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UPLC-MS/MS quantification of quercetin in plasma and urine following parenteral administration
Igor A. Zupanets ORCID: orcid.org/0000-0003-1253-92171,
Yuri V. Pidpruzhnykov1,2,
Valerii E. Sabko2,
Natalia P. Bezugla1 &
Sergii K. Shebeko1
Clinical Phytoscience volume 5, Article number: 11 (2019) Cite this article
Study of pharmacokinetics of quercetin (Qu) and its derivatives after infusion is required in order to better understand mechanisms of therapeutic action of the formulation and development the strategic approaches to treat the underlying disease and pathologically similar syndromes. A pharmacokinetic study of Qu products is a complex analytical problem.
We developed and validated a new method for quantification of Qu and its metabolites in human plasma and urine following intravenous administration of Qu formulation. The method is based on the solid-phase extraction with the help of Oasis® HLB cartridges and ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) quantification. The calibration curves plotted for concentration range of 25–3000 ng/ml for Qu and isorhamnetin in blood plasma and of 100–8000 ng/ml for the same substances in urine were best described by a quadratic function. Qu conjugates underwent preliminary hydrolysis by the mixture of enzymes of sulphatase and β-glucuronidase. The antioxidant L-cysteine mixed with ascorbic acid was used for stabilization of Qu and its metabolites during handling of samples and chromatographic runs with mass detection. The method validation as well as the stability study results confirmed that the developed method meets the established requirements (selectivity, lower limit of quantification, accuracy, precision, recovery).
The main pharmacokinetic parameters of Qu and its metabolites were determined in blood plasma and urine after intravenous administration of Corvitin® (parenteral formulation combining Qu and polyvinylpyrrolidone at a ratio of 1:9) in 12 healthy volunteers.
We showed that the method development for Qu quantification in biological matrixes is valuable and reliable.
Quercetin [3,3′,4′,5,7-pentahydroxyflavone] is a natural bioflavonoid. Oral medical Qu products and dietary supplements containing Qu are used in medical practice, and their pharmacokinetic studies are described [1]. Qu parenteral formulations currently are not widely used, which, most probably, is related to poor solubility of Qu. The original medical Qu formulation Corvitin® manufactured by SIC Borshchahivskiy Chemical-Pharmaceutical Plant CJSC (Ukraine) is a combination of Qu with polyvinylpyrrolidone at a ratio of 1:9. The formulation is produced as a porous lyophilized powder of 500 mg in a sterile vial and administered by intravenous infusion in physiological saline solution. Corvitin® is used as a cardioprotector in therapy of acute coronary circulation failure and myocardial infarction, in therapy and prevention of reperfusion syndrome in surgery of patients with obliterating atherosclerosis of abdominal aorta and peripheral arteries. Pharmacokinetics study of Qu and its derivatives after infusion is required in order to better understand mechanisms of therapeutic action of the formulation and development the strategic approaches to treat the underlying disease and pathologically similar syndromes.
A pharmacokinetic study of Qu products is a complex analytical problem. Qu is an easily oxidized substance, especially in neutral and alkaline media, thus, it is necessary to use effective antioxidants for preparing samples and performing further analysis. Moreover, Qu is rapidly metabolized in the body to methyl derivatives, various glucuronic and sulphate conjugates and their various combinations [2,3,4,5].
Qu and its metabolites are characterised by pronounced pharmacological activity [6,7,8,9,10], thus, in pharmacokinetic studies it is necessary to determine both free Qu and its metabolites. For quantitative determination of conjugated Qu in the form of glucuronide and sulphate derivatives, the method of enzymatic deconjugation is preferred [11]. At this point it is necessary to take into account that a part of Qu is metabolised to the form of methyl derivatives, and especially 3′-О-methylquercetin (isorhamnetin, Ir), 4′-О-methylquercetin (tamarixetin) and their conjugates [12]. That is why during a bioavailability study of Qu products it is required to quantify the deconjugated methyl derivatives of Qu, which is difficult in vitro because of reverse conversion to Qu by fermentation. A pharmacokinetic study of three doses of an oral Qu product, where it was assayed in healthy volunteers' blood plasma by using HPLC with a fluorescent radiation detector is described in [13], and meanwhile both sulphate and glucuronide conjugates of Qu, and its methyl metabolites and their conjugates were determined.
The use of HPLC–MS/MS to determine Qu and its conjugates in blood plasma and urine after oral administration is described in [1]. Bioavailability of Qu and rutin after oral and parenteral administration in rats was studied in [14]. Both pharmacokinetics of free Qu and its conjugates, but not methyl derivatives of Qu and their conjugates, were determined in these two studies.
The first study of Qu pharmacokinetics after intravenous administration at a dose of 100 mg with ethanol vehicle was performed in 1975 [15], however, selective methods (HPLC) for quantitative determination of Qu were not used. According with [16], Qu concentrations in blood plasma and urine after intravenous administration of Qu dissolved in water-dimethylsulfoxide (1:1) mixture was determined by HPLC using UV-detection, and at that point only free Qu (non-conjugated) was assayed. In this study, the solution of Qu in the mixture of water-dimethylsulfoxide was obtained immediately before administration, i.e. 'ex tempore'. Dimethylsulfoxide affects not only the solubility of Qu, but also its bioavailability, and administration of Qu products in the form of water-dimethylsulfoxide solution results in adverse reactions in patients [16], and that, in all likelihood, was the reason for not introducing the investigated drug to market after the study.
In order to carry out the scheduled study, the method of ultra performance liquid chromatography (UPLC) with tandem MS/MS detection was used. This article describes the development and validation of a UPLC–MS/MS method for quantitative determination of Qu and its metabolites in blood plasma and urine of healthy volunteers. We believe that we are the first ones to perform a pharmacokinetic study of the commercial Qu formulation for intravenous administration currently used in medical practice.
Reagents, materials and solvents
Quercetin dihydrate (100.0% purity) and internal standard (IS) fisetin (99.0% purity) were obtained from Sigma, isorhamnetin (99.6% purity) – from Fluka, powder of the mixture of enzymes of sulphatase and β-glucuronidase (source - Helix pomatia), type Н-1 (AS = 20,560 units/g; AG = 2,701,900 units/g) was obtained from Sigma, Corvitin®, lyophilized powder for injection, 0,5 g in vials was provided by SIC Borshchahivskiy Chemical-Pharmaceutical Plant CJSC (Ukraine), batch # 1280408.
Methanol and acetonitrile (AcN) both 99.9% of HPLC-grade were obtained from LAB-SCAN, formic acid 98–100%, puriss. p.a. from Riedel-de-Haen.
All other chemicals were of analytical grade.
Water was purified by Elix-35 (Millipore, USA) with final purification using Milli-Q Gradient (Millipore, USA).
Oasis® HLB 96 well cartridges, 10 mg packing, grain size - 30 μm (Waters, Milford, MA, USA) also were used.
Drug-free human plasma from healthy volunteers was provided by JCC Biofarma, Ukraine, each batch was accompanied by a certificate of analysis and was stored at − 30 °C prior to use.
Chromatography was performed in gradient mode. Solvent А: AcN – water – formic acid (10: 90: 0.2, v/v/v), solvent В: AcN – formic acid (100: 0.2, v/v).
Instrumentation and methods
The LC–MS/MS analyses were performed using a Waters Acquity UPLC® system with a Waters ACQUITY UPLC® BEH С18 columns (50 mm × 2.1 mm, 1.7 μm) (both Milford, MA, USA) in combination with a Quattro Micro API triple quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray interface. MassLynx 4.1 software was used for instruments control and data processing.
Chromatography was performed at 40 °C at a flow rate of 0.4 ml/min with a total run time of 1.4 min. The start mobile phase consisted of 78% solvent A and 22% solvent B and the linear gradient run over 1 min up to 46% A, 54% B. Then composition was set immediately at 100% B and held constant 0.15 min, followed back to 22% B. The autosampler was kept at 5 °C and 40 μl samples were injected.
Data acquisition was performed in the positive ion mode. The capillary voltage was set at 2000 V, the source temperature was set at 100 °C and the desolvation temperature was set at 500 °C. The desolvation gas flow (nitrogen, 99.5%) was set at 750 L/Hr, the collision gas pressure (argon, 99.99%) was set at 3.5 μbar.
Detection of the ions was performed in the multiple reaction monitoring mode (MRM), monitoring the ion combinations of m/z 302.8 ➔ 152.7 amu for Qu, 316.8 ➔ 152.7 amu for Ir and 286.9 ➔ 136.7 amu for fisetin (Fs). The collision energy was set at 34, 40, 30 V and the cone voltage was set at 45, 40, 45 V for Qu, Ir and Fs respectively. These MRM-method parameters were determined by using Autotune option.
Preparation of standard and QC solutions
A reference standards of Qu or Fs (about 5 mg) or Ir (about 2.5 mg) were accurately weighed, put into a 10 ml volumetric flask, dissolved in ethanol containing 0.01% of butylhydroxytoluene in an ultrasonic bath while heating up to 50 °C (stock solution). Shelf life of Qu and Ir solutions was 3 months, for Fs solution shelf life was at least 22 days when kept at + 2 to + 7 °C.
The solution of the Qu and Ir mixture for calibration solutions were prepared by mixing and appropriate dilution of stock solutions by using water-AcN (3:1) mixture containing 0.005% ascorbic acid and 0.2% L-cysteine as solvent. Meanwhile Qu and Ir mixture solutions were obtained with the following concentrations of each analyte: 500; 1000; 1500; 2000; 3000; 5000; 10,000; 15,000; 20,000; 40,000; 60,000 ng/ml. An Fs solution (subsequently was used as IS) with a concentration of 8000 ng/ml was prepared from the Fs stock solution by diluting with the abovementioned solvent. The solutions were kept at a temperature + 2 to + 7 °C.
The calibration solutions and QC solutions based on the respective blank plasma or urine were prepared by adding 23 ml of the antioxidant solution (aqueous solution containing 10% ascorbic acid, 4% L-cysteine and 2% formic acid), 20 μl of respective solution of the Qu and Ir mixture and 40 μl of the IS solution to 400 μl of plasma or 100 μl of urine mixed with 300 μl of sodium-citrate buffer solution рН 5.0 in Eppendorf tubes (1.5 ml). The solutions were vortexed and then the samples were prepared as described in Section 'Preparation of biological samples', beginning with adding 800 μl of cooled 0.5 М solution of H3РО4, containing 1% ascorbic acid.
As a result, the calibration solutions containing Qu and Ir analytes in concentrations of 25; 50; 100; 250; 500; 1000; 2000; 3000 ng/ml and QC solutions of the same analytes in concentrations of 75; 150; 750; 2000 ng/ml, calculated with reference to the content in plasma, as well as the calibration solutions containing Qu and Ir analytes in concentrations of 100; 200; 400; 1000; 2000; 4000; 8000 ng/ml and QC solutions of the same analytes in concentrations of 300; 3000; 6000 ng/ml calculated with the reference to the content in urine, were obtained. The Fs IS concentration in all the solutions based on plasma was 800 ng/ml, and in those based on the urine was 3200 ng/ml, with the reference to a corresponding biological matrix.
Preparation of biological samples
Two equal aliquots were sampled from fresh thawed biological samples; one of them was subjected to enzymatic deconjugation; at the same time, the second aliquot was kept in a freezer at a temperature no higher than - 35 °C. 30 min before the end of enzymatic deconjugation, the frozen aliquots were thawed at room temperature. All the biological samples for analytical run, calibration and QC solutions were simultaneously treated.
In order to analyse routine plasma samples, two 400 μl aliquots were sampled; to analyse urine samples, two 100 μl aliquots were sampled; those were placed into Eppendorf tubes (1.5 ml). 300 μl of sodium-citrate buffer solution рН 5.0 were added to the urine samples and solutions were vortexed. 23 ml of the abovementioned solution of antioxidants and 40 μl of the respective IS solution were added to all the solutions. The solutions were vortexed.
To each test tube with a sample for enzymatic deconjugation the 10 μl of 5% solution of enzyme F H-1 in sodium-citrate buffer solution рН 5.0 were added, and the solution was vortexed. Incubation was performed in a water bath at (+ 37 ± 1) °C for 17 ± 0.1 h. Immediately after incubation 800 μl of cooled to 0 to + 4 °C 0.5 М solution of H3РО4, containing 1% of ascorbic acid was added to these samples, as well as to the second part of the samples, which were kept in a freezer during the incubation and thawed before examination, and the solutions were vortexed.
