Ototoxicity of the three xylene isomers in the rat

Numerous experiments have shown that the aromatic solvents can affect the auditory system in the rat, the cochlea being targeted first. Solvents differ in cochleotoxic potency: for example, styrene is more ototoxic than toluene or xylenes. The goal of this study was to determine the relative ototoxicity of the three isomers of xylene (o-, m- or p-xylene). Moreover, by dosing with the two urinary metabolites of xylene, methylhippuric (MHAs) and mercapturic acids (MBAs), this study points toward a causal relationship between the cochleotoxic effects and potential reactive intermediates arising from the biotransformation of the parent molecules. Separate groups of rats were exposed by inhalation to one isomer following this schedule: 1800 ppm, 6 h/d, 5 d/wk for 3 wk. Auditory thresholds were determined with brainstem-auditory evoked potentials. Morphological analysis of the organ of Corti was performed by counting both sensory and spiral ganglion cells. Among the three isomers, only p-xylene was cochleotoxic. A 39-dB permanent threshold shift was obtained over the tested frequencies range from 8 to 20 kHz. Whereas outer hair cells were largely injured, no significant morphological change was observed within spiral ganglia. The concentrations of urinary p-, o- or m-MHA were greater (p-MHA: 33.2 g/g; o-MHA: 7.8 g/g; m-MHA: 20.4 g/g) than those obtained for MBAs (p-MBA: 0.04 g/g; o-MBA: 6.2 g/g; m-MBA: 0.03 g/g). Besides, there is a large difference between o-MBA (6.2 g/g) and p-MBA (0.04 g/g). As a result, since the cysteine conjugates are not determinant in the ototoxic process of xylenes, the location of the methyl groups around the benzene nucleus could play a key role.     

Capillary gas chromatographic assay of camphor and m-cresol in dermatological creams

Camphor and m-cresol mixtures are used in antiseptic and anti-itching creams. No compendial method exists for these preparations. This paper reports a capillary gas chromatographic method using FID detection with 2,6-di-tert-butyl-4-methylphenol as internal standard on a 30 m×0.32 mm Supelcowax®-10 column (0.25 μm film) with helium as carrier gas. Ramped temperature programming was applied. The method allows simultaneous quantitation of camphor and m-cresol in the presence of o- and p-cresols, calamine and zinc oxide. Overall percent recoveries (±SD, n=9) of camphor, o-, p- and m-cresol from spiked placebo creams, at a labeled amount of 10 (w/w)% were 96.9±0.6, 98.2±0.6, 99.2±0.5 and 101.0±0.9%, respectively, and at a labeled amount of 1% were 96.7±0.6, 97.8±0.9, 97.8±0.6, and 100.3±1.0%, respectively. The recovery studies were carried out at ±30% of the labeled amounts. Linear peak area or height ratios were obtained (r>0.999) for camphor, o-, p- and m-cresol covering a concentration range of 10–200% of the labeled amount. Linearity (r>0.999) was also obtained for m-cresol when the relative concentration of o- and p-cresol was varied from 5 to 100% of the m-cresol concentration. The resolution between the ‘critical pair’ of p- and m-cresol was ≥1.1. The limit of quantitation was 23 pg for m-cresol and 9.3 pg for camphor using an injection split of 1:50. The repeatability (%RSD) for all compounds were <2% for peak area and <1.4% for peak height ratios. System suitability and robustness of the method were established. The method was successively applied to the assay of available commercial products and allows assay of camphor and the three cresol isomers.     

Developmental alterations in murine embryos exposed in vitro to an estrogenic pesticide, o,p'-DDT

Culturing pronuclear embryos from CD-1 mice with o,p′-DDT and p,p′-DDT was examined as a means for directly evaluating toxicant risk and for increasing the speed of screening developmental toxicants. Pronuclear (2PN) embryos from CD-1 mice were cultured 96 h in modified Earle’s balanced salt solution containing 0.1% (v/v) ethanol (control) or 10-fold dilutions of 17β-estradiol, o,p′-DDT, or p,p′-DDT. Compared to control treatment, 96 h incubation of 2PN embryos with 0.1 μg/mL o,p′-DDT significantly reduced embryo development to blastocyst and mean cell number, and increased the percentage of cells undergoing apoptosis. The effects of o,p′-DDT on developmental parameters were dose-responsive. Embryo sexing by multiplex polymerase chain reaction indicated that both sexes were susceptible to toxicant injury with comparable reduction in development to blastocyst (27% and 24%, respectively) in the presence of o,p′-DDT. Results of this study suggest that in vitro exposure of preimplantation embryos to xenobiotics may provide a useful tool for rapidly screening developmental toxicants.     

