New perspectives and lessons learned in the identification of impurities in drug development.

Within the pharmaceutical industry, the rapid identification, elucidation and characterization of synthetic or process impurities or degradants form an intense and a comprehensive undertaking. Advances in laboratory hardware and software are changing the way in which scientists work together to help resolve impurities in a quick and efficient manner. Although the industry trend toward externalization and outsourcing of development tasks provides a cost-effective method, the demand for improved productivity in laboratory workflows in drug development continues to be a high priority. This brings a need for new approaches for communication, collaboration and data management.

Analysis of samples stored as individual plugs in a capillary by electrospray ionization mass spectrometry.

Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry when pumped directly into a fused silica nanospray emitter tip. Using leucine-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was <0.1% and relative standard deviation of signal for a series of plugs was 3%. Detection limits were 1 nM. Sample analysis rates of 0.8 Hz were achieved by pumping 13 nL samples separated by 3 mm long air gaps in a 75 microm inner diameter tube. Analysis rates were limited by the scan time of the ion trap mass spectrometer. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems.

Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery.

Nanospray experiments were performed on an ensemble of drug molecules and their commonly known metabolites to compare performance with conventional electrospray ionization (ESI) and to evaluate equimolar response capabilities. Codeine, dextromethorphan, tolbutamide, phenobarbital, cocaine, and morphine were analyzed along with their well-known metabolites that were formed via hydroxylation, dealkylation, hydrolysis, and glucuronidation. Nanospray exhibited a distinct trend toward equimolar response when flow rate was reduced from 25 nL/min to less than 10 nL/min. A more uniform response between the parent drug and the corresponding metabolites was obtained at flow rates of 10 nL/min or lower. The largest discrepancy was within +/-50% for plasma samples. Nanospray was used as a calibrator for conventional ESI liquid chromatography/tandem mass spectrometry (LC/MS/MS) and normalization factors were applied to the quantitation of an acyl-glucuronide metabolite of a proprietary compound in rat plasma. A nanospray calibration method was developed with the standard curve of the parent drug to generate quantitative results for drug metabolites within +/-20% of that obtained with reference standards and conventional ESI. The nanospray method provides a practical solution for the quantitative estimation of drug metabolites in drug discovery when reference standards are not available.

Automated orthogonal control system for electrospray ionization.

Low-flow electrospray ionization is typically a purely electrostatic method, used without supporting sheath-gas nebulization. Complex spray morphology results from a large number of possible spray emission modes. Spray morphology may assume the optimal Taylor cone-jet spray mode under equilibrium conditions. When coupling to nanobore gradient elution chromatography, however, stability of the Taylor cone-jet spray mode is compromised by the gradient of mobile phase physiochemical properties. The common spray modes for aqueous/organic mobile phases were characterized using orthogonal (strobed illumination) transmitted light and (continuous illumination) scattered light imaging. Correlation of image sets from these complementary illumination methods provides the basis for spray mode identification using qualitative and quantitative image analysis. An automated feedback-controlled electrospray source was developed on a computer capable of controlling electrospray potential using an image-processing based algorithm for spray mode identification. The implementation of the feedback loop results in a system that is both self-starting and self-tuning for a specific spray mode or modes. Thus, changes in mobile phase composition and/or flow rate are compensated in real-time and the source is maintained in the cone-jet or pulsed cone-jet spray modes.