Determination of picogram quantities of zinc in zinc metalloproteins by atomic absorption spectrometry using a graphite furnace atomizer

Optimum operating conditions have been determined for the atomization of zinc from metalloproteins in a graphite furnace. Addition of 50 mm ammonium dihydrogen phosphate to to protein and measurement of the integrated absorbance suppresses or eliminates matrix interference effects. Using a 5-μl sample both the sensitivity and the detection limit are 0.3 ng of Zn/ml, i.e., 1.5 pg of zinc on an absolute basis. For 10 ng/ml of zinc in 5-μl samples of a zinc metalloenzyme, the coefficient of variation is 1.5%. Accuracy has been established by analysis of zinc metalloenzymes of known zinc stoichiometry. The method has been applied successfully to the determination of zinc in several proteins for which zinc stoichiometry had been unknown.     

Low-pressure,microwave-induced plasma spectrometry method for the determination of subnanogram quantities of mercury: Application to mercury-carboxypeptidase analysis

A simple, sensitive, and rapid method for the determination of subnanogram quantities of mercury has been devised using a tantalum-filament vaporization system and low-pressure, microwave-induced emission spectrometry. This method is applied to the analysis of mercury-substituted carboxypeptidase with a sample volume of 5 μl and an analysis time of 3 min. The coefficient of variation for 2 × 10⁻¹⁰ g of mercury in mercury-carboxypeptidase is 6.0%, and the detection limit is 3 × 10⁻¹² g.     

Effect of temperature on the floral scent emission and endogenous volatile profile of Petunia axillaris

The floral scent emission and endogenous level of its components in Petunia axillaris under different conditions (20, 25, 30, and 35 degrees C) were investigated under the hypothesis that floral scent emission would be regulated by both metabolic and vaporization processes. The total endogenous amount of scent components decreased as the temperature increased, the total emission showing a peak at 30 degrees C. This decrease in endogenous amount was compensated for by increased vaporization, resulting in an increase of floral scent emission from 20 degrees C to 30 degrees C. The ambient temperature differently and independently influenced the metabolism and vaporization of the scent compounds, and differences in vapor pressure among the scent compounds were reduced as the temperature increased. These characteristics suggest the operation of an unknown regulator to change the vaporization of floral scent.     

不同利用方式下石灰性紫色土的锌形态剖面分布特征初探

分析对比了4个剖面(稻田、旱地、城墙岩群组林地、蓬莱镇组林地)Zn形态的分布特征,结果表明,稻田各形态Zn的剖面分布比旱地复杂,农地土层深厚,Zn各形态分配在层次间的变化较林地复杂．DTPA-Zn(有效态锌)在表层分配的相对较高,说明作物根系层及林木根系层缺Zn突出．对各种形态在不同剖面中的分配进行了显著性分析,结果表明,农地的碳酸盐结合态锌(3．65％)、紧结有机结合态锌(2．81％)、晶形氧化铁结合态锌(22．04％)显著大于林地(1．86％、0．84％、11．59％)．     

Determination of tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum by liquid chromatography-tandem mass spectrometry

The development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the tetracationic zinc(II) phthalocyanine derivative RLP068 in rabbit serum is described. The dodecadeuterated product (RLP068-D12) was used as co-eluting internal standard. RLP068 was isolated from serum samples by solid-phase extraction using weak cationic exchange cartridges (WCX). An oxidative derivatisation was used in order to simplify the peculiar HPLC and MS behaviour of the analyte and thus increasing sensitivity. Liquid Chromatography was carried out on a Polaris C18 Ether column (50 mm x 2.0 mm) with an isocratic run of 0.5% aqueous TFA/methanol. Detection was achieved by means of a Bruker Esquire 3000+ Ion Trap Mass Spectrometer equipped with an ESI source working in positive mode. A Multiple Reaction Monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 2-65 ng/mL. lower limits of detection (LLOD) and quantification (LLOQ) were respectively 1 and 2 ng/mL. The method is innovative and applicable to pharmacokinetic studies.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Rate of pheromone release by individual spruce budworm moths

The rate and temporal pattern of pheromone emission by single and grouped female spruce budworm moths were measured by combining the trapping of pheromone on Porapak Q with a rapid luciferase bioassay developed for aldehyde pheromone. Pheromone release occurred mainly during the scotophase of a 12:12 L:D cycle in a series of bursts (up to 50 ng/hr) with considerable variability observed between insects. Analysis of over 30 individual females showed that 30% release no detectable pheromone, 60% release between 20 and 100 ng of pheromone and 10% release greater than 100 ng in a 24-hr period. Overall, a single female (1–3 days old) released an average of 60 ± 50 ng (± S.D.) of pheromone per night with a total of 260 ± 210 ng (± S.D.) being released over its life span (～7 days). Grouped females released lower quantities of pheromone. The amount of pheromone in the glands of female moths also displayed a rhythm with the levels beings higher later in the day than at the start of the photophase.     