Simultaneous determination of serum flecainide and its metabolites by using high performance liquid chromatography

Simultaneous determination of serum flecainide and its oxidative metabolites was carried out by using high performance liquid chromatography (HPLC) equipped with conventional octadecylsilyl silica (ODS) column and fluorescence detector. Flecainide and its metabolites, m-O-dealkylated flecainide (MODF) and m-O-dealkylated lactam of flecainide (MODLF) in serum were extracted with ethyl acetate. The recoveries of flecainide, MODF and MODLF were greater than 92, 93, and 60% with the coefficient of variations (CVs) less than 3.2, 5.8, and 5.3%, respectively. The calibration curves were linear at the concentration range of 50–1500 ng/mL for flecainide and 10–500 ng/mL for MODF and MODLF (r>0.999). The CVs for intra-day assay were 2.7–5.3% for flecainide, 3.0–4.2% for MODF, and 3.7–4.3% for MODLF, respectively. The CVs for inter-day assay were 7.0–8.4% for flecainide, 3.3–6.7% for MODF, and 4.4–7.7% for MODLF, respectively. This assay method can be used for assessing the metabolic ability of flecainide in the patients with tachyarrhythmia.     

Dopamine Autoreceptors in Rat Nucleus Accumbens Modulate Prepulse Inhibition of Acoustic Startle

Dopamine and 3,4-dihydroxy phenylacetic acid (DOPAC) levels in discrete regions and apomorphine- or (−)-sulpiride–induced changes in electrically evoked dopamine release from nucleus accumbens slices were assessed after testing prepulse inhibition of acoustic startle (PPI) in rats. Dopamine and DOPAC levels in the nucleus accumbens, but not in the striatum, correlated well with PPI (r = −0.64 for dopamine, r = −0.48 for DOPAC). Evoked dopamine release from the nucleus accumbens did not differ between the high-PPI (more than 60%) and the low-PPI (less than 40%) group. When slices were superfused with 1 μM apomorphine, the S2/S1 ratio in rats showing high PPI was 0.77 ± 0.02 (mean ± SEM, 66% of control), significantly smaller than in the low-PPI group (S2/S1 ratio = 0.97 ± 0.08, 94% of control, p < 0.05). Moreover, (−)-sulpiride–induced increase in evoked dopamine release from the nucleus accumbens in the high-PPI group was inclined to be greater than in the low-PPI group. The results suggest that PPI differences between individuals may reflect the sensitivity of release-modulating dopamine autoreceptors in the nucleus accumbens.     

Enhanced platelet serotonin 5-HT2A receptor binding in anorexia nervosa and bulimia nervosa.

Some evidence exists to suggest that serotonin 5-HT_2A receptor function is altered in anorexia nervosa and bulimia nervosa. In order to further investigate the 5-HT_2A receptor in eating disorders, platelet [³H]lysergic acid diethylamide ([³H]LSD) binding was studied in ten patients with anorexia nervosa, 23 patients with bulimia nervosa and 33 healthy controls. At admission, B_max for platelet [³H]LSD binding was significantly higher both in the anorexia nervosa group (30.6±4.2 fmol/mg protein; mean±S.D.) and in the bulimia nervosa group (30.8±7.6 fmol/mg protein) than in the control group (23.5 ±6.3 fmol/mg protein; p=0.01 and p=0.003, respectively). K_d was borderline significantly higher among anorexics (median 1.45 nM) and significantly higher among bulimics (median 1.66 nM) than among controls (median 0.95 nM; p=0.05 and 0.003, respectively). The Global Assessment of Functioning score and the body mass index were both significantly negatively correlated to K_d (r=−0.40; p=0.03 and r=−0.41 p=0.03, respectively), but not to B_max. The present study indicates that patients with anorexia nervosa as well as patients with bulimia nervosa have an enhanced 5-HT_2A receptor binding and provides further evidence for a serotonergic dysfunction in eating disorders.     

