Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Determination of Vasopressin and Desmopressin in urine by means of liquid chromatography coupled to quadrupole time-of-flight mass spectrometry for doping control purposes.

The anti-diuretic neurohypophysial hormone Vasopressin (Vp) and its synthetic analogue Desmopressin (Dp, 1-desamino-vasopressin) have received considerable attention from doping control authorities due to their impact on physiological blood parameters. Accordingly, the illicit use of Desmopressin in elite sport is sanctioned by the World Anti-Doping Agency (WADA) and the drug is classified as masking agent. Vp and Dp are small (8-9 amino acids) peptides administered orally as well as intranasally. Within the present study a method to determine Dp and Vp in urinary doping control samples by means of liquid chromatography coupled to quadrupole high resolution time-of-flight mass spectrometry was developed. After addition of Lys-Vasopressin as internal standard and efficient sample clean up with a mixed mode solid phase extraction (weak cation exchange), the samples were directly injected into the LC-MS system. The method was validated considering the parameters specificity, linearity, recovery (80-100%), accuracy, robustness, limit of detection/quantification (20/50 pg mL(-1)), precision (inter/intra-day<10%), ion suppression and stability. The analysis of administration study urine samples collected after a single intranasal or oral application of Dp yielded in detection windows for the unchanged target analyte for up to 20 h at concentrations between 50 and 600 pg mL(-1). Endogenous Vp was detected in concentrations of approximately 20-200 pg mL(-1) in spontaneous urine samples obtained from healthy volunteers. The general requirements of the developed method provide the characteristics for an easy transfer to other anti-doping laboratories and support closing another potential gap for cheating athletes.

A reusable smart interface for gas sensor resistance measurement

The advances of the semiconductor industry enable microelectromechanical systems sensors, signal conditioning logic and network access to be integrated into a smart sensor node. In this framework, a mixed-mode interface circuit for monolithically integrated gas sensor arrays was developed with high-level design techniques. This interface system includes analog electronics for inspection of up to four sensor arrays and digital logic for smart control and data communication. Although different design methodologies were used in the conception of the complete circuit, high-level synthesis tools and methodologies were crucial in speeding up the whole design cycle, enhancing reusability for future applications and producing a flexible and robust component.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring.

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000ng/ml for fluoxetine and fluvoxamine, and 2-1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

Cohesive Elements for Shells

A cohesive element for shell analysis is presented. The element can be used to simulate the initiation and growth of delaminations between stacked, non-coincident layers of shell elements. The procedure to construct the element accounts for the thickness offset by applying the kinematic relations of shell deformation to transform the stiffness and internal force of a zero-thickness cohesive element such that interfacial continuity between the layers is enforced. The procedure is demonstrated by simulating the response and failure of the Mixed Mode Bending test and a skin-stiffener debond specimen. In addition, it is shown that stacks of shell elements can be used to create effective models to predict the inplane and delamination failure modes of thick components. The results indicate that simple shell models can retain many of the necessary predictive attributes of much more complex 3D models while providing the computational efficiency that is necessary for design

Phase diagram and complex patterns in the modeling of the bromate-oxalic acid-Ce-acetone oscillating reaction

Simulations have been carried out on the bromate - oxalic acid - Ce(IV) - acetone oscillating reaction, under flow conditions, using Field and Boyd's model (J. Phys. Chem. 1985, 89, 3707). Many different complex dynamic behaviors were found, including simple periodic oscillations, complex periodic oscillations, quasiperiodicity and chaos. Some of these complex oscillations can be understood as belonging to a Farey sequence. The many different behaviors were systematized in a phase diagram which shows that some regions of complex patterns were nested with one inside the other. The existence of almost all known dynamic behavior for this system allows the suggestion that it can be used as a model for some very complex phenomena that occur in biological systems.