Determination of zinc in vegetal tissue microsamples by platinum-wire loop in flame atomization atomic absorption spectrometry

The zinc content of 3 μL of vegetal samples (tree leaves, lichens and grape sap) atomized from a Pt-wire in the methane–air flame has been determined by atomic absorption spectrometry. The effect of gas flow rates and the atomization height in the flame on the absorption of zinc was evaluated at 213.9 nm. The best results were obtained at a height of 5 mm and gas flow rates of 200 L/h air and 26 L/h methane, respectively. The effect of Na, K, Ca, Mg, SO₄²⁻, and PO₄³⁻ on the absorption of zinc was studied too. The detection limit of 0.40 ± 0.21 ng was obtained at a significance level of 0.05, using the two-step Neyman–Pearson criterion. The zinc content of the samples has been determined with continuous nebulization and by atomization from the Pt-wire, using both the standard calibration curve and the standard addition method. The results of the two procedures agree within the determination errors.     

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).     

Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.     

Rapid and sensitive bioanalytical method for measurement of fluvoxamine in human serum using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent: application to a human pharmacokinetic study

A sensitive and rapid high-performance liquid chromatographic method for the analysis of fluvoxamine, a selective serotonin reuptake inhibitor in human serum, is described using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent. The drug and an internal standard (fluoxetine) were extracted from 0.25 mL of serum using ethyl acetate as extracting solvent and subjected to pre-column derivatization by the reagent. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.8) containing 1 mL/L triethylamine (72:28 v/v) was used and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6mm) column. The fluorescence derivatives of the drugs were monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. The calibration curve was linear over the concentration range of 0.5-240 ng/mL with a limit of quantification (LOQ) of 0.5 ng/mL using 0.25 mL serum sample. The method validation was performed for its selectivity, specificity, sensitivity, precision and accuracy. In this method, which was applied in a randomized cross-over bioequivalence study of two different fluvoxamine preparations in 24 healthy volunteers, the sensitivity and run time of analysis were significantly improved.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Analysis of underivatized glycosphingolipids by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Analytical conditions for underivatized glycosphingolipids by using high-performance liquid chromatography atmospheric pressure ionization mass spectrometry (HPLC/API-MS) were investigated. The analysis was performed by using an ordinary reversed-phase column (4.6 X 150 or 4.6 X 250 mm) at a flow rate of 1 ml/min. The glycosphingolipids could be characterized from the HPLC/API-MS in terms of molecular weight, ceramide composition, and partial oligosaccharide sequence. In order to obtain an adequate spectrum the amount of material needed is in the range of a few micrograms of lipid. By selected ion monitoring the sensitivity of the method allowed characterization of only 60 ng of glycosphingolipid. The method will be very useful in the characterization of small quantities of glycosphingolipids from biological samples.     

Interêts du dosage des adénosines 5′-phosphate pour l'étude de la dynamique des populations phytoplanctoniques lacustres (le Pavin - France)

A study on A.T.P., A.D.P. and A.M.P. in the phytoplankton of Lake Pavin was carried out from November 1976 to December 1977. Samples were collected at nine depths, between the surface and 50 m.A.T.P., A.D.P. and ( = A.T.P.+ A.D.P. + A.M.P.) show a similar behaviour in space and time but A.M.P. behaves independently. The A.M.P. concentration is high only when primary production of phytoplankton is high.The energy charge [CE = (ATP + % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaSGaaeaaca% aIXaaabaGaaGOmaaaaaaa!3776!\[{\raise0.7ex\hbox{$1$} \!\mathord{\left/ {\vphantom {1 2}}\right.\kern-\nulldelimiterspace}\!\lower0.7ex\hbox{$2$}}\]ADP)/(ATP + ADP + AMP)] could be used as measure of adaptation in algal populations. Indeed, the energy charge increases as algal cells experience difficulty in maintaining themselves in the biotope.

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Interêts du dosage des adénosines 5-phosphate pour l'étude de la dynamique des populations phytoplanctoniques lacustres (le Pavin - France)

     

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography-tandem mass spectrometry

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm x 2.1mm, 3 microm) with a 10 mm x 2 mm, 5 microm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.     

Visualization of bilin-linked peptides and proteins in polyacrylamide gels

Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.     

