Mass spectrometric detection of trace anions: The evolution of paired-ion electrospray ionization (PIESI).

The negative-ion mode of electrospray ionization mass spectrometry (ESI-MS) is intrinsically less sensitive than the positive-ion mode. The detection and quantitation of anions can be performed in positive-ion mode by forming specific ion-pairs during the electrospray process. The paired-ion electrospray ionization (PIESI) method uses specially synthesized multifunctional cations to form positively charged adducts with the anions to be analyzed. The adducts are detected in the positive-ion mode and at higher m/z ratios to produce excellent signal-to-noise ratios and limits of detection that often are orders of magnitude better than those obtained with native anions in the negative-ion mode. This review briefly summarizes the different analytical approaches to detect and separate anions. It focuses on the recently introduced PIESI method to present the most effective dicationic, tricationic, and tetracationic reagents for the detection of singly and multiply charged anions and some zwitterions. The mechanism by which specific structural molecular architectures can have profound effects on signal intensities is also addressed.

Separation of peptides on superficially porous particle based macrocyclic glycopeptide liquid chromatography stationary phases: consideration of fast separations.

Macrocyclic glycopeptide based liquid chromatography stationary phases are known for their highly selective peptide separations. Fast and ultrafast (t R < 1 min) high-efficiency separations were achieved with superficially porous particle (SPP)-based stationary phases. Separations of pharmaceutically important classes of peptides such as enkephalins and bradykinins have been achieved in less than 5 min in isocratic elution modes. Selectivity for peptides structurally similar to one another was increased with use of teicoplanin-based stationary phases compared with commercial C18 stationary phases. Ultrafast isocratic separations of structurally related peptides were achieved with teicoplanin- and vancomycin-based short SPP columns. Acidic mobile phases produced better separations. Ammonium formate was the optimal mobile phase buffer additive. Use of an appropriate combination of a macrocyclic glycopeptide stationary phase and a mobile phase permits faster and more electrospray ionization mass spectrometry compatible isocratic separations than previous gradient approaches. The tryptic peptide separation characteristics of the teicoplanin stationary phase are demonstrated. Additionally, compared with commercial C18 stationary phases, teicoplanin showed tryptic peptide separations with different selectivities. Graphical Abstract Ultrafast separation of enkephalin peptide epimers.

Enantioseparation of citalopram analogues with sulfated ß-cyclodextrin by capillary electrophoresis.

Capillary electrophoresis methods were developed for the enantiomeric separation of 27 citalopram analogues. Sulfated ß-cyclodextrin was the most broadly selective and useful chiral selector. The separations of most of the citalopram analogue compounds reported in this work have not been reported previously. Excellent enantiomeric separations were obtained for 26 out of 27 compounds, and most of the separations were achieved within 10 min. The effects of chemical parameters such as chiral selector types, buffer types, chiral selector and buffer concentrations, buffer pH and organic modifiers on the separation were investigated. The influence of analyte structure on separation also was examined and discussed.

Gram Scale Conversion of R-BINAM to R-NOBIN.

A mild, operationally simple, and single-step transition-metal-free protocol for the synthesis of enantiomerically pure (R)-(+)-2'-amino-1,1'-binaphthalen-2-ol (R-NOBIN) from (R)-(+)-1,1'-binaphthyl-2,2'-diamine (R-BINAM) is reported. The one-pot conversion proceeds with good yield and shows no racemization. The hydroxyl on the R-NOBIN product was shown to have come from water in the reaction medium via an H2(18)O study. The correct value of the specific rotation of R-NOBIN was reported.

High-Performance Liquid Chromatographic Resolution of Neutral and Cationic Hetero[6]Helicenes.

Cationic hetero[6]helicenes 1+, 2+ and 3+ have been recently disclosed. Herein we report on their enantiomeric separation using high-performance liquid chromatography. Separation of the antipodes can be achieved in preparative scale on neutral adducts with Chiralcel OD-I or Chiralpak ID CSP. Selectivity factors of 1.90, 1.67, and 1.96 were obtained for 1-H, 2-H, and 3-H, respectively. Separation can also be performed on the carbenium ions on regular Chiralpak IA CSP using water-containing eluents, thus allowing for enantiomeric purity determinations in aqueous environments. Resolution of neutral and cationic helicenes is also achieved on more recently developed LARIHC columns. The versatility of the cyclofructan phases allows for baseline separations for both cases and their loading capabilities are demonstrated. Finally, the configurational stability of 1+, 2+, and 3+ was measured. For each replacement of an oxygen atom by an amino group, the racemization barrier increases significantly (ΔG‡ = 29.8, 36.3 and >37 kcal mol(-1) for 1+, 2+, and 3+ respectively).