Co-eletrodeposição oscilatória de cobre e estanho

It is investigated in the present contribution the oscillatory co-electrodeposition of CuSn on a polycrystalline gold surface in the presence of Triton X-100 surfactant and citric acid as additive, in acidic media. The experiments were conducted under potentiostatic control and the system dynamics characterized in terms of the morphology and stability of the current oscillations. Besides modulations in the frequency and amplitude of the current oscillations, several patterned states were observed, including relaxation-like and mixed mode oscillations. The oscillations were found to be very robust and some time series presented regular motions up to about two hours.

Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa

This review first discusses the limitations of many of the supports and stationary phases used in reversed phase high performance liquid chromatography and then describes those, developed more recently, that present better stabilities and more versatile selectivities. Emphases will be given to stationary phases that use higher purity silicas, hybrid silicas, monolithic silicas, metallic oxides and mixed oxides as supports and those that have embedded polar groups or contain phenyl or fluoro groups as the stationary phase as well as the phases used for mixed mode or hydrophilic interaction separations. These modern stationary phases facilitate the analysis of complex mixtures.

Memristive Computing

Memristive computing refers to the utilization of the memristor, the fourth fundamental passive circuit element, in computational tasks. The existence of the memristor was theoretically predicted in 1971 by Leon O. Chua, but experimentally validated only in 2008 by HP Labs. A memristor is essentially a nonvolatile nanoscale programmable resistor — indeed, memory resistor — whose resistance, or memristance to be precise, is changed by applying a voltage across, or current through, the device. Memristive computing is a new area of research, and many of its fundamental questions still remain open. For example, it is yet unclear which applications would benefit the most from the inherent nonlinear dynamics of memristors. In any case, these dynamics should be exploited to allow memristors to perform computation in a natural way instead of attempting to emulate existing technologies such as CMOS logic. Examples of such methods of computation presented in this thesis are memristive stateful logic operations, memristive multiplication based on the translinear principle, and the exploitation of nonlinear dynamics to construct chaotic memristive circuits. This thesis considers memristive computing at various levels of abstraction. The first part of the thesis analyses the physical properties and the current-voltage behaviour of a single device. The middle part presents memristor programming methods, and describes microcircuits for logic and analog operations. The final chapters discuss memristive computing in largescale applications. In particular, cellular neural networks, and associative memory architectures are proposed as applications that significantly benefit from memristive implementation. The work presents several new results on memristor modeling and programming, memristive logic, analog arithmetic operations on memristors, and applications of memristors. The main conclusion of this thesis is that memristive computing will be advantageous in large-scale, highly parallel mixed-mode processing architectures. This can be justified by the following two arguments. First, since processing can be performed directly within memristive memory architectures, the required circuitry, processing time, and possibly also power consumption can be reduced compared to a conventional CMOS implementation. Second, intrachip communication can be naturally implemented by a memristive crossbar structure.

FAT-luokkien määritys hitsatuille levyliitoksille käyttämällä lineaarisen murtumismekaniikan särönkasvun simulointia

Työssä tutkittiin hitsattujen levyliitosten väsymiskestävyyden mitoitusarvoja. Hitsien väsymiskestävyyden mitoitusarvot määritettiin lineaarista murtumismekaniikkaa soveltavalla 2D FEM-laskentaohjelmalla. Murtumismekaanisen laskennan tuloksista määriteltiin, eri liitosgeometrioiden ja kuormitustyyppien mukaisia, nimellisen jännityksen väsymismitoitusmenetelmää vastaavia FAT-luokkia, joissa on huomioitu rakenteellinen jännitys hitsiä vastaan kohtisuorassa suunnassa. Tutkittujen liitosten geometriat olivat pääsääntöisesti poikkeavia mitoitusstandardien ja ohjeiden sisältämistä taulukkotapauksista. Laskennassa otettiin huomioon hitsien liittymiskulma perusaineeseen, rajaviivan pyöristykset ja vajaa hitsautumissyvyys. Kuormitustyyppien vaihtelua tutkittiin rakenteellisen jännityksen taivutusosuuden muutoksilla ja kuormaa kantavien X-liitosten risteävien kuormituksien suhteellisilla suuruuksilla. Väsymiskestävyydet määritettiin kuormituskohtaisille kalvo- ja taivutusjännityksille sekä näiden jännitysjakaumien keskiarvoille. Työssä saatuja FAT-luokkia voidaan hyödyntää vastaavien geometrioiden ja kuormitusten yhteydessä, sekä interpoloimalla myös tuloksien väliarvoissa. Työssä käytetyillä menetelmillä voidaan parantaa nimellisen jännityksen mitoitusmenetelmän tarkkuutta ja laajentaa sitä koskemaan myös taulukkotapausten ulkopuolisia liitoksia. Työn tuloksissa on esitetty FAT-luokkia T-, X- ja päittäisliitoksille ja näiden eri kuormitusyhdistelmille.

