Samuel H. Yang

Restricted access media as a streamlined approach toward on‐line sample preparation: Recent advancements and applications

Restricted access media (RAM) as an alternative to traditional sample preparation strategies are reviewed. RAM comprise chromatographic packing materials that combine, typically, a restrictive outer surface to exclude the retention of large biomolecules, which are common interferences in biological fluids, with retentive inner pores or phases to capture analytes of interest. Through the years, a variety of RAM formats have been created, including internal surface phases, semipermeable phases, and molecularly imprinted polymer phases. Many phases are commercially available through a variety of manufacturers. The use of on-line sample preparation using RAM can increase throughput, recovery, and ease of use for sample preparation of complex biological matrices. The state-of-the-art with respect to production and use of these media for a variety of applications is covered.

Retention behavior of estrogen metabolites on hydrophilic interaction chromatography stationary phases

Estrogens and estrogen metabolites are important biological mediators of the endocrine system. They have also been implicated in detrimental carcinogenesis and beneficial neuroprotective processes. The retention behavior of estrogen metabolites was investigated on five polar stationary phases, used for hydrophilic interaction chromatography, and coupled with ESI-MS. Data were fit to partitioning and surface adsorption models. Retention of the compounds, especially estrogen glucuronides, on the amide- and diol-bonded stationary phases, could be best described by the surface adsorption model; however, mixed modes of retention were observed on most stationary phases. Retention time increased while the peak efficiency decreased proportional to the number of hydroxyl groups in the analytes. The effects of salt concentration and salt type were also investigated. The presence of solvated salt ions, which interact with the stationary phase and the analyte, enhanced retention of the analytes. This was believed to be due to two effects. The increased ionic strength reduced the contribution of secondary electrostatic interactions (mixed-mode effects). It also enhanced hydrogen-bonding and partitioning (hydrophilic interaction) between the analyte and the stationary phase, likely facilitated by the associated solvated salt ions.

Manipulation of protein charge states through continuous flow-extractive desorption electrospray ionization: a new ambient ionization technique.

Manipulation of protein charge states in electrospray ionization-mass spectrometry (ESI-MS) has implications for the study of intact proteins, protein-protein interactions, post-translational modifications, and protein sequencing. Control of these protein charge states is often difficult to achieve with conventional methods of analysis. A novel ambient ionization configuration, continuous flow-extractive desorption electrospray ionization (CF-EDESI), is presented as a means to control the charge state distribution of proteins. A key feature of the CF-EDESI technique is the continuous flow needle, which is a hypodermic needle presented orthogonal to the electrospray source and delivers a solvent flow containing analytes for extractive desorption ionization. With this source design, the successful manipulation of cytochrome c and lysozyme charge states with the use of different additives, such as acetic acid and sulfolane, was demonstrated. Results were compared to data obtained with conventional electrospray ionization. Good agreement with previously reported studies of cytochrome c unfolding/folding studies, performed by conventional ESI-MS, is evident. In addition to the protein analysis presented, the CF-EDESI-MS technique should be applicable for analyzing atypical analyte and solvent systems by mass spectrometry while maintaining optimal electrospray source conditions.

Quantitative determination of Bisphenol A from human saliva using bulk derivatization and trap‐and‐elute liquid chromatography coupled to electrospray ionization mass spectrometry

Endocrine disruptors cause adverse health effects as a result of their ability to shift the hormonal balance that is essential to the body. Bisphenol A (BPA) is an endocrine disruptor that has garnered much attention because of its presence in many consumer materials, which generates a significant risk for exposure. A method is presented for rapid detection of oral exposure to BPA directly from human saliva. Saliva was chosen because it serves as a noninvasive sampling route to detect BPA exposure; however, it is one of many complex biological matrices that have traditionally posed problems in quantitative analysis. Such analyses usually require extensive sample preparation to reduce interferences contributed by the sample matrix. Three validated methods are presented here that feature a streamlined sample-preparation strategy (bulk derivatization) prior to accurate and sensitive analysis by trap-and-elute liquid chromatography coupled to electrospray ionization mass spectrometry. Validated methods include standard addition calibration with variable injection volumes and multiple injection loading, as well as with incorporation of an internal standard. Reported limits of detection reached as low as 49.0 pg/ml (2.9 pg loaded on-column; equivalent to parts per trillion in saliva) among the presented methods with good accuracy and precision throughout. A proof-of-concept study is demonstrated to show that the final validated method has potential application to specific studies for trace-level BPA detection from real samples.

Affinity Mesh Screen Materials for Selective Extraction and Analysis of Antibiotics Using Transmission Mode Desorption Electrospray Ionization Mass Spectrometry

The extraction of active compounds from natural sources has shown to be an effective approach to drug discovery. However, the isolation and identification of natural products from complex extracts can be an arduous task. A novel approach to drug discovery is presented through the use of polymer screens functionalized with an l-lysine-d-alanine-d-alanine (Kaa) peptide to create new affinity capture mesh screen materials. The Kaa sequence is a well-characterized specific binding site for antibiotics that inhibit cell wall synthesis in Gram-positive bacteria. The detailed synthesis and characterization of these novel screen materials are presented in this work. Polypropylene mesh screens were first coated with a poly(acrylic acid) film by pulsed plasma polymerization. The synthesized Kaa peptide was then covalently attached to carboxylic acid groups through a condensation reaction. An analysis of captured compounds was performed in a rapid fashion with transmission-mode desorption electrospray ionization (TM-DESI) mass spectrometry. A proof of principle was demonstrated to show the ability of the novel affinity capture materials to select for a macrocyclic antibiotic, vancomycin, over a negative control compound, spectinomycin. With further development, this method may provide a rapid screening technique for new antibacterial compounds, for example, those extracted from natural product sources having a limited supply. Here, we show that the screen can capture vancomycin preferentially over spectinomycin in a spiked extract of tea leaves.

