Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.     

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.     

Determination of enantiomeric excess of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography

A simple and reliable chiral HPLC method was developed for the determination of enantiomeric excess of a chiral dihydroxy intermediate for the chemoenzymatic synthesis of side chain of statin drugs. After evaluating different columns and conditions, the four stereoisomers of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate were well resolved by a simple gradient elution on OD-RH column, and the enantiomeric excess of the desired 3R,5S-enantiomer was accurately measured. This study provides a simple, rapid, accurate, and reliable method to assess the enantiomeric quality of such important intermediates.     

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.     

Rapid, automated screening method for enzymatic transformations using a robotic system and supercritical fluid chromatography

An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.     

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen-phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30-6000 microg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.     

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.     

A hierarchical screening approach to enantiomeric separation

The screening of a number of chiral stationary phases (CSPs) with different modifiers in supercritical fluid chromatography to find a chromatographic method for separation of enantiomers can be time‐consuming. Computational methods for data analysis were utilized to establish a hierarchical screening strategy, using a dataset of 110 drug‐like chiral compounds with diverse structures tested on 15 CSPs with two different modifiers. This dataset was analyzed using a combinatorial algorithm, principal component analysis (PCA), and a correlation matrix. The primary goal was to find a set of eight columns resolving a large number of compounds, but also having complementary enantioselective properties. In addition to the hereby defined hierarchical experimental strategy, quantitative structure enantioselective models (QSERs) were evaluated. The diverse chemical space and relatively limited size of the training set reduced the accuracy of the QSERs. However, including separation factors from other CSPs increased the accuracies of the QSERs substantially. Hence, such combined models can support the experimental strategy in prioritizing the CSPs of the second screening phase, when a compound is not separated by the primary set of columns.     

Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction

Summary A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the F508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.     

Improving enantiomeric resolutions by avoiding peak distortion effects in on‐line coupled liquid chromatography to gas chromatography

In this work, we study the effect of different variables affecting elution profile distortion on the enantiomeric resolution eventually achievable when working with on‐line coupled liquid chromatography to gas chromatography (LC‐GC). Specifically, the proposed configuration combines achiral reversed‐phase liquid chromatography (RPLC) and chiral gas chromatography (enantio‐GC), with heptakis‐(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin as enantioselective stationary phase to analyse target fractions transferred (from LC to GC) via the through oven transfer adsorption desorption (TOTAD) interface. The high degree of orthogonality resulting from the combination of two chromatographic columns having very different separation mechanisms (and also requiring mobile phases in distinct physical states), as well as integration of the sample preparation step in the first dimension of the system, significantly contributed to exploit the performance of the proposed two‐dimensional approach. Occasional adverse effects, which may result in severe peak distortions during LC‐GC analysis and could be explained by flow instabilities due to viscous fingering, are circumvented by using the outstanding capacity of the TOTAD interface for achieving effective elimination of the eluent arriving from the LC preseparation.     

Direct plasma liquid chromatographic-tandem mass spectrometric analysis of granisetron and its 7-hydroxy metabolite utilizing internal surface reversed-phase guard columns and automated column switching devices