A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

Assay for nipecotic acid in small blood samples by gas chromatography-mass spectroscopy

An analytical method for nipecotic acid quantification in rat blood was developed utilizing a stable isotope internal standard and capillary gas chromatography–mass spectroscopy. The method involves a solid phase extraction step followed by a two-step derivatization. The analytes are separated by capillary gas chromatography and detected by selected ion monitoring of their base peaks at 180 and 185 m/z, respectively. The assay has a limit of detection (LOD) of 10 ng/ml and a limit of quantification of 26 ng/ml in 200 μl of rat whole blood. The linear range of the assay covers from 26 to 6500 ng/ml (r²=0.9996, n=9). The coefficient of variation was less than 10% at concentrations of 50, 1000 and 5000 ng/ml. The assay was used to characterize the pharmacokinetics of R-(-)-nipecotic acid in a rat. R-(-)-nipecotic acid clearance was 4.2 ml/min, its half-life was 1.5 h and its volume of distribution at steady state was 325 ml.     

Psychometric evaluation of the Amphetamine Cessation Symptom Assessment

Testing of a new scale, the Amphetamine Cessation Symptom Assessment (ACSA), in a sample of treatment-seeking amphetamine users (N = 133) showed satisfactory reliability, while factor analysis identified three components explaining 64.7% of the variance in scores. Scores were inversely related to subjective general well-being (r = −.33, p < .01) and directly related to the Beck Depression Inventory (r = .59, p < .01). There were positive relationships between the ACSA and measures of amphetamine dependence (r = .36, p < .01) and the intensity of recent amphetamine use (r = .24, p < .01). The ACSA discriminated between “low-dose” and “high-dose” users, indicating discriminant validity. In inpatients (n = 63), ACSA scores declined significantly over time, while higher scores in inpatient treatment dropouts indicated predictive validity. The ACSA showed satisfactory reliability and validity, with a three-factor solution providing the best fit to the data. The ACSA could play an important role in providing clinical outcome data, particularly in outcome evaluation of new treatment protocols.     

Nephrotoxicity of intravenous colistin: a prospective evaluation

Twenty-one patients who received intravenous colistimethate sodium (CMS) for at least 7 days for the treatment of multidrug-resistant Gram-negative bacterial infections were included in a prospective cohort study at ‘Henry Dunant’ Hospital in Athens, Greece. The mean (± standard deviation) and median daily doses, cumulative doses and duration of treatment of intravenous CMS were, respectively, 5.5 (± 1.9) and 6 million IU, 90.2 (± 52.0) and 72 million IU, and 17.7 (± 11.7) and 15 days (range 7–54 days). Three patients (14.3%) developed nephrotoxicity during treatment with CMS. The cumulative dose of administered CMS was statistically correlated with the difference in values of serum creatinine between the end and start of CMS treatment (r = 0.6, P = 0.004 by Spearman's test).     

Quantitative analysis of analgoantipyretics in dosage form using planar chromatography

In the therapy of pain of weaker genesis, frequently used drugs usually represent a mix of analgoantipyretics of different chemical structures, mostly derivatives of salicylic acid, pyrazolone and p-aminophenol as well as derivatives of propionic and acetylsalicylic acid. For the determination of these drugs, different chromatographic methods have been applied, mostly HPLC, due to the the lower polarity (pyrazolones derivatives) and thermolability, as well as nonvolatility of compounds investigated. TLC method, considering advantages which include simplicity, reasonable sensitivity, rapidity, excellent resolving power and low cost has been successfully explored for the determination of analgoantipyretic compounds.

