William Farrell

Packed Column Supercritical Fluid Chromatography/Mass Spectrometry for High-Throughput Analysis

A supercritical fluid chromatograph was interfaced to a mass spectrometer, and the system was evaluated for applications requiring high sample throughput. Experiments presented demonstrate the high-speed separation capability of supercritical fluid chromatography (SFC) and the effectiveness of supercritical fluid chromatography/mass spectrometry (SFC/MS) for fast, accurate determinations of multicomponent mixtures. A high-throughput liquid chromatography/mass spectrometry (LC/MS) analysis cycle time is reduced 3-fold using our general SFC/MS high-throughput method, resulting in substantial time saving for large numbers of samples. Unknown mixture characterization is improved due to the increased selectivity of SFC/MS compared to LC/MS. This was demonstrated with sample mixtures from a 96-well combinatorial library plate. In this paper, we report a negative mode atmospheric pressure chemical ionization (APCI) method for SFC/MS suitable for most of the components in library production mixtures. Flow injection analysis (FIA) also benefits from this SFC/MS system. A broader range of solvents is amenable to the SFC mobile phase compared with standard LC/MS solvents, and solutes elute more rapidly from the SFC/MS system, reducing sample carryover and cycle time. Finally, our instrumental setup allows for facile conversion between LC/MS and SFC/MS modes of operation.

Comparison of the response of four aerosol detectors used with ultra high pressure liquid chromatography.

The responses of four different types of aerosol detectors have been evaluated and compared to establish their potential use as a universal detector in conjunction with ultra high pressure liquid chromatography (UHPLC). Two charged-aerosol detectors, namely Corona CAD and Corona Ultra, and also two different types of light-scattering detectors (an evaporative light scattering detector, and a nano-quantity analyte detector [NQAD]) were evaluated. The responses of these detectors were systematically investigated under changing experimental and instrumental parameters, such as the mobile phase flow-rate, analyte concentration, mobile phase composition, nebulizer temperature, evaporator temperature, evaporator gas flow-rate and instrumental signal filtering after detection. It was found that these parameters exerted non-linear effects on the responses of the aerosol detectors and must therefore be considered when designing analytical separation conditions, particularly when gradient elution is performed. Identical reversed-phase gradient separations were compared on all four aerosol detectors and further compared with UV detection at 200 nm. The aerosol detectors were able to detect all 11 analytes in a test set comprising species having a variety of physicochemical properties, whilst UV detection was applicable only to those analytes containing chromophores. The reproducibility of the detector response for 11 analytes over 10 consecutive separations was found to be approximately 5% for the charged-aerosol detectors and approximately 11% for the light-scattering detectors. The tested analytes included semi-volatile species which exhibited a more variable response on the aerosol detectors. Peak efficiencies were generally better on the aerosol detectors in comparison to UV detection and particularly so for the light-scattering detectors which exhibited efficiencies of around 110,000 plates per metre. Limits of detection were calculated using different mobile phase compositions and the NQAD detector was found to be the most sensitive (LOD of 10 ng/mL), followed by the Corona CAD (76 ng/mL), then UV detection at 200 nm (178 ng/mL) using an injection volume of 25 μL.

Universal response model for a corona charged aerosol detector

The universality of the response of the Corona Charged Aerosol Detector (CoronaCAD) has been investigated under flow-injection and gradient HPLC elution conditions. A three-dimensional model was developed which relates the CoronaCAD response to analyte concentration and the mobile phase composition used. The model was developed using the response of four probe analytes which displayed non-volatile behavior in the CoronaCAD and were soluble over a broad range of mobile phase compositions. The analyte concentrations ranged from 1μg/mL to 1mg/mL, and injection volumes corresponded to on-column amounts of 25ng to 25μg. Mobile phases used in the model were composed of 0-80% acetonitrile, mixed with complementary proportions of aqueous formic acid (0.1%, pH 2.6). An analyte set of 23 compounds possessing a wide range of physicochemical properties was selected for the purpose of evaluating the model. The predicted response was compared to the actual analyte response displayed by the detector and the efficacy of the model under flow-injection and gradient HPLC elution conditions was determined. The average error of the four analytes used to develop the model was 9.2% (n=176), while the errors under flow-injection and gradient HPLC elution conditions for the evaluation set of analytes were found to be 12.5% and 12.8%, respectively. Some analytes were excluded from the evaluation set due to considerations of volatility (boiling point <400°C), charge and excessive retention on the column leading to elution outside the eluent range covered by the model. The two-part response model can be used to describe the relationship between response and analyte concentration and also to offer a correction for the non-linear detector response obtained with gradient HPLC for analytes which conform to the model, to provide insight into the factors affecting the CoronaCAD response for different analytes, and also as a means for accurately determining the concentration of unknown compounds when individual standards are not available for calibration.