An alternative on-line automated sample enrichment technique useful for the direct determination of various drugs and their metabolites in plasma is described for rapid development of highly sensitive and selective liquid chromatographic methods using mass spectrometric detection. The method involves direct injection of plasma onto an internal surface reversed-phase (ISRP) guard column, washing the proteins from the column to waste with aqueous acetonitrile, and backflushing the analytes onto a reversed-phase octyl silica column using switching valves. The analytes were detected using a tandem mass spectrometer operated in selected reaction monitoring (SRM) mode using atmospheric pressure chemical ionization (APCI). Use of two ISRP guard columns in parallel configuration allowed alternate injections of plasma samples on these columns for sample enrichment and shortened the column equilibration and LCMSMS analysis times, thereby increasing the sample throughput. The total run time, including both sample enrichment and chromatography, was about 6 min. Using this technique, an analytical method was developed for the quantitation of granisetron and its active 7-hydroxy metabolite in dog plasma. Granisetron is a selective 5-HT3 receptor antagonist used in the prevention and treatment of cytostatic induced nausea and vomiting. Recovery of the analytes was quantitative and the method displayed excellent linearity over the concentration ranges tested. Results from a three day validation study for both compounds demonstrated excellent precision (1.3–8.7%) and accuracy (93–105%) across the calibration range of 0.1 to 50 ng/ml using an 80 μl plasma sample. The automated method described here was simple, reliable and economical. This on-line approach using ISRP columns and column switching with LCMSMS is applicable for the quantification of other pharmaceuticals in pharmacokinetics studies in animals and humans which require high sensitivity.     

Development and application of an automated, low-volume chromatography system for resin and condition screening

A small-volume chromatography system was developed for rapid resin and parameter screening and applied to the purification of a therapeutic monoclonal antibody from a key product-related impurity. Accounting for constraints in peripheral volume, gradient formation, column integrity, and fraction collection in microtiter plates, the resulting system employed 2-mL columns and was successfully integrated with plate-based methods for rapid sample analysis (e. g., use of automated liquid handlers, plate readers, and HPLC). Several cation-exchange chromatography resins were screened using automated programs and tailored gradients for the combination of a particular resin and a given antibody feedstock produced during Phase 1 development. Results from the tailored gradient runs were used to select a resin, and to arrive at efficient stepwise elution schedules for the chosen resin. By maintaining a constant residence time, final operating parameters were successfully scaled to representative bed heights and column diameters up to 2.6 cm (106 mL). This approach significantly improved throughput while reducing development time and material consumption.     

Chiral analysis by online coupling of reversed-phase liquid chromatography to gas chromatography and mass spectrometry

Enantiomeric composition of selected chiral compounds present in complex mixtures is determined by using the online coupling of reversed-phase liquid chromatography (LC) to gas chromatography (GC) and mass spectrometry. Integration of sample preparation into GC analysis, in a completely automated way, is achieved by means of the effective clean-up resulting from both the LC fractionation step and the eluent elimination provided by the through oven transfer adsorption desorption system used for LC-GC interfacing. The possibilities of the technique are illustrated through some examples concerning the stereodifferentiation in essential oils of major and minor chiral compounds via LC-GC transfer of different volume fractions, ranging from 0.5 to 1.9 ml, which show the significance of the window size for the determination of enantiomeric profiles.     

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP‐dependent enzymes

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real‐time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler‐assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'‐phosphate‐dependent enzymes is presented using SEC for direct monitoring of enzyme‐bound and free reaction intermediates. Time‐resolved changes of the different cofactor states, e.g. pyridoxal 5'‐phosphate, pyridoxamine 5'‐phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate‐independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP‐dependent enzymes.     

Analysis of strawberry volatiles using comprehensive two-dimensional gas chromatography with headspace solid-phase microextraction

The aims of the current study were to develop an enantioselective multi-dimensional gas chromatography (GC x GC) method for the examination of strawberry volatiles and to use this method to make comparisons between the volatile profiles of different cultivars and between fresh picked and post-harvest berries of the same cultivar. Strawberry volatiles were sampled using solid-phase microextraction (SPME), and the repeatability and reproducibility of this method was examined. Semi-quantitative analysis of the volatiles was conducted using the relatively new technique of comprehensive multi-dimensional gas chromatography, using enantioselective (chiral) columns for the differentiation of analyte enantiomers. Chiral GC x GC facilitated the detection of key enantiomers in strawberry flavour. The (-)-enantiomer of 2,5-dimethyl-4-hydroxy-(2H)-furan-3-one (DMHF) and the S-enantiomer of linalool were tentatively identified as the predominant forms in both the cultivars Selva and Adina. The compounds benzaldehyde and methyl hexanoate were shown to decrease in post-harvest berries, whilst DMHF and nerolidol increased upon storage.     

