Regulation of prostaglandin production by nitric oxide; an in vivo analysis.

1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

HPLC法同时测定盐酸异丙肾上腺素注射液和盐酸肾上腺素注射液的含量及有关物质

目的:建立测定盐酸异丙肾上腺素注射液和盐酸肾上腺素注射液的含量及有关物质的方法。方法:采用高效液相色谱法,色谱柱:Welch Materials C18色谱柱(4.6mm×150mm,5μm);流动相:甲醇-乙腈-庚烷磺酸钠溶液(1:1:8);检测波长:280nm;流速:2.0ml·min-1;柱温:30℃。结果:盐酸异丙肾上腺素在10～40μg.ml-1质量浓度范围内与峰面积呈良好的线性关系,r=0.9999;高、中、低3种浓度的平均回收率为100.5%,RSD为0.76%(n=9)。盐酸异丙肾上腺素的最小检测限为40.66ng·ml-1。盐酸肾上腺素在60～240μg·ml-1质量浓度范围内与峰面积呈良好的线性关系,r=0.9996;高、中、低3种浓度的平均回收率为100.2%,RSD为0.60%(n=9)。盐酸肾上腺素的最小检测限为44.12ng·ml-1。结论:该法简便、快速、准确、灵敏度高,重现性好,适用于盐酸异丙肾上腺素注射液和盐酸肾上腺素注射液的含量测定,也可用于有关物质检查。     

高效液相色谱法同时测定动物血浆中萘哌地尔及其主要代谢产物浓度

为研究大鼠口服萘哌地尔的药代动力学规律,建立了同时测定萘哌地尔及其去甲基代谢产物浓度的高效液相色谱法。以甲醇、乙腈以及0.02mol·L^-1磷酸二氢钾溶液为流动相,RP-C₁₈为固定相,普萘洛尔作内标,紫外定量波长240nm。此条件下内标物、去甲基物和原药的保留时间分别为6.2,7.6和10.3min;绝对回收率分别为97.4%,86.6%和86.1%。去甲基物和原药在10～800ng·ml^-1血浆浓度范围内线性关系良好,γ=0.9998,最低检测浓度分别为8ng·ml^-1和5ng·ml^-1;日内和日间的相对标准偏差(RSD)均分别小于6.3%和11.6%(n=5)。并用此法研究了大鼠单次口服萘哌地尔后两者的药时规律。     

Dexamethasone distribution characteristic following controllable continuous sub-tenon drug delivery in rabbit

Drug delivery systems are required to be safe, minimally invasive and effectively delivery drug to the target tissues. But delivery drugs to the eye has not yet satisfied this need. Here, we focused on examining the distribution of dexamethasone (DEX) in ocular and plasmic samples following controllable continuous sub-Tenon drug delivery (CCSDD) of dexamethasone disodium phosphate (DEXP) in rabbit, and to compare that with two traditional routes: subconjunctival injection and intravenous injection. The DEX concentration was analyzed by Shimadzu LC–MS 2010 system. In CCSDD group, during observed 24?h, the mean DEX level in collected samples from highest to lowest following in order: sclera, cornea, retina/choroid, iris, plasma, aqueous humor, lens and vitreous body. In ocular solid tissue, the DEX level in posterior segment is higher than in anatomic corresponding anterior segment, but it is opposite in ocular fluid tissue. High levels of DEX were maintained at 12?h in the ocular tissue immediately after the administration. Even at 24?h, the mean DEX concentration was 31.72?ng/ml and 22.40?ng/ml in aqueous and vitreous, respectively. In CCSDD group, the ocular DEX exposure (AUC_0-24) is much higher and plasma exposure is much less than IV group, and it is also similar in SC group except iris. The amount of DEX levels are markedly increased in ocular tissues but it yield lower plasma levels indicating reduction of systemic absorption by CCSDD. Thus, CCSDD is an effective method of delivering DEX into anterior and posterior segment of the eye.     

Direct radioimmunoassay procedure for plasma dexamethasone with a sensitivity at the picogram level

A simple radioimmunoassay (RIA) for the direct quantitation of plasma dexamethasone (DEX) at the picogram level has been developed. An antiserum with high specificity and avidity was obtained by the immunization of a carefully synthesized dexamethasone-21-succinyl-thyroglobulin with a high incorporation ratio. As little as 1 pg of DEX in 50 microL of plasma sample can be detected directly by this RIA without extraction and other purification procedures. Intra- and interassay coefficients of variation were 2.1 and 3.3% for plasma levels at 2.93 ng/mL or 2.3 and 7.2% for plasma levels at 0.88 ng/mL. Blank values for plasma of normal or pre-DEX patients were always under the detection limit (20 pg/mL). Excellent linearity (r = 0.9991-0.9999) was demonstrated between the serial dilutions of six plasma samples and their corresponding DEX concentrations. In single-dose DEX (0.25-1 mg) pharmacokinetic studies, plasma DEX was consistently detectable up to 24 h post dose. Compared with existing methods, this direct RIA demonstrates superior performance with regard to simplicity, sensitivity, specificity, and reproducibility. It also enables high sample throughput and has proven robust in our hands. This assay should be readily transferable to other laboratories for clinical or research purposes.     