Gone in seconds: praxis, performance, and peculiarities of ultrafast chiral liquid chromatography with superficially porous particles.

A variety of brush-type chiral stationary phases (CSPs) were developed using superficially porous particles (SPPs). Given their high efficiencies and relatively low back pressures, columns containing these particles were particularly advantageous for ultrafast "chiral" separations in the 4-40 s range. Further, they were used in all mobile phase modes and with high flow rates and pressures to separate over 60 pairs of enantiomers. When operating under these conditions, both instrumentation and column packing must be modified or optimized so as not to limit separation performance and quality. Further, frictional heating results in axial thermal gradients of up to 16 °C and radial temperature gradients up to 8 °C, which can produce interesting secondary effects in enantiomeric separations. It is shown that the kinetic behavior of various CSPs can differ from one another as much as they differ from the well-studied C18 reversed phase media. Three additional interesting aspects of this work are (a) the first kinetic evidence of two different chiral recognition mechanisms, (b) a demonstration of increased efficiencies at higher flow rates for specific separations, and

Combined use of cyclofructans and an amino acid ester-based ionic liquid for the enantioseparation of huperzine A and coumarin derivatives in CE.

Cyclofructans (CFs) and their derivatives have recently been proven to be efficient chiral selectors (CSs) for the enantioseparation of several analytes in CE, HPLC, and GC. In this study, the chiral separation ability of a number of native and derivatized CFs was examined in CE. Particularly, six different CFs, with different derivatization groups and cavity sizes [native CF-6 and CF-7, isopropyl cyclofructan-6 (IPCF-6), IPCF-7, sulfated cyclofructan-6 (SCF-6), and SCF-7] were used as CSs for the enantioseparation of huperzine A, warfarin, and coumachlor. Almost all of the examined CFs, except from SCF-6 & -7, demonstrated relatively low and sometimes no chiral separation ability for huperzine A. In an effort to improve both resolution and efficiency, the chiral ionic liquid D-Alanine tert butyl ester lactate (D-AlaC4Lac) was added into the BGE. In most of the cases, the combination of CF with D-AlaC4Lac resulted in an improvement in peak efficiency and/or resolution. When CF-6 was utilized with D-AlaC4Lac, a resolution of 1.4 was obtained, while the use of IPCF-6/D-AlaC4Lac provided a baseline enantioseparation. Although the combination of SCF-7 and 40 mM D-AlaC4Lac did not affect resolution, it dramatically increased peak efficiency from 24,000 to 117,000. In the case of warfarin and coumachlor, IPCF-6 and IPCF-7 proved to be the most effective CSs. It is, therefore, concluded that the size of the cavity and the CF derivatization are the key parameters for the chiral separation capability. It is also clear from this study that D-AlaC4Lac is necessary for improved peak efficiencies and resolutions.

Superficially Porous Particle Based Hydroxypropyl-ß-cyclodextrin Stationary Phase for High-Efficiency Enantiomeric Separations.

A superficially porous particle (SPP)-based hydroxypropyl-ß-cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)-based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP-based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation.

Enantiomeric separations of ruthenium (II) polypyridyl complexes using HPLC with cyclofructan chiral stationary phases.

The enantiomeric separation of 21 ruthenium (II) polypyridyl complexes was achieved with a novel class of cyclofructan-based chiral stationary phases (CSPs) in the polar organic mode. Aromatic derivatives on the chiral selectors proved to be essential for enantioselectivity. The R-napthylethyl carbamate functionalized cyclofructan 6 (LARIHC CF6-RN) column proved to be the most effective overall, while the dimethylphenyl carbamate cyclofructan 7 (LARIHC CF7-DMP) showed complementary selectivity. A combination of acid and base additives was necessary for optimal separations. The retention factor vs. acetonitrile/methanol ratio plot showed a U-shaped retention curve, indicating that different interactions take place at different polar organic solvent compositions. The separation results indicated that π-π interactions, steric effects, and hydrogen bonding contribute to the enantiomeric separation of ruthenium (II) polypyridyl complexes with cyclofructan chiral stationary phases in the polar organic mode.

Mechanism and sensitivity of anion detection using rationally designed unsymmetrical dications in paired ion electrospray ionization mass spectrometry.

