Development of chiral selectors (SOs) is important both for understanding chiral molecular recognition processes and for their use in the separation of chiral species (selectands). Their evaluation by chromatographic procedures (e.g., as chiral stationary phase) can, however, be time-consuming. In this respect, electrospray ionization-MS (ESI-MS) is tested here as a possible alternative for screening enantioselective binding by SOs. The set of well-characterized cinchona alkaloid SOs are investigated with respect to their enantioselective binding to a set of model enantiomers, dinitrobenzoyl-(R)- and dinitrobenzoyl-(S)-leucine. MS-based enantioselectivity values from normalized gas-phase ion abundances for the diastereomeric complexes are compared empirically to chromatographic (HPLC) enantioselectivity results and shown to be consistent. Investigations into the fundamentals of measuring unbiased enantioselectivity values in the limit of dilute solution by correlation between experimental and modeled theoretical data are shown. Titration experiments are used to extract binding constants and are compared with published calorimetric (ITC) data. Results show that while the magnitude of binding affinities determined for various diastereomeric complexes is attenuated, the relative ranking and stereochemical preference in binding are consistently reproduced. This work represents a fundamental study of solution- versus gas-phase correlation for enantioselective systems by ESI-MS and indicates that, although not all questions and assumptions can be clearly engaged, for these enthalpically driven binding systems, the relative degree of binding affinity and selectivity is preserved.

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A new method for determination of dissociation constants (Kd) using on-line titration by electrospray ionization mass spectrometry is presented. Unlike in common titration experiments, where a set of discrete solutions with a fixed concentration of host and increasing concentration of guest is measured, here a continuous Gaussian concentration profile of guest, formed by band-broadening dispersion during passage through a long tubing, is utilized. An equation allowing access to dissociation constant values from experimental data fit to a 1:1 binding model was derived and incorporated into an in-house-written computer program for automated data processing. The new method is demonstrated for noncovalent complexes of cinchona alkaloid carbamate chiral selectors with N-dinitrobenzoylleucine enantiomers and a series of cyclodextrins with sulfonated azo dyes.

An LC-MS/MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d 4 ) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 μl/min flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d 4 in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d 4 added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

With recent growth in fields such as life sciences and supramolecular chemistry, there has been an ever increasing need for high-throughput methods that would permit determination of binding affinities for noncovalent complexes of various host-guest systems. These are traditionally measured by titration experiments where concentration-dependent signals of species participating in solution-based binding equilibria are monitored by methods such as UV-vis spectrophotometry, calorimetry, or nuclear magnetic resonance spectrometry. Here we present a new titration technique that unifies and allows chromatographic separation of guests with determination of dissociation constants by electrospray mass spectrometry in a multiplexed format. A theoretical model has been derived that describes the complex formation for the guests eluted from a chromatographic column when hosts are admixed postcolumn. The model takes possible competition equilibria into account; i.e., it can deal with unresolved peaks of guests with the possible addition of multiple hosts in one experiment. This on-line workflow makes determination of binding affinities for large libraries of compounds possible. The potential of the method is demonstrated on the determination of dissociation constants for complexes of beta- and gamma-cyclodextrins with nonsteroidal antiinflammatory drugs ibuprofen, naproxen, and flurbiprofen.