Quantitation of amiodarone and N-desethylamiodarone in single HepG2 cells by single-cell printing-liquid vortex capture-mass spectrometry.

Quantitative measure of a drug and its associated metabolite(s) with single-cell resolution is often limited by sampling throughput or other compromises that limit broad use. Here, we demonstrate the use of single-cell printing-liquid vortex capture-mass spectrometry (SCP-LVC-MS) to quantitatively measure the intracellular concentrations of amiodarone (AMIO) and its metabolite, N-desethylamiodarone (NDEA), from thousands of single cells across several AMIO incubation concentrations ranging from 0 to 10 µM. Concentrations obtained by SCP-LVC-MS were validated through comparison with average assays and traditional measurement of cells in bulk. Average of SCP-LVC-MS measurements and aggregate vial collection assay the concentrations differed by < 5%. Both AMIO and NDEA had clear log-normal distributions with similar standard deviation of concentrations in the cell population. The mean of both AMIO and NDEA intracellular concentrations were positively correlated with AMIO incubation concentration, increasing from 0.026 to 0.520 and 0.0055 to 0.048 mM for AMIO and NDEA, respectively. The standard deviation of AMIO and NDEA log-normal distribution fits were relatively similar in value across incubation concentrations, 0.15-0.19 log10 (mM), and exhibited a linear trend with respect to each other. The single cell-resolved conversion ratio of AMIO to NDEA increased with decreasing incubation concentration, 7 ± 2%, 18 ± 3%, and 20 ± 7% for 10.0, 1.0, and 0.1 µM AMIO incubation concentrations, respectively. Association with simultaneously measured lipids had several ions with statistically significant difference in intensity but no clear correlations with AMIO intracellular content was observed.

Metabolic Profiling of Vasorelaxant Extract from Malvaviscus arboreus by LC/QTOF-MS.

We aimed to develop a standardized methodology to determine the metabolic profile of organic extracts from Malvaviscus arboreus Cav. (Malvaceae), a Mexican plant used in traditional medicine for the treatment of hypertension and other illnesses. Also, we determined the vasorelaxant activity of these extracts by ex vivo rat thoracic aorta assay. Organic extracts of stems and leaves were prepared by a comprehensive maceration process. The vasorelaxant activity was determined by measuring the relaxant capability of the extract to decrease a contraction induced by noradrenaline (0.1 µM). The hexane extract induced a significant vasorelaxant effect in a concentration- and endothelium-dependent manner. Secondary metabolites, such as polyunsaturated fatty acids, terpenes and one flavonoid, were annotated by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in positive ion mode. This exploratory study allowed us to identify bioactive secondary metabolites from Malvaviscus arboreus, as well as identify potentially-new vasorelaxant molecules and scaffolds for drug discovery.

Tannic acid, a vasodilator present in wines and beverages, stimulates Ca2+ influx via TRP channels in bEND.3 endothelial cells.

Tannic acid (TA) is a polyphenol compound present in wines and many beverages. Although previous works have shown that TA could cause vasodilation in an endothelial cell (EC)-dependent manner, there is hitherto no report showing whether TA could raise EC cytosolic Ca2+ concentration. In this work we examined the effects of TA on cytosolic Ca2+ of mouse brain bEND.3 EC. TA (1-30 µM) caused a slow elevation in cytosolic Ca2+ level in a concentration-dependent manner. At 30 µM, TA triggered Ca2+ influx without causing intracellular Ca2+ release. TA-triggered Ca2+ influx was suppressed by Ni2+ (a non-specific Ca2+ channel blocker), ruthenium red and SKF 96365 (non-specific TRP channel blockers), CBA (a selective TRPM4 inhibitor) and M 084 (a selective TRPC4/C5 blocker). However, TA-triggered Ca2+ influx pathway was not permeable to Mn2+. Our results suggest TA activated TRP channels, possibly TRPM4 and TRPC4/C5, to promote influx of Ca2+.

Antioxidant and vasorelaxant activities induced by northeastern Brazilian fermented grape skins.