Determination of a catechol-O-methyltransferase inhibitor, nitecapone, in human plasma and urine by liquid chromatography

Methods based on reversed-phase liquid chromatography with amperometric detection have been developed for determination of nitecapone, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione, a COMT inhibitor, in human plasma and urine. Nitecapone was extracted with ethyl acetate-hexane mixtures from plasma after acidification with hydrochloric acid and from urine as the tetrabutylammonium ion-pair of its diphenylborate derivative. The recoveries of both methods exceeded 70% and the relative standard deviations for within-day precision were less than 4% and 8% at 50 ng ml-1 and at the quantitation limits, respectively. The methods are selective, sensitive and precise enough for determination of 4-5 ng ml-1 of nitecapone in plasma and urine and are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.     

Liquid chromatographic determination of a new catechol-O-methyltransferase inhibitor, entacapone, and its Z-isomer in human plasma and urine.

Assay procedures for analysis of entacapone, (E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)-propenamide++ +, and its Z-isomer in human plasma and urine are described. The methods were based on reversed-phase liquid chromatography with amperometric detection. Entacapone and its Z-isomer were extracted with n-hexane-ethyl acetate mixtures after acidification with hydrochloric acid. From urine extracts the analytes were back-extracted into phosphate buffer (pH 7.2). During sample treatment 1-2% of entacapone was changed to the Z-isomer. With recoveries exceeding 75% the relative standard deviations for within-day precision were less than 11% for plasma and less than 6% for urine at the quantitation limit (10 ng ml-1) and less than 6% for both methods at higher concentrations (20-2000 ng ml-1). The assays were specific with respect to all known metabolites and selective, sensitive and precise enough for determination of entacapone and its Z-isomer in plasma and urine down to 10 ng ml-1. The methods are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.     

Sensitive liquid chromatography assay for the determination of amikacin in human plasma

A selective LC method with on-line post-column derivatization is described for the determination of amikacin in biological fluids. Chromatography was performed on a reversed-phase column, using pentane sulphonic acid as an ion-pairing reagent. For the analysis of biological fluids, amikacin and the internal standard tobramycin were extracted using an ion exchanger (Sephadex). Following complete removal of plasma proteins, the aminoglycosides were eluted with alkaline sodium sulphate solution and injected into the chromatograph. After chromatographic separation the eluent was mixed with the derivatization reagent (o-phthalaldehyde and mercaptoethanol in borate buffer pH 10.4) in a reaction coil at 50 degrees C. Detection was performed by fluorescence (excitation: 340 nm, emission: 418 nm). The overall run time was 8 min, at a flow rate of 1.2 ml min-1. The limit of quantification was 25 ng ml-1 for amikacin in plasma.     

气相色谱法测定人血浆中非洛地平浓度及药代动力学

采用气相色谱—电子捕获检测法测定人血浆中非洛地平浓度,研究中国男性正常人口服该药的药代动力学规律,为临床用药提供依据。血浆样品经乙醚—正己烷(2∶1)萃取浓缩后进行测定。结果表明非洛地平浓度在0.5～10ng·ml^-1范围内线性良好(γ=0.9991)。此法简便易行,精密度好,日内、日间的RSD分别小于5.09%及8.62%。回收率平均为97.3%±4.0%。测定了10名健康者单次口服非洛地平10mg后不同时间的血药浓度并计算了相应的药代动力学参数。     

Fluoxetine bioequivalence study: quantification of fluoxetine and norfluoxetine by liquid chromatography coupled to mass spectrometry.

