Fast methods of enantiopurity determination for the Soai reaction: Towards a general enantioenrichment detector?

The Soai reaction system possesses a remarkable combination of characteristics (enantioselective autocatalysis, strong positive nonlinearity leading to asymmetric amplification, ability to be triggered by wide variety of exogenous enantioenriched materials) that make it a potential starting point for a generalized detector for enantioenrichment. In this study we apply standard approaches used in pharmaceutical process research to the problem of developing a rapid method for analysis of the enantiopurity of the Soai reaction product. Several methods for rapid analysis (<1 min per sample) are described, including an approach using chiral supercritical fluid chromatography (SFC) and an approach using achiral chromatography with circular dichroism (CD) detection. Some thoughts on incorporation into a device for generalized enantioenrichment detection are presented.     

超临界流体色谱在药物分析中的应用

本文介绍了超临界流体色变在药物分析中的应用,从药物稳定性试验及代谢产物测定,手性药物分析和各类药物分析三个方面综述了该方法的应用。     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

Improved Chiral SFC Screening for Analytical Method Development

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems. Chirality 25:799–804, 2013. © 2013 Wiley Periodicals, Inc.     

SeMoP: a new computational strategy for the unrestricted search for modified peptides using LC-MS/MS data

A novel computational approach, termed Search for Modified Peptides (SeMoP), for the unrestricted discovery and verification of peptide modifications in shotgun proteomic experiments using low resolution ion trap MS/MS spectra is presented. Various peptide modifications, including post-translational modifications, sequence polymorphisms, as well as sample handling-induced changes, can be identified using this approach. SeMoP utilizes a three-step strategy: (1) a standard database search to identify proteins in a sample; (2) an unrestricted search for modifications using a newly developed algorithm; and (3) a second standard database search targeted to specific modifications found using the unrestricted search. This targeted approach provides verification of discovered modifications and, due to increased sensitivity, a general increase in the number of peptides with the specific modification. The feasibility of the overall strategy has been first demonstrated in the analysis of 65 plasma proteins. Various sample handling induced modifications, such as beta-elimination of disulfide bridges and pyrocarbamidomethylation, as well as biologically induced modifications, such as phosphorylation and methylation, have been detected. A subsequent targeted Sequest search has been used to verify selected modifications, and a 4-fold increase in the number of modified peptides was obtained. In a second application, 1367 proteins of a cervical cancer cell line were processed, leading to detection of several novel amino acid substitutions. By conducting the search against a database of peptides derived from proteins with decoy sequences, a false discovery rate of less than 5% for the unrestricted search resulted. SeMoP is shown to be an effective and easily implemented approach for the discovery and verification of peptide modifications.     

Preparative chromatographic resolution of racemates using HPLC and SFC in a pharmaceutical discovery environment

The preparative chromatographic resolution of racemates has become a standard approach for the generation of enantiomers in pharmaceutical discovery laboratories. This paper will discuss the use of preparative HPLC and SFC to generate individual enantiomers for discovery activities. Analytical HPLC and SFC method development to rapidly screen chiral stationary phases and solvent combinations will be presented. The usefulness of preparative chromatographic resolution of racemates will be demonstrated through the presentation of numerous non-routine case studies from the laboratories at Amgen.     

Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study.     

Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool

A tool for improved tandem column chiral supercritical fluid chromatography (SFC) method development screening was prepared by modification of a commercial analytical SFC instrument with two different software-controllable, six position high-pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument, which has the ability to screen 10 different individual columns and 25 different tandem column arrangements, is a useful tool for facilitating the screening of tandem column SFC arrangements for separation of complex mixtures of stereoisomers or other multicomponent mixtures. Strategies for optimal use of the instrument are discussed, and several examples of the use of the instrument in developing tandem SFC methods for resolution of multicomponent mixtures are presented.     

An optimized differential heat conduction solution microcalorimeter for thermal kinetic measurements

