Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry

Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable "capture and detection" probes, which can be prohibitively time-consuming and expensive in a drug discovery lead-optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass spectrometry (MS) to quantitate oligonucleotides. The challenges include analyte multiple charging and cation adduction (reduced sensitivity), oxidation of analyte on drying and high protein binding (low recovery), ODN affinity to exposed silica (low chromatographic reproducibility and high carryover), nonspecific binding of analyte to containers (low storage stability), and optimization/synthesis of an appropriate internal standard (interference and cross-talk). A buffer (7 mM triethylamine and 3 mM ammonium formate)/methanol, 50:50 (v/v), was used as an ESI-MS infusion solvent and produced a sharp multiple charge-state distribution. The sample extraction method combined a phenol/chloroform liquid-liquid extraction and solid-phase extraction steps, which improved the absolute recovery to >70%. The method was validated in the range of 5-2000 ng/mL and had precision (percent relative standard deviation)<10.1% and accuracy (percent relative error)<11.4%.     

Quantification of VX vapor in ambient air by liquid chromatography isotope dilution tandem mass spectrometric analysis of glass bead filled sampling tubes

An analysis method has been developed for determining low parts-per-quadrillion by volume (ppqv) concentrations of nerve agent VX vapor actively sampled from ambient air. The method utilizes glass bead filled depot area air monitoring system (DAAMS) sampling tubes with isopropyl alcohol extraction and isotope dilution using liquid chromatography coupled with a triple-quadrupole mass spectrometer (LC/MS/MS) with positive ion electrospray ionization for quantitation. The dynamic range was from one-tenth of the worker population limit (WPL) to the short-term exposure limit (STEL) for a 24 L air sample taken over a 1 h period. The precision and accuracy of the method were evaluated using liquid-spiked tubes, and the collection characteristics of the DAAMS tubes were assessed by collecting trace level vapor generated in a 1000 L continuous flow chamber. The method described here has significant improvements over currently employed thermal desorption techniques that utilize a silver fluoride pad during sampling to convert VX to a higher volatility G-analogue for gas chromatographic analysis. The benefits of this method are the ability to directly analyze VX with improved selectivity and sensitivity, the injection of a fraction of the extract, quantitation using an isotopically labeled internal standard, and a short instrument cycle time.     

Electrochemical evaluation of non-electroactive drug erythromycin in trace amount at biological samples by continuous cyclic voltammetry

A fast continuous cyclic voltammetry was used as a detection method for the monitoring of erythromycin in a flow-injection system. A special computer-based numerical calculation method (using Fast Fourier Transformation) is introduced here for enhancing the analyte signal and noise reduction. During the measurements, the potential waveform (consists of potential steps for cleaning, stripping and potential ramp) was continuously applied on an Au disk microelectrode with a 25 μm radius. The objective of the performed experiments was the inspection of the effects of different parameters on the method sensitivity. After the experiments' completion, it was concluded that the method was linear for the concentration range of 7–733,000 pg/ml (r = 0.998) with a limit of detection and quantitation 2.4 and 7 pg/ml, respectively. The stripping time was less than 200 ms. Effects of rest potential, sweep rate, and delay time on the sensitivity and selectivity of the method were investigated.     

Use of dried plasma spots in the determination of pharmacokinetics in clinical studies: validation of a quantitative bioanalytical method

A novel approach has been developed for the quantitative determination of circulating drug concentrations in clinical studies using dried plasma spots (DPS) on paper substrates, rather than conventional plasma samples. A quantitative bioanalytical high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay has been validated using paroxetine as a tool compound (range 0.2-200 ng/mL human plasma). The assay employed simple solvent extraction of a punched disk taken from the DPS sample, followed by reversed phase HPLC separation, combined with multiple reaction monitoring mass spectrometric detection. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (precision, accuracy, linearity, sensitivity, selectivity), experiments are included to assess the effect of the volume of plasma spotted and the use of an indicating paper. The validated DPS approach was successfully applied to a clinical study utilizing pooled samples and a direct comparison of DPS and plasma was made (single oral dose of 37.5 mg of paroxetine).     

