Endothelium-dependent hypotensive and vasorelaxant effects of the essential oil from aerial parts of Mentha x villosa in rats.

Cardiovascular effects of an essential oil from the aerial parts of Mentha x villosa (OEMV) were tested in rats using a combined in vivo and in vitro approach. In non-anesthetized normotensive rats, OEMV (1, 5, 10, 20, 30 mg kg(-1) body wt., i.v.) induced a significant and dose-dependent hypotension (-3 +/- 1.8%; -6 +/- 0.7%; -40 +/- 6.7%; -58 +/- 3.8%; -57 +/- 2.1%, respectively) associated with decreases in heart rate (-1 +/- 0.3%; -9 +/- 0.9%; -17 +/- 3.2%; -72 +/- 3.1%; -82 +/- 1.4%, respectively). The hypotensive and bradycardic responses evoked by OEMV were attenuated and blocke by pre-treatment of the animals with atropine (2 mg kg(-1) body wt., i.v.). In isolated rat atrial preparations, OEMV (10, 100, 300, 500 microg ml(-1)) produced concentration-related negative chronotropic and inotropic effects (IC50 value = 229 +/- 17 and 120 +/- 13 microg ml(-1), respectively). In isolated rat aortic rings, increasing concentrations of OEM (10, 100, 300, 500 microg ml(-1)) were able to antagonize the effects of phenylephrine (1 microM), prostaglandin F2alpha (10 microM) and KCl (80 mM)-induced contractions (IC50 value = 255 +/- 9, 174 +/- 4 and 165 +/- 14 microg ml(-1), respectively). The vasorelaxant activity induced by OEMV was attenuated significantly by either endothelium removal (IC50 value = 304 +/- 9 microg ml(-1)), NG-nitro L-arginine methyl ester (L-NAME) 100 microM (IC50 value=359 +/- 18 microg ml(-1)), L-NAME 300 microM (IC50 value = 488 +/- 20 microg ml(-1)) or indomethacin 10 microM (IC50 value = 334 +/- 18 microg ml(-1)). However, it was not affected by atropine 1 microM (IC50 value = 247 +/- 12 microg ml(-1)). Furthermore, the hypotensive response induced by OEMV was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg kg(-1) body wt., i.v.), while bradycardia was not altered. The results suggest that the hypotensive effect induced by OEMV is probably due to its direct cardiodepressant action and peripheral vasodilation, which can be attributed to both endothelium-dependent (via EDRFs, at least NO and prostacyclin) and endothelium-independent mechanisms (such as Ca2+ channel blockade).     

Endothelium-derived nitric oxide is involved in the hypotensive and vasorelaxant responses induced by the aqueous fraction of the ethanolic extract of the leaves of Albizia inopinata (Harms) G. P. Lewis in rats.

The acute cardiovascular effects of an aqueous fraction of the ethanolic extract of the leaves (AFL) of Albizia inopinata (Harms) G. P. Lewis (Leguminosae) were studied in rats using a combined in vivo and in vitro approach. In conscious, unrestrained rats, AFL (5, 10 and 20 mg/kg(-1) body wt. i.v., randomly) produced a significant and dose-dependent hypotension associated with increases in heart rate and cardiac output, and with a strong reduction in total peripheral resistances. The hypotensive response to AFL (20 mg/kg(-1) body wt.) was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg/kg(-1) body wt. i.v.). Furthermore, under these conditions, the associated tachycardia was inhibited completely. In isolated rat aortic rings, increasing concentrations of AFL (10, 20, 40 and 80 microg/ml(-1)) were able to antagonize the effects of phenylephrine- (1 microM) and KCl- (80 mM) induced contractions (IC50 value 65 +/- 4 and 54 +/- 6 microg/ml(-1), respectively). The smooth muscle-relaxant activity of AFL was inhibited similarly either removal of the vascular endothelium or by L-NAME (10 and 100 microM), but was not affected significantly by atropine (1 microM) or indomethacin (10 microM). In isolated rat atrial preparations, AFL (30, 100, 300 and 500 microg/ml(-1)) produced concentration-related negative inotropic and chronotropic effects (IC50 value = 274 +/- 53 and 335 +/- 23 microg/ml(-1), respectively). These results suggest that in rats, the hypotensive effect of AFL is due to a peripheral vasodilation, at least partly secondary to the release of NO by the vascular endothelium. The direct cardio-depressant actions of AFL are of little importance in the systemic effects of the extract.     

