Development of HPLC-CAD stability indicating assay method for polyethylene glycol-conjugated phospholipid (DMPE-PEG 2000) and identification of its degradation products.

The study introduces first report on a liquid chromatographic method for the quantification of 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] ammonium salt (DMPE-PEG 2000), which is an important constituent of lipid-based nanoparticles. It involves an HPLC-CAD stability-indicating assay method development for DMPE-PEG 2000 and structure elucidation of its degradation products. Hypersil Gold™ PFP column (150 mm × 4.6 mm, 3.0 µm) was used to achieve the separation among DMPE-PEG 2000 and its degradation products using 0.0025% formic acid in water: methanol (80:20 v/v) as mobile phase A and methanol: acetonitrile (60:40 v/v) as mobile phase B in a gradient elution mode. The method was validated for precision, linearity, sensitivity, solution stability and robustness. Relative standard deviations for the intra-day precision, inter-day precision and sensitivity were 1.6%, 0.6% and 3.8%, respectively. The method was linear in the range from 210 µg/mL to 390 µg/mL with R2 value of 0.996. Further, the solution stability of DMPE-PEG 2000 was evaluated under different stressed and storage conditions to understand the impact of any excursion to its regular storage temperature of -20 °C. The observed degradation products were identified through liquid chromatography high resolution mass spectrometry and a tentative pathway was proposed for the generation of these degradants.

Development of simple isocratic HPLC methods for siRNA quantitation in lipid-based nanoparticles.

This paper describes the development of simple and user-friendly HPLC methods that can quantitate the amount of small interfering RNA (siRNA) in lipid-based nanoparticle (LNP) formulations. The methods have been used as alternative chromatographic approaches to the size exclusion chromatography in order to perform "fit for purpose" analysis such as determining the amount of released siRNA from LNP formulations as a part of in-vitro release testing. Two HPLC conditions were optimized using reversed phase (a 250 × 4.6 mm Waters XSelect CSH column) and cation exchange columns (a 250 × 4.6 mm Zorbax SCX-300 column) maintained at 30 °C with a mobile phase of 0.1 M ammonium bicarbonate aqueous solution containing 20-30% acetonitrile. All the siRNA variants were excluded from pores in stationary phase and led to a single peak eluted earlier than the solvent front. The impact of chromatographic conditions and column configurations on method specificity, accuracy, and sensitivity have been investigated. Validation data for both methods in terms of precision, linearity, accuracy and sensitivity are also presented.

Determination of polymeric impurities in asunaprevir drug substance and product using size exclusion effect of reversed-phase columns.

This paper describes the development of a simple reversed-phase HPLC method that can quantitate trace amounts of a polymeric degradants (BMT-041910) in asunaprevir drug substance and formulated drug product with quantitation limits of ∼0.05% w/w. The method has overcome several challenges of polymer quantitation such as band broadening, peak coeluting and low sensitivity. The hydrophobic function group (BOC) of BMT-041910 is removed to increase its aqueous solubility by a simple sample treatment procedure (des-BOC). The des-BOC polymer (BMT-052076) is excluded from stationary phase pores and eluted as a single peak before solvent front, and then its peak area response can be used to determine BMT-041910 amount. The HPLC conditions were optimized using a 250 × 4.6 mm Waters XSelect CSH column maintained at 30 °C with a mobile phase of water-acetonitrile-trifluoroacetic acid (20:80:0.1 v/v/v). The feasibility of other HPLC approaches including size exclusion chromatography and normal phase chromatography were also investigated and found to be less suitable for this particular application. Validation data for this method in terms of precision, linearity, accuracy and sensitivity are also presented.

Interactions between a poorly soluble cationic drug and sodium dodecyl sulfate in dissolution medium and their impact on in vitro dissolution behavior.

In the pharmaceutical industry, in vitro dissolution testing ofsolid oral dosage forms is a very important tool for drug development and quality control. However, ion-pairing interaction between the ionic drugand surfactants in dissolution medium often occurs, resulting in inconsistent and incomplete drug release. The aim of this study is toevaluate the effects ofsodium dodecyl sulfate (SDS) mediated medium onthe dissolution behaviors of a poorly soluble cationic drug (Drug B). The study was carried out by measuring solubility of Drug B substance and dissolution rate of Drug B product in media containing SDS.Desolubilization of Drug B substance was observed at pH 4.5 in the presence of SDS at concentrations below critical micelle concentration (CMC) which is attributed to the formation of an insoluble di-dodecyl sulfate salt between SDS and Drug B. This ion-pairing effect is less significant with increasing medium pH where Drug B is less ionized and CMC of SDS is lower. In medium at pH 4.5, dissolution of Drug B product was found incomplete with SDS concentration below CMC due to the desolubilization of Drug B substance. In media with SDS level above CMC, the dissolution rate is rather slower with higher inter-vessel variations compared to that obtained in pH 4.5 medium without SDS. The dissolution results demonstrate that the presence of SDS in medium generates unexpected irregular dissolution profiles for Drug B which are attributed to incompatible dissolution medium for this particular drug. Therefore, non-ionic surfactant was selected for Drug B product dissolution method and ion-pairing effect in SDS mediated medium should be evaluated when developing a dissolution method for any poorly soluble cationic drugs.

Separation of atropisomers by chiral liquid chromatography and thermodynamic analysis of separation mechanism.