基于伏安型离子选择性电极的pH检测研究

将含有电活性物质的离子选择性膜直接滴涂在玻碳电极上,制成一种新型伏安型离子选择性电极(VISE)。通过在电极上施加循环伏安信号,提取电活性物质的还原峰电位可实现待测离子的定量分析。将这种VISE应用于pH检测,分别考察VISE的pH响应特性、离子选择性、实际样检测能力以及对积累电荷的抗干扰能力。结果表明:基于循环伏安法的VISE在pH检测中具有较好的准确性、灵敏度、重复性和离子选择性,可用于实际样品的检测。相比于基于电位分析法的传统离子选择性电极(ISE),可不受电荷积累的影响,在水质的实时在线监测中具有较好的应用前景。     

超声波提取—气相色谱法测定土壤中有机氯农药残留

本文采用超声波萃取-气相色谱法测定土壤中八种有机氯类农药残留,方法简单、快速、灵敏度高,检测限在0．70-2．4μg/L之间,回收率在81．4—105．5％之间,精密度在4．08—8．52％之间。     

气相色谱法测定电子清洗液中混合醇的含量

建立了气相色谱法测定电子清洗液中混合醇含量的方法.此法具有良好的重现性和线性关系,相对标准偏差小于5%,回收率在89%～110%之间,是一种简便、快速、准确、灵敏的测定方法.     

电弧等离子体法制备纳米Ce-NiO的气敏特性

用Ｈ２＋Ａｒ电弧等离子体法制备纳米Ｃｅ－ＮｉＯ并研究其气敏特性。实验结果表明：与通常用作氧敏传感器的Ｐ型ＮｉＯ半导体材料相比，Ｈ２＋Ａｒ电弧等离子体法制备的纳米Ｃｅ－ＮｉＯ气敏材料表现出一定的ｎ型导电性，对乙炔具有很好的选择性和较高的灵敏度；其气敏特性与纳米Ｃｅ－ＮｉＯ晶粒的显微结构有关。     

Bioelectronic sniffer device for trimethylamine vapor using flavin containing monooxygenase

A bioelectronic sniffer device for trimethylamine (TMA) in the gas phase "fish-odor substance" was constructed using a flavin-containing monooxygenase 3 (FMO3, one of xeno-biotic metabolizing enzymes) and a reaction unit with both gas and liquid cells separated by a porous poly(tetrafluoroethylene) diaphragm membrane (pore size: 30-60 /spl mu/m, thickness: 0.20 mm). A substrate regeneration cycle was applied to the FMO3 immobilized device in order to amplify the output signal by coupling the monooxygenase with a reducing reagent system of ascorbic acid (ASA) in phosphate buffer. The sniffer device with 10.0 mmol/l AsA could be used to measure TMA vapor from 0.52 to 105 ppm; this covers the maximum permissible concentration in the work place (5.0 ppm of time weighted average concentration) and the sensing level-5 of smell in humans (3.0 ppm). Since the application of the substrate regeneration cycle was possibly successful, it improved the sensitivity of the FMO3 immobilized device. The sniffer device possessed high selectivity for TMA being attributable to the FMO3 substrate specificity, continuous measurability, and good reproducibility in the repeatedly measurements (coefficient of variation = 2.41%, n=10).     

A novel deep etching technology for Si and quartz materials

A disadvantage of ICP type plasma etching method compared with CCP one is low selectivity to photo resist. So a novel etching method was developed to obtain high selectivity to photo resist by incorporating a sputtering method, in which a target was placed on the opposite side of the substrate. Satisfactory results were obtained for a continuous gas feeding treatment and also for a gas modulating treatment.     

SnO_2/C复合材料的制备及NO_x气敏研究

为制备在室温条件下对NOx的检测具有低检测线、响应迅速、灵敏度高的材料,通过高压静电纺丝法制备了多孔SnO2/C复合材料,采用TEM、SEM、XRD等手段研究了复合材料形貌及结构.研究表明：SnO2/C复合材料表面存在丰富的微孔、介孔和大孔,且以10 nm左右的介孔居多.室温下（16～18℃）对SnO2/C复合材料NO...     

