Determination of Ochratoxin-A in the brain microdialysates and plasma of awake, freely moving rats using ultra high performance liquid chromatography fluorescence detection method.

In this study, a simple, efficient and rapid Ultra High Performance Liquid Chromatography method with fluorescence detection (UHPLC-FLD) has been developed and validated for the determination of Ochratoxin-A (OTA) in rat brain microdialysates and plasma samples. Six adult male wistar rats were used in the study and a single dose (5 mg/kg b.w.) of OTA was given by intraperitoneal (i.p.) injection. Rat blood and microdialysate samples were collected simultaneously after i.p. injection in awake freely moving rats, over a twelve-hour period. An UHPLC analysis was performed on a Zorbax Eclipse Plus C8 (150 mm × 3.0 mm ID × 1.8 µm particles) column with a mobile phase of acetonitrile:water:phosphoric acid (50:50:0.1, v/v) using a flow rate of 0.6 mL/min. The fluorescence detector was set at 330 nm excitation and 460 nm emission wavelengths. Diflunisal (DIF) was used as an internal standard (IS). OTA and IS were separated within 5 min under these conditions. The method was validated in terms of linearity, precision, accuracy, limit of detection, limit of quantification, and stability. Calibration curves obtained with spiked biological matrices show good linearity with high correlation coefficients. The intra- and inter-day assay variability was <5% for the OTA. The limit of detection and the limit of quantification values were found to be 0.490 ng/mL and 1.48 ng/mL for plasma; 0.0900 ng/mL and 0.270 ng/mL for microdialysate samples, respectively. This method was successfully applied for the monitoring of OTA levels in the rat brain and plasma samples.

A validated capillary electrophoretic method for the determination of indacaterol and its application to a pharmaceutical preparation.

Indacaterol is a new inhaled ultra-long acting ß2-agonist. It has been recently approved in the European Union for the treatment of chronic obstructive pulmonary disease. This paper reports, for the first time, a method for the determination and validation of Indacaterol (IND) using an internal standard in capsules. Capillary electrophoretic separation was performed on an uncoated fused-silica capillary (50 cm effective length, 75 µm i.d.) and background electrolyte composed of 20 mmol L-1 of sodium tetraborate buffer, 15% (v/v) methanol (pH = 10.0) with the application of 20 kV of potential; 10 s at 5 × 103 N m-2 (50 mbar) of injection time; and wavelength of 200 nm and 25 °C of temperature. The linearity was evaluated in the range of 4.90 × 10-6 mol L-1 (2.50 µg mL-1) and 3.94 × 10-5 mol L-1 (20.00 µg mL-1), with R = 0.9993 for inter-day. LOD and LOQ values were 2.18 × 10-8 mol L-1 (0.011 µg mL-1) and 7.25 × 10-8 mol L-1 (0.037 µg mL-1) for inter-day, respectively. The precision values were 0.50-1.06% for intra-day and 2.12% for inter-day as RSD%. The accuracy was tested by the standard addition method with the recovery values being between 98.79 and 99.09 as percentages with RSD% interval of 0.01-0.80. The developed method was validated according to ICH guidelines. Indacaterol was successfully determined in Arcapta® capsule dosage form by the validated CE method with a relative error of 0.28%. The result was within the requirements of the USP 34-NF29. Therefore, the validated method may be used for the determination of Indacaterol in its capsules in quality control laboratories.

A Validated Capillary Electrophoretic Method for the Determination of Olopatadine and Its Application to a Pharmaceutical Preparation of Eye Drops.