The tubes with the samples were centrifuged at 28600 g for 10 min (the centrifuge was pre-cooled to 10 °C). The content of each tube was transferred by an automatic pipette to a respective cell of an Oasis® HLB cartridge prewashed with 500 μl of 0.5 M solution of H3PO4 and, by creating "slight" vacuum, the solution was passed through the sorbent at a rate of about 0.2 ml/min. 500 μl of 5% solution of methanol in 0.5 M H3PO4 was added with the help of the automatic pipette to the same cells, the cells were kept for 1 min and drained under "slight" vacuum. Then 200 μl of 50% solution of methanol in 0.5 M H3PO4 was added to the same cells by the automatic pipette, the cells were kept for 1 min and drained under vacuum till complete exsiccation of the sorbent. In the apparatus for vacuum filtration, the collection tray for washing solutions was replaced with a collector dish. Into each used cell of the Oasis® HLB cartridge, 300 μl of methanol was poured, and they were kept for 1 min and then eluted into the collector dish, creating "slight" vacuum. The elution procedure was repeated one more time under the above conditions. To each used cell of the collector dish, 800 μl of water solutions of ascorbic acid, L-cysteine and formic acid with concentrations of 0.005, 0.002, and 0.2%, respectively, were added. The solutions in the cells were stirred by pipetting, and the air-tightly closed dish was taken for recording chromatograms.
The validation of the developed method for determination of Qu and Ir, as well as its sulphate and glucuronide conjugates in blood plasma and urine was performed in accordance with [17]. The validation covered the following parameters: selectivity, lower limit of quantification, calibration curve, within-run and between-run precision and accuracy, recovery, dilution procedure of biological samples of blood plasma, and stability.
In order to determine the selectivity, we used three different batches of respective blank biological matrices added with Qu and Ir in amounts corresponding to the LLOQ (25 ng/ml calculated with the reference to the content in plasma or 100 ng/ml calculated with the reference to the content in urine), and IS at concentrations that were mentioned above in Section 'Preparation of standard and QC solutions'.
In order to determine the lower limit of quantification (LLOQ), we prepared 5 different solutions based on respective blank biological matrices added with Qu and Ir in amounts corresponding to the LLOQ and Fs IS, obtaining Fs concentrations of 800 ng/ml in plasma, and 3200 ng/ml in urine. The chromatograms of the obtained solutions, as well as solutions of blank plasma and urine, prepared as described above, were recorded and the peak areas in the respective chromatograms were compared.
In order to determine parameters of the calibration curve, while determining analytes in plasma and urine, respectively 8 and 7 calibration solutions with concentrations specified in Section 'Preparation of standard and QC solutions' were used.
In order to assess the parameters Precision and Accuracy five QC-solutions of each concentration were prepared; meanwhile four concentrations in plasma and three concentrations in urine were used (see Section 'Preparation of standard and QC solutions'). The experiment was carried out on three separate occasions. Within-run and between-run precisions were calculated by using the formula %R.S.D. = (S.D./M)(100), where M is the mean of experimentally determined concentrations and S.D. is the standard deviation of M. Accuracy was calculated using the formula %RE = |(E − T)(100/T)|, where E is an experimentally determined concentration and T is a theoretical concentration.
Recovered Qu and Ir were assessed at three concentration levels for plasma and at four concentration levels for urine. The analyte and IS concentrations of the replicate QC samples (n = 3) are given in Section 'Preparation of standard and QC solutions'. The peak areas of extracted samples with the blank biological matrices extracts fortified with the drug post extraction were compared.
The validity of the fivefold dilution procedure for the model biological samples with blank plasma was assessed by Precision and Accuracy parameters [17]. Three different concentrations of Qu and Ir (about 2500, 5000 and 10,000 ng/ml for each analyte in plasma) were evaluated by analyzing five aliquots of each concentration.
Using three and four concentration levels, each type of stability specific for bioanalytical methods was studied [17]: short-term stability, freeze-thaw stability, bench top stability, long-term stability. The analytes and IS concentrations of the replicate QC samples (n = 3) are given in Section 'Preparation of standard and QC solutions'. All the samples for stability studies were prepared on the basis of blank biological matrix spiked with respective amounts of the analytes in a mixture of water–AcN (3:1), containing 0.005% ascorbic acid and 0.2% L-cysteine. All samples were analyzed immediately after preparation and after the applied storage conditions. The short-term stability was examined following sample defrosting and 4-h incubation at room temperature. The freeze-thaw stability was examined after three freeze-thaw cycles.
The long-term stability was determined by analyzing three samples at each concentration level stored at − 70 °C for 108 days. The stability of the samples processed in autosampler was examined after 18 h. Stability of the stock analytes solutions was assessed after 3 month storage and of the stock IS solution – for 22 days when kept at temperature of + 2 to + 7 °C.
During the development of the method, we investigated the time required to sufficiently complete enzymatic deconjugation of glucuronide and sulphate derivatives of Qu. Meanwhile, the completeness and stability of formed aglycones were evaluated using biosamples obtained from the subjects loaded with a dose of an active substance. An enzyme solution was added to two routine samples collected 2 h after the administration of the formulation, and they were incubated at 37 °C for 17 h, 20 h and 23 h. The further preparation of samples and their analysis were carried out in accordance with the method which was described above.
Application for pharmacokinetic study
The pharmacokinetic study of injectable medicinal product Corvitin® was carried out (March–April, 2009) in accordance with the Declaration of Helsinki, GCP and GLP requirements. The trial was registered in the Ukrainian national registry (Resolution of State Pharmacological Center of Ministry of Healh of Ukraine #741/2.2.1–5 from 10.02.2009). Before the study the sponsor monitoring on GCP requirements was held, also the State Expert Center of Ministry of Health of Ukraine audits are conducted on regular basis (The audit before the trial was conducted in November, 2008). The protocol and associated informed consent forms were reviewed and approved by The Ethical Committee and the informed consent forms were signed by the volunteers prior to the study initiation. Twelve healthy volunteers (aged 18–45) years of both sexes were enrolled in the study. In order to carry out the study, each volunteer was given the formulation (combination of Qu and polyvinylpyrrolidone at the ratio of 1:9 in form of porous lyophilized powder of 500 mg), which was diluted with 50 ml of sterile physiological solution 2 h before administration, by using infusion pumps ensuring the same speed of intravenous injection. The dose was calculated individually on the basis of a body surface area of volunteers at a rate of 250 mg of the formulation per square meter of body surface. Thus, administered doses of the product were 365 to 528 mg (37 to 53 ml of the solution).
Thirteen blood samples were drawn from each subject during the study, 5.5 ml of blood at each study time point. The blood sampling was conducted according to the following schedule: 0 h (pre-dose), 15, 30 and 45 min, and 1, 1.5, 2, 4, 5, 6, 8, 12 and 24 h post-dose.
The samples were put into 6 ml plastic vacuum tubes pre-treated with sodium heparin and centrifuged within 3 min after collection. The centrifugation was conducted in the temperature-stabilized rotary chamber at 10 °C for 5 to 6 min at 4000 g. The obtained plasma was acidified (1 ml of plasma was added with 0.05 ml of 0.25 М solution of acetic acid). The plasma was divided into two aliquots (the first one was for the analysis, the second one was for the back-up) but the volume was not less than 1.2 mL for each aliquot, which were transferred into the pre-labeled cryotubes. The plasma was kept at a temperature below − 70 °C at the study site and at the bioanalytical laboratory as well. The time period between blood collection and plasma transfer into − 70 °C freezer did not exceed 15 min.
The sampling of urine was performed as follows. The volunteers were given a labelled container for urine collection for each period of time (0–3 h, 3–6 h, 6–9 h, 9–12 h, 12–16 h, 16–24 h). The urine collection was done 'on volunteer's demand'. Before receiving an aliquot, a container was kept at temperature + 4 °С. The volume of total urine obtained over the specified period was measured by using a graduated cylinder. Then the urine was mixed, and two 5 ml aliquots were taken as soon as possible (the first aliquot was for the analysis, the second one was for the back-up), then they were immediately frozen and kept at a temperature below − 70 °C.
From the clinical site to the bioanalytical laboratory, the frozen samples were transferred in secondary containers put in cryoboxes filled with "dry ice" (solid carbon dioxide).
The developed method was used for quantification of total Qu (QuTot), obtained after enzymatic hydrolysis, unconjugated (non-hydrolyzed) Qu (Qu2), conjugated Qu1 = QuTot – Qu2, and methyl metabolites of Qu with reference to Ir: total one (IrTot), obtained after enzymatic hydrolysis, unconjugated Ir (Ir2) and conjugated Ir1 = IrTot – Ir2 in biological matrixes. Additionally, we determined the amounts of total bioavailable Qu, i.e. QuSum = QuTot + 0.95567*IrTot in order to assess bioavailability of Qu from the formulation of Corvitin®. 0.95567 is a conversion factor of Ir to Qu, taking into account the difference between their molecular masses.
The pharmacokinetic profiles were obtained for every volunteer. The findings of Cmax and Tmax were obtained by applying the common statistics.
The data analysis of pharmacokinetic parameters for Qu in the blood plasma was performed with the help Win-Nonlin professional software version 5.2 (Pharsight, Mountain View, CA, USA) using non-compartmental approach to the model assessment of NCA Model 202 (continuous infusion). The infusion time 15 min was taken into account. Area under the plasma concentration curve (AUC0–24) values (t being the time of the last plasma concentration measured) were calculated by the linear trapezoidal rule.
The AUC from 0 to infinity (AUC0–∞) was calculated by the software. The first-order rate constant Kel was assessed by linear regression of time vs log of the concentration. Terminal half-life (T1/2) was also calculated.
The pharmacokinetic data on determination of Qu in the urine were treated by using the model assessment of NCA Model 212 of the same software.
Using the least-squares method and log-transformed data of excretion rate (R) of the substance in the urine, the elimination constant Kel.(R) and half-life T1/2(R) were assessed. The excretion rate of the test substance in the urine was calculated for each of n time periods (time of the beginning of a period – Tn-1, time of the end of a period – Tn) by the formula: R = C*V/(Tn - Tn-1), where С is a concentration of analyte in the urine, in ng/ml, V is a volume of urine in the specified time period in ml. Also the weight (М) in μg of eliminated Qu within this time period in the urine was calculated: M (μg) = С × V/1000.
It should be noted that according to the study protocol, the first time period of sampling urine was stated from 0 to 3 h. However, the infusion of the formulation continued as per protocol for 15 min (0.25 h). Thus, the actual first sampling period was from 0.25 h to 3 h, which was taken into account in the further calculations (the mean of the first period was 1.63 h). For plotting graphs of concentration vs time for Qu in urine, as well as the excretion rate of Qu in urine, midpoints of urine sampling periods were used.
The other pharmacokinetic parameters: Rmax, ТRmax, AURC0—t, AURC0–∞, cumulative excretion of the substance in the urine Ае, and mass of Qu excreted in the urine were assessed also.
We used fisetin (Fs) as IS for this study. Fs (3,3′,4′,7-tetrahydroxyflavone) differs from Qu by one hydroxyl group, which is missing in position 5. It has similar physical and chemical properties, chromatographic behaviour of the analytes, and is absent in the human plasma at the endogenous level. Fs was successfully used earlier as an IS for HPLC MS/MS determination of Qu and its sulphate and glucuronide conjugates [1]. The structural formulae of the specified substances are shown in Fig. 1.
Chemical structures of quercetin, its metabolite isorhamnetin and fisetin
A gradient: methanol-water solution of trifluoroacetic acid was used for chromatographic separation of glucuronides of Qu in paper [3]. The gradient based on acetonitrile-water solution of formic acid was used for chromatographic separation of Qu metabolites in the paper [5]. We used a similar to the latter gradient for chromatographic separation because a gradient elution mode demonstrated better results than an isocratic mode. The chromatographic conditions and parameters of detection are given in Section 'Instrumentation and methods'.
The time of data registration was 1.4 min. A chromatographic run continued for 1.9 min, including the time of sampling. The short time of recording chromatograms allowed injection of each test sample twice; the result was calculated as a mean of two parallel injections. The UPLC chromatography allowed significant reduction of time for recording chromatograms comparing with traditional НРLC.
The chromatograms of blank plasma, blank urine and LLOQ standards for plasma and urine are shown in Fig. 2.
Chromatograms: (a) blank plasma (Qu, Ir and IS free); (c) blank plasma spiked with 25 ng/ml Qu (LLOQ) and 25 ng/ml Ir (LLOQ); (b) blank urine (Qu, Ir and IS free); (d) blank urine spiked with 100 ng/ml Qu (LLOQ) and 100 ng/ml Ir (LLOQ)
Qu can be easily oxidized during preparation of samples, storage of the solutions, and MS-detection as mentioned in Section 'Introduction'.