BACKGROUND: In northeastern Brazil, grape pomace has become a potential alternative byproduct because of the recover phenolic compounds from the vinification process. Comparative analyses were performed between lyophilized extract of grape skins from pomace, described as fermented (FGS), and fresh, unfermented (UGS) grape skins to show the relevant brand's composition upon the first maceration in winemaking. METHODS: The use of in vitro testing such as Folin-Ciocalteu's, DPPH free radical scavenger and HPLC methods were performed to evidence antioxidant effect and phenolic compounds. Additionally, vascular reactivity studies were performed in third-order branches of rat superior mesenteric arteries, which were obtained and placed in organ baths containing Krebs-Henseleit solution, maintained at 37 °C, gassed with a mixture of 95% O2 and 5% CO2, and maintained at pH 7.4. The in situ formation of reactive oxygen species (ROS) was evaluated in small mesenteric rings using oxidative fluorescent dihydroethidium dye. RESULTS: We found higher phenolic content and antioxidant activity in FGS when compared to UGS. HPLC analyses identified a significant number of phenolic compounds with antioxidant potential in both samples. The vasorelaxant effect induced by FGS was more potent than that induced by UGS, and the activity was attenuated after removal of vascular endothelium or by blockade of endothelium-derived relaxing factors, such as NO and EDHF. CONCLUSIONS: The FGS extract may be a great source of natural polyphenol products with potent antioxidant effects and endothelium-dependent vasodilatory actions involving NO and EDHF pathways.

Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract.

We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.

Mucoadhesive elementary osmotic pump tablets of trimetazidine for controlled drug delivery and reduced variability in oral bioavailability.

The objectives of this work was preparation and evaluation of the mucoadhesive elementary osmotic pump tablets of trimetazidine hydrochloride to achieve desired controlled release action and augmentation of oral drug absorption. The drug-loaded core tablets were prepared employing the suitable tableting excipients and coated with polymeric blend of ethyl cellulose and hydroxypropyl methylethylcellulose E5 (4:1). The prepared tablets were characterized for various quality control tests and in vitro drug release. Evaluation of drug release kinetics through model fitting suggested the Fickian mechanism of drug release, which was regulated by osmosis and diffusion as the predominant mechanism. Evaluation of mucoadhesion property using texture analyzer suggested good mucoadhesion potential of the developed osmotic systems. Solid state characterization using Fourier-transform infrared spectroscopy, differential scanning calorimetry and powder X-ray diffraction spectroscopy confirmed the absence of any physiochemical incompatibilities between drug and excipients. Scanning electron microscopy analysis showed the smooth surface appearance of the coated tablets with intact polymeric membrane without any fracture. In vivo pharmacokinetic studies in rabbits revealed 3.01-fold enhancement in the oral bioavailability vis-à-vis the marketed formulation (Vastarel MR®). These studies successfully demonstrate the bioavailability enhancement potential of the mucoadhesive elementary osmotic pumps as novel therapeutic systems for other drugs too.

Simultaneous determination of four components in Baige capsule by HPLC: application to pharmacokinetics and tissue distribution of normal and middle cerebral artery occlusion rats.

A sensitive and specific high-performance liquid chromatography (HPLC) method was developed and applied to the pharmacokinetics for simultaneous identification and quantification of four components - puerarin, daidzein, imperatorin and isoimperatorin - in the plasma and tissues of normal and middle cerebral artery occlusion rats after oral administration of Baige capsule. Ferulic was used as the internal standard. The extraction procedure was composed of two independent steps. The plasma was prepared by liquid-liquid extraction with light petroleum-acetic ether (1:1, v/v) first and then protein was precipitated with methanol. The tissue samples were weighted and homogenated with normal saline, then the homogenate was prepared by liquid-liquid extraction and protein precipitation. The parameters of pharmacokinetics were calculated using DAS 2.1.1 software. The calibration curves of all four components in the plasma and tissue homogenates were in good linearity in the measured range with R(2) ≥ 0.9921. The relative standard deviation of the intra- and inter-day accuracy at different levels was less than ±18.0%. In conclusion, the established method was a simple and effective one to simultaneously detect all four components in the plasma and tissues of rats, and was successfully applied in the pharmacokinetics of Baige capsule.

Stability-indicating determination of trimetazidine dihydrochloride inthe presence of two of its related substances using a direct GC/MS method.

A novel, simple, direct, and selective stability- indicating GC/MS procedure was developed for the determination of the anti-ischemic drug trimetazidine dihydrochloride (TMZ) in the presence of two of its related substances (potential impurities), namely, 2,3,4-trimethoxybenzyl alcohol (T1) and 2,3,4-trimethoxybenzaldehyde (T2). The method involved resolution of the undeilvatized compounds using a 100% dimethylpolysiloxane (Rtx-1) capillary column, and MS detection was carried out in the electron-impact mode. The peaks of the three compounds eluted at retention times 11.69, 11.92, and 15.47 min for T1, T2, and TMZ, respectively. Quantification of the parent drug TMZ was based on measuring its peak area. The reliability and analytical performance of the proposed method, including linearity, range, precision, accuracy, selectivity, detection, and quantification limits, were statistically validated. The calibration curve of TMZ was linear over the range 100-600 µg/mL. The proposed method was successfully applied to the assay of TMZ in several commercially available pharmaceutical formulations with recoveries not lessthan 96.2%.

