theory (DFT) calculations. It was found that, at low collision energy, all protonated tyrosines exhibit common fragmentation pathways, including the competitive eliminations of NH3 and the concomitant loss of H2O and CO, but there are significant differences in relative abundances, depending on the combined electron-donating abilities of the substituents in the phenyl ring. The ions initially formed by loss of NH3 are phenonium ions, but subsequent fragmentation is most easily understood in terms of the isomeric benzyl cation structures. These [M + H − NH3]+ ions fragment at relatively low collision energies, mainly by loss of ketene; by contrast, the [M + H − H2O − CO]+ ions are more stable towards dissociation. At higher collision energies, losses of one, two and even three iodine atoms were observed. DFT calculations (at the B3LYP/DZVP level of theory) were performed on protonated 3-iodotyrosine to compare the reaction profiles for the fragmentation mechanisms. The iodo-substituent in the 3-position is weakly electron-withdrawing and this results in a barrier (27.5 kcal/mol at 0 K) that is slightly higher than that for protonated tyrosine (26.8 kcal/mol). The phenoxy group PhO– is a weaker electron-donor than HO– and protonated 3,5-diiodo-4-phenoxytyrosine has an even higher barrier (31.1 kcal/mol) to NH3 loss than protonated 3,5-diiodotyrosine (28.8 kcal/mol). Linear free energy plots for Δ H 0 ° ‡ and Δ G 298 ° ‡ against σ+ for the four protonated tyrosine derivatives show good correlations. More importantly, as the products of the dissociation are higher in energy than the transition states to their formation, the plots of Δ H 0 ° and Δ G 298 ° for the overall reaction for NH3 loss also correlate very well with σ+ (correlation coefficients of 0.99 and 0.98, respectively). The positive slopes of these Hammett plots show that the barriers to the loss of NH3 by the neighboring-group mechanism are increased by the presence of electron-withdrawing groups in the phenyl rings.

Collision-induced dissociation of complexes, Ag+/NH2CH2(CH2) nCH2NH2, where n has values of 0, 1, 2 and 3, show loss of one H2 molecule at low energies. For the largest complex ( n = 3), a second H2 molecule is also lost. For complexes with n = 0 and 1, loss of AgH (the only pathway observed in the fragmentation of Ag+/monoamine complexes) also occurs at low collision energies and this becomes the dominant fragmentation pathway at centre-of-mass energies above 2 eV. For complexes with n = 2 and 3, negligible amounts of product ions resulting from loss of only AgH were formed. However, for these complexes, the major products at higher collision energies result from loss of either AgH plus NH3 or, more likely, H2 plus AgNH2; the product ions are postulated to be cyclic iminium ions. Density functional theory (DFT) calculations (B3LYP with a DZVP basis set) on Ag+/NH2CH2(CH2)2CH2NH2 showed the loss of H2 followed by AgNH2 to have a slightly lower barrier than loss of AgH plus NH3. For complexes with n = 2 and 3, there are minor losses of various combinations of AgH, H2, 2H2 and NH3. Reaction profiles for the losses of H2 and AgH from all four Ag+/α,ω-diamine complexes have been examined computationally using DFT calculations. The second step on the reaction profile, loss of either H2 or AgH from the complex between Ag+ and α-amino-γ-imino-propane, Ag+/NH2CH2CH2CHNH, has also been calculated. The highest energy transition state on the profiles to loss of both H2 and AgH from the smallest complex ( n = 0) are identical; on all the other profiles, the barrier for H2 loss is lower than that for AgH loss (by 2.5 kJ mol−1 when n = 1, by 13.4 kJ mol−1 when n = 2 and by 33.0 kJ mol−1 when n = 3). Fragmentation of the ligand backbone occurs most extensively for ions derived from the Ag+/NH2CH2CH2CH2NH2. After loss of H2, the product ion, Ag+/NH2CH2CH2CH = NH fragments via a transition state in which the CH2–CH2 bond is breaking, while a 1,5-hydrogen shift from one nitrogen to the other is occurring. Cleavage of the carbon chain of NH2CH2CH2CH = NH2+ occurs at relatively low energy; DFT calculations provide a mechanism by which H2C = NH plus CH3CH = NH2+ are produced.

The room-temperature reactivities of complexes of Cu + and Ag + with glycine have been investigated using an inductively coupled plasma-selected ion flow tube (ICP-SIFT)/multicollision-induced dissociation (CID) tandem mass spectrometer. These complexes were produced in a flow tube, collisionally thermalized and then allowed to react with CO, D 2 O or NH 3 . The measured reactivities of the CuGly + and AgGly + complexes have been compared with those of the bare metal cations towards the same neutral reagents. All observed reactions resulted in adduct formation, with helium buffer gas presumably acting as a stabilizing agent. Reaction rate enhancements of up to three orders of magnitude and lower extents of ligation were the main characteristics of the reactions initiated by the metal cation–glycine adducts as compared with those initiated by the bare metal cations. The kinetic information combined with equilibrium analyses and CID results suggest that, irrespective of the reagent, ligation to CuGly + is stronger than to AgGly + and, in both instances, ligation of carbon monoxide and water has comparable strengths, while much stronger coordination is achieved with ammonia. The structures of the complexes have been investigated computationally using density functional theory (DFT) at the B3LYP level employing the DZVP basis set. Optimized structures and the free energy changes associated with ligation have been computed. A good correlation was obtained between the experimental reaction efficiencies and the calculated free energies of bonding. Also, results are presented for the potential energy surfaces for the conversion of the “charge solvated” forms into the “metal salt” forms of CuGly + and AgGly + and of their adducts with CO or NH 3 . The computations indicate a modest catalytic effect of the CO and NH 3 on this conversion.