Solvent Molecules Undergo Homolytic Cleavage and Radical Recombination Processes during Negative-Mode Electrospray Ionization: Adduct Formation with Antimony(III)-Tartrate Dianion

Negative-ionization mode electrospray ionization-mass spectrometry (ESI-MS) analysis of antimony(III)-tartrate in frequently used solvent systems, ACN/H(2)O and MeOH/H(2)O, revealed that the antimony(III)-tartrate dianion associates to solvent reaction products generated by radical formation and their subsequent recombination during the negative-mode electrospray process. A systematic increase and decrease in negative spray capillary voltage (SCV) from normal operational voltage ranges of a conventional quadrupole ion trap instrument during these analyses showed initially unobserved adduct ions to correspondingly increase and diminish in relative ion intensity. The identity of the adducted species, including products such as H(2)O(2), NCCH(2)CH(2)CN, and CH(2)(OH)(2), were confirmed by performing similar experiments with deuterated and nondeuterated solvent mixtures. Relative intensity dependence of these adducted ions was monitored as the volume composition of each solvent system was changed. It was clearly observed that the relative intensity of {[Sb(2)-tar(2)][H-O-O-H]}(2-) and {[Sb(2)-tar(2)][NC-CH(2)-CH(2)-CN]}(2-) adduct ions increased with the volume percent of H(2)O and CH(3)CN, respectively. Similarly, an increase in volume percent of CH(3)OH increased the relative intensity of {[Sb(2)-tar(2)][H-O-CH(2)-O-H]}(2-) adducted ions. On the basis of this evidence, it was proposed that homolytic cleavage of C-H bonds for CH(3)CN and CH(3)OH molecules, and O-H bonds for H(2)O molecules, produces a series of radicals during negative-ionization mode ESI, and subsequent self-recombination or cross-recombination of these radicals then occurs to form the neutral solvent products, which are observed in the mass spectra as [Sb(2)-tar(2)]-adducted ionic species. These findings provide new insight into processes, which are relevant to understanding the mechanism of electrospray ionization, a widely used technique.

ESI‐MS investigation of solvent effects on the chiral recognition capacity of tartar emetic towards neutral side‐chain amino acids

The effect of solvent systems on previously-reported ESI-MS based proton-assisted enantioselective molecular recognition phenomena of tartar emetic, L-antimony(III)-tartrate, was evaluated. This was achieved by carrying out a series of competitive binding experiments using chiral selectors, bis(sodium) D- and -L-antimony(III)-tartrates with chiral selectands, neutral side-chain amino acid enantiomeric isotopomers of alanine (Ala), valine (Val), leucine (Leu) and phenylalanine (Phe), in three different solvent systems, ACN/H(2)O (75/25 v/v), H(2)O (100%) and H(2)O/MeOH (25/75 v/v). Observations from these experiments suggest that the effect of solvent systems on previously reported proton-assisted chiral recognition capacity of D,L-antimony(III)-tartrates is small, but not negligible. It was observed that an ACN/H(2)O (75/25 v/v) solvent system facilitates and enhances the chiral discrimination capacity of protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species. Further, amino acid enantiomers showed a general trend of increasing selectivity order, Val ≤ Ala < Leu ≈ Phe towards the protonated {[D,L-Sb(2)-tar(2)][H]}(-) ionic species which was independent of the solvent system employed. The lack of enantioselective binding for {[D,L-Sb(2)-tar(2)]}(2-) ionic species was consistently recorded in respective mass spectra from all performed experiments, which suggests that ESI-friendly solvent systems have no effect and do not influence this phenomenon.

Reversed phase liquid chromatography hyphenated to continuous flow-extractive desorption electrospray ionization-mass spectrometry for analysis and charge state manipulation of undigested proteins.

The application of continuous flow–extractive desorption electrospray ionization (CF-EDESI), an ambient ionization source demonstrated previously for use with intact protein analysis, is expanded here for the coupling of reversed phase protein separations to mass spectrometry. This configuration allows the introduction of charging additives to enhance detection without affecting the chromatographic separation mechanism. Two demonstrations of the advantages of CF-EDESI are presented in this work. First, a proof-of-principle is presented to demonstrate the applicability of hyphenation of liquid chromatography (LC) to CF-EDESI. LC-CF-EDESI-MS has good sensitivity compared to LC–electrospray ionization (ESI)–mass spectrometry. Second, the supercharging mechanism investigated in CF-EDESI provides an insight into a highly debated supercharging process in ESI. The results indicate that the mechanism of protein charging seen in HPLC-CF-EDESI is different from supercharging phenomena in conventional ESI. The surface tension mechanism and binding mechanism may both contribute to protein supercharging in ESI.