Multiple poisonings with sodium azide at a local restaurant.

BACKGROUND: Sodium azide is a chemical with a mechanism similar to cyanide. There is concern that it could be used as a chemical warfare agent. OBJECTIVES: We report a cluster of poisonings that occurred at a public restaurant and the subsequent investigation that identified iced tea contaminated with sodium azide (NaN3) and hydrazoic acid, as the foodborne vehicle and agents, respectively. CASE REPORT: Five patients became ill within minutes of drinking iced tea at a restaurant. They all presented to the same Emergency Department with similar symptoms, and improved with fluids, antiemetics, and supportive care. A joint investigation by the Dallas County Department of Health and Human Services, the Texas State Health Department, the Dallas County Southwestern Institute of Forensic Sciences, and the medical toxicologists at the University of Texas Southwestern School of Medicine identified iced tea, contaminated with sodium azide (NaN3) and hydrazoic acid, as the foodborne vehicle and agents, respectively. CONCLUSION: The recurrence, and seriousness, of these events suggests a need for continued education of emergency providers. Emergency physicians should consider exposures to toxic chemicals in their differential when a cluster of patients presents with similar symptoms over a short period of time.

Quality control certification of RNA aptamer-based detection.

Aptamers are single-stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer-based biosensors are prone to producing false-positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false-positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA-DNA duplexes with 1) a quencher-labelled DNA strand (Q(1)DNA), 2) a dual-fluorophore-labelled DNA strand (F(1)DNAF(2)) and 3) another quencher-labelled DNA strand (Q(2)DNA). The addition of a target releases Q(2)DNA from the duplex assembly, and produces the expected positive signal from F(2). However, the authenticity of target recognition is validated only if no signal is generated from F(1). We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high-quality detection when RNA aptamers are employed as molecular-recognition elements.

Coated hydralazine hydrochloride beads for sustained release after oral administration.

Hydralazine hydrochloride is an antihypertensive used alone or in combination with isosorbide nitrate for the treatment of congestive heart failure. Since control of blood pressure should be continuous, sustained release delivery of this drug is considered therapeutically beneficial. Core beads for oral administration of this drug were prepared by extrusion-spheronization. Using experimental design to define the coat that was applied, the core beads were coated using a fluid bed coater to different coat thickness with combinations of two commercially available products dissolved in a hydroalcoholic solvent. The coat is a film with a combination of ethylcellulose and hydroxypropylcellulose that can provide desirable release profiles. Visually spherical and rugged bead products were obtained. Two products were identified that exhibited essentially a zero order release profile following a 2-h lag time with release of greater than 70% of the drug over the next 10 h in simulated intestinal fluid.

Physicochemical stability study of a new Trimix formulation for treatment of erectile dysfunction.

Currently, severe erectile dysfunction can be treated by intracavernous injections of solutions containing three active ingredients: prostaglandin E1 (PGE1), papaverine and urapidil. Very few data exist on this mixture where phentolamine has been replaced by Urapidil because Phentolamine is not used for this indication in France. The aim of our study was to assess the stability of this formulation and to extend its expiration to permit preparation of batches. Three batches of the preparation containing 15µg/mL PGE1, 15mg/mL of papaverine and 2mg/mL urapidil were made aseptically and then packed in polypropylene syringes stored at 4°C. The physico-chemical stability has been tested as follows: HPLC stability-indicating method, visual observation, measurement of pH and osmolarity. We found that the limiting factor was PGE1 and we exceeded the threshold of 10% loss after 55 days. Replacement of Urapidil by Phentolamine seems to have a slight detrimental effect on stability. Nevertheless, these results allow us to consider the advance preparation of this formulation and provide quality treatment to these patients by avoiding too frequent visits to the hospital.

Purification and partial characterization of a novel lectin from Dioclea lasiocarpa Mart seeds with vasodilator effects.