In this study, the authors assessed the bioequivalence of two fluoxetine tablet formulations in 24 healthy volunteers of both sexes who received a single 20 mg dose of each fluoxetine formulation, and a new sensitive method for the quantification of fluoxetine and norfluoxetine in human plasma was developed. The study was conducted using an open, randomized, two-period crossover design with a 4-week washout interval. Plasma samples were obtained over a 672-hour period. Plasma fluoxetine and norfluoxetine concentrations were analyzed by combined liquid chromatography coupled to mass spectrometry (LC-MS) with positive ion electrospray ionization using selected ion recording (SIR). Kolmogorov-Smirnov's test, histograms, probit plots, and the correlation between norfluoxetine AUC(0-infinity) and fluoxetine AUC(0-infinity) were used to analyze the population distribution. The limit of quantification was 0.15 ng.ml-1 and 0.50 ng.ml-1 for both fluoxetine and norfluoxetine, respectively. Within- and between-run imprecision was less than 13% and 17%, respectively. The pharmacokinetic parameters obtained for fluoxetine and norfluoxetine after the administration of each formulation included AUC(0-672 h), AUC(0-infinity), Cmax, Cmax/AUC(0-672 h), tmax, t1/2, and Ke. The AUC values for fluoxetine were not consistent with a normal distribution, reflecting the existence of two different populations (poor and extensive metabolizers). The mean pharmacokinetic parameters for extensive fluoxetine metabolizers were 27.0 ng ml-1 for Cmax, 2064.0 ng h ml-1 for AUC(0-infinity), and 85.4 h t1/2. The mean pharmacokinetic parameters for norfluoxetine (in extensive metabolizers only) were 2532.0 ng h ml-1 for AUC(0-infinity) and 8.4 ng ml-1 for Cmax. For fluoxetine bioequivalence, the 90% CI of the individual ratio geometric mean for Psiquial/Prozac (including both extensive and poor metabolizers) was 101.6% to 121.1% for AUC(0-672 h) and 86.1% to 102.6% for Cmax. For norfluoxetine, the 90% CI of the individual ratio geometric mean for Psiquial/Prozac (including both extensive and poor metabolizers) was 90.3% to 108.3% for AUC(0-672 h) and 84.5% to 106.3% for Cmax. The new method developed (LC-MS) presented high sensitivity, specificity, and short chromatographic run for the quantification of both fluoxetine and norfluoxetine in human plasma. Since both 90% CI for AUC and Cmax geometric mean ratios were included in the 80% to 125% interval proposed by the U.S. Food and Drug Administration, Psiquial was considered bioequivalent to Prozac according to both the rate and extent of absorption. The finding that there were no significant differences in the bioequivalence assessed by either fluoxetine or norfluoxetine pharmacokinetic parameters indicates that future bioequivalence trials may be performed by quantifying fluoxetine only.     

液相色谱-串联质谱测定米格列奈血药浓度的不确定度评价

目的:对采用液相色谱-串联质谱联用法测定米格列奈的血药浓度进行不确定度评价,以找到影响不确定度的因素,为评价检测报告提供科学依据。方法:根据《测量不确定度评定与表示》,并参考《化学分析中不确定度的评估指南》,对血浆中米格列奈浓度测定进行测量不确定度的分析与评定。结果:10 ng.ml-1米格列奈的标准不确定度为0.289 ng.ml-1,扩展不确定度为0.579 ng.ml-1,浓度测定结果可表示为(9.275±0.579)ng.ml-1。结论:建立的不确定评估方法可用于液相色谱-串联质谱联用法测定血浆中米格列奈浓度的不确定度评估。标准曲线拟合和样品前处理是主要的不确定度来源。测量不确定度的评定方法的确立对于血浆中米格列奈浓度测定方法标准的研究具有重要意义。     

Fast-LC determination of 1-methyl-1H-tetrazole-5-thiol in human plasma with column-switching sample clean-up

A simple, fast and sensitive method for the quantitative determination of 1-methyl-1H-tetrazole-5-thiol using HPLC is reported. Samples are deproteinated by plasma water filtration and injected into a HPLC system capable of column switching and backflushing the analytical column. The limit of quantitation was determined to be 70 ng ml-1 and the limit of detection 22 ng ml-1. Between-day precision (expressed as percent coefficient of variation) of standard curve slopes was +/- 3.5% with a range of within-day percent coefficient of variations from 0.28 to 1.4%. Recovery and precision of spiked control samples was 96 +/- 7% over a concentration range of 630-6300 ng ml-1.     