A validated rapid and sensitive capillary zone electrophoretic method for the determination of olopatadine hydrochloride (OLO) is described. Optimum conditions were found: 20 mmol/L sodium tetraborate buffer, acetonitrile 15% (v/v), 10 mmol/L NaCl at pH 9.5, with 25 kV of applied potential, injection time of 10 s at 5 × 103 N/m2, at a wavelength of 205 nm, and fixed temperature of 30°C. The calibration curve was linear in the range of 1.13 × 10-5 mol/L (4.22 µg/mL) to 5.65 × 10-5 mol/L (21.12 µg/mL), with R = 0.9995 for interday precision. LOD and LOQ values were 1.58 × 10-6 (0.58 µg/mL) and 4.78 × 10-6 mol/L (1.75 µg/mL), respectively. Precision values were 1.10-1.97% for intraday and 1.41% for interday RSDs. Accuracy was tested by preparing a synthetic mixture whose composition was similar to the pharmaceutical preparation for Patanol. The RSDs of the recovery values (98.2%) were between 0.42 and 0.65% and the amount of OLO found was 1.09 mg/mL. The result was within the requirements of USP 31-NF 26. Therefore, this validated method is suggested for routine analysis for the determination of OLO in laboratories.

Determination of leflunomide in tablets by high performance liquid chromatography.

In the present study, a reverse phase high performance liquid chromatography (HPLC) method was validated and applied for the determination of leflunomide in tablets. Chromatographic separation of leflunomide and oxazepam as an internal standard was carried out on a C(18) column (50 mm, 3 mm i.d.) using a mobile phase, consisting of methanol and water (60:40, v/v), at a flow rate of 0.5 ml min(-1) and UV detection at 260 nm. The retention times for oxazepam and leflunomide were 2.6 and 5.2 min, respectively. The validated quantification range of the method was 2.7 x 10(-6) to 5.5 x 10(-5) M for leflunomide. The results of the developed procedure in tablets were compared with those of UV spectrophotometry to assess active leflunomide content.

Passage of vasoactive intestinal peptide across the blood-brain barrier.

We investigated the ability of vasoactive intestinal peptide (VIP) to cross the blood-brain barrier (BBB), the interface between the peripheral circulation and central nervous system (CNS). VIP labeled with 131I (I-VIP) and injected intravenously into mice was taken up by brain as determined by multiple-time regression analysis. Excess unlabeled VIP was unable to impede the entry of I-VIP, indicating that passage is by nonsaturable transmembrane diffusion. High pressure liquid chromatography (HPLC) showed the radioactivity entering the brain to be intact I-VIP. After intracerebroventricular (i.c.v.) injection, I-VIP was sequestered by brain, slowing its efflux from the CNS. In summary, VIP crosses the BBB unidirectionally from blood to brain by transmembrane diffusion.

Determination of lansoprazole in pharmaceutical capsules by flow injection analysis using UV-detection.

The direct determination of lansoprazole by using a flow injection analysis (FIA) with UV-detection and its application to the pharmaceutical capsules is described, in this study. The best carrier solvent was found to be 0.01 M NaOH and it was determined at optimum conditions such as flow rate of 1 ml min(-1) and wavelength of 292 nm. Examining the repeatability of the method that was found to be 1.72% for intra-day and 2.13% for inter-day precision using the 8.01 x 10(-6) M lansoprazole concentration has validated the method. The linear range of the method was 5.4 x 10(-6) to 5.4 x 10(-5) M. The limit of detection and quantification was found to be 5.8 x 10(-7) and 1.7 x 10(-6) M, respectively. The proposed method was applied to the pharmaceutical capsules and very good results obtained. Thus, the FIA method for the quantification of lansoprazole can be proposed as a cheap, rapid, easy, accurate, and precise method for the routine determination in pharmaceutical preparations.

Determination of Amlodipine in Pharmaceutical Formulations by Differential-Pulse Voltammetry with a Glassy Carbon Electrode.