To prevent oxidation of Qu we tried various concentrations of ascorbic acid, which was added to Qu and Ir solutions. Increasing concentrations of ascorbic acid, however, did not give any positive effect to stabilize of Qu. At the same time using L-cysteine mixed with ascorbic acid as antioxidant led to the stabilization of Qu and Ir both during handling of samples and chromatographic runs with mass detection. Higher efficiency of L-cysteine as antioxidant can be explained by greater negative reduction potential of the cysteine/cystine system then in the ascorbic acid/dehydroascorbic acid pair [18]. Antioxidant concentrations used at each step of handling of samples are given in Section 'Preparation of standard and QC solutions'. According to our data, the effective method proposed to stabilize of Qu and Ir in solutions was used for the first time.
We used a procedure of solid phase extraction by using Oasis® HLB cartridge after pre-dilution of samples with 0.5 М solution of phosphoric acid [19] to improve the separation of the analytes.
As mentioned in Section 'Introduction' investigation of pharmacokinetics of Qu products requires determination of both free Qu and its metabolites – methylated derivatives and sulphate and glucuronic conjugates.
In order to assay total glucuronide and sulphate metabolites they were enzymatically deconjugated.
Since methylated Qu derivatives are resistant to enzymes, calibration curves were built with Ir standards for quantification of this kind of compounds.
It was found that enzymatic deconjugation of Qu and methylquercetin derivatives lasted for 17 h and this time interval was used in all the further experiments for deconjugation.
Results of method validation
It was established that LLOQ of Qu and Ir were 25 ng/ml in blood plasma and 100 ng/ml in urine for each analyte. At that the signal/noise ratios in plasma were: for Qu > 8.2, for Ir > 7.6; in urine: for Qu > 45.4, for Ir > 100.4. These results meet the acceptance criteria of ≥5 [17]. Qu and Ir concentrations at the LLOQ level were determined at accuracy (RE < 14%) and precision (RSD < 17%), which meet the established criteria [17] for both parameters (≤20%).
The method selectivity was demonstrated by using 3 different batches of respective blank biological matrices spiked with Qu and Ir at LLOQ amounts, and with IS Fs with appropriate concentration. The ratios of peak areas in the chromatograms of solutions of blank biological matrix and solutions for determining selectivity were the following: for plasma samples: Qu < 0.161, Ir < 0.149; for urine samples: Qu < 0.259, Ir < 0.224, which meet the criteria established by our laboratory (< 0.5).
The calibration curves for plasma and urine are shown in Fig. 3. The results of blank and zero samples were not taken into consideration when constructing the calibration function. The most adequately all the calibration curves are described by using a quadratic model with the weighting factor of 1/X (i.e., 1/concentration).
Calibration curves for Qu (a, c) and Ir (b, d) in plasma (a, b) and urine (c, d)
The values of coefficients of the regression equation of the calibration curve Y = a∙X2 + b∙X + c that were determined during the method validation of assay of Qu and Ir in plasma and urine are given in Table. 1.
Table 1 The coefficients of the regression equation of calibration curve (Y = a∙X2 + b∙X + c) and coefficients of determination
The table shows that the coefficients of determination (R2) were greater than 0.990 in all the determinations. The back calculated concentrations of the calibration standards complied with the acceptance criteria [17].
When assessing the parameters Precision and Accuracy for quantification of Qu and Ir in plasma, it was established that the R.S.D. (both intra- and inter-run) for Qu was not greater than 14.5%, and for Ir – 11.6% at the value of accuracy for Qu and Ir not greater than 6.8 and 9.7%, respectively. It was established that R.S.D. (both intra- and inter-run) for Qu was not greater than 9.4%, and for Ir – 6.9% at the value of accuracy for Qu and Ir not greater than 7.5 and 6.5%, respectively for quantification of Qu and Ir in urine.
The recovery values of Qu and Ir from plasma were of 61.8–65.5% and of 73.5–76.9%, respectively. The recovery values of Qu and Ir from urine were of 68.5–73.3% and of 75.1–77.4%, respectively.
The level of expected concentrations after intravenous administration of Qu was a priori rather complicated to establish. The procedure of fivefold dilution of biosamples with blank plasma was validated in order to prepare a routine analysis. Validation was performed according to Precision and Accuracy parameters. The obtained results of assessment of these parameters were as follows: R.S.D. for both analytes was not greater than 6.3%, RE for Qu was less than 14.4, and for Ir < 14.8%, which confirmed the validity of the dilution procedure [17].
The stability study was performed using model biosamples containing Qu and Ir in respective blank biological matrix. It was established that analytes are stable after undergoing three freeze-thaw cycles, as well as after defrosting the samples and keeping them at room temperature for 4 h. Additionally, it was shown that analytes are stable when the abovementioned biosamples were stored at the temperature below minus 70 °C for 108 days. Storage time of the real biosamples did not exceed 103 days.
The stability of solutions in an autosampler for 18 h was also demonstrated.
The mean RE values for all the concentration levels and above listed stabilities tested were not greater than 15%, i.e. they met the established acceptance criteria [17]. The stability of the stock solutions of analytes for 3 months, and IS solutions for 22 days that were stored at the temperature of + 2 to + 7 oС was also demonstrated with RE < 3.7%.
Pharmacokinetic study
Figure 4 shows the mean plasma concentration/time profiles of free Qu and conjugated metabolites of Qu, free Ir and conjugated metabolites of Ir, and summarised: free Qu and Ir and conjugated metabolites of Qu and Ir.
Mean plasma concentration - time profile following intravenous administration of 250 mg/m2 of human body surface area. a - free Qu and conjugated metabolites of Qu, b - free Ir and conjugated metabolites of Ir, c – sum of free and conjugated metabolites of Qu and Ir (+ S.D.)
The basic pharmacokinetic parameters of these curves are shown in Table 2. The table shows that Qu is rapidly metabolized upon administration (the infusion time was 15 min, this was the first time point to sample) to form both glucoronide and sulphate conjugates, and, in a less degree, methyl metabolites and their conjugates. Over this time about 45% of Qu is metabolized; at that, about 28% of Qu forms sulphate and glucuronide metabolites, about 5% turns into metabolites being methylated Qu, and 12% – into sulphate and glucuronide conjugates of methylated metabolites.
Table 2 Mean pharmacokinetic parameters of Qu in plasma for 12 volunteers after intravenous administration of Corvitin® (average dose of anhydrous Qu was 44 mg)
Free Qu was not detected in plasma of any volunteer in 1.5 h after infusion. T1/2 of free Qu and free Ir are 0.13 and 0.18 h, respectively, which indicates a very rapid metabolism of aglycones, which also is confirmed by high elimination constants of these substances (6.08 h− 1 and 4.02 h− 1, respectively). At the same time, the parameter T1/2 for conjugated Qu, conjugated Ir, and total Qu are 6.90, 4.40 and 5.80 h, respectively, indicating their relatively slow excretion from the body (elimination constants of these compounds are 0.11, 0.19 and 0.13, respectively). The highest value of Cmax was observed for the total Qu at Tmax = 0.25 h, i.e. immediately after infusion that is natural for injectable drugs.
When determining Qu and its methyl metabolites in terms of Ir in urine, it was found that free Qu and Ir in urine were absent at all time points. This is natural, taking into account the rapid metabolism of these compounds to the sulphate form and glucuronide conjugates. Figure 5 shows the elimination rate/time curves for conjugated Qu, conjugated Ir, and total Qu. Table 3 demonstrates the basic elimination pharmacokinetic parameters of these substances in urine. For the points where the volume of urine samples was equal to zero and which were not end points, the mean elimination rate equal to the mean elimination rate at the previous and following time points was taken. The cumulative weight of total Qu eliminated in urine over 24 h was 4.57 μg, which is about 10.2% of the infused Qu.
Mean rate of Qu excretion in urine – time profiles following intravenous administration of a dose with reference to 250 mg/m2 of human body surface area for conjugated metabolites of Qu, conjugated metabolites of Ir and for total conjugated metabolites of Qu and Ir
Table 3 Mean pharmacokinetic parameters of Qu in urine for 12 volunteers after intravenous administration of Corvitin® (average dose of anhydrous Qu was 44 mg)
Thus, the obtained data on the pharmacokinetics of Corvitin represent the full characteristic behaviour of Qu and its metabolites in the human blood beginning from its intravenous administration.
We developed a new quantification method for quercetin and its metabolites in plasma and urine by using L-cysteine as an antioxidant. The method is based on the solid-phase extraction of quercetin and products of its enzymatic hydrolysis followed by the analysis of an extract by the UPLC-MS/MS method. The method was successfully applied in the pharmacokinetic study of the parenteral formulation of quercetin used in medical practice.
Wang L, Morris ME. Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine. J Chromatogr B. 2005;821:194–201.
Day AJ, Mellon F, Barron D, Sarrazin G, Morgan MRA, Williamson G. Human metabolism of dietary flavonoids: identification of plasma metabolites of quercetin. Free Radic Res. 2001;35:941–52.
Wittig J, Herderich M, Graefe EU, Veit M. Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry. J Chromatogr B. 2001;753:237–43.
Hong YJ, Mitchell AE. Metabolic profiling of flavonol metabolites in human urine by liquid chromatography and tandem mass spectrometry. J Agric Food Chem. 2004;52:6794–801.
Manach C, Morand C, Crespy V, Demigne C, Texier O, al e. Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett. 1998;426:331–6.
Justino GC, Santos MR, Canario S, Borges C, Florencio MH, al e. Plasma quercetin metabolites: structure–antioxidant activity relationships. Arch Biochem Biophys. 2004;432:109–21.
Larson AJ, Symons JD, Jalili T. Quercetin: a treatment for hypertension? – a review of efficacy and mechanisms. Pharmaceuticals. 2010;3:237–50.
Manach C, Williamson G, Morand C, Scalbert A, Remesy C. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr. 2005;81:230S–42S.
Lodi F, Jimenez R, Moreno L, Kroon PA, Needs PW, al e. Glucuronidated and sulfated metabolites of the flavonoid quercetin prevent endothelial dysfunction but lack direct vasorelaxant effects in rat aorta. Atherosclerosis. 2009;204:34–9.
Boesch-Saadatmandi C, Loboda A, Wagner AE, Stachurska A, Jozkowicz A, al e. Effect of quercetin and its metabolites isorhamnetin and quercetin-3-glucuronide on inflammatory gene expression: role of miR-155. J Nutr Biochem. 2011;22:293–9.
Manach C, Morand C, Demigne C, Texier O, Regerat F, Remesy C, al e. Bioavailability of rutin and quercetin in rats. FEBS Lett. 1997;409:12–6.
Mullen W, Boitier A, Stewart AJ, Crozier A. Flavonoid metabolites in human plasma and urine after the consumption of red onions: analysis by liquid chromatography with photodiode array and full scan tandem mass spectrometric detection. J Chromatogr A. 2004;1058:163–8.
Egert S, Wolffram S, Bosy-Westphal A, Boesch-Saadatmandi C, Wagner AE, al e. Daily quercetin supplementation dose-dependently increases plasma quercetin concentrations in healthy humans. J Nutr. 2008;138:1615–21.
Yang CY, Hsiu SL, Wen KC, Lin SP, Tsai SY, al e. Bioavailability and metabolic pharmacokinetics of Rutin and quercetin in rats. J Food Drug Anal. 2005;13:244–50.
Gugler R, Leschik M, Dengler HJ. Disposition of quercetin in man after single oral and intravenous dose. Eur J Clin Pharmacol. 1975;9:229–34.
Ferry DR, Smith A, Malkhandi J, Fyfe DW, deTakats PG, al e. Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and evidence for in Vivo tyrosine kinase inhibition. Clin. Cancer Res. 1996;2:659–68.
U.S. Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER), Centre for Veterinary Medicine (CVM) (May 2001). Guidance for Industry: Bioanalytical Method Validation.
Burton K (1961) Biochemists' Handbook. E and FN Spon Ltd., London, 90.
Ishii K, Furuta T, Kasuya Y. High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection. J Chromatogr B. 2003;794:49–56.
We thank to SIC 'Borshchahivskiy Chemical-Pharmaceutical Plant CJSC', Ukraine for funding the study.
SIC 'Borshchahivskiy Chemical-Pharmaceutical Plant CJSC', Ukraine.
National University of Pharmacy, 27 Pushkinska Street, Kharkiv, 61057, Ukraine
Igor A. Zupanets
, Yuri V. Pidpruzhnykov
, Natalia P. Bezugla
& Sergii K. Shebeko
Bioanalytical laboratory Clinfarm, 3 Shevchenko Street, Irpin, Kiev Oblast, 08200, Ukraine
Yuri V. Pidpruzhnykov
& Valerii E. Sabko
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All authors read and approved the final manuscript.
Correspondence to Igor A. Zupanets.
The study was approved by the Central Ethics Committee of Ministry of Health of Ukraine (resolution #5.12–194/KE from 04.03.2099) and by the Local Ethics Committee (resolution #01/19 from 10.03.2009) with minor amendments to the trial protocol (resolution #02/19 from 27.03.2009).
Zupanets, I.A., Pidpruzhnykov, Y.V., Sabko, V.E. et al. UPLC-MS/MS quantification of quercetin in plasma and urine following parenteral administration. Clin Phytosci 5, 11 (2019) doi:10.1186/s40816-019-0107-1
UPLC-MS/MS


