复方缬沙坦片在健康人体的药代动力学和生物等效性

目的研究国产复方缬沙坦片和胶囊在健康人体的药代动力学及其生物等效性。方法20名男性健康志愿者单次口服复方缬沙坦片和胶囊160mg/25mg,用反相高效液相色谱-荧光及紫外检测法测定血浆中缬沙坦、氢氯噻嗪浓度,据血药浓度—时间数据进行有关参数计算。结果受试与参比制剂的药代动力学参数如下。缬沙坦tmax分别为(3.1±0.7)和(3.0±0.7)h,Cmax分别为(4932.82±1557.19)和(4975.94±1472.51)mg·L-1,AUC0-t分别为(30.54±10.73),(29.54±10.68)mg·h·L-1;氢氯噻嗪tmax分别为(2.2±0.9)和(2.2±0.6)h,Cmax分别为(196.04±13.95)和(185.77±16.35)μg·L-1,AUC0-t分别为(1.29±0.23)和(1.34±0.28)mg·h·L-1。复方缬沙坦片中缬沙坦、氢氯噻嗪相对生物利用度分别为(104.8±13.0)%,(98.3±14.7)%。结论2制剂生物等效。

Determination of Diphenytriazol (DL111-IT) in Rabbit Plasma by High-Performance Liquid Chromatography with Fluorescence Detection

Aim To develop an HPLC method with fluorescence detection for the assay ofDL111-IT in rabbit plasma. Methods DL111-IT and internal standard glybenzcyclamide in rabbit plasmawere extracted with chloroform. The determination was performed on a Diamonsil ODS-C_(18) column(150 mm x 4.6 mm, 5 μm) with a mobile phase of acetonitrile and 0.025 mol·L^(-1) diammoniumhydrogen phosphate buffer (pH 5.0, adjusted by phosphoric acid) (60:40, V/V) at a flow-rate of 1.0mL·min^(-1) . Fluorescence detector was operated at excitation wavelength of 250 nm and emissionwavelength of 332 nm. Results The calibration curve in plasma was linear from 1.00 - 20.00ng·mL^(-1) ( r = 0.999 6, n = 5). The method afforded average extracting recoveries of 85.3% ±1.3%, 84.9% ± 2.7% and 85.8% ± 1.8%, and the average method recoveries were 99.5% ±0.4%, 102.1%± 1.8% and 101.3% ± 2.4% for the high (20.00 ng·mL^(-1)) , middle (10.00 ng·mL^(-1)) and low (1.00 ng·mL^(-1)) check samples, respectively. The intra-day (n = 5) and inter-day (n = 5) precisions(RSD) were less than 3.0% and 7.0%, respectively. The limit of detection and quantitation for themethod were 0.3 ng·mL^(-1) (S/N = 3) and 1 ng·mL^(-1) (S/N = 10, RSD<7.0%) plasma sample,respectively. Conclusion The developed method was accurate, sensitive, simple and could be used forpharmacokinetic study of DL111- IT.

Validation of Analytical Method of Irbesartan Plasma in Vitro by High Performance Liquid Chromatography-Fluorescence

Irbesartan is an antihypertensive drug whose concentration in blood is very small so it requires a sensitive method of analysis, selective and valid for analysis. In this study, it is carried out optimization of analytical conditions and validation for the analysis of irbesartan in plasma. Chromatography was performed on a C 18 column （250 × 4.6 mm, 5 μm） under isocratic elution with acetonitrile-0.1% formic acid （46：54 v/v）, pH 3.75. Detection was made at excitation 250 nm and emission 370 nm and analyses were run at a flow-rate of 1.0 mL/min at a temperature of 40 ℃. Losartan potassium was used as internal standard. Plasma extraction was done by deproteination with acetonitrile, mixed with vortex for 30 seconds, then centrifuged it at 10,000 rpm for 10 rain. In plasma validation, the recovery was 96.22%, and the lower limit of quantification （LLOQ） in plasma was 2 ng/mL. The method also fulfill the criteria for accuracy and precision intra and inter day by normal values （%Diff） not exceed ± 15%. On the stability study, irbesartan in plasma temperature -20 ℃has been stable for 28 days.

Relationship Between Leaf Structure and Aloin Content in Six Species of Aloe L.