Evaluation of stationary phases packed with superficially porous particles for the analysis of pharmaceutical compounds using supercritical fluid chromatography

Superficially porous particles (SPP), or core shell particles, which consist of a non-porous silica core surrounded by a thin shell of porous silica, have gained popularity as a solid support for chromatography over the last decade. In the present study, five unbonded silica, one diol, and two ethylpyridine (2-ethyl and 4-ethyl) SPP columns were evaluated under SFC conditions using two mixtures, one with 17 drug-like compounds and the other one with 7 drug-like basic compounds. Three of the SPP phases, SunShell™ 2-ethylpyridine (2-EP), Poroshell™ HILIC, and Ascentis(®) Express HILIC, exhibited superior performances relative to the others (reduced theoretical plate height (hmin) values of 1.9-2.5 for neutral compounds). When accounting for both achievable plate count and permeability of the support using kinetic plot evaluation, the Cortecs™ HILIC 1.6μm and Ascentis(®) Express HILIC 2.7μm phases were found to be the best choices among tested SPPs to reach efficiencies up to 30,000 plates in the minimum amount of time. For desired efficiencies ranging from 30,000 to 60,000 plates, the SunShell™ 2-EP 2.6μm column clearly outperformed all other SPPs. With the addition of a mobile phase additive such as 10mM ammonium formate, which was required to elute the basic components with sharp peaks, the Poroshell™ HILIC, SunShell™ Diol and SunShell™ 2-EP phases represent the most orthogonal SPP columns with the highest peak capacities. This study demonstrates the obvious benefits of using columns packed with SPP on current SFC instrumentation.

Investigation of polar organic solvents compatible with Corona Charged Aerosol Detection and their use for the determination of sugars by hydrophilic interaction liquid chromatography.

A range of organic solvents (ethanol, isopropanol and acetone) has been investigated as alternatives to acetonitrile and methanol when used in conjunction with Corona Charged Aerosol Detection (Corona CAD). These solvents have been evaluated with regard to their effect on the response of the Corona CAD. Three dimensional response surfaces were constructed using raw data showing the relationship between detector response, analyte concentration and percentage of organic solvent in the mobile phase, using sucralose or quinine as the test analyte. The detector response was non-linear in terms of analyte concentration for all solvents tested. However, detector response varied in an approximately linear manner with percentage of organic solvent over the range 0-40% for ethanol or isopropanol and 0-80% for acetone and methanol. The chromatographic performance of the various solvents when used as aqueous-organic mobile phases was evaluated for isocratic and gradient separations of sugars and sugar alcohols by hydrophilic interaction liquid chromatography (HILIC) using an Asahipak NH2P-504E column coupled with Corona CAD detection. It was found that whilst acetonitrile provided the highest column efficiencies and lowest detection limits of the solvents studied, acetone also performed well and could be used to resolve the same number of analytes as was possible with acetonitrile. Typical efficiencies and detection limits of 5330 plates m(-1) and 1.25 μg mL(-1), respectively, were achieved when acetone was used as the organic modifier. Acetone was utilised successfully as an organic modifier in the HILIC separation of carbohydrates in a beer sample and also for a partially digested dextran sample.

A platform for automated nanomole-scale reaction screening and micromole-scale synthesis in flow