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The need for vision
I am personally grateful that this raw and painful week is coming to a close. And that it will end with some precious time for us to take refuge in silence, song and community. Wherever you are, I encourage you to shut off the feed and find a way to nourish your soul. While none of this racist violence is new, it is nonetheless exhausting to encounter face to face (or on Facebook as the case may be).
For those who were able to be at The People's Baptist Church last night (and for those of you that did childcare so others could be), thank you. It was a very powerful experience to position ourselves in multiracial, multi-faith community and together denounce white supremacy, antisemitism, islamophobia, sexism and homophobia. More so, it was powerful to meet new people, sing out and receive the powerful insights of the Pastor Isaiah Banks.
If I were bold enough to title his sermon I would call it, "We need to have vision."* Which felt like the fulfillment of Divine grace in the room, knowing two things. First, that he did not know he would be speaking. And secondly, that this week's Torah portion, Re'eh, can literally be translated as "See!"
His call to all of us, whether we were in that room or not, is that we need to behold a vision of transformation and liberation. That peace and justice are not just practices of action but also radical imagination. That we must have the courage to see ourselves and each other more clearly. That we must be able to envision a world free of white supremacy.
I have been sitting with this teaching all day, trying to understand how to make it real and tangible. In this moment, what feels most true is that liberation is a creative act of the imagination. Let me explain.
It is said about our creation story that for each of the plants and animals, they are created min b'mino, with many kinds. Meaning that there are species of plants and animals. But not so with humans. We are all one. We are the only living thing created by the Holy One that is not named as min b'mino. There are not essential "kinds" of people. Said another way. The rabbis ask, Why was it so important that the first human being was created singularly, that there was only one Adam? To which they reply: So that no person could ever say to another, "My ancestors are better than yours!"
A belief in the oneness of all things and the dignity of every human being is at the core of my own theology embodied in these teachings. Creation itself, and our creativity is a product of imagination. How do we see the world? How do we see each other? Pastor Banks made it clear last night, we must have a vision of a world where no human being is better than another. Where all living are one and holy.
It is my prayer as we move into this Shabbat, that each of you find sanctuary. That you connect to your essential wholeness. That you allow yourself to imagine a world that is whole and just and entirely loving. And from that place, may we all emerge better able to envision how to get there.
I offer you all the closing words of a beloved poem by Marge Piercy that I shared last night.
Let us lift each other on our shoulders and carry each other along.
Let holiness move in us.
Let us pay attention to its small voice,
Let us see the light in others and honor that light.
Remember the dead who paid our way here dearly, dearly
and remember the unborn for whom we build our houses.
Praise the light that shines before us, through us, after us, Amen.
*The story the Pastor told from the New Testament was about a blind beggar asking for vision. I want to name the ableist nature of this story and reclaim vision here in the way I think the Pastor intended it - as metaphor - rather than the ability to see, which I do not believe is a prerequisite for our liberation.