A differential-pulse voltammetric method was developed for the determination of amlodipine based on the oxidation of the dihydropyridine group on the surface of glassy carbon electrode under stationary and rotating conditions. The experiments were conducted in a supporting electrolyte consisting of 0.2 MKCl, 0.1 Mphosphate buffer, and 10 % (v/v) methanol during investigation of initial potential and pH effects. No adsorption effect was observed on using an initial potential of 0 mVand the supporting electrolyte solution at pH 5.5 under both stationary and rotating conditions. The factor affecting the voltammetric current was diffusional in the range of 200-1000 rpm for rotating, and 2-40 mV s(-1) for stationary conditions up to a concentration of 0.04 mg mL(-1) amlodipine. The limit of detection (LOD) and the limit of quantitative (LOQ) for the rotating and stationary techniques were found to be 0.004 and 0.0072 mg mL(-1) (for S/N = 3.3) and LOQ 0.012 and 0.022 mg mL(-1) (for S/N = 10), respectively. The proposed method was applied to the tablets containing amlodipine and according to the statistical evaluations acceptable results were obtained at the 95 % probability level.

Determination of pravastatin in tablets by capillary electrophoresis.

Pravastatin (PRA) is an inhibitor of HMG-CoA reductase enzyme, which is clinically used as a hypolipidemic agent to reduce cholesterol level. A capillary electrophoretic method for the determination of PRA in pharmaceutical tablet formulations is described. PRA and lansoprazole as an internal standard (IS) were well migrated in the background electrolyte of 10 mM borate buffer (pH 8.5) and 10% acetonitrile using a fused silica capillary. The separation was achieved by applying 27.5 kV, detecting at 200 nm and injecting the sample for 0.5 s and with an average migration time (tm) for PRA and IS of 4.7 and 3.9 min, respectively, at ambient temperature. The results were precise and repeatable for areas of the peaks and peak normalization ratio (PNPRA/PNIS). Linearity was found in the concentration range of 1.56-7.78 x 10(-5) M. Intra-day and Inter-day assays were performed and reliable results were obtained. Limit of detection and limit of quantitation were 8 x 10(-6) and 2.4 x 10(-5) M, respectively. The proposed method was successfully applied for the analysis of PRA in the pharmaceutical tablet formulation. The method proved simple, precise and fast since the analysis can be performed in less than 5 min.

Effects of the vasoactive intestinal peptide on stress-induced mucosal ulcers and modulation of methylation of histamine in gastric tissue of the rats.

Vasoactive intestinal peptide (VIP) prevents stress-induced gastric ulcers, inhibits mast cell degranulation and protects gastric tissue from lipid peroxidation. Histamine has an important role in the development of gastric ulcers and mast cell derived histamine might be essential in this process.

A validated method development for ketoprofen by a flow-injection analysis with UV-detection and its application to pharmaceutical formulations.

A simple, sensitive and fast flow-injection analysis method with UV-detection method was developed for the determination of ketoprofen in pharmaceuticals. The standard and sample solutions were dissolved in a 10% ethanol which was suitable for this study. A flow-rate of 0.6 ml min(-1) was used and the analyte was monitored at 260 nm. Variables such as concentrations, flow rate of reagents and other flow injection parameters were optimized to produce the most sensitive and reproducible results. Linear calibration curves were obtained in the range of 1.6 x 10(-6) and 1.7 x 10(-4) M. Limit of detection and limit of quantification were 1.7 x 10(-6) M (S/N=3.3) and 5.3 x 10(-6) M (S/N=10), respectively. The method was applied successfully to the analysis of ketoprofen pharmaceutical tablets. The recoveries were 102.75% for peak area and 98.42% for peak height. The proposed method is fast, precise, sensitive and easy to use for the determination of ketoprofen in pharmaceuticals.

A rapid determination of patulin using capillary zone electrophoresis and its application to analysis of apple juices.

This study describes a capillary zone electrophoretic method for the determination of patulin. An optimum run buffer was found to be 25 mM of sodium tetraborate and 10% acetonitrile (v/v) at pH 10. Optimum conditions were determined to be: an applied voltage of 25 kV (normal polarity), temperature of 25°C and injection time of 10 s at 50 mbar; the signals of patulin and phenobarbital as internal standard were detected at 276 nm. The method was highly reproducible, with relative standard deviations of 0.02-0.85 for intra-day and 0.04-0.42 for inter-day for standard patulin. Acceptable linearity [y = 0.0020C (µg/L) - 0.0680 (r = 0.9999)] was obtained over a concentration range of 0.25 to 4.99 µg/mL of patulin. The limits of detection and quantification were calculated to be 5.9 × 10(-3) and 1.79 × 10(-2) µg/mL, respectively. Recovery was 68.0%. The proposed method was applied to 21 apple juice samples purchased from different Turkish markets, and two were found to contain higher than the limits of the European Union Directive for patulin.

