Analysis of PCB's in Water Using High Resolution Gas Chromatography/High Resolution Mass Spectrometry

Polychlorinated biphenyls (PCBs) in water samples are generally analyzed by high resolution gas chromatography/low resolution mass spectrometry (HRGC/LRMS) or high resolution gas chromatography with electron capture detection (HRGC/ECD). The detection limits reported using these techniques are on the order of 50-500 parts per trillion (ppt) per sample for the Mono-Deca PCBs (HRGC/LRMS). High resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) is routinely used for the analysis of polychlorinated dibenzodioxins and dibenzofurans (PCDDs/PCDFs) in water samples, with detection limits as low as 10 parts per quadrillion (ppq). This HRGC/HRMS technique has been utilized for the analysis of PCBs in water/wastewater samples and the results indicate that the detection limits of these species are at least two orders of magnitude lower (100 ppq range) than achieved using the low resolution mass spectrometric technique. Using this technique, PCBs are reported as totals for each isomer group as well as isomer specific analysis for eleven isomers, seven of which are quantified by isotope dilution mass spectrometry. The technique was validated using blank reagent water samples and then it was applied to measure PCBs concentration in samples collected from a river.Master of Science in Public Healt

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, “S-XL6,” was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A’s in vivo half-life; and that S-XL6 crosses the blood–brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS

A Comparison of LC-MS/MS and a Fully Integrated Autosampler/Solid Phase Extraction System for the Analysis of Protein Binding Samples

A new analysis approach was evaluated for plasma protein binding (PPB) of small molecules using the Agilent RapidFire™ High-Throughput System coupled with a Sciex API 4000 Mass Spectrometer (RF-MS/MS). Thirty three proprietary and 12 literature compounds were subjected to rapid equilibrium dialysis (RED) and evaluated in parallel using RF-MS/MS at 20 seconds/sample and traditional LC-MS/MS at 3.5min/sample, thus making the RF-MS/MS analysis 12 times faster than LC-MS/MS. The developed high throughput analysis method showed excellent correlation with the traditional LC-MS/MS analysis method with an r2 value of 0.95. This RF-MS/MS analysis method was developed and implemented to increase sample throughput, decrease turnaround time for PPB data and time burden on existing LC-MS/MS instruments

Investigation of Dried Blood Spot Card-Induced Interferences in Liquid Chromatography/Mass Spectrometry

Unique and remarkable interferences were observed when dried blood spot (DBS) sampling was used in conjunction with liquid chromatography/mass spectrometry (LC/MS) assays. In particular, chromatographic retention time shifting and chromatographic peak shape distortion were observed, along with a severe suppression of MS signal intensity. The type of DBS cards, and chromatographic conditions were investigated using the same set of test compounds to gain insight into these interferences. It was determined that a constituent of the DBS cards, namely sodium dodecyl sulfate (SDS), was responsible for the interferences by means of an ion-pairing mechanism. SDS formed ion pairs with compounds containing basic amine groups, which resulted in increased retention on a C18 stationary phase, peak shape distortion and ion suppression. These interferences were greatly alleviated and/or completely overcome with non-acidic mobile phases and/or DBS cards with no SDS coating. To the best of the authors’ knowledge, this is the first in-depth report of interferences induced by DBS cards

High-Throughput Analysis of In-Vitro Cytochrome P450 Inhibition Samples Using Mass Spectrometry Coupled with an Integrated Liquid Chromatography and Autosampler System

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A novel diclofenac-hydrogel conjugate system for intraarticular sustained release: Development of 2-pyridylamino-substituted 1-phenylethanol (PAPE) and its derivatives as tunable traceless linkers

A local sustained-release drug delivery system, or depot, for intra-articular injection offers the opportunity to release a therapeutic agent directly to the joint with limited need for reinjection. A successful system would provide more consistent efficacy and minimize systemic side effects. In this paper, we explore the potential use of diclofenac, a non-steroidal anti-inflammatory drug, for use in a polymer-conjugate depot system. During the course of our exploration it was determined that “conventional ester” conjugates of diclofenac were not appropriate as upon incubation in buffer (pH 7.4) or in bovine synovial fluid, a considerable amount of undesired diclofenac-lactam was released. Thus we developed a novel linker system for diclofenac in order to minimize the production of the lactam. This new linker enables a diclofenac conjugate system with tunable release rates and minimizes the production of undesired lactam side-products