redpajama_set_name: RedPajamaCommonCrawl


Memory Tapes gives us a full stream of his upcoming third album, Grace/Confusion. The album comes featuring Analog synths coupled with dance beats. Grace/Confusion drops on December 4 via Carpark Records, preview below.


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Posted on December 24, 2015 December 24, 2015 Written by MaudComments Off on New Interview with the LA Times
New Interview with the LA Times
LA Times : When historians look back at this decade of moviemaking their first instinct may be to crown Alejandro Iñárritu's "Birdman" as the definitive statement on the culture of celebrity of this era. If they are smart, they'll present another film that is arguably more spot on in how it portrays the movie industry's inherent narcissism: Olivier Assayas' "Clouds of Sils Maria." Critics have been simmering on the drama since it debuted at the 2014 Cannes Film Festival and surprised many by remembering to shower one of its stars, Kristen Stewart, with honors over the past month.
On one Sunday this month, Stewart won the supporting actress prize from both the New York Film Critics Circle and the Boston Society of Film Critics. A few days later, she was the official runner-up for the same honor from the Los Angeles Film Critics Assn. These were unforeseen kudos even when you consider that Stewart became the first American woman to win a César Award, France's equivalent of the Academy Award, for her performance in "Clouds" in February. Because it was released in the United States this calendar year, Assayas' drama was eligible for most critic honors this awards season, but Stewart says she wasn't expecting any of the adoration she's receiving.
"For that movie to get any sort of attention it's a huge surprise considering it's not the most commercial movie and especially considering everyone else who was considered," Stewart says. "It blew my head off, to be honest."
"Clouds" finds Stewart playing Valentine, the young assistant to a famous Hollywood actress, Maria (Juliette Binoche), who is trying to come to terms with her place in the global celebrity microcosm after agreeing to star in the revival of a play that launched her career two decades earlier. Valentine and Maria's relationship straddles the line between professional and personal, allowing Stewart to showcase some of the most nuanced work of her career.
"Initially, I looked at it as something that moved me. It was very emotional, but it was kind of interesting banter between two women," Stewart says. "It was an interesting role for me to play considering my position in the business and my awareness of how potentially superficial and strange it can be and how funny it would be to hear certain words come out of my mouth."
Binoche and Stewart formed a strong bond while shooting the picture that still resonates. One of the more memorable moments involved a scene where Valentine and Marie jumped into a freezing river. Assayas wanted the actors to figure out the scene together, and Stewart jokes that her reaction was the "epitome of a young American."
"It's literally glacial runoff this … lake, and I looked up and before I knew it [Binoche] was completely … naked. And I'm like, 'I'm not doing that.' And then we just jumped in," Stewart recalls. "We had such a big adrenaline rush, and we'd both been through so much already. All the air was out of our lungs. We couldn't breathe. And that sort of exemplified that experience."
Frankly, Stewart earning recognition for her work in "Clouds" now isn't that surprising. She's coming off a year and a half during which she also impressed in the Sundance drama "Camp X-Ray" and Oscar-winning tearjerker "Still Alice." Of course, those sorts of accolades are the furthest things from her mind. Stewart always attempted to find projects that pushed her during her breaks during the four years she was filming and promoting the "Twilight" franchise but says she "feels lucky" so many interesting films presented themselves afterward.
"Now people know I'm a weirdo and open to doing stuff down the line that isn't completely commercial," Stewart notes. "It's a strange feeling, because I've always liked things that were a bit more diagonal in terms of how people consume them."
Stewart shot five films in 2015, including Kelly Reichardt's "Certain Women," which will debut at the Sundance Film Festival next month, Woody Allen's untitled 2016 picture, Ang Lee's "Billy Lynn's Long Halftime Walk" and "Personal Shopper," a drastically different reunion with Assayas that she describes as "entirely existential and lonely, really poetic and really, really cool." Or, in even simpler terms, "super French."
"The person I'm playing now is a nut bag, so it's kind of working in my favor," Stewart says of her nonstop 12 months. "I'm lucky it's OK for me to look a little tired right now."
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'Billy Lynn's Long Halftime Walk' to be released Nov 11, 2016
NEW Interview with the Los Angeles Times + NEW Portrait
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I rarely use makeup or put anything on my skin unless it's necessary. But our skin goes with our age, too. That's why we have to take care of our body and choose a healthy lifestyle. Being a female, it's important to maintain our skin, especially the face. Wrinkles start to appear as we age.
So while looking for a product that I could safely use, I saw Pond's Age Miracle Wrinkle Corrector Night Cream. It is contained in a small white and red box and costs Php99.00 only. Given it's brand, this is something affordable and it's available in major drugstores and supermarket.
The product claims that it has the most advanced anti-aging technology called RP-C24 recovery complex. It releases retinol (Vitamin A1) which works non-stop to help reduce wrinkles from appearing. I opened the box and it contains a small bottle containg 10 grams of pink unscented cream. I must say it's like their usual cold cream and I'm liking it already.
I applied this cream every night after cleansing my face. So I just apply dots of this cream on my face and massage it using my ring and middle fingers only in circular motion.
The package comes with warnings that says to store away from direct sunlight and heat, which is almost a rule to all products. Another warning is to avoid using it on the eyelids. If ever contact occurs, you need to rinse the eyes with water immediately. And I'm careful when massaging the cream near the eye area. The third warning says that you may experience mild redness or flaking, which are signs of exfoliation. It claims that this indicates that the formula is working. But if this effect persists or cause discomfort, stop using it and consult a doctor.
So far, after two weeks of using it, I haven't experienced the redness or flaking. I don't know when the effects will really show. I've been taking care of my health, eating more vegetables, and taking my daily doses of vitamins C and E, so that's another factor to consider. When I attended a seminar recently, someone guessed my age ten years younger than my actual. Oh well, ever since I've reached my 30's I've said to myself that my age will be a guessing game and I'm aging gracefully.
I'm going to rate this product as 5 (I'm loving it, personally).