Heat conduction calorimeters are widely used in biological sciences, but baseline instability, low resolution, electrical noise and motion artifacts have limited their utility. Two main sources of noise, baseline fluctuation or drift and a motion artifact, were traced to amplifier drift, a small (0.015°C) gradient within the constant temperature cylinder, and the method of installing the thermopiles. The addition of heaters to the top and bottom of the cylinder reduced the gradient to approximately 0.003°C and greatly reduced the slow component of the motion artifact. The drift error was reduced by proper mounting of the amplifier and its external components and the enclosure of the calorimeter in a temperature-controlled box.An R-C model of the heat flow in the calorimeter was developed which was employed to discover several means of increasing sensitivity without increasing the rise-time of the calorimeter. Analysis, also based on the model, showed that variations in the air gap between the cell holder can be a major source of error when the calorimeter is used to investigate the kinetics of a chemical reaction. This analysis also showed that the time for the heat to flow through the solution through the solution in the cell can be the dominant factor in determining the rise-time of the instrument.The heat conduction calorimeter described here has improved characterics: a baseline stability of 200 nJ · s^?1 (peak-to-peak) over a 48 h period; a resolution of 200 nJ · s^?1; a sensitivity of 6.504 ± 0.045 J · V^?1 · s^?1 referred to the sensor output; and a rise-time of 122 s for the 10–90% response.     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.     

Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.     

Past and present concepts in flow cytometry: a European perspective

The development of flow cytometric instrumentation, methods and research concepts in Europe has been a continuous driving force for the general scientific advancement in this area over the years. This review addresses early European concepts of continuing interest with regard to instrumentation, data analysis, clinical and eperimental DNA analysis, cell function and microbiology at their worldwide first appearence while flow cytometric immunology and immunophenotyping will be covered separately. Flow cytometry represents an efficient approach to the enormous complexity of molecular cell architecture and cell function by the analysis of apparent molecular cell phenotypes in heterogeneous cell samples. The present merger of flow and image cytometry into the method independent cytomics discipline increases the potential of cell analysis very significantly. It opens the way for predictive medicine as well as for predictive cytopathology and predictive cytology in everyday clinical and medical practice. Current progress is driven by joint advances in molecular fluorescence technologies and instrument development. This complements the analysis of genome sequence information in an efficient way.     

Particle classification from light scattering with the scanning flow cytometer.

BACKGROUND:The differential light-scattering pattern, an indicatrix, provides the most complete characterization of the optical properties of a particle. Particle classification can be performed on the basis of particle parameters retrieved from the indicatrices. This classification extends the ability of flow cytometry in particle recognition. METHODS:The scanning flow cytometer (SFC) permits an acquisition of traces of light scattering signals, i.e., native SFC traces, from single particles. The acquired native SFC traces are transformed into indicatrices. The performance of the SFC in measurements of indicatrices has been demonstrated for the following particles: lymphocytes, erythrocytes, polystyrene particles, and milk-fat particles. RESULTS:The structure and profile of the indicatrix for each particle type have been found to be unique. Classification of polystyrene particles has been performed on the basis of the map formed by particle refractive index and size. The polystyrene particles were classified using this map into different size categories ranging from 1.4-7 microm, with a size deviation of 0.07 microm. CONCLUSIONS:The method based on analysis of native SFC traces shows better performance in particle classification than the method based on the particle refractive index and size map. The classification performance of the SFC will be useful, for example, for particle sorting and particle identification, and with additional fluorescent measurements may have applications in multiparameter particle-based immunoassay.     

Enantioseparation of napropamide by supercritical fluid chromatography: Effects of the chromatographic conditions and separation mechanism

Supercritical fluid chromatography (SFC) is already used for enantioseparation in the pharmaceutical industry, but it is rarely used for the separation of chiral pesticides. Comparing with high performence liquid chromatography, SFC uses much more environmnetal friendly and economic mobile phase, supercritical CO₂. In our work, the enantioseparation of an amide herbicide, napropamide, using three different polysaccharide‐type chiral stationary phases (CSPs) in SFC was investigated. By studying the effect of different CSPs, organic modifiers, temperature, back‐pressure regulator pressures, and flow rates for the enantioseparation of napropamide, we established a rapid and green method for enantioseparation that takes less than 2 minutes: The column was CEL2, the mobile phase was CO₂ with 20% 2‐propanol, and the flow rate was 2.0 mL/min. We found that CEL2 demonstrated the strongest resolution capability. Acetonitrile was favored over alcoholic solvents when the CSP was amylose and 2‐propanol was the best choice when using cellulose. When the concentration of the modifiers or the flow rate was decreased, resolutions and analysis times increased concurrently. The temperature and back‐pressure regulator pressure exhibited only minor influences on the resolution and analysis time of the napropamide enantioseparations with these chiral columns. The molecular docking analysis provided a deeper insight into the interactions between the enantiomers and the CSPs at the atomic level and partly explained the reason for the different elution orders using the different chiral columns.     