银杏提取物及其羟丙基-β-环糊精包合物在家兔体内的药物动力学

目的：建立测定家兔血浆中总黄酮的高效液相色谱法,研究银杏提取物-羟丙基-β-环糊精包舍物（GBE—HP-β—CD）及市售银杏提取物（GBE）在家兔灌胃后的体内药物动力学行为。方法：流动相为甲醇-0．4％磷酸溶液（50：50）,色谱柱为Diamonsil C18（250mm×4．6mm,5um）。6只家兔随机分为两组,分别灌胃市售GBE及GBE—HP-β-CD后,检测血药浓度。采用PKSolver1．0药物动力学程序处理。结果：GBE和GBE—HP—B—CD的Cmax分别为（196．73±11．71）ng·ml-1和（324．12±20．22）ng·ml-1;AUC0-∞。分别为（3013．14±230．25）ng·h·ml-1和（6336．94±167．52）ng·h·ml-1,两者差异有统计学意义（P〈0．05）。结论：该法准确、灵敏,适用于GBE血浆浓度的测定;GBE—HP-β—CD与市售GBE相比吸收显著增加。     

高效液相色谱-质谱-质谱联用法测定人血浆中艾司西酞普兰浓度及其药代动力学研究

目的建立测定人血浆中艾司西酞普兰浓度的高效液相色谱-质谱-质谱联用法.方法血浆样品经甲醇沉淀后进行分析.色谱柱Lichrospher CN柱150 mm×4.6 m I.D.5μm,流动相甲醇水(含15 mmol·L-1乙酸铵)甲酸(72∶28O.1,v/v/v),流速1.0ml·min-1,电喷雾离子化三重四极杆串联质谱检测,以选择离子反应监测(SRM)扫描方式进行检测,采用选择离子反应监测(SRM)方式进行定量分析,用于监测的离子为m/z 325.0→234.0(西酞普兰)和m/z 409.1→238.1(氨氯地平,内标).结果线性范围为0.20～50.00ng·ml-1,最低定量浓度为0.20 ng·ml-1,应用此法测试了10名健康受试者口服草酸艾司西酞普兰片(10 mg)后不同时间的血药浓度,得到艾司西酞普兰药动学参数,Cmax为9.21±2.10 ng·ml-1,Tmax为3.75±1.04h,AUC0-t为514.6±152.3 ng·h·ml-1,AUC0-∞为540.5±162.3 ng·h·ml-1,t1/2为34.06±7.71 h及Ke为0.021±O.004h-1.结论该法专属、灵敏、简便、快速,适用于人血浆中艾司西酞普兰浓度的测定.     

Liquid chromatographic-mass spectrometric method for quantitative determination of lisinopril in human plasma

A validated liquid chromatographic-mass spectrometric (LC/MS) method for the determination of lisinopril in human plasma is presented. Enalapril was used as an internal standard. After the addition of internal standard, solid phase extraction was used as a cleaning step. To separate lisinopril and enalapril from interfering endogenous plasma substances, the analysis was performed using column switching valve. The quantitative determination was performed using selected ion monitoring (+)-electrospray LC-MS. A combination of an acidic mobile phase and a reverse phase column was used. A precision in the linear range from 10 to 500.0 ng/mL plasma, absolute recovery of 91.69% for lisinopril and 90.26% for enalapril, stability for 3.5 months at -20 degrees C have been achieved. Limit of quantitation (LOQ) was 10 ng/mL while limit of detection (LOD) was about 1 ng/mL.     

Protective action of hydroxyethyl rutosides on singlet oxygen challenged cardiomyocytes.

1. The effect of a standardized mixture of beta-hydroxyethyl rutosides against oxidative damage in singlet oxygen-challenged isolated cardiac myocytes from adult rats was investigated. The morphology of the myocytes was evaluated as an indicator for cell viability (elongated, rod shaped cells vs. hypercontracted, rounded cells). The determination of the production of thiobarbituric acid reactive substances served as an indicator for lipid peroxidation. 2. Exposure to singlet oxygen which was generated by photo-excitation of rose bengal (10(-7) M) reduced the number of rod shaped (vital) cardiomyocytes by 78.5 +/- 2.5% and increased the production of thiobarbituric acid reactive substances by 1180 +/- 150% in comparison to incubation with control buffer. 3. Coincubation of the cells with beta-hydroxyethyl rutosides (concentration range: 6.7 pg ml-1 to 670 micrograms ml-1) increased the number of rod shape cardiomyocytes after exposure to singlet oxygen in a dose-dependent bell-shaped manner. A significant protective effect was observed at beta-hydroxyethyl rutosides concentrations ranging from 0.67 ng ml-1 to 67 ng ml-1. 4. In spite of their protective action, beta-hydroxyethyl rutosides did not reduce the accumulation of thiobarbituric acid reactive substances, used as an indicator for lipid peroxidation. 5. The data suggest that beta-hydroxyethyl rutosides exert a protective action against oxygen radical-induced damage of cardiac myocytes at very low concentrations without interfering with lipid peroxidation.     

