David Semin

Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry.

A novel strategy for rapid chiral method development has been implemented using sample pooling and supercritical fluid chromatography-mass spectrometry (SFC-MS) on four chiral stationary phases, namely Chiralpak AD and AS, and Chiralcel OJ and OD, and eight different modifier concentrations (5 to 40% methanol-0.2% isopropylamine). The screening is performed under an outlet pressure of 110 bar at 35 degrees C, and at a flow-rate of 2.5 ml/min for the initial 20 min and then ramped up to 4 ml/min and held for 4.5 min to elute all solutes from the column. The entire process is fully automated from injection to data processing, and operates unattended for 15 h overnight to obtain optimal chiral separation for multiple compounds. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interferences from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than UV and much shorter analysis times compared to normal-phase/reversed-phase liquid chromatography.

Stability-indicating hydrophilic interaction liquid chromatography method for highly polar and basic compounds

A hydrophilic interaction liquid chromatography (HILIC) method was developed for the analysis of very polar and basic 4-(aminomethyl)pyridine (4-AMP) and its related compounds. Separation parameters such as stationary phase, buffer pH, buffer ionic strength, organic modifier, and column temperature were evaluated. The retention mechanisms were explored through the evaluation of the common chromatographic parameters in the method development. The data indicated the existence of surface adsorption phenomena for 4-AMP and its positional isomers (2-AMP, 3-AMP). For two degradants, different retention mechanisms might be involved when compared to 4-AMP. The selectivity of two critical pairs 3-/4-AMP isomer and Degradant-1/-2 diastereomer changed through isoelution temperature with reversal of elution order. The validation results indicated that the HILIC method is a sensitive, reproducible, and robust method suitable for the analysis of 4-AMP and its related compounds.

FLUORESCENCE PHOTON ANTIBUNCHING FROM SINGLE MOLECULES ON A SURFACE

Abstract Fluorescence correlation spectroscopy of individual Rhodamine 6G (R6G) molecules immobilized on a silica surface is performed in air at room temperature using confocal laser scanning optical microscopy (CLSM). The high excitation irradiance in CLSM is used to saturate single R6G molecules, and to observe fluorescence photon antibunching. An experimental arrangement is described that improves the overall coincidence counting efficiency at high irradiance. Photophysical parameters obtained from the saturation data are used to model the antibunching behavior. This is the first reported example of photon antibunching from individual quantum systems on a surface.

Application of Single Molecule Detection to Dna Sequencing

Abstract A flow cytometric, single molecule approach to DNA sequencing is described. A single, fluorescently labeled DNA fragment is suspended in a flow stream. An exonuclease is added to sequentially cleave the end base into the flow stream where it is detected and identified by laser-induced fluorescence.

Short solution phase preparation of fused azepine-tetrazoles via a UDC (Ugi/de-Boc/cyclize) strategy ☆

Abstract A novel application of the TMSN 3 modified Ugi 4-component reaction is disclosed for the solution phase synthesis of fused azepine-tetrazole libraries. The reaction of a N -Boc-α-amino aldehyde, secondary amine, methyl isocyanoacetate and trimethylsilylazide in methanol, followed by acid treatment, proton scavenging and reflux affords bicyclic azepine-tetrazoles. This efficient protocol, producing products with three diversity points, can be used to generate arrays of biologically relevant small molecules for general and targeted screening.

An Automated Screening Assay for Determination of Aqueous Equilibrium Solubility Enabling SPR Study During Drug Lead Optimization

Aqueous solubility is one of the most critical physicochemical properties to be determined in the process of drug lead optimization. Particularly, an equilibrium solubility method is highly valuable to the study of structure property relationship (SPR), while meeting the needs of analytical sensitivity, reproducibility, and throughput. In this report, an automated solubility assay in a 96-well library format was designed and developed by means of robotic liquid handling, centrifugal separation, and HPLC-UV quantification. Requiring 1 mg of solid compound, this assay was used to determine the equilibrium solubility in three user-selected media, that is, 0.01 N HCl, phosphate buffer saline (PBS), and fasted state simulated intestinal fluid (SIF), with a throughput of up to 192 compounds a week. The assay parameters, including the equilibration time and the separation technique, were optimized to ensure that the thermodynamic solubility was measured at the presence of excess solid compound. A fast gradient HPLC method was developed with single-point on-plate calibration for each compound, followed by a customized 96-well chromatographic data analysis. The reporting solubility range was 1–200 μg/mL, appropriate for oral drug candidate selection at the stage of discovery lead optimization. Based on the test results obtained on the commercially available drugs and Amgen research compounds, this assay was considered to be equivalent to the conventional shake-flask methods. Examples were given to demonstrate that the thermodynamic solubility determined by this assay enabled the SPR study to support drug lead optimization.

