A microtube screening approach affords simple and convenient assessment of the selective adsorption of metal impurities by a variety of different process adsorbents. This approach is helpful in identifying rapid solutions to metal impurity problems in pharmaceutical process research. Several examples illustrating the utility of the approach are presented.

Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with sub/supercritical fluid chromatography (SFC) moving toward these new applications beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at the preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein, we overcome these problems by introducing chaotropic effects in SFC separations using a nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3 –). In contrast to other salts, HCO3 – was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen–deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.

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Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 μm fully porous and sub-3 μm superficially porous particles) with nine mobile phase eluent choices. This automated setup operates under a vast selection of reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, polar-organic mode, and polar-ionic mode conditions with minimal manual intervention and high success rate. Examples of highly efficient enantioseparations are illustrated from the integration of chiral screening conditions and computer-assisted modeling. Furthermore, we describe the nuances of in silico method development for chiral separations via second-degree polynomial regression fit using LC simulator (ACD/Labs) software. The retention models were found to be very accurate for chiral resolution of single and multicomponent mixtures of enantiomeric species across different types of CSPs, with differences between experimental and simulated retention times of less than 0.5%. Finally, we illustrate how this approach lays the foundation for a streamlined development of ultrafast enantioseparations applied to high-throughput enantiopurity analysis and its use in the second dimension of two-dimensional liquid chromatography experiments.

α,α-Diaryl primary amines are highly important chemical building blocks in agrochemical and pharmaceutical active ingredients. One of the limitations of current chromatographic methodologies for these molecules is the poor greenness factor as a result of high solvent consumption to reach the necessary productivity, as well as the lack of systematic approaches. Herein, we overcome these challenges by introducing a simple and highly efficient enhanced sub/supercritical fluid chromatography (eSFC) approach that enables analysis and purification of over 40 α,α-diaryl primary amine mixtures. This approach translates into several highly desirable features from a simplicity and greenness perspective including (1) improved chromatographic performance and productivity, (2) augmented MS detection that allows for analytical eSFC−MS and preparative MS-directed purifications, and (3) efficient and direct solvent removal. This strategy represents a move toward modernized greener and sustainable separations as demonstrated by a 30-fold improvement of the analytical method greenness score (AMGS) compared to currently available chromatographic approaches.

Recent developments in the fields of organic synthesis, process research, and biopharmaceuticals are leading to increasingly complex mixtures of closely related species that often prove challenging for analysis, separation, and characterization. Herein, computer-assisted modeling using LC Simulator (ACD/Labs) software is introduced as an initial analytical framework to isolation and purification workflows, enabling the rapid increase of scale-up productivity (kkD: kilograms of purified analyte per kilogram of stationary phase per day) of target pharmaceuticals in multicomponent mixtures. This approach allows us to achieve dramatic increases of kkD while minimizing solvent consumption and hazardous waste by accomplishing three main goals: (1) selectively improving the resolution of only target analytes for the maximum loading while also reducing the cycle time, (2) changing the elution order of the target peaks to prevent coelution caused by undesirable tailing components while increasing sample loading, and (3) enabling the generation of three-dimensional (3D) resolution maps that serve as a database to reduce preparative optimization when similar reaction mixtures are encountered, as typically occurs in the development and manufacturing of new drug substances. Chromatographic simulations served to generate 3D resolution maps with robust separation conditions that matched the outcome of subsequent experimental data (overall relative standard deviation (RSD) of retention times <3% between simulated and experimental conditions). The optimal separation procedures generated through this strategy were successfully applied to the preparative isolation and purification of multicomponent mixtures of closely related species using readily available reversed-phase liquid chromatography (RPLC) and ion-exchange chromatography (IEC) instrumentation, resulting in a substantial increase of purification workflow efficiency in all cases. This computer-assisted modeling approach enables more efficient, cost-effective, and greener preparative chromatography workflows.