Gas chromatographic determination of verapamil in plasma and urine

A gas-liquid chromatographic method for the determination of verapamil in body fluids has been developed. Verapamil and internal standard are extracted from alkalized fluid with heptane and then back extracted into 1 N HCl. After re-extraction into heptane, verapamil and internal standard are analysed by gas-liquid chromatography using a nitrogen-specific detector (N-FID). The method is specific for verapamil. Concentrations can be measured down to 4 ng ml-1 plasma. The coefficients of variation in plasma samples were 5--8% (within-day; range of concentration 5--30 ng ml-1). Recovery in this range was 100 +/- 6.2% during several weeks.     

Inhibition by dexamethasone of Langerhans cell migration: influence of epidermal cytokine signals.

The influence of dexamethasone (DEX), a synthetic glucocorticoid, on the induction in mice of Langerhans cell (LC) migration has been investigated. Systemic treatment of mice with DEX was found to inhibit significantly the ability of a topically applied contact allergen (oxazolone) to induce the migration of LC from the epidermis and their subsequent accumulation as dendritic cells (DC) in draining lymph nodes. The stimulation of LC migration during skin sensitization is dependent upon signals provided by the epidermal cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). It was found that treatment with DEX was unable to inhibit either LC migration or DC accumulation induced by the intradermal injection of TNF-alpha. In contrast, LC migration provoked by similar exposure of mice to IL-1beta (the action of which is dependent upon the de novo synthesis of TNF-alpha) was inhibited by DEX as was the arrival of DC in draining lymph nodes induced by this cytokine. Taken together, the data reported here indicate that DEX is able to inhibit very markedly the stimulation of LC migration during skin sensitization and it is proposed that such inhibition may represent an important aspect of the immunosuppressive properties of glucocorticoids and of their proven utility in the treatment of cutaneous inflammatory disorders. The results also indicate that DEX does not inhibit LC migration secondary to direct effects on cell motility. The proposal is that impaired LC migration results from the regulation by DEX of the de novo synthesis and/or release of TNF-alpha, an inducible epidermal cytokine that provides one important signal for LC to traffic from the skin.     

Determination of benzylpenicillin in plasma and lymph at the ng ml-1 level by reversed-phase liquid chromatography in combination with digital subtraction chromatography technique

A method for the determination of benzylpenicillin (Pc-G) at very low levels in plasma and lymph is described. Detection at 325 nm of the mercuric mercaptide of benzylpenicillinic acid was made by liquid chromatography via a pre-column derivatization method. By using a digital subtraction chromatography technique, the bioanalytical method could be applied to different kinds of samples whether interfering peaks were present or not. Two different clean-up steps were used for two concentration ranges 0.1-100 micrograms and 1-1000 ng Pc-G ml-1; namely, precipitation of a 100-microliters sample with acetonitrile, or precipitation of a 1- or 2-ml sample followed by concentration via liquid-liquid extraction. The pre-column derivative of Pc-G was achieved using mercury(II)chloride in the presence of imidazole. The blank samples required for the digital subtraction chromatography technique were obtained by penicillinase treatment. Standard curves were made in the two concentration ranges. The relative standard deviation (RSD) at 5 ng Pc-G ml-1 plasma was 4.9% and at 5 micrograms Pc-G ml-1 lymph it was 2.8%. The stability of Pc-G, including the problems with non-sterile samples, was studied.     

Pharmacokinetics and bioequivalence of two trimebutine formulations in healthy volunteers using desmethyl-trimebutine levels

Trimebutine tablets (dimethylamino-2-phenyl-2-n-butyl-3,4,5- trimethoxybenzoate maleate, CAS 34140-59-5, reference) and a new tablet formulation (Eurogalena, test) were administered in 24 healthy volunteers of both sexes according to a cross-over design, in a single dose of one 100 mg tablet of each formulation. Blood samples were drawn off over a 24-h period, before (time 0) and after each administration at specific intervals. Trimebutine and its main active metabolite, desmethyl-trimebutine, were measured in plasma using a validated HPLC method with UV detection. For both compounds, the sensitivity was 20 ng.ml-1 and the analytical method was proved to be linear for concentrations between 20 ng.ml-1 and 5000 ng.ml-1, with a variability less than 11%. The non-compartmental method was used for pharmacokinetic analysis. The confidence interval approach was used for comparison of the formulations according to the EU guidance note on bioavailability and bioequivalence on Cmax, AUC0-t and AUC0-infinity, log transformed. Tmax values were statistically compared using the Friedman non-parametric test. No trimebutine concentration was measured in the plasma samples. The obtained data with desmethyl-trimebutine proved the bioequivalence of the two tested formulations.