Développement d’une nouvelle méthode d’analyse multi-résidus par LDTD/APCI-MS/MS pour la quantification de pesticides et de produits pharmaceutiques dans les eaux usées

Une nouvelle méthode d'extraction en phase solide (SPE) couplée à une technique d'analyse ultrarapide a été développée pour la détermination simultanée de neuf contaminants émergents (l'atrazine, le déséthylatrazine, le 17(béta)-estradiol, l'éthynylestradiol, la noréthindrone, la caféine, la carbamazépine, le diclofénac et le sulfaméthoxazole) provenant de différentes classes thérapeutiques et présents dans les eaux usées. La pré-concentration et la purification des échantillons a été réalisée avec une cartouche SPE en mode mixte (Strata ABW) ayant à la fois des propriétés échangeuses de cations et d'anions suivie d'une analyse par une désorption thermique par diode laser/ionisation chimique à pression atmosphérique couplée à la spectrométrie de masse en tandem (LDTD-APCI-MS/MS). La LDTD est une nouvelle méthode d'introduction d'échantillon qui réduit le temps total d'analyse à moins de 15 secondes par rapport à plusieurs minutes avec la chromatographie liquide couplée à la spectrométrie de masse en tandem traditionnelle (LC-MS/MS). Plusieurs paramètres SPE ont été évalués dans le but d'optimiser l'efficacité de récupération lors de l'extraction des analytes provenant des eaux usées, tels que la nature de la phase stationnaire, le débit de chargement, le pH d'extraction, le volume et la composition de la solution de lavage et le volume de l'échantillon initial. Cette nouvelle méthode a été appliquée avec succès à de vrais échantillons d'eaux usées provenant d'un réservoir de décantation primaire. Le recouvrement des composés ciblés provenant des eaux usées a été de 78 à 106%, la limite de détection a été de 30 à 122 ng L-1, alors que la limite de quantification a été de 88 à 370 ng L-1. Les courbes d'étalonnage dans les matrices d'eaux usées ont montré une bonne linéarité (R2 > 0,991) pour les analytes cibles ainsi qu’une précision avec un coefficient de variance inférieure à 15%.

An investigation into the mechanics of cutting using data from orthogonally cutting Nylon 66

Cutting force data for Nylon 66 has been examined in terms of various different models of cutting. Theory that includes significant work of separation at the tool tip was found to give the best correlation with experimental data over a wide range of rake angles for derived primary shear plane angle. A fracture toughness parameter was used as the measure of the specific work of separation. Variation in toughness with rake angle determined from cutting is postulated to be caused by mixed mode separation at the tool tip. A rule of mixtures using independently determined values of toughness in tension (mode 1) and shear (mode 11) is found to describe well the variation with rake angle. The ratio of modes varies with rake angle and, in turn, with the primary shear plane angle. Previous suggestions that cutting is a means of experimentally determining fracture toughness are now seen to be extended to identify the mode of fracture toughness as well.