Nanoparticles of antimony doped tin dioxide as a liquid petroleum gas sensor: effect of size on sensitivity

The gas sensitivity exhibited by nanoparticles of 1 wt% Pd catalysed antimony doped tin dioxide (ATO) prepared by a citrate-nitrate process is reported here. The reduction of particle size to <3 nm, a dimension smaller than double the thickness of the charge depletion layer, has resulted in an exceptionally high butane sensitivity and selectivity. The sensitivity and selectivity of ATO particles of different sizes unequivocally proved that reducing the size of particles to below twice the Debye length dimension produces materials with exceptionally high sensitivity and selectivity for sensor applications. The sensitivity of the samples towards 1000 ppm butane varied in the order 98%>55%>47%, for CNP>SP>CP samples having crystallite sizes of the order of 2.4 nm to 18 nm to 25 nm, respectively. The ATO nanoparticles exhibited not only a remarkable increase in gas sensitivity of around 98% towards 1000 ppm butane at 350?°C, but also a preferential selectivity to butane compared to other gases such as CO, CO2, SO2, CH4 and H2. In addition to the exceptionally high sensitivity and selectivity, the developed sensors also exhibited an improved response time and long term stability, which are of paramount importance for practical device development.     

Study for amorphous silicon etching process using dielectric barrier discharge

Characteristics of amorphous silicon (a-Si) etching using atmospheric pressure plasma discharge had been studied. Dielectric barrier discharge (DBD) plasma with nitrogen gas was employed for the study. The active chemical agent for etching was generated by mixing a small quantity of sulfur hexafluoride (SF₆) gas into the plasma. The two distinguishable plasma zones are generated with the specially designed DBD plasma generator. The one is the main discharge zone generated between the two parallel plate electrodes. And the other one is downstream plasma zone extracted from the main discharge zone through the holes perforated on the bottom electrode. A test specimen was etched located at the plasma zone and moved the zone several times for etching on a temperature controlled stage. The etch rate of a-Si and the selectivity to silicon nitride (SiNx) were improved by addition of hydrogen (H₂) or methane (CH₄) gas into the plasma. However, when the specimen temperature was lower than 100 °C with H₂ or CH₄ gas added plasma condition, a-Si layer was not etched at all, but in the range of 100-140 °C of specimen temperature, the a-Si layer started to be etched while the influence of the specimen temperature on etching of a-Si was ignorable in that temperature range. At the optimized condition, the a-Si etch rate was up to 3000 A/min in the downstream plasma zone with the 3 mm of the distance between the surface of the specimen and the bottom side of the DBD plasma generator module. And the etch rate ratio between a-Si and SiNx was more than 100:1.     

Supercritical fluid chromatography and high-performance liquid chromatography/tandem mass spectrometric methods for the determination of cytarabine in mouse plasma

The separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by packed-column supercritical fluid chromatography (pSFC) was achieved on bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate. SFC is commonly assumed to be only applicable to nonpolar and relatively low-polarity compounds. In this work, a broader range of compound polarities amenable to pSFC with appropriate mobile-phase modifiers and additives under normal-phase retention mechanism was demonstrated. The pSFC was integrated with an atmospheric pressure chemical ionization source and a tandem mass spectrometer (MS/MS) to enhance the sensitivity, selectivity, and speed of the assay. The influence of mobile-phase components on chromatographic performance and ionization efficiency of the test compounds was investigated for improving the sensitivity and separation for the analyte and the internal standard. The pSFC-MS/MS approach requiring approximately 2.5 min/sample for the determination of ara-C at nanograms per milliliter in mouse plasma was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C obtained by the pSFC-MS/MS method were found to be consistent with those determined by various reversed-phase, high-performance liquid chromatography methods using a porous graphite carbon column, a mixed-mode column, or a C18 column in conjunction with an ion-pairing agent coupled to a tandem mass spectrometer.     

Determination of total sulfur at microgram per gram levels in geological materials by oxidation of sulfur into sulfate with in situ generation of bromine using isotope dilution high-resolution ICPMS

We have developed a new, simple, and accurate method for the determination of total sulfur at microgram per gram levels in milligram-sized silicate materials with isotope dilution high-resolution inductively coupled plasma mass spectrometry equipped with a flow injection system. In this method, sulfur can be quantitatively oxidized by bromine into sulfate with achievement of isotope equilibrium between the sample and spike. Detection limits for 32S+ and 34S+ in the ideal solution and silicate samples were 1 and 6 ng mL(-1) and 0.07 and 0.3 microg g(-1), respectively. The total blank was 46 ng, so that a 40-mg silicate sample containing 10 mirog g(-1) sulfur can be measured with a blank correction of < 10%. This total blank can be lowered to 8 ng if a low-blank air system is used for evaporations. To evaluate the applicability of this method, we analyzed not only silicate reference materials with sulfur content of 5.25-489 microg g(-1) and sample sizes of 13-40 mg but also the Allende meteorite with a sulfur content of 2%. The reproducibility for various rock types was < 9%, even though blank corrections in some samples of low sulfur content were up to 24%. This method is suitable for analyzing geological samples as well environmental samples such as soils, sediments, and water samples.     

