Development and Validation of a HPLC-UV Method for Urea and Related Impurities.

Urea is used in biopharmaceutical manufacturing processes for the purification of therapeutic proteins, for cleaning columns, and for refolding proteins after purification. The urea used for such purposes is typically USP grade material obtained from commercial sources and further characterization is required prior to use, such as determination of purity and identity. For this purpose, a robust analytical method is needed that can characterize the known organic impurities of urea.

Urinary human chorionic gonadotropin isoform concentrations in doping control samples.

Anti-doping laboratories routinely use immunoassays to measure urinary concentrations of human chorionic gonadotropin (hCG). To minimize immunoassay differences and false positive screen results from inactive isoforms (free β-subunit (hCGβ), β-subunit core fragment (hCGβcf)) laboratories now use intact hCG instead of total hCG immunoassays to measure hCG. To determine the distribution of hCG isoforms in urine, we determined the concentrations of intact hCG, hCGβ, and hCGβcf in male urine samples based on immunoassay total hCG concentrations using a sequential immunoextraction and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.

Estimating the hCGβcf in urine during pregnancy.

Objective: Elevated urine concentrations of hCG beta core fragment (hCGβcf) are known to cause false negative qualitative point-of-care hCG test results, but limited information is available regarding urine hCGβcf. In this study, we evaluate the relationship between serum and urine hCG concentrations and the frequency of elevated urine hCGβcf concentrations.

Design And Methods: Paired serum and urine specimens were obtained from 60 women at various stages of pregnancy and hCG was measured using the Abbott Architect and Roche Cobas e602 assays.

The Urine Marker Test: An Alternative Approach to Supervised Urine Collection for Doping Control.

Background: Urine sample collection for doping control tests is a key component of the World Anti-Doping Agency's fight against doping in sport. However, a substantial number of athletes experience difficulty when having to urinate under supervision. Furthermore, it cannot always be ensured that athletes are actually delivering their own urine.

Evaluation of a semi-quantitative pregnancy device for susceptibility to interference caused by hCGβcf.

Objective: Previous work has documented the ability of the Clearblue Advanced Test with Weeks Estimator, a new over-the-counter (OTC) urine hCG device, to accurately estimate weeks since ovulation in early pregnancy. In this study, the performance of this device in more advanced pregnancy was assessed.

Methods: The Clearblue Advanced Test with Weeks Estimator device was used to test solutions containing purified intact hCG and hCGβcf at concentrations consistent with early, middle and late pregnancy.

Immunoextraction-tandem mass spectrometry method for measuring intact human chorionic gonadotropin, free β-subunit, and β-subunit core fragment in urine.

Background: Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine.

Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry.

Background: Insulin-like growth factor 1 (IGF-1)(7) is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported.

In-cell fluorescence activation and labeling of proteins mediated by FRET-quenched split inteins.

Methods to visualize, track, and modify proteins in living cells are central for understanding the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins.

Manganese(III)-salens induce tumor selective apoptosis in human cells.

In order to explore the apoptotic and anti-tumor activities of metallo-salens, we synthesized several Mn(III)-salen derivatives (compds. 1-9) and analyzed their effects on cultured human cancer and non-cancer cells. Our results demonstrated that Mn(III)-salen derivatives affect cell viability, induce nuclear condensation and fragmentation in breast cancer cells (MCF7).

Iron(III)-salen complexes with less DNA cleavage activity exhibit more efficient apoptosis in MCF7 cells.

To understand the relationship between DNA damage potential and biochemical activities, we synthesized nine different Fe(III)-salen derivatives with varying substituents, and analyzed their in vitro DNA cleavage properties and biochemical effects on cultured human cells. Our results demonstrated that Fe(III)-salen complexes affect cell viability, induce nuclear fragmentation, and activate caspases and apoptosis in cultured human cells. The nature and the position of the substituents in the Fe(III)-salen complexes play critical roles in determining their apoptotic efficiencies.

Iron(III)-salen damages DNA and induces apoptosis in human cell via mitochondrial pathway.

We synthesized a water soluble Fe(III)-salen complex and investigated its biochemical effects on DNA in vitro and on cultured human cells. We showed that Fe(III)-salen produces free radicals in the presence of reducing agent dithiothreitol (DTT) and induces DNA damage in vitro. Interestingly, upon treatment with Fe(III)-salen at concentration as low as 10microM, HEK293 human cells showed morphological changes, nuclear fragmentation, and nuclear condensation that are typical features of apoptotic cell death.