Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin.

USP's peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Isolation and characterization of novel degradation products of Doxofylline using HPLC, FTIR, LCMS and NMR.

Forced degradation of Doxofylline (DFL) in different stress (base and peroxide) conditions gave rise to two potential unknown impurities. These unknown degradation products DFL DEG-I and DFL DEG-II were evaluated using a new-reverse-phase high performance liquid chromatography (HPLC), where it was eluted at 0.44 and 1.09 relative retention times to DFL peak. DFL DEG-I and DFL DEG-II were isolated using preparative HPLC from degradation mixtures. The structure of DFL DEG-I and DFL DEG-II were elucidated using high resolution MS, multi-dimensional NMR and FTIR spectroscopic techniques, and characterized. The stereochemistry of the enantiomers in DFL DEG-II has further been investigated using computational techniques. To the best of our knowledge, DFL DEG-I and DFL DEG-II are novel impurities and not reported elsewhere.

Isolation and structural characterization of novel photolytic degradation impurities of Deflazacort using Q-TOF, 2D-NMR and FTIR.

Forced Degradation of Deflazacort drug substance in ultraviolet light condition resulted into a number of significant degradation products. Two of these degradation products were found to be unknown during the study and marked as DD-I and DD-II. Thus, the objective of this work is to investigate and identify these two novel degradation products of DFZ. The isolation method for these new degradation products were developed using a new reverse-phase high performance liquid chromatography (HPLC). DD-I and DD-II, eluting at 0.53 and 1.57 relative retention times with respect to Deflazacort (DFZ) peak respectively, were isolated from reaction mass using preparative HPLC and their structures were elucidated using high resolution MS, multidimensional NMR and FTIR spectroscopic techniques. To best of our knowledge, these two degradation products are novel impurities which are not discussed in any form of publication yet.

Investigation of Fat Metabolism during Antiobesity Interventions by Magnetic Resonance Imaging and Spectroscopy.

The focus of current treatments for obesity is to reduce the body weight or visceral fat, which requires longer duration to show effect. In this study, we investigated the short-term changes in fat metabolism in liver, abdomen, and skeletal muscle during antiobesity interventions including Sibutra mine treatment and diet restriction in obese rats using magnetic resonance imaging, magnetic resonance spectroscopy, and blood chemistry. Sibutramine is an antiobesity drug that results in weight loss by increasing satiety and energy expenditure. The Sibutramine-treated rats showed reduction of liver fat and intramyocellular lipids on day 3. The triglycerides (TG) decreased on day 1 and 3 compared to baseline (day 0). The early response/nonresponse in different fat depots will permit optimization of treatment for better clinical outcome rather than staying with a drug for longer periods.

Slow relaxation of longitudinal multispin orders in weakly and strongly coupled two-spin systems.

Longitudinal multispin order (LOMO) corresponds to a nonequilibrium population distribution in spin systems that exhibit scalar (J), dipolar, or quadrupolar coupling. We investigated the relaxation of longitudinal two-spin order (2-LOMO) in systems that had either weakly or strongly J-coupled spins. Our results indicated longer relaxation times for the 2-LOMO state compared with the corresponding longitudinal single-spin state (1-LOMO). Accessing nuclear spin states that have relaxation times longer than T(1), without the use of external contrast agents, is potentially useful for in vivo imaging and also for studying systems using dynamically hyperpolarized nuclear spins where longer life times are sought to increase the time available to study (bio)chemical events.