A sensitive and rapid determination of ranitidine in human plasma by HPLC with fluorescence detection and its application for a pharmacokinetic study.

A novel precolumn derivatization reversed-phase high-performance liquid chromatography method with fluorescence detection is described for the determination of ranitidine in human plasma. The method was based on the reaction of ranitidine with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole forming yellow colored fluorescent product. The separation was achieved on a C(18) column using methanol-water (60:40, v/v) mobile phase. Fluorescence detection was used at the excitation and emission of 458 and 521 nm, respectively. Lisinopril was utilized as an internal standard. The flow rate was 1.2 mL/min. Ranitidine and lisinopril appeared at 3.24 and 2.25 min, respectively. The method was validated for system suitability, precision, accuracy, linearity, limit of detection, limit of quantification, recovery and robustness. Intra- and inter-day precisions of the assays were in the range of 0.01-0.44%. The assay was linear over the concentration range of 50-2000 ng/mL. The mean recovery was determined to be 96.40 ± 0.02%. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of ranitidine.

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine.

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 µm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.

Assessment of phenolic acid content and in vitro antiradical characteristics of hawthorn.

The infusions and extracts obtained from leaves with flowers, fruit peel, and seed from hawthorn (Crataegus monogyna Jacq., Family Rosaceae) were subjected to evaluation as potential sources of antioxidant phytochemicals on the basis of their total content of phenolics, levels of phenolic acids, and in vitro antiradical activity. Total phenolic content of extracts was determined using the modified Folin-Ciocalteau method. Antioxidant activity was determined for phenolic extracts by a method involving the use of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Phenolic acids containing extracts and infusions from hawthorn leaves, fruit peel, and seeds were obtained using different polarity solvents and separated by reverse-phase high-performance liquid chromatography, which enabled improved separation by the use of a C(18) column, an acidic mobile phase, and gradient elusion. The highest total phenolic content (343.54 mg of gallic acid equivalents/g) and the highest DPPH radical scavenging activity as the inhibition percentage (60.36%) were obtained in ethyl acetate extract from hawthorn leaves with flower. Also, the highest phenolic acid content was measured in the extracts of hawthorn leaves with flowers: protocathechuic (108-128 mg/100 g), p-hydroxy benzoic (141-468 mg/100 g), caffeic (137-3,580 mg/100 g), chlorogenic (925-4,637 mg/100 g), ferulic (3,363-3,462 mg/100 g), vanillic (214 mg/100 g), and syringic (126 mg/100 g) acids. The results indicate that hawthorn is a promising plant because of its high antioxidant activity.

Vasoactive intestinal peptide (VIP) treatment of Parkinsonian rats increases thalamic gamma-aminobutyric acid (GABA) levels and alters the release of nerve growth factor (NGF) by mast cells.