The aim of this work was to develop a simple and rapid HPTLC method for the determination of acetylsalicylic acid, paracetamol, caffeine and phenobarbitone in dosage form. The determination of analgoantipyretics were performed on pre-coated HPTLC silica gel plates (10×20 cm²) by development in the mobile phase dichlormethane–ethyl acetate–cyclohexane–isopropanol–0.1 M HCL–formic acid (9:8:3:1.5:0.2:0.2 v/v/v/v/v/v). Migration distances (68.6+0.2 mm, 54.1+0.1 mm, 36.4+0.14 mm and 85.9+0.11 mm for acetylsalicylic acid, paracetamol, caffeine and phenobarbitone, respectively) with low RSD values (0.13–0.39%) showed a satisfactory reproductivity of the chromatographic system. TLC scanner was used for direct evaluation of the chromatograms in the reflectance/absorbance mode. Established calibration curves (r>0.999), precision (0.3–1.02%) and detection limits, as well as recovery values (96.51–98.1%) were validated and found to be satisfactory. The method was found to be reproducible and convenient for the quantitative analysis of compounds investigated in their dosage forms.     

Lactobacillus rhamnosus strain GG restores alkaline phosphatase activity in differentiating Caco-2 cells dosed with the potent mycotoxin deoxynivalenol

Deoxynivalenol (DON) contamination of cereal crops occurs frequently, and may cause acute exposure at high levels or chronic more moderate exposure. DON has proven toxicity including restriction of enterocyte differentiation, which may play a part in DON induced gastroenteritis. The probiotic bacteria Lactobacillus rhamnosus strain GG (GG) can bind DON, and therefore potentially restrict bioavailability of this toxin. Binding efficacy is not significantly altered by heat treatment, and therefore this in vitro study evaluated whether heat inactivated GG could restore the differentiation process in Caco-2 cells, using alkaline phosphatase (ALP) activity as a marker of differentiation. DON (200 ng/mL) caused a significant (p < 0.001) 36% reduction in ALP activity (1598 ± 137 U/mg protein) compared to untreated cells (2502 ± 80 U/mg). A dose dependant restoration of ALP activity was observed where DON treated cells were co-incubated with heat inactivated GG (1719 ± 84; 2007 ± 142; 2272 ± 160 U/mg for GG at 1 × 10⁴ (p > 0.9), 1 × 10⁷ (p < 0.001), and 1 × 10¹⁰ CFU/mL (p < 0.001), respectively). Co-incubation of the non-binding strain, LC-705 (1 × 10¹⁰ CFU/mL), with DON did not significantly restore the ALP (1841 ± 97 U/mg, p < 0.077) compared to DON only treated cells. When viable GG were co-incubated with DON a similar restoration of ALP activity was observed as seen for heat inactivated GG. These combined data suggest that the major effect of GG on restoring ALP activity, and therefore Caco-2 cell differentiation, was due to specific binding of DON, with possibly a more minor role of non-specific bacterial interference.     

Effect of injection routes on pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin in rats

Pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin (9-NC) were compared after intravenous (i.v.) and intramuscular (i.m.) injection at a dose of 1.5 mg/kg 9-NC solution. The concentrations of three different forms of 9-NC, namely lactone, carboxylate and total 9-NC, were measured by HPLC analysis. Injection routes were demonstrated to have significant effect on pharmacokinetics of 9-NC. Compared with i.v. injection route, mean residence time (MRT) of 9-NC three forms was significantly prolonged following i.m. route (p < 0.05). The AUC_0–∞ ratios of i.m. to i.v. route were calculated to be 102 ± 43%, 273 ± 221% and 150 ± 62% for lactone, carboxylate and total 9-NC, respectively. Compared with i.v. injection route, although AUC_0–∞ was barely changed, MRT of lactone 9-NC was dramatically prolonged 4.5-fold after i.m. injection, which may account for the reported improved antitumor efficacy. However, the results of the present study also demonstrated that i.m. injection route increased both AUC_0–∞ and MRT of carboxylate 9-NC more significantly. Since the carboxylate form of CPT analogs including 9-NC is associated with their unwanted toxicity, i.m. injection route might lead to severe toxicity compared with i.v. route. Lactone/carboxylate equilibrium was also significantly influenced by injection routes. Based on the AUC_0–∞ measurements, the lactone 9-NC constituted 50 ± 8% and 32 ± 7% of circulating total 9-NC after i.v. or i.m. administration, respectively (p < 0.01).     

