The Degradation Chemistry of Farglitazar and Elucidation of the Oxidative Degradation Mechanisms.

The chemical degradation of farglitazar (1) was investigated using a series of controlled stress testing experiments. Farglitazar drug substance was stressed under acidic, natural pH, basic, and oxidative conditions in solution. In the solid state, the drug substance was stressed with heat, high humidity, and light. Farglitazar was found to be most labile toward oxidative stress. A series of mechanistic experiments are described in which the use of 18O-labelled oxygen demonstrated that oxidative degradation of farglitazar is caused primarily by singlet oxygen formed under thermal conditions. Major degradation products were isolated and fully characterized. Mechanisms for the formation of degradation products are proposed. Drug product tablets were also stressed in the solid state with heat, high humidity, and light. Stressed tablets afforded many of the same degradation products observed during drug substance stress testing, with oxidation again being the predominant degradation pathway. Evidence for the activity of singlet oxygen, formed during thermal stress testing of the solid oral dosage form, is presented. The degradation pathways observed during stress testing matched those observed during long-term stability trials of the drug product.

Comparative analysis of a neurotoxin from Calliostoma canaliculatum by on-line capillary isotachophoresis/1H NMR and diffusion 1H NMR.

NMR spectroscopy has been coupled on-line to capillary isotachophoresis (cITP) to enhance structural analyses of dilute charged species through separation and sample concentration. Microcoils, the most mass-sensitive NMR probes available, provide optimal detection for cITP/NMR. To evaluate the utility of cITP/NMR for natural product analysis, a homogenate of the hypobranchial gland from the marine snail Calliostoma canaliculatum containing a cationic neurotoxin (1, a disulfide-bonded dimer of 6-bromo-2-mercaptotryptamine) was studied. For comparison, hypobranchial gland homogenate was also examined by diffusion-NMR, an alternative approach for NMR mixture analysis. cITP/NMR concentrated the neurotoxin by almost 20-fold and isolated it from some of the other components present in the matrix. However, a minor component, likely a precursor or degradant, co-migrated with compound 1. Diffusion-NMR also did not resolve the two, indicating that the compounds possessed similar diffusion coefficients and electrophoretic mobilities. The strengths and limitations of the two approaches for NMR mixture analysis are discussed.

Retention characteristics of protonated mobile phases injected into deuterated mobile phases in capillary liquid chromatography (LC) using on-line nuclear magnetic resonance (NMR) detection.

The behavior of protonated binary solvents injected into deuterated binary mobile phases in capillary LC is studied with NMR. Specifically, the solvent elution is followed on-flow with a capillary LC coupled to a 900 nL volume microcoil NMR probe. A range of identical composition 5% protonated (and 95% deuterated) solvents is injected into composition-matched deuterated mobile phases of CD(3)CN/D(2)O and CD(3)OD/D(2)O. The protonated components separate for all solvent combinations except at 80% CD(3)CN/20% D(2)O and similar to 72% CD(3)OD/28% D(2)O where only a single retention time is observed. The more hydrophilic protonated component, HOD, elutes first with higher percentages of hydrophilic solvent, D(2)O, in the mobile phase whereas retention is reversed with the higher percentage of the more hydrophobic solvent (CD(3)CN and CD(3)OD) in the mobile phase. The hydrophilic/hydrophobic nature of the chromatographic system as a function of mobile phase composition is characterized by following the retention times of protonated solvents.

Mobile phase compensation to improve NMR spectral properties during solvent gradients.

A solvent compensation method based on flow injection analysis is used to obtain high quality nuclear magnetic resonance (NMR) spectra during solvent gradients. Using a binary solvent system containing D2O and CD3OD, NMR line broadening and chemical shift changes are observed with a 10% methanol per min solvent composition gradient. However, by creating a second equal but reverse gradient and combining the two solvent gradients before the NMR detector, the composition of solvent reaching the NMR flow cell is kept constant. We demonstrate a system using flow injection analysis of combining solvent gradients and show constant NMR spectral performance as a function of time as the combined flow has a constant solvent composition irrespective of the initial solvent gradient. Using this approach, methods can be developed to measure high quality NMR spectra during on-flow gradient LC-NMR experiments. The ultimate ability of this approach depends on the ability to compensate for the disturbance of the solvent gradient and reverse gradient by a pair of LC columns (the analytical and reverse gradient columns).

Characterization of a novel gastropod toxin (6-bromo-2-mercaptotryptamine) that inhibits shaker K channel activity.