The ventral anterior nucleus of the thalamus (VATh) gathers motor information from the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr) of the basal ganglia and projects directly to motor areas of cortex. GPi/SNpr send their tonically active gamma-aminobutyric acid (GABA)ergic outputs to VATh. The abnormal firing patterns of GABAergic neurons in GPi/SNpr lead to motor deficits. In Parkinson's disease, the spontaneous firing pattern of GPi/SNpr neurons is abnormal due to the degeneration of the nigrostriatal dopaminergic pathway. In a previous study, we found that systemically administered vasoactive intestinal peptide (VIP) was effective at reversing the motor deficits (but not the decline in striatal dopamine levels) in a rat model of Parkinson's disease (6-hydroxydopamine (6-OHDA) exposure). In addition to the beneficial effects on the motor response, VIP could also attenuate both neuronal cell death and the characteristic loss of the myelin sheath that is associated with 6-OHDA administration into the rat striatum. VIP was thought to preserve neurons by inducing native brain mast cells to adopt a nondegranulating phenotype that had the ability to secrete numerous neuroprotective substances, such as nerve growth factor (NGF) and heparin. In the present study, the effect of systemically administered VIP (25 ng/kg i.p.) was investigated on GABA levels of the VATh, dopamine/3,4-dihydroxyphenylacetic acid (DOPAC) levels in the corpus striatum, and the NGF, rat mast cell protease II (RMCPII), serotonin, and heparin content of brain mast cells in 6-OHDA-lesioned rats. Extracellular concentrations of GABA, dopamine, and DOPAC were measured by microdialysis and high-performance liquid chromatography. NGF, RMCPII, serotonin, and heparin levels were examined by immunohistochemical staining techniques. A total of 48 young adult Sprague-Dawley rats were used in the study, and these were assigned to one of six groups. Unilateral injection of 6-OHDA, 2 microl (6 mg/microl), was made into the right corpus striatum. VIP-treated animals received 25 ng/kg VIP i.p. at 2-day intervals for a period of 15 days. The present results demonstrated that VIP significantly increased the levels of GABA in the VATh that were reduced by 6-OHDA application and increased the number of NGF-immunoreactive mast cells but did not alter dopamine metabolism. Therefore, the protective effect of VIP on motor function is possibly related to the increased levels of GABA in the VATh, and its neuroprotective actions may be mediated by the release of NGF from brain mast cells.

Time and temperature dependent microbiological and mycotoxin (ochratoxin-A) levels in boza.

This study describes the examination of microbiological tests and the determination of OTA in boza temperature and time dependently. Prior to the analysis, physicochemical properties of the boza samples such as moisture, total acidity as lactic acid, pH, protein amount and viscosity were investigated. The incidence of total aerobic bacteria (TAB), lactic acid bacteria (LAB), coliforms, E.coli, Salmonella, S. aureus, B. cereus, yeast and moulds were examined. E.coli, Salmonella, S. aureus and B. cereus were not found in all boza samples. Initially, Aspergillus fumigatus; Acremonium sp.; Geotrichum candidum and Geotrichum capitatum were identified in the samples. Certain extraction techniques such as direct injection, liquid-liquid and solid phase (SP) were tried for the OTA analysis. The most available way was found to be direct injection among them and the recovery was 70.56%+/-9.80 (13.89 RSD). OTA amounts were determined in all boza samples utilizing an isocratic HPLC analysis with an ODS column. OTA was detected in only one sample as 3.58 microg/kg and this amount is above the limits of European Commission Regulations. Time and temperature-dependent changes were investigated and insignificant variation was observed.

Determination of formoterol by capillary electrophoresis and its application to inhaler capsules.

A capillary electrophoretic (CE) method for the determination of formoterol (FOR) in a pharmaceutical preparation is described. Analysis was made in a background electrolyte consisting of 20 % acetonitrile and 50 mM phosphoric acid at pH 2.5, using fused silica capillary (86 cm x 75 microm ID), 27 kV potential, and detecting at 200 nm. Under these electrophoretic conditions 3, 4-dihydroxybenzylamine used as an internal standard (IS) and FOR showed symmetrical peaks at 6.1 and 8.3 min., respectively. The inter-day and intra-day precision was examined in the concentration range of 2.98 x 10(-6) M to 8.94 x 10(-6) M. Good correlation and accuracy were obtained. Limit of detection, (LOD) and limit of quantitation (LOQ) values were 3.71 x 10(-7) M and 1.11 x 10(-6) M, respectively. The method was applied for the analysis of FOR in pharmaceutical inhaler capsules. The proposed method is reliable, precise, accurate, fast, and cost effective.

A modified HPLC method for the determination of ochratoxin A by fluorescence detection.