The leaf structure, content and the storage location of aloin in the leaves of six species of Aloe L. were studied by means of semi-thin section, high performance liquid chromatography (HPLC) and fluorescent microscope. Results showed that all leaves consisted of epidermis, chlorenchyma, aquiferous tissue and vascular bundles. The leaves had the xeromorphic characteristics, including thickened epidermal cell wall, thickened cuticle, sunken stomata and well-developed aquiferous tissue. With the exception of thus, there were remarkable differences in leaf structure among the six species. The chlorenchyma cells were similar to palisade tissues in Aloe arborescens Mill. and A. mutabilis Pillans, but isodiametric in A. vera L., A. vera L. var. chinensis Berg., A. saponaria Hawer and A. greenii Bali. A. arborescens, A. mutabilis, A. very and A. vera var. chinensis included large parenchymatous cells at the vascular bundles, whereas no such cells were observed at the vascular bundles of A. saponaria and A. greenii. In A. arborescens, A. mutabilis and A. vera, the aquiferous tissue sheaths were present and composed of a layer of small parenchymatous cells without chloroplasts around the aquiferous tissue. While there were no aquiferous tissue sheaths in A. vera var. chinensis, A. saponaria and A. greenii. The HPLC revealed that the content of aloin was high in A. arborescens, low in A. vera, and very low in A. saponaria among the six species. The fluorescent microscopy showed that the yellow-green globule only appeared in the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath, but not in the chlorenchyma and aquiferous tissue. Consequently, the large parenchymatous cells of vascular bundles, vascular bundle sheath and aquiferous tissue sheath were the storage location of aloin. They were positively correlated with the content of aloin.

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells

Objective To investigate whether hypertonic saline （HS） can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. Methods Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat （1 day）. The astrocytes were randomly divided into five groups： isotonic （IS） and HS groups, astrocytes were incubated by IS and HS （320 mosM NaCl） medium, respectively, for 1, 3, 5, 10 or 15 rain; carbenoxolone （CBX） ＋IS and CBX＋HS groups, astrocytes were pre-treated with CBX （100 mmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2＋＋HS group, astrocytes were pre-incubated with Ca2＋ （1 000 μmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein （GFAP） and anti-glutamate. The C6 cells were divided into four groups： IS, HS, CBX＋IS and CBX＋HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry （FCM）. Results （1） Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. （2） The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 rain （P 〈 0.01 vs the 5 min of HS group）. In CBX＋HS group, the glutamate intensity was higher than that in CBX＋IS and HS groups. （3） The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min （P 〈 0.01 vs 1 min and 3 min）. On the contrary, the medium glutamate concentrations in the CBX＋HS or Ca2＋＋HS group were significant lower than that in the HS group （P 〈 0.01）. （4） FCM showed HS and CBX＋HS induced glutamate increase in C6 cells. Conclusion HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

Determination of fleroxacin in human plasma by HPLC with fluorescence detection and the pharmacokinetic study

Aim To develop a sensitive and accurate HPLC method for the determination of fleroxacin in human plasma, and study its pharmacokinetics in healthy subjects. Methods The analytes were isolated fi＇om plasma by simple protein precipitation with methanol, separated on a Diamonsil C18 column by isocratic elution with the mobile phase consisted of 1% triethylamine at pH 4.8 （adjusted with phosphoric acid） and acetonitrile （80/20, V/V） at a flow rate of 1.0 mL.min^-1 and analyzed by fluorescence detector with an excitation at 290 nm and emission 458 nm. The pharmacokinetic study of fleroxacin was performed according to a double period crossover design. Results The weighted （1/x） calibration curve was linear over the plasma concentration range of 0.025 - 8.00 μg.mL^-1 The inter- and intra-day precisions （RSD/%） were no more than 5.16%, and the method accuracies and extraction recoveries at three concentrations ranged firom 99.1% to 100.9%, and 86.7% to 92.0%, respectively. Following oral administration at a dose of 400 mg fleroxacin, the main pharmacokinetic parameters for test and reference capsules were Cmax5.08 ± 0.78 and 5.38 ± 1.40 μg.mL^-1, tmax 1.72 ±0.79 and 1.82 ± 0.78 h, t1/2 11.68 ± 1.27 and 11.38 ± 1.51 h^-1, AUC0-∞ 78.44 ± 11.44 and 76.53 ± 13.24 μg.mL^-1.h, respectively. Conclusion The method is sensitive and accurate, and suitable for human pharmacokinetic study of fleroxacin.