Simple protocols for NMR analysis of the enantiomeric purity of chiral diols

A three-component chiral derivatization protocol for determining the enantiopurity of chiral diols by (1)H NMR spectroscopic analysis is described here. The present approach involves the derivatization of 1,2- 1,3- and 1,4-diols with 2-formylphenylboronic acid and enantiopure alpha-methylbenzylamine. This method affords a mixture of diastereoisomeric iminoboronate esters whose ratio can be determined by integration of well-resolved diastereotopic resonances in their (1)H NMR spectra, thus enabling the determination of the enantiopurity of the parent diol. The protocol as described takes less than 90 min to complete.     

Factors governing the flow cytometric analysis and sorting of large biological particles

We have investigated the suitability of large flow cell tips for the flow cytometric analysis and sorting of large biological particles, including plant cells (pollen) and protoplasts. Using flow tips ranging in diameter from 79-204 micron, we have optimized conditions for the establishment of a stable hydrodynamic flow leading to accurate droplet production. We describe instrument modifications required for large particle sorting and demonstrate the use of these experimental conditions for the sorting to high purity of pollen and viable plant protoplasts possessing diameters as large as 95 micron. Our experiments have revealed a complex interaction among sorting efficiency, particle diameter, flow cell tip diameter and bimorphic crystal drive frequency. This interaction can be satisfactorily explained in terms of interference effects owing to phase differences between the particle-induced disturbance and the undulation driven by the bimorphic crystal.     

Revisiting flow‐chemiluminescence techniques: pharmaceutical analysis

The state of the art in flow‐chemiluminescence (flow‐CL) technique for automated pharmaceutical analysis is reviewed. Flow‐CL approaches have become powerful and promising tools for pharmaceutical screening in recent years due to their simplicity, low cost and high sensitivity. Because of these advantages, these methods have been widely used for pharmaceutical analysis in recent years. The literature reviewed covers papers of analytical interest that appeared between 2007 and mid‐2012 and have been divided into several sections based on fundamental types of CL systems employed. Furthermore, entries have been summarized alphabetically in tabular form giving details of analytical figures of merit of the methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of confocal microscopy system performance

BACKGROUND: The confocal laser scanning microscope (CLSM) has been used by scientists to visualize three-dimensional (3D) biological samples. Although this system involves lasers, electronics, optics, and microscopes, there are few published tests that can be used to assess the performance of this equipment. Usually the CLSM is assessed by subjectively evaluating a biological/histological test slide for image quality. Although there is a use for the test slide, there are many other components in the CLSM that need to be assessed. It would be useful if tests existed that produced reference values for machine performance. The aim of this research was to develop quality assurance tests to ensure that the CLSM was stable while delivering reproducible intensity measurements with excellent image quality. METHODS: Our ultimate research objective was to quantify fluorescence using a CLSM. To achieve this goal, it is essential that the CLSM be stable while delivering known parameters of performance. Using Leica TCS-SP1 and TCS-4D systems, a number of tests have been devised to evaluate equipment performance. Tests measuring dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, laser stability, photomultiplier tube (PMT) reliability, and system noise were either incorporated from the literature or derived in our laboratory to measure performance. These tests are also applicable to other manufacturer's systems with minor modifications. RESULTS: A preliminary report from our laboratory has addressed a number of the QA issues necessary to achieve CLSM performance. This report extends our initial work on the evaluation of CLSM system performance. Tests that were described previously have been modified and new tests involved in laser stability and sensitivity are described. The QA tests on the CLSM measured laser power, PMT function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance, and laser stability. Laser power stability varied between 3% and 30% due to various factors, which may include incompatibility of the fiber-optic polarization with laser polarization, thermal instability of the acoustical optical transmission filter (AOTF), and laser noise. The sensitivity of the system was measured using a 10-microm Spherotech bead and the PMTs were assessed with the CV concept (image noise). The maximum sensitivity obtainable on our TCS-SP1 system measured on the 10-microm Spherotech beads was approximately 4% for 488 nm, 2.5% for 568 nm, 20% for 647 nm, and 19% for 365 nm laser light. The values serve as a comparison to test machine sensitivity from the same or different manufacturers. CONCLUSIONS: QA tests are described on the CLSM to assess performance and ensure that reproducing data are obtained. It is suggested strongly that these tests be used in place of a biological/histological sample to evaluate system performance. The tests are more specific and can recognize instrument functionality and problems better than a biological/histological sample. Utilization of this testing approach will eliminate the subjective assessment of the CLSM and may allow the data from different machines to be compared. These tests are essential if one is interested in making intensity measurements on experimental samples as well as obtaining the best signal detection and image resolution from a CLSM. Published 2001 Wiley-Liss, Inc.