A high-performance liquid chromatographic method (HPLC) with fluorescent detector is described for the determination of ochratoxin A (OTA). A mobile phase consisting of acetonitrile:water:acetic acid (99:99:2, v/v/v) was used for the resolution of the compound on a C(18) Hypersil column. The retention time for OTA and diflunisal which was used as an internal standard (IS) were 11.7 and 12.8 min, respectively. The method is selective, reliable, reproducable with relative standard deviation (RSD) of 1.70 and linear in the range of 2.5 x 10(-9)-1.5 x 10(-8) M OTA. The limit of detection (LOD) and limit of quantification (LOQ) were 2.5 x 10(-10) M corresponding to 0.1 ng mL(-1) and 8.2 x 10(-10) corresponding to 3.3 ng mL(-1), respectively. Recovery studies were 81.2 +/- 1.9 (SD). The method was applied for analysis of OTA in wheat, corn, red pepper, cheese and wine. The proposed method can be used for the routine analysis of OTA in food and animal feed.

Determination of amlodipine in pharmaceutical formulations by differential-pulse voltammetry with a glassy carbon electrode.

A differential-pulse voltammetric method was developed for the determination of amlodipine based on the oxidation of the dihydropyridine group on the surface of glassy carbon electrode under stationary and rotating conditions. The experiments were conducted in a supporting electrolyte consisting of 0.2 M KCl, 0.1 M phosphate buffer, and 10% (v/v) methanol during investigation of initial potential and pH effects. No adsorption effect was observed on using an initial potential of 0 mV and the supporting electrolyte solution at pH 5.5 under both stationary and rotating conditions. The factor affecting the voltammetric current was diffusional in the range of 200-1000 rpm for rotating, and 2-40 mV s-1 for stationary conditions up to a concentration of 0.04 mg mL-1 amlodipine. The limit of detection (LOD) and the limit of quantitative (LOQ) for the rotating and stationary techniques were found to be 0.004 and 0.0072 mg mL-1 (for S/N = 3.3) and LOQ 0.012 and 0.022 mg mL-1 (for S/N = 10), respectively. The proposed method was applied to the tablets containing amlodipine and according to the statistical evaluations acceptable results were obtained at the 95% probability level.

Protective effect of vasoactive intestinal peptide on testicular torsion-detorsion injury: association with heparin-containing mast cells.

OBJECTIVES: To elucidate the action of vasoactive intestinal peptide (VIP) on detorsion injury and the heterogeneity of mast cells in the testes of rats. METHODS: Prepubertal male Sprague-Dawley rats were used in six groups. Group 1 was the control group (sham operation); group 2 had 2 hours of torsion; group 3, 2 hours of torsion and 1 hour of detorsion after administration of saline; group 4 had 2 hours of torsion and 4 hours of detorsion after administration of saline; group 5, 2 hours of torsion and 1 hour of detorsion after administration of intraperitoneal VIP (25 ng/kg); and group 6, 2 hours of torsion and 4 hours of detorsion after intraperitoneal VIP. The 2 hours of torsion was created by rotating the right testis 720 degrees in a clockwise direction. VIP (25 ng/kg) was injected intraperitoneally 1 minute before the 1 and 4 hours of detorsion. At the end of the experiment, catalase enzyme activity was measured polarographically, and superoxide dismutase, malondialdehyde, and protein were measured spectrophotometrically. Nitric oxide was measured by capillary electrophoresis in the testicular tissue. Routine histologic examination of testicular mast cells was done under light microscopy; the histochemistry was also analyzed. RESULTS: Torsion significantly induced oxidative stress, mast cell degranulation, and tissue damage. Detorsion attenuated oxidative stress without any diminution of the histologic damage to the tissue. VIP significantly protected the testicular tissue from detorsion injury. It also inhibited mast cell activity while increasing the heparin content. CONCLUSIONS: VIP can protect testicular tissue from detorsion injury. Heparin-containing mast cells seem to be important mediator cells for this protection.