Cocaine concentrations in fetal C57BL/6 mouse brain relative to maternal brain and plasma

Cocaine concentrations in maternal plasma and brain and fetal brain of mice were evaluated as a model for fetal brain exposure during maternal cocaine use. On days 12–18 of gestation, mice (C57BL/6; N = 5–7/group) received SC cocaine-HCl: 20 or 40 mg/kg. Maternal plasma and brain (accumbens and caudate nuclei removed), and fetal brain were collected at 0.5, 1, and 2 h following the last injection. Analysis was by GC-MS. Brain cocaine levels in the dams declined from 9.6 to 3.4 and 20.9 to 12.5 mg/g during the 0.5–1-h period after the low and high doses, respectively, and were 7.5–14.3 times greater than plasma levels. The corresponding fetal brain concentrations changed from 1.6 to 1.3 and 2.9 to 3.4 mg/g. By 2 h, brain cocaine concentrations in dams declined to approximately 10% of their 0.5-h values, with a slower drug decay occurring in fetal brain. Maternal plasma cocaine concentrations correlated with those of maternal brain (r = 0.94, p < 0.01) and fetal brain (r = 0.69, p < 0.01). The present results indicate that cocaine accumulates to a lesser extent in fetal brain than in maternal brain of C57BL/6 mice; however, the duration of exposure appears to be more sustained in the fetus, a phenomenon that may have toxicological implications for human in utero cocaine exposure.     

Development and validation of two chromatographic methods for the quantification of E-6087 and one of its metabolites, E-6132, in rat plasma

E-6087 is a nonsteroidal anti-inflammatory compound under development that selectively inhibits cyclooxygenase-2. In vitro studies have shown that one of its metabolites, E-6132, also inhibits this enzyme. Due to chromatographic reasons, two reverse phase HPLC methods were developed and validated in order to elucidate which compound is responsible for the pharmacological activity in vivo. Chromatographic separation of E-6087 was achieved using acetonitrile–phosphate buffer (pH 2.5; 25 mM) (60:40, v/v) as mobile phase and two 4.6×150 mm×5 μm Inertsil ODS-2 columns. For E-6132, two Inertsil ODS-3 columns and 52% of acetonitrile were used instead. Internal standards and fluorescence detection differed between both methods. The same on-line solid-phase extraction method was used. Mean retention times for E-6087 and E-6132 were 15.2 (±1.3) and 36.1 (±0.6) min, respectively. The methods were selective and linear over the concentration range of 10–500 ng ml⁻¹ (r²>0.996) for E-6087 and 5–200 ng ml⁻¹ (r²>0.997) for E-6132. The limits of quantitation were 10 ng ml⁻¹ (E-6087) and 5 ng ml⁻¹ (E-6132) with a precision and accuracy <16% (E-6087) and <11% (E-6132). Mean recoveries from plasma were 43.2–61.9% (E-6087) and 60.4–65.2% (E-6132). For both compounds, both inter-assay and intra-assay precision and accuracy were within acceptable limits (<15%). As an example of the suitability of these methods, the results from a pharmacokinetic study are reported. After single oral administration of 5 mg kg⁻¹ of E-6087 to rats, plasma concentrations of E-6087 at peak time were higher than those of E-6132, suggesting that activity is mainly due to E-6087.     

The effect of DHEA complementary treatment on heroin addicts participating in a rehabilitation program: A preliminary study

We evaluated the effect of DHEA complementary treatment in opiate addicts undergoing detoxification. DHEA (100 mg/day) or placebo was added to the routine medication protocol in a randomized, double blind controlled study. Follow-up for 12 months was conducted. Two separate DHEA-treated subgroups were identified by the Fuzzy clustering method: one showed statistically significant improvement in the severity of withdrawal symptoms, depression and anxiety scores (n = 34; p < 0.001 for all) and the other subgroup deteriorated in all measures (n = 15). DHEA at the end of the detoxification program showed a tendency towards correlation with the duration of abstinence (r = 0.6843; p > 0.05; n = 6), while a negative correlation was obtained with the cortisol level (r = − 0.900; p = 0.005, n = 8). The completion-rate of the DHEA-improved subgroup was greater than in the DHEA-deteriorated subgroup (64.7% vs. 33.3%, respectively).