A lectin from seeds of Dioclea lasiocarpa (DLL) was purified in a single step by affinity chromatography in a Sephadex G-50 column. DLL haemagglutinated rabbit erythrocytes showing stability even after 1 h of exposure to a different pH values (optimal between pH 6.0 and 8.0) but was inhibited after incubation with D-mannose and D-glucose. The pure protein possessed a molecular weight of 25 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 25,410Da by mass spectrometry. The results analyzed by the software SELCON 3 indicate that ß-sheet secondary structures are predominant in DLL (approximately 40.2% antiparallel ß-sheet, 4.6% parallel ß-sheet, 7.2% α-helices, 17.3% turns, and 28.7% unordered structures). Mechanical activity of isolated aorta from rat measured by cumulative concentration curves of DLL, performed at the contraction plateau induced by phenylephrine in either endothelium-intact or denuded aorta. DLL (IC(50) = 34.12 ± 3.46 µg/ml) relaxed precontracted endothelized aortic rings by 34.61 ± 9.06%, 55.19 ± 11.9%, and 81.33 ± 14.35%, respectively, at 10 µg/ml (initial concentration), 30 µg/ml, and 100 µg/ml (maximum effect). All effects occurred via interaction with lectin domains and participation of nitric oxide.

Sodium azide poisoning at a restaurant - Dallas County, Texas, 2010.

In April 2010, Dallas County Health and Human Services (DCHHS) staff members investigated reports of acute-onset dizziness among patrons in a local restaurant. Symptoms, which included fainting resulting from low blood pressure, occurred within minutes of consuming food from the restaurant and were consistent with chemical poisoning. Toxicologic and epidemiologic investigations were begun to determine the cause of the poisonings and identify potentially exposed persons. This report summarizes the results of those investigations, including a case-control study that identified iced tea as the likely contaminated food or drink (odds ratio [OR] = 65; 95% confidence interval [CI] = 2.4-3,292). Approximately 5 months after the incident, extensive laboratory testing identified sodium azide (NaN3) and hydrazoic acid (formed when sodium azide contacts water) as the toxic agents in the iced tea. All five ill restaurant patrons recovered from their symptoms. For rapid-onset foodborne illnesses, chemical poisons should be considered as a potential cause, regardless of negative initial toxicologic screening tests. Although unusual chemicals can be challenging to detect, a multidisciplinary approach involving public health officials and forensic and medical toxicologists can lead to appropriate testing. In the absence of an identified agent, epidemiologic tools are valuable for active case-finding and confirming suspected contaminated food vehicles.

Exposure to isoflavone-containing soy products and endothelial function: a Bayesian meta-analysis of randomized controlled trials.

BACKGROUND AND AIMS: To determine whether and to what degree exposure to isoflavone-containing soy products affects EF. Endothelial dysfunction has been identified as an independent coronary heart disease risk factor and a strong predictor of long-term cardiovascular morbidity and mortality. Data on the effects of exposure to isoflavone-containing soy products on EF are conflicting. METHODS AND RESULTS: A comprehensive literature search was conducted using the PUBMED database (National Library of Medicine, Bethesda, MD) inclusively through August 21, 2009 on RCTs using the keywords: soy, isoflavone, phytoestrogen, EF, flow mediated vasodilation, and FMD. A Bayesian meta-analysis was conducted to provide a comprehensive account of the effect of isoflavone-containing soy products on EF, as measured by FMD. A total of 17 RCTs were selected as having sufficient data for study inclusion. The overall mean absolute change in FMD (95% Bayesian CI) for isoflavone-containing soy product interventions was 1.15% (-0.52, 2.75). When the effects of separate interventions were considered, the treatment effect for isolated isoflavones was 1.98% (0.07, 3.97) compared to 0.72% (-1.39, 2.90) for isoflavone-containing soy protein. The models were not improved when considering study-specific effects such as cuff measurement location, prescribed dietary modification, and impaired baseline FMD. CONCLUSIONS: Cumulative evidence from the RCTs included in this meta-analysis indicates that exposure to soy isoflavones can modestly, but significantly, improve EF as measured by FMD. Therefore, exposure to isoflavone supplements may beneficially influence vascular health.

Simultaneous screening and determination of 18 illegal adulterants in herbal medicines and health foods for male sexual potency by ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry.

An ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry method was developed and validated to simultaneously screen, confirm, and determine 18 illegal adulterants in herbal medicines and health foods for male sexual potency. The separation was achieved on a Shim-Pack XR-ODS II column (2.0 × 100 mm, 2.2 µm) with acetonitrile and aqueous solution (12 mmol/L ammonium formate, 0.01% acetic acid) as mobile phase at a flow rate of 0.4 mL/min with a gradient elution. The column temperature was maintained at 40°C and the run time was within 18 min. The 18 illegal adulterants were detected in electrospray ionization positive mode by multiple-reaction monitoring. All the calibration curves showed good linearity with correlation coefficient (r) higher than 0.996 within the tested concentration ranges. The extraction recoveries and relative recoveries were in the range of 79.5-114% and 82.0-120%, respectively. The RSD of repeatability and intermediate precision was all less than 18% and the accuracy was in the range of 81.7-118%. The intra-day and inter-day stability was in the range of 86.8-110%. The validated method was successfully applied to screen, confirm, and determine 16 samples. Nine products were confirmed to contain illegal adulterants and the contents of adulterants were related to the therapeutic dosages.

Quantitation of transdermal tadalafil in human skin by reversed-phase high-performance liquid chromatography.

A reliable and sensitive HPLC method was developed for the quantitation of tadalafil transdermal permeation through human skin. An RP column with UV detection at 290 nm was used for chromatographic separation at ambient temperature. The mobile phase was acetonitrile-water containing 20 mM pH 7 phosphate buffer (35/65, v/v) with a flow rate of 1.0 mL/min. The LOQ achieved was 1 ng/mL, and the calibration curve showed good linearity over the concentration range of 5-2000 ng/mL for tadalafil, with a determination coefficient (R2) of 0.998. The RSD values of intraday and interday analyses were all within 7%. Parameters of validation proved the precision of the method; this validated method was applied for the determination of tadalafil in transdermal permeation and drug deposition in human skin studies.

The hospital pharmacist: an important contributor to improved patient safety in the hospital.

Injectable drugs are high-risk products and their reconstitution in hospital wards is a potential source of errors. Thus, in order to secure the reconstitution process and thereby improve safety, the pharmacy department of Lausanne University Hospital is focusing on developing ready-to-use forms (CIVAS). These preparations are compounded in controlled clean rooms and are analyzed prior to release. In the intensive care unit, amiodarone 12.5 mg/mL in glucose 5% is one of the high-risk preparations, which has led the pharmacy to develop a ready-to-use solution. To this end, a one-year stability study was initiated, and the preliminary results (after six months) are illustrated here. A stability-indicating HPLC method was developed and validated for monitoring the concentration of amiodarone. Batches were stored at 5 °C and 30 °C, which were analyzed immediately after preparation, after one, two, four and six months of storage. The pH and osmolality values were monitored at the respective time intervals. It was observed that after six months, all the results were within specifications. However, the pH values started to decrease after two months when amiodarone was stored at 30 °C. After six months, a degradation peak appeared on the chromatogram of these solutions, which suggested that amiodarone is more stable at 5 °C. The preliminary results obtained in this study indicated that injectable amiodarone solutions are stable for six months under refrigerated storage conditions. The study is ongoing.

In depth analysis on the carbohydrate-binding properties of a vasorelaxant lectin from Dioclea lasiophylla Mart Ex. Benth seeds.

Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose-specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high-mannose N-glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors.Communicated by Ramaswamy H. Sarma.

Comparative study of Puerariae lobatae and Puerariae thomsonii by HPLC-diode array detection-flow injection-chemiluminescence coupled with HPLC-electrospray ionization-MS.

An on-line HPLC-diode array detection-flow injection chemiluminescence (HPLC-DAD-FICL) method was applied to estimate the difference of Puerariae lobatae and Puerariae thomsonii. Their chemical and active profiles could be obtained by HPLC-DAD-FICL in one run. Seventeen compounds in two species were tentatively identified by HPLC-electrospray ionization-MS (HPLC-ESI-MS) method. The main antioxidants were rapidly screened by active fingerprints coupled with MS data. Similarity and Hierarchical clustering analysis (HCA) were used to distinguish different samples. The results suggested that the chemical fingerprints of 16 batches of samples were similar by similarity evaluation, while HCA could discriminate the two species. The active fingerprints of Puerariae lobatae and Puerariae thomsonii were significantly different. More antioxidants were found in Puerariae lobatae than in Puerariae thomsonii. Main antioxidants, including 3'-hydroxypuerarin, genistein 8-C-glycoside-xyloside, puerarin, 6″-O-xylosylpuerarin, mirificin and daidzein in two species, may be reasonable markers for the discrimination of the two species. The integrated fingerprint based on the chemical and active characteristics may provide an objective quality evaluation for Puerariae lobatae and Puerariae thomsonii.