Determination of amino acid neurotransmitters in mouse brain tissue using high-performance liquid chromatography with fluorescence detection

Amino acid neurotransmitters facilitate the transmission of nerve messages across the synapses and play essential roles in the control and regulation of a variety of functions in the central and peripheral nervous system. In this study, we developed a sensitive and efficient method using high-performance liquid chromatography （HPLC） with fluorescence detection for the assay of five important amino acid n After derivatization with o-phthaldialdehyde （OPA）, aspartate （Asp）, glutamic acid （Glu）, glycine （Gly）, taurine （Tau） and ）,-aminobutyric acid （GABA） were simultaneously detected in the presence of the internal standard homoserine （Hse）. Precise separation of these five amino acids was achieved using isocratic elution within 24 min. Good linearity was found over the concentration range with correlation coefficients （r2） not less than 0.9998. The limit of detection （LOD） values were no more than 10 nmol/L. The intra- and inter-day reproducibility was adequate with the relative standard deviation （RSD） of 10.5% or below. This method has also been applied to the analysis of amino acids in the substantia nigra and striatum samples obtained from C57BL/6 mice.

A high-performance liquid chromatography with fluorescence detection method for the simultaneous quantitation of monoamine neurotransmitters and their metabolites in subregions of rat brain

Abstract： In the presem study, we simultaneously quantified the levels of monoamine neurotransmitters （MANTs） and their metabolites （levodopa, norepinephrine, epinephrine, dopamine, 5-HT, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid） in different brain subregions of rats using a newly developed simple, sensitive and selective high-performance liquid chromatography with fluorescence detection （HPLC-FLD） method. In this new HPLC-FLD method, analytes were directly extracted and separated without deriveatization step within 20 min. The FLD wavelength was set at 280 nm and 330 nm for excitation and emission, respectively. The analytes were separated on an Agilent Eclipse Plus Cls column （4.6 mm×150 mm, 5.0 μm） equipped with an Agilent XDB-C18 security guard column （4.6 mm×12.5 mm, 5.0 lam）, and the column temperature was maintained at 35 ℃. The mobile phase for elution was isocratic. The mobile phase consisted of citric acid buffer （50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L octane sulfonic acid sodium salt, 0.5 mmol/L Na2EDTA and 5 mmol/L triethylamine, pH 3.8） and methanol （90：10, v/v） at a flow rate of 1.0 mL/min. The detection limit （DL） was 0.9-23 nM for all the MANTs and their metabolites with a sample volume of 50 μL. The method was shown to be highly reproducible in terms of peak area （intraday, 0.08%-1.85% RSD, n = 5）. The simultaneous measurement of these MANTs and their metabolites improved our understanding of the neurochemistry in the central nervous system （CNS） in relation to different addictive drugs （methamphetamine, heroin and their mixture） in drug-addicted rat models.

A system for screening agonists targeting β_2-adrenoceptor from Chinese medicinal herbs

In order to develop a model for screening the agonists of human β2-adrenoceptor from Chinese medicinal herbs extracts, we used a cell-based functional assay based on a common G protein-coupled receptor （GPCR） regulation mechanism and destabilized enhanced green fluorescent protein （d2EGFP） reporter gene technique. The positive cell clone was confirmed by real-time polymerase chain reaction （PCR） and imaging analysis. To assess the value of this model, we screened over 2000 high performance liquid chromatography （HPLC）-fractionated samples from the ethanol extracts of Chinese medicinal herbs. Six fractions （isolated from Panax japonicus, Veratrum nigrum, Phellodendron amurense, Fructus Aurantii lmmaturus, Chaenomeles speciosa, and Dictamnus dasycarpus） showed significant effects on active reporter gene expression, three of which （isolated from Phellodendron amurense, Fructus Aurantii lmmaturus, and Chaenomeles speciosa） were selected for further concentration response analysis and the half maximal effective concentration （EC1/2 max） values were 4.2, 2.7, and 4.8 μg/ml, respectively. Therefore, this reporter gene assay was suitable for screening β2-adrenoceptor agonists. The results suggest that the six herbal extracts are the possible agonists of β2-adrenoceptor.