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.     

Chiral 3D Open‐Framework Material Ni(D‐cam)(H2O)2 Used as GC Stationary Phase

Metal‐organic frameworks (MOFs) have been explored for analytical applications because of their outstanding properties such as high surface areas, flexibility and specific structure features, especially for chromatography application in recent years. In this work, a chiral MOF Ni(D‐cam)(H₂O)₂ with unusual integration of molecular chirality, absolute helicity, and 3‐D intrinsic chiral net was chosen as stationary phase to prepare Ni(D‐cam)(H₂O)₂‐coated open tubular columns for high‐resolution gas chromatographic (GC) separation. Two fused‐silica open tubular columns with different inner diameters and lengths, including column A (30 m × 250 µm i.d.) and column B (2 m × 75 µm i.d.), were prepared via a dynamic coating method. The chromatographic properties of the two columns were investigated using n‐dodecane as the analyte at 120 °C. The number of theoretical plates (plates/m) of the two metal–organic framework (MOF) columns was 1300 and 2750, respectively. The racemates, isomer and linear alkanes mixture were used as analytes for evaluating the separation properties of Ni(D‐cam)(H₂O)₂‐coated open tubular columns. The results showed that the columns offered good separations of isomer and linear alkanes mixture, especially racemates. Chirality 26:27–32, 2013. © 2013 Wiley Periodicals, Inc.     

Fully-automated systematic toxicological analysis of drugs,poisons, and metabolites in whole blood,urine, and plasma by gas chromatography–full scan mass spectrometry

The availability of automated, rapid and reliable methods for the systematic toxicological analysis (STA) of drugs and poisons in biosamples is of great importance in clinical and forensic toxicology laboratories. Gas chromatography–continuous scan mass spectrometry (GC–MS) possesses a high potential in STA because of its selectivity and identification power. However, in order to develop a fully automated STA method based on GC–MS two main obstacles have to be overcome: (a) sample preparation is rather sophisticated owing to the need to isolate analytes from the aqueous matrix and to allow a correct GC repartition of polar analytes; (b) the large amount of information collected within a single analysis makes it difficult to isolate relevant analytical information (mass spectra of analytes) from the chemical noise. Using a bench-top GC–MS system equipped with a laboratory robot for sample preparation (the Hewlett-Packard 7686 PrepStation) and an original method for mass spectral purification, a fully automated STA procedure was developed involving isolation of drugs from the sample (whole blood with minimal pretreatment, plasma, urine) by means of solid-phase extraction, derivatization (trimethylsilylation) of the acidic–neutral and of the basic extracts, GC–MS analysis, processing of data, and reporting of results. Each step of the procedure, and the method for data analysis in particular, can be easily integrated with other existing STA methods based on GC–MS.     

Development and optimization of a process for automated recovery of single cells identified by microengraving

Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost‐effective manner, but connecting these systems to the existing platforms used in routine high‐throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody‐producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high‐throughput, single‐cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099‐01) were sorted with 100% accuracy (50 clones of each) in ～2 h, and the cells retained viability. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Simple and rapid screening methods for microorganisms with phospholipase A1, A2 and C activities

Summary A simple and rapid screening method for microorganisms with phospholipase A₁, A₂ and C activities using agar plate and gas chromatography (GC) method was successfully carried out. In agar plate method, soy bean lecithin and taurocholic acid were used as carbon source and emulsifier, respectively. In this agar plate method, microorganisms with phospholipase A₁ and A₂ or C activity produce a halo around the colony and two kinds(A's and C) of microorganisms are clearly distinguished by turbidity of the halo. Microorganisms with phospholipase A₁ and A₂ activity is simply distinguished by GC using a synthetic phospholipid containing different fatty acid at sn-1 and sn-2 position.