An Effective Prodrug Strategy to Selectively Enhance Ocular Exposure of a Cannabinoid Receptor (CB1/2) Agonist

Glaucoma is a leading cause of vision loss and blindness, with increased intraocular pressure (IOP) a prominent risk factor. IOP can be efficaciously reduced by administration of topical agents. However, the repertoire of approved IOP-lowering drug classes is limited, and effective new alternatives are needed. Agonism of the cannabinoid receptors CB1/2 significantly reduces IOP clinically, and experimentally. However, development of CB1/2 agonists has been complicated by the need to avoid cardiovascular and psychotropic side effects. 1 is a potent CB1/2 agonist that is highly excluded from the brain. In a phase I study, compound 1 eyedrops were well tolerated and generated an IOP-lowering trend, but were limited in dose and exposure due to poor solubility and ocular absorption. Here we present an innovative strategy to rapidly identify compound 1 prodrugs that are efficiently metabolized to the parent compound, for improved solubility and ocular permeability, while maintaining low systemic exposures

Metabolically Stable tert-Butyl Replacement

Susceptibility to metabolism is a common issue with the tert-butyl group on compounds of medicinal interest. We demonstrate an approach of removing all the fully sp3 C-H’s from a tert-butyl group: replacing some H’s with F’s and increasing the s-character of the remaining C-H’s. This approach gave a trifluoromethylcyclopropyl group which increased metabolic stability. Trifluoromethylcyclopropyl-containing analogs had consistently higher metabolic stability in vitro and in vivo compared to their tert-butyl-containing counterparts

Design of thioether cyclic peptide scaffolds with passive permeability and oral exposure

Advances in the design of permeable peptides and in the synthesis of large arrays of macrocyclic peptides with diverse amino acids have evolved on parallel, but independent tracks. Less precedence combines their respective attributes, in turn limiting potential to identify permeable peptide ligands for key protein targets. Herein, we present one strategy for focusing the powerful ligand-finding capability of DNA- or RNA-templated peptide synthesis within permeability-biased property space. Despite higher than standard molecular weights (from 774 to 1076 g·mol-1), the 6-, 7-, and 8-mer cyclic peptides of the present contribution are partially N-methylated to achieve low energy conformations with low desolvation penalties. The N-methylation patterns were selected using in silico methods, then experimentally validat-ed with high passive permeability and oral exposure. Further, the present work shows that chemical structures that overlap the synthetic capabilities of DNA- or RNA-templated peptide syntheses in water, can be both permeable and orally exposed. We envision that, by retaining the backbone N-methylation pattern and consequent bias toward per-meability, one can generate large peptide arrays with sufficient side chain diversity to identify permeability-biased lig-ands to a variety of protein targets

An Effective Prodrug Strategy to Selectively Enhance Ocular Exposure of a Cannabinoid Receptor (CB_1/2) Agonist

Glaucoma is a leading cause of vision loss and blindness, with increased intraocular pressure (IOP) a prominent risk factor. IOP can be efficaciously reduced by administration of topical agents. However, the repertoire of approved IOP-lowering drug classes is limited, and effective new alternatives are needed. Agonism of the cannabinoid receptors CB_1/2 significantly reduces IOP clinically and experimentally. However, development of CB_1/2 agonists has been complicated by the need to avoid cardiovascular and psychotropic side effects. <b>1</b> is a potent CB_1/2 agonist that is highly excluded from the brain. In a phase I study, compound <b>1</b> eyedrops were well tolerated and generated an IOP-lowering trend but were limited in dose and exposure due to poor solubility and ocular absorption. Here we present an innovative strategy to rapidly identify compound <b>1</b> prodrugs that are efficiently metabolized to the parent compound for improved solubility and ocular permeability while maintaining low systemic exposures