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SIGHTING, UK: Another big cat spotted in Britain as 'Labrador sized' feline is filmed prowling outside a Cotswolds pub Inn
A "Labrador-sized" cat has been filmed prowling a field near a Cotswolds pub.
The video was taken by pub landlord Ed Simpson after he spotted the beast out of a first floor window of The Fox Inn, in the village of Broadwell.
It follows police and the RSPCA confirming a hamlet in Cornwall where pets have vanished is being stalked by a large panther-like animal.
Ed said: "We were upstairs and saw something out of the window and said 'look at that!'
"There was this big cat. It paraded across the field quite happily then it jumped over a wall and vanished."
Posted by Liv McCarthy at 18:20 No comments:
SIGHTING, UK: Big cat fears grow in South West as gym boss spots large feline in Devon
Britain's big cat outbreak could be wider than first feared - after a man claims he has spotted one twice in a second county.
Steve Swatton, 63, claims he has also seen a large fawn cat and a large black cat on the outskirts of his home city of Plympton, Devon.
He spoke out after reading about the recent sightings and incidents in Harrowbarrow and Callington in Cornwall
CARL WRITES: 31/3/19 - Dunmow, Essex
Read the original story here.
"I think it is entirely possible that Tim Davis observed a genuine big cat behind his village home in Bardfield End Green in Dunmow. I presume the gym located above his garage provided a high observational vantage point allowing him to first spot the mystery animal from a window, and then he subsequently identified it when magnified through his binoculars. It's just a shame he didn't have time to also get a photograph.
Essex has a rich history of reports of both panther and puma-like animals; some of which are highly credible, and this area In the Uttlesford district would potentially provide a wild roaming big cat with many of the necessary ecological resources needed to survive.
I can completely understand the factory worker becoming wary of leaving his house after dark, especially if he feels convinced he has seen a large predator. However, if this truly was a leopard or a puma, the chances of it spending prolonged periods of time near human habitation is unlikely to say the least, It would be far more likely stalking nearby woodlands that border open farmland. Therefore, I doubt Mr Davies has much to worry about as both these species are highly secretive and shy animals; especially when they are surviving in low numbers. In most cases you wouldn't even know if one was residing in the area.
From a distance, and during a brief observation, it might be difficult to differentiate between a melanistic leopard and a darkly pigmented puma; therefore it's probably wise not to suggest a specific identity based on pigmentation until further corroborating evidence is acquired.
I would suggest Mr Davis purchase a reasonably priced motion activated night vision camera and secretly position it facing the trail where the beast was last seen, and to leave the camera untouched for at least a month for a better chance of results.
The animal, whatever it is, will have left trace evidence of its movements in the area, therefore a thorough and prolonged search for spoor, faecal remains, and snagged hairs could prove positive.
As for ghostly black dog traditions, I doubt the local legends of Black Shuck have much to do with modern reports of big cats in Essex.
Going by the shape and dimensions of the muddy footprint in the inserted photograph, and the presence of what appears to be claw marks, all indicate this is probably the track of a Canid most likely a large domestic dog.
Further evidence is required such as clear video footage or preferably some sort of biological sample for analysis would be ideal.
Anyone living in this area should remain vigilant. However, human attacks are unlikely to occur as both pumas and leopards have large ranges and would be very unlikely to stay in the same area for long. In Britain these animals appear to avoid human activity at all costs and would therefore be extremely unlikely to cause any problems. The large numbers of wild deer species, rabbits, and ground dwelling game birds, along with the occasional farmers lamb or sheep, would be ample for large predatory Felids to survive without bothering risking hunting human beings."
NEWSLINK: Tiger attack sends keeper to the hospital, US zoo pledges 'nothing will happen to the tiger'
A keeper at a zoo in Topeka, Kansas, has been attacked by one of the facility's tigers.
The city confirmed the attack on Saturday and noted that the keeper was alert when she was transported to the hospital.
City spokeswoman Molly Hadfield told The Washington Post that the incident happened at 9:15 Saturday morning while the zookeeper was in the same space as Sanjiv, the zoo's male Sumatran tiger.
The zookeeper is currently in stable condition and the tigers are being kept in holding, she said.
ARTICLE: Big cats on the prowl
Last week another 'big cat' sighting was reported in Cornwall. A Labrador was attacked by a 'panther like' creature, with suspicious footprints also found in the mud.
We will be examining the possibility of big cats roaming the British countryside. Whilst this is often laughed out of contention, there is a body of evidence that's fascinating if nothing else.
Over the last decade Police Forces across the UK logged over five hundred Big Cat sightings.
The South West has been a hotspot for sightings since the nineties but since then there has been an increase in sightings in Norfolk, Suffolk and the Forest of Dean.
VIDEO: Rare White Lion Cubs Born at Crimean Safari Park
Two rare white lion cubs have been born in a Crimean safari park. The male and female cubs arrived in the Taygan lion park near Belogorsk several days ago but haven't been named yet. Their mother, Milady, became a mother for the second time, after giving birth last year as well. There are about 300 white lions in the world – many of them in captivity. The Taygan park, the first lion park in Europe, was established by enthusiast Oleg Zubkov in 2006 and now houses 60 big cats.
CARL WRITES: 10/4/19 - Broadwell, Cotswolds
"A so-called "Labrador-sized" cat filmed prowling a field near a Cotswold pub by Landlord Ed Simpson from the first floor window of his village pub in Broadwell, has been causing quite a stir on social media.
Admittedly, it is very difficult to see what species has been recorded in the grainy video, as the animal situated in the background is somewhat distorted by foliage in the foreground, blocking a good clear view of the beast.
However, when comparing the dimensions of the mystery cat with the tree in the video, one must admit the creature does look larger than one might expect from a domestic moggie. It is often said that the morphology and movements of domestic cats observed or filmed at great distances can easily be mistaken for those of genuine big cats such as leopards, but it must also be said the same applies vice versa. If specific features are unclear, it must also be possible (however unlikely) that black leopards observed or filmed from such distances, might well be mistaken for large moggies, especially when there are no comparable objects in the video.
Unfortunately, the animal in this video is just too blurry to specifically identify and is probably nothing more than a reasonably large domestic cat. It's gracile movements also imply this to be likely true, however, it is indeed impossible to be certain from the video alone. It would be a good idea for someone to measure the approximate dimensions of the tree in the video and measure the distance between the tree, the animal and the observation point.
Ed Simpson said:
"We were upstairs and saw something out of the window and said 'look at that'!"
Bar manager Kerry Hogg, 56, first spotted the animal out of the first floor window while she was in the room talking with Ed and his wife. Kerry agreed that the animal was approximately the size of a Labrador, but clearly moved in the manner of a cat.
There have been other, equally unreliable reports coming in over the last few weeks. Steve Swatton, 63, claims to have observed a large black and white cat (sounds like a domestic) on the outskirts of Plymouth, Devon and Becky Abrey filmed a black felid in Praa Sands near Penzance, which, based on the details of the image, was undoubtedly that of a black domestic cat.
Are there big cats still stalking these areas of the country? Possibly!
Are these examples indicative of their existence?
Probably not!"
ARTICLE: Tracking big cats on safari through India's 'Tiger State' of Madhya Pradesh
I'm swaddled in a mess of blankets to fight the pre-dawn chill as I bump down the dusty dirt roads of India's Kanha National Park in an open-air safari jeep. My guide, Prabhat Verma of PureQuest Adventures, is brimming with optimism. But it's my third trip past the park's green gates, and I've yet to lock eyes with the creature I flew halfway around the world to meet. You can go to Africa for cheetahs or lions, but for Bengal tigers, your best bet is the wildlife reserves at the heart of the Indian subcontinent in the so-called "Tiger State" of Madhya Pradesh. Somewhere up ahead are nearly 100 of these regal cats, who prowl 932 square kilometres of pristine Indian wilderness. Even so, there are no guarantees I'll see one.
NEWSLINK: How the lion has been weakened: More than 100 years of hunting has seen big cats lose their strength and put their survival at risk
Still hailed as the king of the jungle, the lion species has seen an alarming rapid decline in their genetic fitness from their ancestors who lived more than 100 years ago. Following a study conducted by researchers at the Zoological Society of London, the impact of hunting the big cats has seen a worrying decline in their genetic status. According to the research, the decline in their physical strength over the course of a century is likely to have been impacted by hunting of the wild animals in Africa.
SIGHTING, UK: Dunmow man won't go out after dark after he spotted 'a panther' strolling near home
An Essex resident has claimed to have spotted a 'massive panther' just yards from his home. 50-year-old Tim Davies, from Bardfield End Green in Dunmow, was looking out of his garage window when he saw a "big black thing" strolling across the open field behind his house. You might think it's quite rare to see a panther roaming the fields of Essex, but there have been big cat sightings across the county for decades. The so-called 'Beast of Essex' is thought to look like a black panther or puma.
NEWSLINK: Male tiger to be relocated from Ranthambore to Sariska
After a gap of six years, the Rajasthan forest department is preparing to shift a big cat from Ranthambore Tiger Reserve (RTR) to Sariska Tiger Reserve (STR) soon. This time a male tiger will be relocated. Skewed sex ratio has been hampering the repopulation of big cats in Sariska. The National Tiger Conservation Authority (NTCA) had approved the relocation of a tiger in June 2017, with an aim to increase the population.
PHOTOS: Hikers stunned by rare sighting of a caracal on Table Mountain
Now this is why you strap your hiking shoes on and make the journey up Table Mountain: Hikers were treated to an amazing sight this week when they came across an extremely rare caracal which had made it to the top.
The wild cats are dotted around the area between the mountain and Lion's Head. They are anti-social creatures and they do not interact with humans. So catching one on camera is a pretty rare event. The magic of Table Mountain Petra van der Berg and Lars Hurenkamp were the lucky people behind the lens this time. They crossed paths with this majestic animal as it slinked between rocks at the summit of the mountain. In the background, the stunning city backdrop and Cape Town Stadium can be spotted – maybe he was trying to catch the Ed Sheeran concert?
NEWSLINK: Man is not leopard's normal prey
With several instances of leopards in human settlements and attacks on humans in recent times, deputy conservator of forests (DCF) West, Shivaji Phule talked with Sumita Sarkar to clear myths about this big cat. Incidents of leopards attacking people are being reported more frequently. What causes that? The natural habitat of leopards is forest. Forest cover has reduced. Whatever encroachment was there has been regularised. There are patches of human settlement, like honeycombs, with little scope for isolated animal habitats.
NEWSLINK: Southern California's Mountain Lions May Soon Go Extinct
Southern California's mountain lions have faced serious threats for some time, but a new study highlights the urgent need to act before we lose these incredible big cats forever. According to wildlife scientists, who just published their findings in the journal "Ecological Applications," two populations of mountain lions in the Santa Monica and Santa Anita Mountains could go extinct — possibly within the next 50 years.
VIDEO: The ex-poachers saving big cats in Russia
In Sailugemsky National Park, the snow leopard population has found an unlikely ally - ex-poachers. Their knowledge of the animal's movements and trap-setting was once used for hunting, but has now been reclaimed to monitor snow leopard populations thanks to a World Wildlife Fund project which began in 2015. Camera traps are used to monitor the snow leopard, which have seen a rise in population.
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We are a friendly group of people who enjoy meeting to share ideas, experiences and gain knowledge about all subjects related to gardens. New members are cordially welcomed.
We meet every first Thursday of the month from 7.30pm to 9.30pm in the Jessop Room at Leasingham Village Hall, unless otherwise stated on the programme. Membership is by subscription of £20 [£30 family] per year; with an additional £1 per evening when there is a speaker; visitors are welcome with a charge of £3 per evening. The group supports the Leasingham Horticultural Show and we are planning visits/walks later in the year.