Tandem mass spectrometry performed on a pool of 18 oligopeptides shows that the product ion spectra of argentinated peptides, the [b n + OH + Ag]+ ions and the [y n − H + Ag]+ ions bearing identical sequences are virtually identical. These observations suggest strongly that these ions have identical structures in the gas phase. The structures of argentinated glycine, glycylglycine, and glycylglycylglycine were calculated using density functional theory (DFT) at the B3LYP/DZVP level of theory; they were independently confirmed using HF/ LANL2DZ. For argentinated glycylglycylglycine, the most stable structure is one in which Ag+ is tetracoordinate and attached to the amino nitrogen and the three carbonyl oxygen atoms. Mechanisms are proposed for the fragmentation of this structure to the [b2 + OH + Ag]+ and the [y2 − H + Ag]+ ions that are consistent with all experimental observations and known calculated structures and energetics. The structures of the [b2 − H + Ag]+ and the [a2 − H + Ag]+ ions of glycylglycylglycine were also calculated using DFT. These results confirm earlier suggestions that the [b2 − H + Ag]+ ion is an argentinated oxazolone and the [a2 − H + Ag]+ an argentinated immonium ion.

The [M − nH + mNa](m−n)+ and [M − nH + mK](m−n)+ ions are common in the electrospray mass spectra of proteins and peptides. The feasibility of forming these ions in the gas phase via collision activation and/or ion-molecule reaction is investigated. Sodium and potassium affinities of the N-methylacetamide anion, the acetate anion, and the 1-propanamide anion have been calculated using density functional theory at the B3LYP/6-311+ +G(d,p) level of theory. These anions were chosen as models for the functional groups on a protein or peptide. These affinity values are then used to calculate reaction enthalpies of alkali hydroxides, chlorides, and hydrates with N-methylacetamide, acetic acid, the acetate anion, and 1-propanamine, model reactions that may lead to formation of the [M − nH + mNa](m−n)+ and [M − nH + mK](m−n)+ ions. It is found that a number of these reactions are exothermic or slightly endothermic (ΔH 0 < + 20 kcal/mol) and are accessible after collision activation in the lens region. The potential energy hypersurfaces of model reactions between NaOH and formamide as well as NaCl and formamide show relatively flat surfaces devoid of significant barriers.

Density functional calculations at B3LYP/6-31+G(d) and B3LYP/DZVP are reported for Mg(NH 3 ) n +· , where n = 1–6 and for some solvated ions Mg(NH 3 ) n +· … NH 3 ( n = 1–3, 6). After correction for basis set superposition errors, the enthalpies for sequential addition of NH 3 to Mg +· resulting from direct coordination to the metal are 38.1, 26.6, 21.2, 13.7, 12.1, and 11.3 kcal mol −1 . The free energies for these same addition reactions are all negative, although for complexes with n ≥ 4 the values are very small. Attempts at optimising structures with higher coordination numbers all resulted in the formation of solvated octahedral complexes. Enthalpies for solvation through hydrogen bonding to one of the ligated NH 3 molecules are all less than 16 kcal mol −1 and decrease rapidly as the number of ligated NH 3 molecules increases. Molecular orbital calculations at B3LYP/DZVP have been used to optimise structures for ions Ag(NH 3 ) n + , where n = 1–6. The five-coordinate and six-coordinate structures have very small binding enthalpies (4.3 and 2.6 kcal mol −1 ) and the free energies for formation of these ions are positive. The binding energies for the addition of the first and second NH 3 molecules added to Ag + are 40.1 and 36.1 kcal mol −1 , while those for the third and fourth additions are much smaller (15.1 and 11.0 kcal mol −1 ). Adducts up to n = 3 have been detected in electrospray experiments. The first three adducts of Ag + with NH 3 have been formed in the selected ion flow tube apparatus and multicollision induced dissociation experiments show Ag(NH 3 ) 3 + to have a lower binding enthalpy than both Ag(NH 3 ) 2 + and Ag(NH 3 ) + .

Collision-induced dissociation experiments on the Ag+–phenylalanine complex using several collision energies were shown to yield ten different fragment ions. Unambiguous assignment of these fragment ions were made by careful analysis of deuterium labeling experiments. The losses of H2O, CO, CO2, and AgH were commonly observed; also encountered were the losses of H2, Ag, and H. Deuterium labeling experiments and density functional calculations have been employed to probe fragmentation mechanisms that account for all experimental results.