Chemical vapor detection using a capacitive micromachined ultrasonic transducer

Distributed sensing of gas-phase chemicals using highly sensitive and inexpensive sensors is of great interest for many defense and consumer applications. In this paper we present ppb-level detection of dimethyl methylphosphonate (DMMP), a common simulant for sarin gas, with a ppt-level resolution using an improved capacitive micromachined ultrasonic transducer (CMUT) as a resonant chemical sensor. The improved CMUT operates at a higher resonant frequency of 47.7 MHz and offers an improved mass sensitivity of 48.8 zg/Hz/μm(2) by a factor of 2.7 compared to the previous CMUT sensors developed. A low-noise oscillator using the CMUT resonant sensor as the frequency-selective device was developed for real-time sensing, which exhibits an Allan deviation of 1.65 Hz (3σ) in the presence of a gas flow; this translates into a mass resolution of 80.5 zg/μm(2). The CMUT resonant sensor is functionalized with a 50-nm thick DKAP polymer developed at Sandia National Laboratory for dimethyl methylphosphonate (DMMP) detection. To demonstrate ppb-level detection of the improved chemical sensor system, the sensor performance was tested at a certified lab (MIT Lincoln Laboratory), which is equipped with an experimental chemical setup that reliably and accurately delivers a wide range of low concentrations down to 10 ppb. We report a high volume sensitivity of 34.5 ± 0.79 pptv/Hz to DMMP and a good selectivity of the polymer to DMMP with respect to dodecane and 1-octanol.     

复杂转子-轴承-汽封耦合系统的非线性振动分析

基于非线性动力学和转子动力学理论,综合考虑Muszynska非线性汽封力、非线性油膜力和转子不平衡量的耦合作用,建立了双叶轮-轴承交错布置的复杂转子-轴承-汽封系统动力学模型。采用有限元法（FEM）推导系统运动微分方程,编程计算了系统转速、圆盘偏心量、汽封长度和汽封间隙等参数对系统动力特性的影响,并利用分岔图、频谱图、相轨迹和Poincare映射图表征了系统的运动性态。研究表明：耦合系统具有高度非线性,随着参数的变化系统呈现出周期运动、倍周期运动、准周期运动和混沌运动等复杂动力学行为。通过减小圆盘偏心,增加系统汽封长度,选取合适的汽封间隙有利于提高转子-轴承-汽封系统的稳定性,改善系统的运动特性。     

Selected reaction monitoring LC-MS determination of idoxifene and its pyrrolidinone metabolite in human plasma using robotic high-throughput, sequential sample injection

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format. Method validation included determination of intra- and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy +/- 12.0% precision, 95.1 +/- 4.9%, and 90.3 +/- 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QC1 (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 +/- 14.4%, 91.1 +/- 13.0%, and 90.8 +/- 12.2%, respectively). The validated method was applied to the analysis of 613 human clinical plasma samples. An average run time of 23 s/sample (approximately 37 min/ 96-well plate or over 3,700 sample/day) was achieved. The successful validation presented indicates that rapid methods of analysis can efficiently and reliably contribute to the fast sample turnaround required for high sample number generating processes.     

采用简便方法制备快速响应的聚吡咯复合薄膜传感器

在叉指碳电极上采用原位制备的方法构筑了聚吡咯复合薄膜气敏传感器,对其在三甲基胺气氛中的电响应性能进行了研究.结果表明,对5.14×10-7mol/mL的三甲基胺,其敏感性可达3～6个数量级的变化, 远高于同等实验条件下酞菁、卟啉等及其氟代衍生物的气敏性.该薄膜传感器具有好的重现性和选择性,在室温下可用氮气完全恢复.