text:
DIRECTING FILM
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Cold and precise, An Dan-Te is the no-nonsense director of Yoo Seong CRC, a company which specializes in the restructuring and resurrecting of companies on the brink of failure. Sent to Joybuster, a game company on the brink of bankruptcy, Dan-Te takes on the role of CEO and wastes no time in trying to get this company back on its feet. As head of game development at Joybuster, Shin Yeon-Hwa teams up with her new CEO, in an attempt to save their dying company. Tasked with developing a new game which satisfies all of Dan-Te's demands, Yeon-Hwa soon finds herself wavering somewhere between having a nervous breakdown and committing murder. Completely at odds with each other, Yeon-Hwa and Dan-Te never seem to stop fighting; but everyone knows opposites always find a way to attract. Can the poker-faced boss with zero emotions and the hot-tempered game developer really find love when they can't even agree on a way to save their dying company?
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MBN, Dramax,
Sung Hoon, Han Bo-Reum, Danny Ahn, Baro, Kang Byul, Ryu Seung-su, Lee Byung-joon
An Dan-Te
Sung Hoon
Shin Yeon-Hwa
Han Bo-Reum
Park Gil-Woo
Danny Ahn
Kwak Han-Cheol
Bae Ya-Chae
Kang Byul
Jo Tae-Koo
Ryu Seung-su
Yeon-Hwa's father
Lee Byung-joon
Level Up Season
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fuel / lord of the flies / panic / selfish
I live in a country populated by idiots and run by buffoons
by Sean · 31 March 2012
Last year parts of the UK were blighted by riots and looting.
Last week the more feeble-minded of the country's car-driving population started panic-buying petrol and diesel. This was sparked by concerns over a possible strike by lorry drivers, and was exacerbated by remarks made by Francis Maude – a government minister – who advised people should stock up on fuel for their cars. This was stupid and dangerous advice, driven by an idealogical desire to thwart the impact of a potential fuel strike.
Our less-than-impressive Prime Minister, David Cameron, echoed these sentiments.
What happened next was an explosion of the same attitudes and outlook we witnessed in last August's wave of arson and theft – a complete breakdown of perspective and an overwhelming sense of utter selfishness. I'm all right Jack is the attitude du jour.
To begin with there was fear over a shortage of fuel at the pumps over the Easter weekend, brought on by striking tanker drivers. But it soon began to sink in to these fretful idiots, many of whom had queued for hours at petrol stations to fill their cars and their many portable containers, that there would not be a strike-prompted shortage of fuel over Easter. After all, no strike had been called. Added to which, in the event a strike is called, seven days notice would be given prior to the actual strike.
On Thursday 29 March, sales of petrol and diesel were up 172%.
The queuing and panicky behaviour extended into Friday, with long queues, petrol stations running out, tempers fraying and people generally carrying on like confused, frighted children.
Why continue to panic-buy fuel when there is no impending problem?
Perhaps because if you don't go and buy as much as you can there won't be any left when you need it, because other people will have bought it all.
In the wake of last August's riots, David Cameron said: "For me, the root cause of this mindless selfishness is the same thing that I have spoken about for years."
He was right and his home-spun truism works well here too.
Back then I wrote:
He went on, as politicians often do: "It is a complete lack of responsibility in parts of our society. People allowed to feel that the world owes them something, that their rights outweigh their responsibilities and that their actions do not have consequences."
You can read the full post here.
All of this has left me more convinced than ever that there is a bitter streak of unpleasantness that runs through people in this country. We do not stand together. We squabble and complain. We are mistrustful of others. We put our own short-term wants and needs ahead of everything.
The UK gets more like a Lord of the Flies LARP with each passing year.
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Stephen Collins, quite obviously, has a lot on his self-inflicted plate right now. On top of his worldwide spread scandal for being accused of child molestation, he and has long-time wife, Faye Grant, are divorcing. According to TMZ, Collins is demanding a million dollars for Grant supposedly recording his guilty confession during a therapy appointment. Collins' demands this amount because he says that he cannot get any work after TMZ and other news sources have posted videos of his confession.
Is this an unreasonable request from Collins? Going through a divorce where Collins could potentially be convicted for many crimes, can the divorce go through its normal process? What are the legal worries in this divorce? Collins and Grant have a community property that is valued approximately $14million dollars - what will most likely happen to that value throughout the time of their divorce?
We touched base with Kaine Fisher, local attorney and Chairman of the Family Law Department at Rose Law Group, and asked him to weigh in on high profile celebrity divorce settlement.
"It appears Stephen Collins' million-dollar claim is intended to be a damage claim related to defamation-like legal principals. I am interested to know how he and his attorneys came up with the million-dollar figure. What is based on and how did they come up with such specific amount? It feels like the number is a bit arbitrary to me - could be reasonable if his reputation is ruined for good but again seems arbitrary.
Also, in order to prevail on such a claim, my understanding is that there would at least be some discussion about whether the molestation allegations are true. This is dangerous territory if you ask me. Although I have not heard the recording, he apparently admitted it so not real sure I like his chances there. He will also have to prove his wife was the one who leaked the story to TMZ. Not sure how easy that will be either.
Perhaps the most intriguing aspect of Collins' claim is that he will have to testify and present evidence about the molestation allegations in order to pursue his damage claim in Family Court. I am surprised this is something his criminal lawyer is comfortable with. The way I see it either Stephen waives his 5th Amendment right against self-incrimination to pursue his million-dollar claim or he invokes his 5th Amendment right while at the same time then potentially sacrificing his ability to pursue his damage claim. I don't think he can have it both ways.
For advice from Rose Law Group, call 480.505.3936 or visit their site here.


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, né le  à Dax et mort le  dans la même ville, est un joueur français de rugby à XV qui évolue au poste de pilier. Il effectue l'intégralité de sa carrière au sein de l'effectif du club français de l'US Dax.

Biographie
Né le  à Dax, Jean Bachelé fait partie, aux côtés d'André Berilhe et de Léon Berho, de la première ligne dacquoise des « Trois B ». S'il remporte deux fois le challenge Yves du Manoir, son palmarès reste vierge de tout Bouclier de Brennus, disputant deux finales avec l'US Dax en 1956 et 1961.

À l'issue de cette carrière de joueur, « Nono » reste intégré au sein de la famille de l'US Dax et se reconvertit au poste d'entraîneur, tout d'abord en tant qu'éducateur pour l'école de rugby du club, puis en tant qu'entraîneur de l'équipe fanion en tandem avec Claude Dufau pendant la saison 1978-1979. Alors à la tête des équipes en catégorie junior, il est sacré plusieurs fois champion de France, particulièrement avec les cadets.

Il meurt dans la nuit du 5 au .

Palmarès
 Championnat de France de première division :
 Vice-champion (2) : 1956 et 1961.
 Challenge Yves du Manoir :
 Vainqueur (2) : 1957 et 1959.

Notes et références

Notes

Références

Annexes

Bibliographie

Liens externes

Joueur français de rugby à XV
Joueur de l'US Dax
Pilier (rugby à XV)
Naissance en juin 1932
Naissance à Dax
Décès en mars 2015
Décès à 82 ans
Décès à Dax

redpajama_set_name: RedPajamaWikipedia


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Tesla Adds 'Sentry Mode' Safety Feature to Its Electric Cars
By Reuters | Updated: 14 February 2019 15:56 IST
Elon Musk's Tesla on Wednesday launched a safety feature called "sentry mode" for its electric cars, as it attempts to make its vehicles more attractive to buyers.
The feature will be compatible with US Model 3 vehicles, followed by Model S and Model X vehicles that were manufactured after August 2017, the electric carmaker said.
When enabled, the "sentry mode" monitors the environment around an unattended car and uses the vehicle's external cameras to detect potential threats, according to Tesla's blog.
A minimal threat will be detected if anyone leans on the car, triggering a message on the touchscreen and warning that its cameras are recording.
For a more severe threat, like someone breaking a window, the mode activates the car alarm, increases the brightness of the centre display, plays loud music and alerts owners on their Tesla mobile app.
The United States had 773,139 motor vehicles stolen in 2017 - the highest since 2009, according to data from the US Federal Bureau of Investigation.
Last week, Tesla lowered the price of its Model 3 sedan for the second time this year to make its cars more affordable for US buyers. The Palo Alto, California-based company has been cutting costs as it looks to turn in profit this year.
The starting cost of the Model 3 is now about $35,000 (roughly Rs. 25 lakhs), after about $8,000 of credits and fuel savings, Tesla Chief Executive Officer Elon Musk had tweeted.
In a reply to a Twitter user asking about when Tesla would drop the Model 3 base price to $35,000, without credits, Musk said, "We're doing everything we can to get there. It's a super hard grind."
Tesla ended its customer referral incentive plan on February 1 after Musk tweeted that the referral program was "adding too much cost to the cars, especially Model 3".
Further reading: Tesla
MIUI 10 Global Beta ROM Brings Dark Mode to System Apps


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Hello stamping friends! Today I wanted to share a fun fold birthday card with you. This fold is called a "Z-Fold" and it has been around for quite a while. I wouldn't even know who to give credit to for creating it, but thank you to the person who did because it's pretty cool! I've shared the measurements below in case you would like to make one as well.
For my card, I wanted to use some products from the Occasions Catalog that have been a little neglected in my stamp studio. I paired the Picture Perfect Birthday stamp set with the gorgeous Sweet Soirée Specialty DSP. All of the paper in that pack is foiled on one side which makes it extra fancy.
Another item that I think is completely underrated in the Occasions Catalog is the Sweet Soirée Embellishment kit. There are SO many items in the kit, including lots of die cuts (vellum, numbers and some with foil detail), ribbon, washi tape, clothes pins, tassels. I used the floral die cuts on my card. They have gorgeous foiled accents on them and (at first glance kind) of look like you stamped, colored and fussy cut them out. So easy and so beautiful!
Marina Mist base: 5-1/2" x 8-1/2", scored at 2-1/8" and 4-1/4"
One thing I am ALL about right now is decorating the insides of my cards. I love adding a little more detail other than just the sentiment. Since this fold really allows you to showcase the inside of the card as well, I used quite a few of the stamps from the Picture Perfect Birthday stamp set along with the silver washi tape from the Sweet Soirée Embellishment Kit. Did I mention how much I like that kit?
I liked this card so much I decided to use it for one of my team swaps this month! The only thing I really changed was how I tied the ribbon. I found the bow made the card a little tough to mail and the simple knot corrected that. Plus it was a lot easier to knot the ribbon when producing 10 of these cards. I actually made 2 different cards this month for our team swap. I'll be sharing the other one really soon!
I hope you enjoyed my card today! All of the products I used to create the card are listed below. You can click on any of those products to purchase them at my online Stampin' Up! store. I'm probably going to pick up another one of those Sweet Soirée Embellishment Kits myself. :) Also, don't forget that this is the LAST week for Sale-A-Bration where you can earn free product with your qualifying order. Click HERE for more details. Thank you so much for stopping by!


This edition DVD includes the first time shared information about the crystal consciousness grids that surround the Earth, mankind's evolution out of Atlantis, their development globally by many cultures over the last 13,000 years, and the amazing science that has been discovered around the pyramids and temples worldwide that has even been presented to the United Nations.
Also presented is the true story of one particular grid, often called THE UNITY CONSCIOUSNESS GRID, and how it has finally come to birth since February 2008. The birth of this new living consciousness grid is for certain the most important story since Atlantis, and yet hardly anyone on Earth is aware that it has happened or what it means for humanity.
One thing is clear; the outcome of this global consciousness transition is going to affect every single last person on this planet. If you know what has now become history, your heart can rest knowing that what is to follow in our lives is one of the greatest gifts that Source has given to mankind.
Drunvalo Melchizedek is the author of four books including The Ancient Secrets of the Flower of Life, Volumes I and II, Living in the Heart and his newest one, Serpent of Light. These books have been published in 29 languages and reach out to over one hundred countries throughout the world.
In 1993 he founded the Flower of Life Workshops with now over 300 trained and certified facilitators teaching in over sixty countries.
Drunvalo is a world traveler and has given workshops, seminars and lectures on sacred geometry, human energy fields, spirituality, meditation and living in the heart in 45 countries.
Drunvalo majored in physics and minored in mathematics at the University of California at Berkeley.