In the pharmaceutical industry, the analysis of atropisomers is of considerable interest from a scientific and regulatory perspective. The compound of interest contains two stereogenic axes due to the hindered rotation around the single bonds connecting the aryl groups, which results in four potential configurational isomers (atropisomers). The separation of the four atropisomers is achieved on a derivatized ß-cyclodextrin bonded stationary phase. Further investigation shows that low temperature conditions, including sample preparation (-70 °C), sample storage (-70 °C), and chromatographic separation (6 °C), were critical to preventing interconversion. LC-UV-Laser Polarimetric analysis identified peak 1/2 as a pair of enantiomers and peak 3/4 as another. Thermodynamic analysis of the retention data indicated that the separation of the pairs of enantiomers is primarily enthalpy controlled as indicated by the positive slope of the van't Huff plot. The difference in absolute Δ (Δ H), ranged from 2.20 kJ/mol to 2.42 kJ/mol.

Spatial and temporal variability of contaminants within estuarine sediments and native Olympia oysters: A contrast between a developed and an undeveloped estuary.

Chemical contaminants can be introduced into estuarine and marine ecosystems from a variety of sources including wastewater, agriculture and forestry practices, point and non-point discharges, runoff from industrial, municipal, and urban lands, accidental spills, and atmospheric deposition. The diversity of potential sources contributes to the likelihood of contaminated marine waters and sediments and increases the probability of uptake by marine organisms. Despite widespread recognition of direct and indirect pathways for contaminant deposition and organismal exposure in coastal systems, spatial and temporal variability in contaminant composition, deposition, and uptake patterns are still poorly known. We investigated these patterns for a suite of persistent legacy contaminants including polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) and chemicals of emerging concern including pharmaceuticals within two Oregon coastal estuaries (Coos and Netarts Bays). In the more urbanized Coos Bay, native Olympia oyster (Ostrea lurida) tissue had approximately twice the number of PCB congeners at over seven times the total concentration, yet fewer PBDEs at one-tenth the concentration as compared to the more rural Netarts Bay. Different pharmaceutical suites were detected during each sampling season. Variability in contaminant types and concentrations across seasons and between species and media (organisms versus sediment) indicates the limitation of using indicator species and/or sampling annually to determine contaminant loads at a site or for specific species. The results indicate the prevalence of legacy contaminants and CECs in relatively undeveloped coastal environments highlighting the need to improve policy and management actions to reduce contaminant releases into estuarine and marine waters and to deal with legacy compounds that remain long after prohibition of use. Our results point to the need for better understanding of the ecological and human health risks of exposure to the diverse cocktail of pollutants and harmful compounds that will continue to leach from estuarine sediments over time.

Using a harm reduction approach with clients who have alcohol/drug dependencies in a spinal cord rehabilitation program.

BACKGROUND: Dissatisfied with the frequently adversarial nature of relationships with clients who use alcohol or drugs while rehabilitation inpatients, and the often less than optimal outcomes for these individuals, the Spinal Cord Program at the G.F. Strong Rehabilitation Center in Vancouver, BC, decided to pilot a new approach. OBJECTIVE: The goal of the pilot project is to promote successful rehabilitation, including less conflict in rehabilitation, a completed rehabilitation program, and continued connection after discharge if needed. METHOD: A dedicated team was formed and trained to work with these clients using harm reduction principles. PARTICIPANTS: From its inception in December 2000, through May 2001, the team worked with 6 inpatients, 12% of admissions to the Spinal Cord Program during that period. RESULTS: Outcomes based on the above goals have been positive. There have been no discharges against a client's will or instances of significant conflict with the team. Several clients have returned to the center for assistance or to visit post-discharge. Only 1 client left rehabilitation prematurely.

Evaluation of captopril for the management of hypertension in autonomic dysreflexia: a pilot study.

OBJECTIVE: To evaluate the efficacy of captopril for management of hypertensive urgencies in autonomic dysreflexia. DESIGN: A 1-year, prospective, open-label pilot study. SETTING: Rehabilitation hospital. PATIENTS: Twenty-six consecutive patients older than 15 years with spinal cord injury above T6. INTERVENTIONS: During an autonomic dysreflexia episode, captopril 25mg was administered sublingually if systolic blood pressure (SBP) was at or above 150mmHg despite the use of nondrug measures. If SBP remained elevated 30 minutes after captopril administration, 1 dose of immediate-release nifedipine 5mg was given as rescue by the bite and swallow method and repeated, if necessary, in 15 minutes. MAIN OUTCOME MEASURE: SBP 30 minutes after captopril administration at initial autonomic dysreflexia episode. RESULTS: A total of 33 autonomic dysreflexia episodes were documented, of which 18 episodes in 5 patients were treated with drug therapy. Captopril alone was effective in 4 of 5 initial episodes (80%). Mean SBPs at baseline and 30 minutes after captopril were 178+/-18mmHg and 133+/-28mmHg, respectively. There were no cases of reactive hypotension. The addition of nifedipine successfully reduced SBP in the remaining patient. Of the combined 18 initial and repeat autonomic dysreflexia episodes, 94% were successfully treated with our protocol. CONCLUSION: Captopril appears to be safe and effective for autonomic dysreflexia management.

Pain during spinal cord injury rehabilitation: client perspectives and staff attitudes.

Pain after spinal cord injury (SCI) is well documented in the literature. Effective treatment for pain after SCI remains elusive and treatment protocols have not been well researched. Staff on a 32-bed SCI rehabilitation unit designed and implemented a descriptive research study to improve pain management outcomes. An interdisciplinary plan to improve pain management practices was developed as a result of the study. Guidelines of the American Academy of Pain Medicine and the American Pain Society (1997) and the Agency for Healthcare Policy and Research (1992 [now called Agency for Healthcare Research and Quality]) provided the framework for the interdisciplinary management of pain.