Paired ion electrospray ionization (PIESI) mass spectrometry was developed as a useful technique that provides sensitive detection for anions in the positive ion mode. The ion-pairing reagent (IPR) utilized plays an essential role affecting the detection limits. This work describes the design and synthesis of two novel dications with unsymmetrical structures and their utilization for anion detection and mechanistic insights. The performance of dications was evaluated for seven selected anions in both single ion monitoring (SIM) mode and selected reaction monitoring (SRM) mode. The unsymmetrical dications allowed sensitive detection for these anions with down to subpicogram limits of detection (LOD) and an improved sensitivity from 1.5 to 12 times compared to the corresponding symmetrical dications. The enhanced sensitivity could be attributed to the surface activity of the unsymmetrical dications, which results in a concurrent strong partitioning of the anion to the aerosol droplet surface. Surface activity measurements of the anion/IPR complex were conducted, and a correlation between the observed ESI responses and the surface activity of the complex was found. The mechanism was further explored and explained based on the concepts of the equilibrium partitioning model (EPM).

Development and evaluation of gas and liquid chromatographic methods for the analysis of fatty amines.

In contrast to the plethora of publications on the separation of fatty acids, analogous studies involving fatty amines are scarce. A recently introduced ionic-liquid-based capillary column for GC was used to separate trifluoroacetylated fatty amines focusing on the analysis of a commercial sample. Using the ionic liquid column (isothermal mode at 200 °C) it was possible to separate linear primary fatty amines from C12 to C22 chain length in less 25 min with MS identification. The log of the amine retention factors are linearly related to the alkyl chain length with a methylene selectivity of 0.117 kcal/mol for the saturated amines and 0.128 kcal/mol for the mono-unsaturated amines. The sp2 selectivity for unsaturated fatty amines also could be calculated as 0.107 kcal/mol for the ionic liquid column. The commercial sample was quantified by GC with flame ionization detection (FID). An LC method also was developed with a reversed phase gradient separation using acetonitrile/formate buffer mobile phases and ESI-MS detection. Native amines could be detected and identified by their single ion monitoring chromatograms even when partial coelution was observed. The analysis of the commercial sample returned results coherent with those obtained by GC-FID and with the manufacturer's data.

Cleavage of DNA by proton-coupled electron transfer to a photoexcited, hydrated Ru(II) 1,10-phenanthroline-5,6-dione complex.

Visible light irradiation of a ruthenium(II) quinone-containing complex, [(phen)(2)Ru(phendione)](2+) (1(2+)), where phendione = 1,10-phenanthroline-5,6-dione, leads to DNA cleavage in an oxygen independent manner. A combination of NMR analyses, transient absorption spectroscopy, and fluorescence measurements in water and acetonitrile reveal that complex 1(2+) must be hydrated at the quinone functionality, giving [(phen)(2)Ru(phenH(2)O)](2+) (1H(2)O(2+), where phenH(2)O = 1,10-phenanthroline-6-one-5-diol), in order to access a long-lived (3)MLCT(hydrate) state (τ ∼ 360 ns in H(2)O) which is responsible for DNA cleavage. In effect, hydration at one of the carbonyl functions effectively eliminates the low-energy (3)MLCT(SQ) state (Ru(III) phen-semiquinone radical anion) as the predominant nonradiative decay pathway. This (3)MLCT(SQ) state is very short-lived (<1 ns) as expected from the energy gap law for nonradiative decay, (1) and too short-lived to be the photoactive species. The resulting excited state in 1H(2)O(2+)* has photophysical properties similar to the (3)MLCT state in [Ru(phen)(3)](2+)* with the added functionality of basic sites at the ligand periphery. Whereas [Ru(phen)(3)](2+)* does not show direct DNA cleavage, the deprotonated form of 1H(2)O(2+)* does via a proton-coupled electron transfer (PCET) mechanism where the peripheral basic oxygen sites act as the proton acceptor. Analysis of the small molecule byproducts of DNA scission supports the conclusion that cleavage occurs via H-atom abstraction from the sugar moieties, consistent with a PCET mechanism. Complex 1(2+) is a rare example of a ruthenium complex which 'turns on' both reactivity and luminescence upon switching to a hydrated state.

Use of sulfated cyclofructan 6 and sulfated cyclodextrins for the chiral separation of four basic pharmaceuticals by capillary electrophoresis.

Sulfated cyclofructan 6 (S-CF6) and sulfated cyclodextrins (S-α-, ß-, γ-CDs) are highly selective chiral selectors for the enantioseparation of basic solutes. In this study, S-CF6 was introduced for the enantiomeric separation of four basic pharmaceuticals (including tamsulosin, tiropramide, bupivacaine, and norephedrine) by capillary electrophoresis (CE), and the enantiomeric separation performance was compared with S-α-, ß-, γ-CDs. The effects of the chiral selector type, chiral selector concentration, operating voltage, and column temperature were examined and optimized. Excellent resolutions were obtained for all solutes on these chiral selectors.