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6-6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.

Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation.

Time-resolved NMR spectroscopy is used to studychanges in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of two capillary flows are mixed rapidly, and then the NMR spectra of the combined flow are recorded at precise time points. The distance after mixing the two fluids and flow rates define the solvent-protein interaction time; this method allows the measurement of NMR spectra at precise mixing time points independent of spectral acquisition time. Integration of a micromixer and a microcoil NMR probe enables low-microliter volumes to be used without losing significant sensitivity in the NMR measurement. Ubiquitin, the model compound, changes its conformation from native to A-state at low pH and in 40% or higher methanol/water solvents. Proton NMR resonances of the His-68 and the Tyr-59 of ubiquitin are used to probe the conformational changes. Mixing ubiquitin and methanol solutions under low pH at microliter per minute flow rates yields both native and A-states. As the flow rate decreases, yielding longer reaction times, the population of the A-state increases. The micromixer-NMR system can probe reaction kinetics on a time scale of seconds.

NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis.

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.

Microscale NMR.

NMR spectroscopy is increasingly being used to characterize microliter and smaller-volume samples. Substances at picomole levels have been identified using NMR spectrometers equipped with microcoil-based probes. NMR probes that incorporate multiple sample chambers enable higher-throughput NMR experiments. Hyphenation of capillary-scale separations and microcoil NMR has also decreased analysis time of mixtures. For example, capillary isotachophoresis/NMR allows the highest mass sensitivity nanoliter-volume flow cells to be used with low microliter volume samples because isotachophoresis concentrates the microliter volume sample into the nanoliter volume NMR detection probe. In addition, the diagnostic capabilities of NMR spectroscopy allow the physico-chemical aspects of a capillary separation process to be characterized on-line. Because of such advances, the application of NMR to smaller samples continues to grow.

Insights into the cITP process using on-line NMR spectroscopy.

Recently, capillary isotachophoresis (cITP) has been coupled on-line with nuclear magnetic resonance (NMR) to enhance analysis of dilute charged analytes through sample concentration and separation. This study focuses on the unique detection capabilities of NMR to noninvasively examine the cITP process and obtain diagnostic information. With their enhanced mass sensitivity, microcoil NMR probes provide optimal detection for cITP/NMR. Whereas previous studies used deuterated buffers, a 1H NMR observable leading electrolyte, tetramethylammonium acetate, is employed here to better track cITP progression. Fortuitously, the 1H chemical shift of the acetate methyl resonance depends on pD. Hence, by using a calibration curve, the solution pD can be determined on-line during cITP. Similarly, intracapillary temperature can be measured in cITP/NMR by observing the HOD chemical shift. To obtain accurate chemical shift measurements, charge-neutral tert-butyl alcohol is added to all cITP electrolyte solutions as an internal reference. As an ancillary benefit, line width measurements of the ubiquitous tert-butyl alcohol enable NMR spectral resolution to be examined throughout the experiment. Capable of providing quantitative results, NMR simultaneously determines the concentrations of the leading ion, sample, and counterion over the course of the cITP experiment.

Capillary isotachophoresis/NMR: extension to trace impurity analysis and improved instrumental coupling.

Building upon its promising initial performance, the online coupling of capillary isotachophoresis (cITP) to nuclear magnetic resonance (NMR) is extended to trace impurity analysis. By simultaneously concentrating and separating dilute charged species on the basis of their electrophoretic mobility, cITP greatly facilitates NMR structural elucidation. cITP/NMR appears particularly attractive for identifying trace charged synthetic and natural organic compounds obscured by large excesses of other components. A 9.4 microL injection of 200 microM (1.9 nmol) atenolol in a 1000-fold excess of sucrose (200 mM) is analyzed by cITP/NMR. A microcoil, the most mass sensitive NMR probe, serves as the detector as it provides optimal NMR observation of the capillary-scale separation. cITP successfully isolates the atenolol from the sucrose while concentrating it 200-fold to 40 mM before presentation to the 30 nL observe volume microcoil, thereby enabling rapid 1H NMR spectral acquisition of atenolol (experimental time of 10 s) without obstruction from sucrose. For this particular probe and sample, the stacking efficiency is near the theoretical limit as 67% of the sample occupies the 1 mm long microcoil during peak maximum. A multiple-coil probe with two serial 1 mm long microcoils arranged 1 cm apart has been developed to facilitate peak trapping and sample band positioning during cITP/NMR.