Solvent-induced morphology changes in thin silver films

Abstract Thin Ag films supported on dielectric materials have been extremely popular as surface-enhanced Raman scattering (SERS) substrates. In an effort to characterize SERS-active films under the conditions of typical SERS experiments, we have extended our previous work to elucidate the effect of solvent exposure on thin Ag film optical properties and surface morphology. When thin Ag films are dipped in a solvent, a common method for analyte application, the optical extinction spectra blue-shift more than 20 nm with a significant reduction in optical density. Over a time period of 60 min, the optical spectra gradually shift back toward the original pre-dipped profiles, but do not return completely. While it is known that the solvent modifies the local dielectric, the effect of solvent on the Ag surface morphology has only briefly been addressed. In this study, atomic absorbance spectrometry was employed to rule out significant loss of Ag from the substrate during the dipping process. A numerical algorithm was developed for automated analysis of atomic force microscopy images. With this algorithm, for a typical 1 μm × 1 μm scan, approximately 2000 Ag islands can be evaluated for height, radius, and the distribution in both these values. In addition, radial distribution functions are easily generated. Based on an atomic force microscopic investigation of Ag films before and after solvent exposure, the dominant morphological change in Ag islands appears to be an overall increase in aspect ratio. This increase in aspect ratio is consistent, according to the electromagnetic theory, with observed trends in the optical spectra.

Time‐dependent morphology changes in thin silver films on mica: A scaling analysis of atomic force microscopy results

The postdeposition evolution of the morphology of a thin Ag film on a mica substrate was studied using a combination of experimental and theoretical techniques. Atomic force microscopy (AFM) was used to follow the surface morphology as a function of time at temperatures in the range 30–56 °C. The AFM images were numerically processed to obtain the distribution function of island sizes, defined as island height (h), as a function of time, f(h,t). The Ag films were observed to coarsen, i.e., small Ag islands disappeared while larger Ag islands increased in size. The island height distribution function was of a scaling form, f(h,t)∼f[h/h(t)], where h(t), the mean island height, increased monotonically as a power law h(t)∼tβh up until a crossover time t×. The experimental results for this low temperature annealing process are most consistent with a mechanism whereby the film coarsens through an island–island coalescence process. From the temperature dependence of the annealing kinetics, it was found that t...

Efficient detection of single molecules eluting off an optically trapped microsphere

We demonstrate efficient detection of single fluorescent molecules eluting off a polystyrene microsphere optically trapped in a flowing sheath stream. A 1μm diameter analyte doped microsphere was positioned ∼20 μm upstream of a 16 μm diameter probe laser without significant degradation of the detection signal-to-noise ratio due to scattered laser light and fluorescence from the microsphere. In comparison to more standard capillary sample introduction, the microsphere causes only small perturbations to the sheath fluid flow. The small diameter of the analyte stream eluting from the microsphere results in a greater than 90% detection efficiency for single rhodamine-6G molecules, limited primarily by the photostability of the dye.

Applicability of total reflection X-ray fluorescence (TXRF) as a screening platform for pharmaceutical inorganic impurity analysis

Palladium (Pd) is extensively used in pharmaceutical small molecule drug substance processes, however it must be removed prior to release of the active pharmaceutical ingredient (API). Evaluation of four TXRF instruments and configurations were compared to ICP-MS instrumentation for trace metal analysis, most importantly for Pd. Standards and six pharmaceutical drug substances, triprolidine HCl, diphenhydramine HCl, chlorpheniramine maleate, pseudoephedrine HCl, ephedrine sulfate, and scopolamine HBr, were analyzed to determine linearity, sensitivity, accuracy, and precision for Pd plus Cr, Fe, Cu, Rh, and Pt versus interferences, particularly from Cl, S, and Ar, on the various X-ray fluorescence lines. Irrespective of instrument platform, in general X-ray sources capable of accessing Pd-K lines were found to be most effective in determination of Pd in APIs.