基于Cyt b基因和形态学的鼠兔属系统发育研究及鼠兔属1新亚属5新种描述

本研究扩增了169号鼠兔属样本的cyt b序列,代表13个种11亚种及5个未分类单元,下载了66条分类地位确定而完整的Cyt b序列,包括25种及9个亚种。构建了Bayesian系统发育树,计算了遗传距离,开展了物种界定分析;对13个种4亚种及5个未分类单元174号完整头骨和外形的20个形态学量度数据开展了主成分分析、判别分析和T检验;对头骨和身体外部特征进行了比较研究。结果显示：鼠兔属包含5个亚属,分别是Ochotona, Conothoa, Pika, Lagotona 和一新亚属;5个亚属间的遗传距离在12.8%-16.0%之间;高黎贡鼠兔（Ochotona gaoligongensis）是灰颈鼠兔（O. forresti）的亚种;喜马拉雅鼠兔(O. himalayana)是灰鼠兔(O. roylei)的亚种;木里鼠兔(O. muliensis)是川西鼠兔(O. gloveri)的亚种;宁夏鼠兔(O. argentata)应为蒙古鼠兔(O. pallasi)的亚种;西伯利亚鼠兔（O. turuchanensis） 为高山鼠兔(O. alpina)的同物异名。藏鼠兔循化亚种（O. thibetana xunhuaensis）应提升为种：循化鼠兔(O. xunhuaensis);东北鼠兔长白山亚种(O. hyperborea coreana)应该提升为种：长白山鼠兔(O. coreana);藏鼠兔峨眉亚种（O. thibetana sacraria）应提升为种：峨眉鼠兔（O. sacraria）; 藏鼠兔锡金亚种（O. thibetana sikimaria）应提升为种：锡金鼠兔（O. sikimaria）。通过分子系统学和形态学研究,发现了鼠兔属1 新亚属和5个新种。新亚属命名为异耳鼠兔亚属(Alienauroa subgen. n.),属模为黄龙鼠兔（O. huanglongensis sp. n.）,成员包括黄龙鼠兔新种、峨眉鼠兔、循化鼠兔,以及在龙门山和大巴山发现的2个新种,共计5个种。新亚属的鉴定特征是：耳上前缘内侧有一个密被短毛的异耳屏（Congenial tragus）;头骨扁平,颅面平直;眼小,眼眶长和眼眶宽均小于其他亚属;被毛粗长而无光泽;腹毛多为灰白色。5个新种分别发现于四川黄龙自然保护区、四川北部龙门山、四川北部大巴山、四川西部邛崃山、西藏雅鲁藏布江中游的朗县。我们分别将其命名为黄龙鼠兔（Ochotona huanglongensis sp. n.）,扁颅鼠兔（Ochotona flatcalvariam sp.n.）,大巴山鼠兔（Ochotona dabashanensis sp. n.）,雅鲁藏布鼠兔（Ochotona yarlungensis sp. n.）和邛崃鼠兔（Ochotona qionglaiensis sp. n.）。黄龙鼠兔的鉴定特征是：门齿孔前段平行;脑颅扁平,颅高为颅全长的33.5%;耳大,平均20mm以上（不含耳基部的管）,异耳屏三角形,但顶端圆形;毛长而粗糙,无光泽,背部毛长约20mm;腹面毛色以灰白为主;前后足背面灰白色,腹面黑灰色,指（趾）垫大,橘黄色,露出毛外,爪黄白色半透明。扁颅鼠兔的鉴定特征是：脑颅异常扁平,颅高约11mm,颅高仅为颅全长的31%;眼眶很小,平均6.7mm×5.6mm;个体小,体长140mm以下;体毛长而粗糙无光泽,背部毛长22mm;耳小,平均17mm左右,异耳屏阔圆型,相对较小;背毛沙黄色,腹毛黄白。大巴山鼠兔的鉴定特征是：异耳屏镰刀状;耳相对较小,均在17mm以下,和峨眉鼠兔、扁颅鼠兔差不多,比该亚属循化鼠兔和黄龙鼠兔小。颅高相对较大,颅面更隆突,颅高平均12.45mm, 眼眶相对较大,平均8.52mm×7.13mm。雅鲁藏布鼠兔的鉴定特征是：与黑唇鼠兔（O. curzoniae）和努布拉克鼠兔拉萨亚种（O. nubrica lhasaensis）有很近的亲缘关系;唇缘有很窄的深灰色圈,深灰色圈外侧为灰白色;前后足底部毛浓密,趾端毛多而长,趾垫和爪隐于毛中;个体中等,体长150mm左右,比黑唇鼠兔和努布拉克鼠兔拉萨亚种小得多;颅面较隆突,但与黑唇鼠兔相比,颅面相对平直;被毛黑褐色。邛崃鼠兔的鉴定特征是：大小及头骨和藏鼠兔很相似,但该种和藏鼠兔的显著不同是眶间宽狭窄,平均4.05mm（3.64-4.19mm）,而藏鼠兔眶间宽一般超过4.2mm,平均为4.45mm;该种毛色为沙色带黄色调,毛粗长;藏鼠兔为黑褐色调,毛相对较短;邛崃鼠兔前后足背面为显著的草黄色,而藏鼠兔前后足背面黄白色。