A reaction screen in flowing solvent Chemists charged with manufacturing pharmaceuticals have recently been exploring the efficiency advantages of continuous flow techniques. Perera et al. now show that a flow apparatus can also accelerate reaction optimization earlier in the drug discovery process. They modified a high-performance liquid chromatography system to screen a wide variety of solvent, ligand, and base combinations to optimize carbon-carbon bond formation. Injecting stock solution aliquots of the catalyst and reactants into a carrier solvent stream let the authors vary the main solvent efficiently and scale up the optimal conditions for product isolation. Science, this issue p. 429 Chromatographic, flow-based screening of reaction conditions is demonstrated for Suzuki coupling in pharmaceutical research. The scarcity of complex intermediates in pharmaceutical research motivates the pursuit of reaction optimization protocols on submilligram scales. We report here the development of an automated flow-based synthesis platform, designed from commercially available components, that integrates both rapid nanomole-scale reaction screening and micromole-scale synthesis into a single modular unit. This system was validated by exploring a diverse range of reaction variables in a Suzuki-Miyaura coupling on nanomole scale at elevated temperatures, generating liquid chromatography–mass spectrometry data points for 5760 reactions at a rate of >1500 reactions per 24 hours. Through multiple injections of the same segment, the system directly produced micromole quantities of desired material. The optimal conditions were also replicated in traditional flow and batch mode at 50- to 200-milligram scale to provide good to excellent yields.

Structure-based design, SAR analysis and antitumor activity of PI3K/mTOR dual inhibitors from 4-methylpyridopyrimidinone series.

PI3K, AKT and mTOR, key kinases from a frequently dysregulated PI3K signaling pathway, have been extensively pursued to treat a variety of cancers in oncology. Clinical trials of PF-04691502, a highly potent and selective ATP competitive kinase inhibitor of class 1 PI3Ks and mTOR, from 4-methylpyridopyrimidinone series, led to the discovery of a metabolite with a terminal carboxylic acid, PF-06465603. This paper discusses structure-based drug design, SAR and antitumor activity of the MPP derivatives with a terminal alcohol, a carboxylic acid or a carboxyl amide.

Advances in High Throughput Supercritical Fluid Chromatography

Abstract Supercritical fluid chromatography (SFC) has long been touted as the separation of choice for high throughput applications and, after nearly a decade, is finally considered mainstream in the analytical chemistry community. Applications range from microscale analysis of complex mixtures to macroscale purification of chiral enantiomers in a variety of industries, such as pharmaceuticals, foods, cosmetics, agrochemicals, petrochemical and natural products. The inherent speed, efficiency, and versatility of SFC have transformed the perceptions of the technology from novelty to integral tool for the modern analytical lab, especially those labs wanting to maximize throughput. However, maneuvering SFC through the transition from single samples to array-like libraries has strained the development of instrumentation to meet the challenging demands. This review presents the major developments and applications useful to those embarking on using SFC for high throughput applications in other fields.

Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity

Driven by the ever-increasing pace of drug discovery and the need to push the boundaries of unexplored chemical space, medicinal chemists are routinely turning to unusual strained bioisosteres such as bicyclo[1.1.1]pentane, azetidine, and cyclobutane to modify their lead compounds. Too often, however, the difficulty of installing these fragments surpasses the challenges posed even by the construction of the parent drug scaffold. This full account describes the development and application of a general strategy where spring-loaded, strained C–C and C–N bonds react with amines to allow for the “any-stage” installation of small, strained ring systems. In addition to the functionalization of small building blocks and late-stage intermediates, the methodology has been applied to bioconjugation and peptide labeling. For the first time, the stereospecific strain-release “cyclopentylation” of amines, alcohols, thiols, carboxylic acids, and other heteroatoms is introduced. This report describes the development, synthesis, scope of reaction, bioconjugation, and synthetic comparisons of four new chiral “cyclopentylation” reagents.

Comparing the selectivity and chiral separation of D- and L- fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography : evaluating throughput, economic and environmental impact

The chiral separation of d- and l- FMOC amino acids was undertaken using the Lux Cellulose-1 polysaccharide based chiral column in HPLC (normal phase and reverse phase) and SFC conditions. This was done to compare the relative selectivity and separation between the three separation modes and to evaluate the potential benefits of SFC separations with regards to resolution, throughput, economic and environmental impact. It was established that the separation of d- and l- FMOC amino acids in SFC displayed behaviours that were similar to both normal phase and reversed phase, rather than distinctly one or the other. Additionally, although reversed phase conditions yielded significantly higher resolution values between enantiomers across the range of amino acids studied, improvements in selectivity in SFC via the introduction of higher concentrations of formic acid in the mobile phase allowed for better resolution per unit of time. Moreover since the SFC mobile phase is composed mostly of recyclable CO2, there is a reduction in organic solvent consumption, which minimises the economic and environmental costs.