Characterization of High Molecular Weight Multi-Arm Functionalized PEG-Maleimide for Protein Conjugation by Charge-Reduction Mass Spectrometry Coupled to Two-Dimensional Liquid Chromatography.

A current trend in drug development involves the use of high molecular weight, branched, and functionalized polymers for protein conjugation and drug delivery. Accurately characterizing these polymers is critical to control the product quality, to monitor the stability, and ultimately to ensure the drug efficacy and patient safety. However, due to the heterogeneity in size, the multiplicity of functional groups, and the highly convoluted charge-distribution profile in mass spectra, the characterization of these polymers is highly challenging from both chromatography and mass spectrometry perspectives. To overcome these challenges, we developed a strategy utilizing charge-reduction mass spectrometry (CRMS) coupled with two-dimensional HPLC (2D-LC). We then applied the workflow to characterize a 40 kDa 8-arm polyethylene glycol (PEG) functionalized with a maleimide terminal group for protein conjugation. The development was carried out in stages, where first we focused on the development of a CRMS method to simplify the charge profile of the polymers and then coupled it to HPLC to obtain discernible mass spectra of key impurities and degradants. Second, the CRMS method was applied to an investigation of the size-variant impurity resolved by reversed-phase size-exclusion 2D-LC. Finally, a separate size-exclusion reversed-phase 2D-LC-CRMS method was developed to capture a wider range of process-related impurities and reaction intermediates from the PEG-maleimide polymers throughout the conjugation process. The combination of these experiments using the 2D-LC-CRMS strategy enables the sensitive characterization of the entire impurity profile of the high molecular weight multifunctionalized PEG-maleimide conjugation intermediate.

Validation of a two-dimensional liquid chromatography method for quality control testing of pharmaceutical materials.

Despite the advantages of 2D-LC, there is currently little to no work in demonstrating the suitability of these 2D-LC methods for use in a quality control (QC) environment for good manufacturing practice (GMP) tests. This lack of information becomes more critical as the availability of commercial 2D-LC instrumentation has significantly increased, and more testing facilities begin to acquire these 2D-LC capabilities. It is increasingly important that the transferability of developed 2D-LC methods be assessed in terms of reproducibility, robustness and performance across different laboratories worldwide. The work presented here focuses on the evaluation of a heart-cutting 2D-LC method used for the analysis of a pharmaceutical material, where a key, co-eluting impurity in the first dimension (1D) is resolved from the main peak and analyzed in the second dimension (2D). A design-of-experiments (DOE) approach was taken in the collection of the data, and the results were then modeled in order to evaluate method robustness using statistical modeling software. This quality by design (QBD) approach gives a deeper understanding of the impact of these 2D-LC critical method attributes (CMAs) and how they affect overall method performance. Although there are multiple parameters that may be critical from method development point of view, a special focus of this work is devoted towards evaluation of unique 2D-LC critical method attributes from method validation perspective that transcend conventional method development and validation. The 2D-LC method attributes are evaluated for their recovery, peak shape, and resolution of the two co-eluting compounds in question on the 2D. In the method, linearity, accuracy, precision, repeatability, and sensitivity are assessed along with day-to-day, analyst-to-analyst, and lab-to-lab (instrument-to-instrument) assessments. The results of this validation study demonstrate that the 2D-LC method is accurate, sensitive, and robust and is ultimately suitable for QC testing with good method transferability.

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.

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.

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.

Continuous flow-extractive desorption electrospray ionization: analysis from "non-electrospray ionization-friendly" solvents and related mechanism.

Due to their low polarities and dielectric constants, analytes in solvents such as hexane, chloroform, and ethyl acetate exhibit poor electrospray ionization (ESI) efficiency. These are deemed to be "non-ESI-friendly" solvents. Continuous flow extractive desorption electrospray ionization (CF-EDESI) is a novel ambient ionization technique that was recently developed in our group to manipulate protein charge distributions. Here we demonstrate its potential for ionizing analytes from non-ESI-friendly solvents. This feature makes CF-EDESI attractive to the general analytical community due to its apparent potential in lipidomics, normal phase separations, and hyphenation of mass spectrometry with HPLC-NMR systems. In this context, interest was subsequently initiated to discern mechanistic aspects of CF-EDESI. To achieve this, mechanistic experiments associated with a seemingly similar ambient ionization technique, extractive electrospray ionization (EESI), were emulated to compare CF-EDESI and EESI. Analysis of a series of fatty acids in multiple solvents in the negative ionization mode revealed differences between the two techniques. Whereas EESI has been previously shown to operate via extraction of analytes into the spray solvent, data presented here for CF-EDESI point toward a liquid-liquid mixing process to facilitate ionization. Further, a partial factorial design experiment was performed to evaluate the effects of different experimental variables on signal intensity. Sample flow rate was confirmed to be among the most significant factors to affect sensitivity. As a whole, the work presented provides greater insight into a new ambient ionization process, which exhibits expanded capabilities over conventional ESI; in this case, for direct analysis from non-ESI-friendly solvents.

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.

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.

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.

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.

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.