The influence of supplementary DHEA treatment was mostly effective in heroin addicts who had not previously used either cocaine or benzodiazepines and who had experienced only few withdrawal programs.     

Rapid quantification of nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple, sensitive and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric method was developed and validated for the quantitation of nebivolol in human plasma. The method involved a simple single-step liquid–liquid extraction with diethyl ether/dichloromethane (70/30). The analyte was chromatographed on Waters symmetry^® C₁₈ reversed-phase chromatographic column by isocratic elution with water:acetonitrile:formic acid (30:70:0.03, v/v) and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 406.4–151.5 and m/z 409.1–228.1 were used to measure the analyte and the internal standard (I.S.), respectively. The chromatographic runtime was 2 min and the weighted (1/x²) calibration curves were linear over the range 50–10,000 pg/mL. The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in human plasma were 10 and 50 pg/mL, respectively. The within- and between-batch accuracy and precision were found to be well within acceptable limits (<10%). The analyte was stable after three freeze-thaw cycles (deviation <10%). The average absolute recoveries of nebivolol and tamsulosin, used as an internal standard, from spiked plasma samples were 73.4 ± 3.7 and 72.1 ± 2.0%, respectively. The assay method described here was applied to study the pharmacokinetics of nebivolol.     

Functional characterization of adenosine transport across the BBB in mice

We investigated transport characteristics of adenosine across the blood–brain barrier (BBB) in mice. Uptake clearance across the BBB was measured by using an in situ mouse brain perfusion technique and cultured mouse brain capillary endothelial cell line (MBEC4 cells). Nucleoside transporter was cloned by RT-PCR and expressed on Xenopus laevis oocyte. Both in situ and in vitro studies revealed that the adenosine uptake is concentration-dependent, Na⁺-independent and S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive. The K_t values of in situ and in vitro studies were 31.7 ± 13.8 μM and 11.9 ± 2.84 μM, respectively. A good correlation was found for the inhibitory effects of nucleoside analogs to adenosine uptake between in situ and in vitro studies. RT-PCR revealed the expression of RNA of mouse equilibrative nucleoside transporter (mENT1) in mouse brain capillary and MBEC4 cells. In mENT1 expressed on X. laevis oocyte, K_t value of adenosine transport was 6.9 ± 2.7 μM (and comparable to those in situ and in vitro studies). In conclusion, we characterized the adenosine transport across the BBB in mice by using in situ brain perfusion technique and MBEC4 cells and found that these transports share common characteristics with mENT1-mediated transport. Transport of adenosine across the BBB in mice may be attributable to mENT1.     

基于指纹图谱及多组分定量分析对金荞麦止咳膏质量评价方法的研究

目的:建立金荞麦止咳膏指纹图谱定性分析和8个主要成分定量分析的测定方法,为金荞麦止咳膏质量控制方法的建立提供科学依据。方法:采用高效液相色谱法(HPLC)建立13批金荞麦止咳膏的指纹图谱,标定共有峰,并进行相似度评价,对采用对照品比对确认的8种组分进行定量分析。结果:金荞麦止咳膏标定共有峰21个,其中原儿茶酸、原儿茶醛、芦丁、柚皮苷、黄芩苷、汉黄芩苷、黄芩素、汉黄芩素8种成分采用对照品比对确认,各成分的线性范围分别0.011 75~1.174 8 mg·mL^-1(r=1.000 0),0.016 92~1.691 8 mg·mL^-1(r=1.000 0),0.015 88~1.588 0 mg·mL^-1(r=0.999 9),0.030 34~3.034 mg·mL^-1(r=0.999 9),0.017 24~1.724 2 mg·mL^-1(r=0.999 9),0.016 86~1.686 4 mg·mL^-1(r=0.999 9),0.026 58~2.658 mg·mL^-1(r=0.999 9),0.017 04~1.704 mg·mL^-1(r=0.999 9),平均回收率在97.4%~102.3%范围内,RSD<1.1%(n=3)。结论:建立的金荞麦止咳膏指纹图谱和含量测定方法准确可靠、快速高效,可用于金荞麦止咳膏的定性、定量分析。