The enantiomeric separation of tetrahydrobenzimidazoles cyclodextrins- and cyclofructans.

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were used to examine the enantiomeric separation of a series of 17 racemic tetrahydrobenzimidazole analytes. These compounds were prepared as part of a synthetic program directed towards a select group of pyrrole-imidazole alkaloids. This group of natural products has a unique framework of pyrrole- and guanidine-containing fused rings which can be constructed through the intermediacy of a tetrahydrobenzimidazole scaffold. Several bonded cyclodextrin- (both native and derivatized) and derivatized cyclofructan-based chiral stationary phases were evaluated for their ability to separate these racemates via HPLC. Similarly, several cyclodextrin derivatives and derivatized cyclofructan were evaluated for their ability to separate this set of chiral compounds via CE. Enantiomeric selectivity was observed for the entire set of racemic compounds using HPLC with resolution values up to 3.0. Among the 12 different CSPs, enantiomeric recognition was most frequently observed with the Cyclobond RN and LARIHC CF6-P, while the Cyclobond DMP yielded the greatest number of baseline separations. Fifteen of the analytes showed enantiomeric recognition in CE with resolution values as high as 5.0 and hydroxypropyl-γ-cyclodextrin was the most effective chiral additive.

Unified chromatography in drug development: Exploiting chaotropic/kosmotropic salts for an accelerated method development.

Recent trends in supercritical fluid chromatography (SFC) introduced an innovative gradient profile called Unified Chromatography (UC), which pushes the amount of liquid modifier up to 80-100 % of the total mobile phase composition. These new conditions allow the full transition from a supercritical to a liquid state, unifying the benefits of both SFC and liquid chromatography. However, to facilitate the use of UC for industrial drug development, a stronger effort is needed to streamline and simplify its method development and optimization. In this work, a quick and novel method development procedure for UC is introduced, enabled by the first-time use of novel additives in SFC/UC that exploit chaotropic/kosmotropic properties. A comprehensive view on some fundamental properties, such as the amount of liquid modifier blended with supercritical CO2 (scCO2) and the percentage of water added in the mobile phase is given, to clarify the benefits of using either a chaotropic salt (NaClO4), kosmotropic (HCOONa) or salt with mixed properties (NaOMs - sodium methanesulfonate). With this expanded knowledge, challenging separations of nucleosides, nucleotide, indoles, triazoles and related derivates have been accomplished with UC. Finally, we provide an example of UC delivering a faster and better method for an AbbVie pipeline compound under accelerated stability study. The combined use of scCO2-based chromatography and the novel additive NaClO4 ensures the retention and elution of all degradation species generated at different conditions, where RP-HPLC failed to provide satisfactory performance.

Separation of multiply charged anions by capillary electrophoresis using alkyl phosphonium pairing agents.

Two newly developed UV transparent phosphonium-based cationic reagents were evaluated as background electrolyte additives for capillary electrophoresis for the separation of multiply charged anions, including several complex anions. These cationic reagents showed moderate suppression of the electroosmotic flow, interacted with the analytes to improve their separation and often improved the peak shape. The effects of the additives and their concentration on the separation were studied, as well as the buffer type, pH, and voltage. The dicationic reagent effectively separated eight divalent anions within 17 min and the tetracationic reagent best separated nine trivalent anions, as well as a mixture of all the anions.

CE-ESI-MS separation of divalent organic and inorganic anions using a tricationic complexing reagent.

A tricationic ion-pairing reagent, 1,3,5-1-butyl-3-methyl-1H-imidazol-3-ium-2,4,6-trimethylbenzene, was used to form complexes with doubly charged anions for their subsequent analysis by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in the positive-ion mode. This methodology offers the advantages of greater versatility and sensitivity relative to direct detection of the anions in negative-ion mode, and it can be realized by a number of possible complexation strategies, including pre-column, on-column, and post-column modes. Three model anions, sulfate [SO4²â»], thiosulfate [TSFA, S2O3²â»], and benzenedisulfonate [BZDS, C6H4(SO3)2²â»], were amenable to complexation with the tricationic reagent, yielding singly charged cations with greater mass-to-charge (m/z) ratios than the native analytes. By utilizing optimized parameters obtained through previous work with dicationic reagents and singly charged anions, including the CE separation buffer composition and pH, the concentration of the dicationic reagent, the mode of complexation, the nebulizing gas pressure, and the sheath liquid composition, it was possible to develop a robust CE-ESI-MS method appropriate for the analysis of divalent anions in a mixture.