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

Development and validation of a liquid chromatographic method for Casiopeina IIgly in rat plasma

A sensitive and specific liquid chromatographic method using solid-phase extraction with Sep-pak cartridges has been developed for the determination of Casiopeina IIgly and validated over the linear range 2.5-50 microg/ml in rat plasma. The analysis was performed on a Symetry C(18) (5 microm) column with a Phenomenex C(18) precolumn. The mobile phase was methanol-water (58:42, v/v). The column effluent was monitored at 273 nm. The results showed that the assay is sensitive at 2.5 microg/ml. Maximum intra-day coefficient of variation was 11.47%. The recovery based upon addition of internal standard to rat plasma was 80.98%. The method was used to perform preclinical pharmacokinetic studies in rat plasma and was found to be satisfactory.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Solid-phase extraction-liquid chromatographic method for the determination and pharmacokinetic studies of albiflorin and paeoniflorin in rat serum after oral administration of Si-Wu decoction

A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-Clean cartridges, and the extracts were analyzed by HPLC on a reversed-phase C(18) column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230 nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450 ng/ml for albiflorin and 10-2000 ng/ml for paeoniflorin. The intra- and inter-day precisions (R.S.D.) were 相似文献   

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Stereoselective pharmacokinetics of tetrahydropalmatine after oral administration of (-)-enantiomer and the racemate

Tetrahydropalmatine (THP) is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). THP is a racemic mixture which contains 50% of the (+) and 50% of (-) enantiomer. The (-) enantiomer accounts for most of the analgesic effects. Plasma concentrations of THP enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OJ column with quantification by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of rac-THP or (-)-THP. The pharmacokinetic profiles of the two enantiomers after dosing with rac-THP were significantly different both in rats and dogs. The mean C(max) and AUC(0-infinity) values in rats were 1.93 +/- 0.36 microg/ml and 6.65 +/- 2.34 microg x h/ml for the (-) enantiomer, and 1.11 +/- 0.25 microg/ml and 2.03 +/- 0.45 microg x h/ml for the (+) enantiomer. The mean C(max) and AUC(0-infinity) in dogs were 1.60 +/- 0.81 microg/ml and 9.88 +/- 2.58 microg x h/ml for the (-) enantiomer, while 0.36 +/- 0.21 microg/ml and 1.22 +/- 0.40 microg x h/ml for the (+) enantiomer. rac-THP at 40 mg/kg and (-)-THP at 20 mg/kg had very similar plasma concentration-time profiles, and C(max), AUC(0-infinity), and t(1/2) of the (-) enantiomer in both rats and dogs, indicating that the two treatments were equivalent with respect to the pharmacokinetic properties of the (-) enantiomer.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) assay for biperiden in bulk form and pharmaceutical dosage forms

Current compendial (USP) methods of assay for the analysis of biperiden in bulk form and pharmaceutical dosage forms involve the use of titrimetric and spectrophotometric procedures, respectively. These are non-selective and non-stability-indicating techniques. In this work, a stability-indicating high performance liquid chromatographic assay procedure has been developed and validated for biperiden. The liquid chromatographic separation was achieved isocratically on a symmetry C8 column (150 mm x 3.9 mm i.d., 5 microm particle size) using a mobile phase containing methanol-buffer (50:50, v/v, pH 2.50) at a flow rate of 1 ml/min and UV detection at 205 nm. The buffer was composed of sodium dihydrogen phosphate (50 mM) and 1-heptanesulfonic acid sodium salt (5 mM). The method was linear over the concentration range of 0.5-25 microg/ml (r=0.9998) with a limit of detection and quantitation 0.03 and 0.1 microg/ml, respectively. The method has the requisite accuracy, selectivity, sensitivity and precision to assay biperiden in bulk form and pharmaceutical dosage forms. Degradation products resulting from the stress studies did not interfere with the detection of biperiden and the assay is thus stability-indicating.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography

A novel assay for measuring the free leptin fraction was developed and validated against a chromatographic technique. The assay used acid-ethanol extraction (AEE) for separation of bound/free leptin moieties. The interassay coefficient of variation was 3.9%. The specificity for leptin binding was confirmed by incubation with 1 microg of unlabeled rat leptin that effectively competed with radiolabeled leptin whereas human growth hormone and interleukin-6 were ineffective in competing with radiolabeled leptin binding. Scatchard analysis of competitive binding experiments with rat plasma demonstrated a linear relationship with a binding affinity of 0.3-0.6 x 109 M-1. This novel assay was used to determine if age-related insensitivity to leptin action is secondary to altered serum leptin binding. Rats at various age groups were studied for changes in body adiposity and serum total and free leptin concentrations. Serum free leptin concentrations (ng/ml mean +/- SEM) were significantly increased in 24-month-old rats (5.56 +/- 0. 21) compared with 18-month-old rats (4.76 +/- 0.17) (P < 0.01) despite similar body weight and adiposity of the two age groups. The increase in plasma free leptin concentrations in 12-month-old rats (3.86 +/- 0.28) and 6-month-old rats (2.05 +/- 0.06) relative to 3-month-old rats (1.37 +/- 0.06) (P < 0.001) was out of proportion to the increase in body adiposity in aging rats. It is concluded that aging in rats is associated with relative insensitivity to leptin. This change cannot be attributed to increased plasma binding or to a reduction in the leptin free fraction.     

Determination of a potent non-competitive AMPA receptor antagonist in rat brain

N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic-lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 degrees C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 microg/ml for all studied compounds (n=6). In the within-day assay (n=4), the accuracy ranged from 87.5% determined with 0.05 microg/ml of PS3 to 110.1% determined with 0.2 microg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 microg/ml of PS3 to 9.7 determined with 0.2 microg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 microg/ml to 94.3 for PS3Ac at 0.5 microg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.     

Genotoxic effects of estradiol-17beta on human lymphocyte chromosomes

The cytogenetic effect of a hormonal steroid, estradiol-17beta, was assessed in peripheral blood human lymphocyte culture. Sister chromatid exchanges (SCE) and chromosome aberrations (CA) were scored as genetic end points. Significant induction of CA was observed at 25 microg/ml and 50 microg/ml concentrations of estradiol-17beta in the absence of microsomal activation. The drug was effective in all treatments in the presence of rat liver S(9) microsomal fraction (S(9) mix) and exhibited increased frequency of chromosomal aberrations. The drug was effective in increasing the SCE frequency which was found to be maximum at the dose of 50 microg/ml concentration (i.e., 4.34+/-1.22) both with and without metabolic activation. It was found that estradiol-17beta itself and possibly its metabolites are potent mutagens beyond a particular dose in human lymphocytes.     

Measurement of total mirtazapine and normirtazapine in plasma/serum by liquid chromatography with fluorescence detection

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean +/- S.D.) was 79 +/- 2, and 64 +/- 3%, respectively. The LOD was estimated as 0.5 microg/l, LLOQ was 1 microg/l, with a linear response over the concentration range 4-1000 microg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at -20 degrees C. Intra- and inter-day accuracy were in the range 91-107 and 93-103%, respectively. In clinical samples (n = 14, median mirtazapine dose 45 mg per day, range 15-45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8-40) and 21 (8-32) microg/l, respectively.