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W /
Webster, Paul /
Statement of Allegations: In the Matter of Paul Webster
STATEMENT OF ALLEGATIONS
(Subsections 127(1) and 127(10) of the Securities Act, RSO 1990 c S.5)
1. Staff of the Enforcement Branch (Staff) of the Ontario Securities Commission (the Commission) elect to proceed using the expedited procedure for inter-jurisdictional proceedings as set out in Rule 11(3) of the Commission's Rules of Procedure.
A. ORDER SOUGHT
2. Staff request that the Commission make the following inter-jurisdictional enforcement order, pursuant to paragraphs 4 and 5 of subsection 127(10) of the Ontario Securities Act, RSO 1990 c S.5 (the Act):
(a) against Paul Webster (Webster) that:
until the later of July 24, 2030, or the date on which the amount in paragraph 2(3) of the Order of the British Columbia Securities Commission (BCSC) dated July 24, 2018 (the BCSC Order) has been paid:
i. pursuant to paragraphs 2 and 2.1 of subsection 127(1) of the Act, trading in any securities or derivatives, or acquisition of any securities, by Webster cease, except that he may trade in securities or derivatives through a registrant in one non-registered account, one RRSP account and one TFSA account held in his own name, if he first provides copies of the BCSC Order and the order of the Commission in this proceeding, if granted, to the registrant;
ii. pursuant to paragraph 3 of subsection 127(1) of the Act, any exemptions contained in Ontario securities law do not apply to Webster;
iii. pursuant to paragraphs 7, 8.1 and 8.3 of subsection 127(1) of the Act, Webster resign any positions that he holds as a director or officer of any issuer or registrant;
iv. pursuant to paragraphs 8, 8.2 and 8.4 of subsection 127(1) of the Act, Webster be prohibited from becoming or acting as a director or officer of any issuer or registrant, except that Webster may become or act as an officer or director of a company of which he owns all the shares; and
v. pursuant to paragraph 8.5 of subsection 127(1) of the Act, Webster be prohibited from becoming or acting as a registrant or promoter;
(b) such other order or orders as the Commission considers appropriate.
B. FACTS
Staff make the following allegations of fact:
3. On July 24, 2018, Webster entered into the Settlement Agreement with the BCSC.
4. Pursuant to the Settlement Agreement, Webster admitted to breaching British Columbia securities legislation, and agreed to be made subject to sanctions, conditions, restrictions or requirements within the province of British Columbia.
5. Webster is subject to an order of the BCSC, which imposes sanctions, conditions, restrictions or requirements upon him.
(i) The BCSC Proceedings
Agreed Statement of Facts
6. In the Settlement Agreement, Webster agreed with the following facts:
(a) Webster is a resident of British Columbia. Between July 2011 and April 2014, he was a director and officer or de facto director and officer of Titan-West Explorations Inc. (Titan-West).
(b) In 2012, Titan-West and Webster attempted to mine for gold in the Yukon. Webster relocated his family to the Yukon for this purpose. Based on information provided to him from the people who sold the claims, he understood there was a substantial amount of gold on the property. Titan-West's 2012 financial statements showed gold revenue of $41,307 and an operating loss of $566,050.
(c) Titan-West is dissolved.
(d) Titan-West did not mine for gold in 2013. However, between October 2013 and February 2014, Titan-West and Webster raised capital for Titan-West.
(e) Representatives of Titan-West provided some of the prospective investors promotional material, including a business plan and executive summary that Webster provided to the representatives.
(f) The business plan provided to prospective investors did not disclose the 2012 operating loss of $566,050 on gold revenue of $41,307. Instead, the business plan disclosed an outdated 2012 "pro forma" financial statement with gross revenue of $12 million and net operating income of $6,959,418, which Webster ought to have known would be relied on by the investors.
(g) Four prospective investors who received the business plan invested $172,000.
(h) On February 13, 2014, the Executive Director of the BCSC issued an Order under section 164(1) of the British Columbia Securities Act, RSBC 1996 c 418 (the BC Act), ordering that all trading in the securities of Titan-West cease (Cease Trade Order).
Misrepresentations
(i) Titan-West made misrepresentations to investors by failing to disclose the 2012 operating loss, and by disclosing the 2012 pro forma financial statements, contrary to section 50(1)(d) of the BC Act. As a director and officer of Titan-West, Webster authorized, permitted or acquiesced in Titan-West's misrepresentations.
Breach of Cease Trade Order
(j) After the Cease Trade Order was issued:
1. Titan-West representatives emailed and telephoned prospective investors, soliciting investment in Titan-West; and
2. Titan-West issued two promissory notes for $27,000 to two previous Titan-West investors. The $27,000 comprises part of the $172,000 mentioned above.
(k) The foregoing conduct contravened the Cease Trade Order. As a director and officer of Titan-West, Webster authorized, permitted or acquiesced in Titan-West's contraventions of the Cease Trade Order.
(l) Webster has advised the BCSC's Executive Director that he received legal advice that Titan-West could borrow money despite the Cease Trade Order. As a result, Webster did not think Titan-West was contravening the Cease Trade Order by issuing promissory notes.
(ii) BCSC Settlement and Undertaking
Disgorgement
(m) Webster agreed to an order under section 161(1)(g) of the BC Act to pay $48,000 to the BCSC.
(n) Webster undertook to not directly or indirectly make an application for restoration of Titan-West.
(iii) The BCSC Order
7. The BCSC Order imposed the following sanctions, conditions, restrictions or requirements upon Webster:
(a) under section 161(1)(d)(i) of the BC Act, Webster resign any position he holds as a director or officer of an issuer or registrant;
(b) Webster is prohibited for the later of 12 years from the date of the Settlement Agreement and the date that the amount set out in sub-paragraph (c) below is paid:
1. under section 161(1)(b)(ii) of the BC Act, from trading in or purchasing any securities or exchange contracts, except that he may trade securities through a registrant in one non-registered account, one RRSP account and one TFSA account held in his own name if he first provides a copy of the BCSC Order to the registrant;
2. under section 161(1)(c) of the BC Act, from relying on any of the exemptions set out in the BC Act, the regulations or a decision;
3. under section 161(1)(d)(ii) of the BC Act, from becoming or acting as a director or officer of any issuer or registrant except that Webster may become or act as an officer or director of a company of which he owns all the shares;
4. under section 161(1)(d)(iii) of the BC Act, from becoming or acting as a registrant or promoter;
5. under section 161(1)(d)(iv) of the BC Act, from acting in a management or consultative capacity in connection with activities in the securities market; and
6. under section 161(1)(d)(v) of the BC Act, from engaging in investor relations activities;
(c) Webster pay to the BCSC $48,000 pursuant to section 161(1)(g) of the BC Act.
Consent to Regulatory Orders
8. Webster consented to regulatory orders made by any provincial or territorial securities regulatory authority in Canada containing any or all of the orders set out in paragraph 2 of the Settlement Agreement.
C. JURISDICTION OF THE ONTARIO SECURITIES COMMISSION
9. Pursuant to the Settlement Agreement, Webster agreed to be made subject to sanctions, conditions, restrictions or requirements within the province of British Columbia.
10. Webster is subject to an order of the BCSC imposing sanctions, conditions, restrictions or requirements upon him.
11. Pursuant to paragraphs 4 and 5, respectively, of subsection 127(10) of the Act, an order made by a securities regulatory authority, derivatives regulatory authority or financial regulatory authority, in any jurisdiction, that imposes sanctions, conditions, restrictions or requirements on a person or company, or an agreement with a securities regulatory authority, derivatives regulatory authority or financial regulatory authority, in any jurisdiction, that a person or company is to be made subject to sanctions, conditions, restrictions or requirements may form the basis for an order in the public interest made under subsection 127(1) of the Act.
12. Staff allege that it is in the public interest to make an order against Webster.
13. Staff reserve the right to amend these allegations and to make such further and other allegations as Staff deem fit and the Commission may permit.
DATED at Toronto this 27th day of November, 2018.
Kai Olson
Litigation Counsel
Enforcement Branch
Email: mailto:kolson@osc.gov.on.ca


"Review: True Religion Fall 2017 Preview"
In 2002, Los Angeles based denim brand True Religion emerged onto the fashion scene with its one-of-a-kind collection. Fast forward to 2017, the company has expanded with collections in several mass retailers, high volume merchandise sales, ads in high-end fashion magazines, and international runway shows. The stitch of True Religion's denim has not only garnered a lot of fame, but the attention of some of the most respected designers. I recently attended their fall preview collection at Milk Studios located in the Meatpacking District.
Located on the 8th fl of Milk studios, the event was filled with several media and fashion outlets socializing and previewing the women and men's collection. Guests were served with cocktails and refreshments throughout the evening. The preview line was a small collection featuring bomber jackets, camouflage denimwear, construction/distress denim, and contemporary sneakers. For product inquiry or store locations, visit www.truereligion.com.


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Morrison feels at home
metrowebukmetroWednesday 4 Apr 2007 3:15 pm
New Bradford star Glenn Morrison is glad he has settled in well with the Bulls – because opponents are making life on the field far from easy.
The former Parramatta second row has made an immediate impact at the Grattan Stadium to help the Bulls to the top of the engage Super League table.
"It's been great so far," the 30-year-old said of his first two months in Super League. "We've got a good bunch of blokes, a good coach and everyone is positive and enjoying themselves, me included."
He added: "It's made it easier for me moving over here. I've fitted in well and we're winning games, which makes it easier for me, and I've got a couple of tries too.
"We've got good strength across the park and I couldn't really pinpoint one weapon. Everyone's working together as a side and pushing in the right direction.
"I don't think we did too much wrong against Catalans. We weren't on our best game but with the level of competition this year everyone is pretty equal.
"If you just slacken off and one or two players aren't on the money you're going to lose the game. We know we've got a few things to improve but nothing major.
"Leeds are going to be tough. It's a big derby and both sides want to win. We're both at the top and we want to stay there."
Glenn MorrisonSuper League
Snowboarder films himself getting swallowed by avalanche
Home › Sport


Police are currently looking for a missing person.
George Martin, 30, was last seen around 1:00 a.m. on Monday, March 11 in the area of Sandalwood Parkway West and Brisdale Drive.
Martin is described as black, 6'0, 150 pounds, with a medium build, short black hair, black goatee, and brown eyes.
He was last seen wearing blue jeans, a white t-shirt, a brown jacket, a black toque, and running shoes.
"Police and family are concerned for his well-being as he does not have medication that he requires," police say.
Anyone with information on Martin's whereabouts is asked to call police at 905-453-2121, ext. 2233.
UPDATE: Martin has been located in good health.


Beloved children's novel protagonist Tugger Nutts is off on his latest adventure: braving the crowds at PAX West 2016. Inspired by an episode of Qwerpline, this pin by Featherweight is the first Pinny Arcade pin to feature our ruminant friend, but it might not be the last!
This is an official Pinny Arcade pin and thus can be traded for other Pinny Arcade pins, if that's something that interests you. You can look here for the general rules.