Metal cation detection in positive ion mode electrospray ionization mass spectrometry using a tetracationic salt as a gas-phase ion-pairing agent: evaluation of the effect of chelating agents on detection sensitivity.

RATIONALE: The detection of metal cations continues to be essential in many scientific and industrial areas of interest. The most common electrospray ionization mass spectrometry (ESI-MS) approach involves chelating the metal ions and detecting the organometallic complex in the negative ion mode. However, it is well known that negative ion mode ESI-MS is generally less sensitive than the positive ion mode. To achieve greater sensitivity, it is necessary to examine the feasibility of detecting the chelated metal cations in positive ion mode ESI-MS. METHODS: Since highly solvated native metal cations have relatively low ionization efficiency in ESI-MS, and can be difficult to detect in the positive ion mode, a tetracationic ion-pairing agent was added to form a complex with the negatively charged metal chelate. The use of the ion-pairing agent leads to the generation of an overall positively charged complex, which can be detected at higher m/z values in the positive ion mode by electrospray ionization linear quadrupole ion trap mass spectrometry. RESULTS: Thirteen chelating agents with diverse structures were evaluated in this study. The nature of the chelating agent played as important a role as was previously determined for cationic pairing agents. The detection limits of six metal cations reached sub-picogram levels and significant improvements were observed when compared to negative ion mode detection where the metal-chelates were monitored without adding the ion-pairing reagent (IPR). Also, selective reaction monitoring (SRM) analyses were performed on the ternary complexes, which improved detection limits by one to three orders of magnitude. CONCLUSIONS: With this method it was possible to analyze the metal cations in the positive ion mode ESI-MS with the advantage of speed, sensitivity and selectivity. The optimum solution pH for this type of analysis is 5-7. Tandem mass spectrometry (MS/MS) further increases the sensitivity. Speciation is straightforward making this a broadly useful approach for the analysis of metal ions.

Mechanisms of ESI-MS selectivity and sensitivity enhancements when detecting anions in the positive mode using cationic pairing agents.

Recently, we have shown that dilute multivalent cationic reagents can be paired with analyte anions in ESI-MS, thereby allowing them to be detected in the positive mode at very low limits of detection. However, there can be differences in the efficiency of this technique depending on the nature of the cationic pairing agent and the anion being analyzed. In this study, three dicationic ion-pairing agents and four singly charged anionic species were examined in a series of experiments to elucidate the mechanism of action that allows for such sensitive detection and the profound differences in the selectivity of this ion-pairing method. The binding constants for the dication/anion complexes were determined by NMR and ESI-MS. The results indicated that the binding of these species is greatly enhanced as they move from the solution phase to the gas phase. Furthermore, surface tension measurements for the complexes were performed. This test revealed that, as the dication pairs with the anion, it creates a surface-active species within the ESI droplet. This is determined to be one of the major factors that leads to the overall sensitivity enhancement. This has led to a better understanding of how this ion-pairing technique produces unprecedented limits of detection for anions and why there are selectivity differences in pairing agents of different structures.

Measurement of nitrophenols in rain and air by two-dimensional liquid chromatography-chemically active liquid core waveguide spectrometry.

We report a novel system to analyze atmospheric nitrophenols (NPs). Rain or air sample extracts (1 mL) are preconcentrated on a narrow bore (2 mm) aliphatic anion exchanger. In the absence of strong retention of NPs exhibited by aromatic ion exchangers, retained NPs are eluted as a plug by injection of 100 microL of 0.1 M Na(2)SO(4) on to a short (2 x 50 mm) reverse phase C-18 column packed with 2.2 mum particles. The salt plug passes through the C-18 column unretained while the NPs are separated by an ammonium acetate buffered methanol-water eluent, compatible with mass spectrometry (MS). The eluted NPs are measured with a long path Teflon AF-based liquid core waveguide (0.15 x 1420 mm) illuminated by a 403 nm light emitting diode and detected by a monolithic photodiode-operational amplifier. The waveguide is rendered chemically active by suspending it over concentrated ammonia that permeates into the lumen. The NPs ionize to the yellow anion form (lambda(max) approximately 400 nm). The separation of 4-nitrophenol, 2,4-dinitrophenol, 2-methyl-4-nitrophenol, 3-methyl-4-nitrophenol, and 2-nitrophenol (these are the dominant NPs, typically in that order, in both rain and air of Houston and Arlington, TX, confirmed by tandem MS) takes just over 5 min with respective S/N = 3 limits of detection (LODs) of 60, 12, 30, 67, and 23 pg/mL compared to MS/MS LODs of 20, 49, 11, 20, and 210 pg/mL. Illustrative air and rain data are presented.