A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N′-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

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.

Proactive motivation and engagement in career behaviors: Investigating direct, mediated, and moderated effects

Proactive career behaviors become increasingly important in today's career environment, but little is known about how and when motivational patterns affect individual differences. In a six-month longitudinal study among German university students (Study 1; N = 289) it was demonstrated that motivation in terms of "can do" (self-efficacy and context beliefs), "reason to" (autonomous career goals), and "energized to" (positive affect) significantly predicted career behaviors. Contrary to expectation, negative context beliefs had a positive effect when combined with other motivational states. Study 2 replicated and extended those results by investigating whether "can do" motivation mediates the effect of proactive personality and whether those effects are conditional upon the degree of career choice decidedness. We tested a moderated multiple mediation model with a unique sample of 134 German students, assessed three times, each interval being 6 weeks apart. The results showed that effects of proactivity were partially carried through higher self-efficacy beliefs but not context beliefs. Supporting a moderation model, indirect effects through self-efficacy beliefs were not present for students with very low decidedness.

Simultaneous determination of antidementia drugs in human plasma: Procedure transfer from HPLC-MS to UPLC-MS/MS.

A previously developed high performance liquid chromatography mass spectrometry (HPLC-MS) procedure for the simultaneous determination of antidementia drugs, including donepezil, galantamine, memantine, rivastigmine and its metabolite NAP 226-90, was transferred to an ultra performance liquid chromatography system coupled to a tandem mass spectrometer (UPLC-MS/MS). The drugs and their internal standards ([(2)H(7)]-donepezil, [(13)C,(2)H(3)]-galantamine, [(13)C(2),(2)H(6)]-memantine, [(2)H(6)]-rivastigmine) were extracted from 250μL human plasma by protein precipitation with acetonitrile. Chromatographic separation was achieved on a reverse phase column (BEH C18 2.1mm×50mm; 1.7μm) with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile at a flow rate of 0.4mL/min and an overall run time of 4.5min. The analytes were detected on a tandem quadrupole mass spectrometer operated in positive electrospray ionization mode, and quantification was performed using multiple reaction monitoring. The method was validated according to the recommendations of international guidelines over a calibration range of 1-300ng/mL for donepezil, galantamine and memantine, and 0.2-50ng/mL for rivastimgine and NAP 226-90. The trueness (86-108%), repeatability (0.8-8.3%), intermediate precision (2.3-10.9%) and selectivity of the method were found to be satisfactory. Matrix effects variability was inferior to 15% for the analytes and inferior to 5% after correction by internal standards. A method comparison was performed with patients' samples showing similar results between the HPLC-MS and UPLC-MS/MS procedures. Thus, this validated UPLC-MS/MS method allows to reduce the required amount of plasma, to use a simplified sample preparation, and to obtain a higher sensitivity and specificity with a much shortened run-time.

iLOGP: A Simple, Robust, and Efficient Description of n-Octanol/Water Partition Coefficient for Drug Design Using the GB/SA Approach.

The n-octanol/water partition coefficient (log Po/w) is a key physicochemical parameter for drug discovery, design, and development. Here, we present a physics-based approach that shows a strong linear correlation between the computed solvation free energy in implicit solvents and the experimental log Po/w on a cleansed data set of more than 17,500 molecules. After internal validation by five-fold cross-validation and data randomization, the predictive power of the most interesting multiple linear model, based on two GB/SA parameters solely, was tested on two different external sets of molecules. On the Martel druglike test set, the predictive power of the best model (N = 706, r = 0.64, MAE = 1.18, and RMSE = 1.40) is similar to six well-established empirical methods. On the 17-drug test set, our model outperformed all compared empirical methodologies (N = 17, r = 0.94, MAE = 0.38, and RMSE = 0.52). The physical basis of our original GB/SA approach together with its predictive capacity, computational efficiency (1 to 2 s per molecule), and tridimensional molecular graphics capability lay the foundations for a promising predictor, the implicit log P method (iLOGP), to complement the portfolio of drug design tools developed and provided by the SIB Swiss Institute of Bioinformatics.

Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies.

Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include a dagger(9)-tetrahydrocannabinol (THC), 11-hydroxy-a dagger(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), a dagger(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-a dagger(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and a dagger(9)-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal (TM) device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex (TM) column packed with 2.6-mu m core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000 (TM) triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection

¿Favorece la publicación en abierto el impacto de los artículos científicos? Un estudio empírico en el ámbito de la medicina intensiva

Peer-reviewed

Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry.

A highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of buprenorphine and its major metabolite norbuprenorphine in human plasma. In order to speed up the process and decrease costs, sample preparation was performed by simple protein precipitation with acetonitrile. To the best of our knowledge, this is the first application of this extraction technique for the quantification of buprenorphine in plasma. Matrix effects were strongly reduced and selectivity increased by using an efficient chromatographic separation on a sub-2μm column (Acquity UPLC BEH C18 1.7μm, 2.1×50mm) in 5min with a gradient of ammonium formate 20mM pH 3.05 and acetonitrile as mobile phase at a flow rate of 0.4ml/min. Detection was made using a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The procedure was fully validated according to the latest Food and Drug Administration guidelines and the Société Française des Sciences et Techniques Pharmaceutiques. Very good results were obtained by using a stable isotope-labeled internal standard for each analyte, to compensate for the variability due to the extraction and ionization steps. The method was very sensitive with lower limits of quantification of 0.1ng/ml for buprenorphine and 0.25ng/ml for norbuprenorphine. The upper limit of quantification was 250ng/ml for both drugs. Trueness (98.4-113.7%), repeatability (1.9-7.7%), intermediate precision (2.6-7.9%) and internal standard-normalized matrix effects (94-101%) were in accordance with international recommendations. The procedure was successfully used to quantify plasma samples from patients included in a clinical pharmacogenetic study and can be transferred for routine therapeutic drug monitoring in clinical laboratories without further development.

Comprehensive Study of Crystal Growth from Solution

Crystal growth is an essential phase in crystallization kinetics. The rate of crystal growth provides significant information for the design and control of crystallization processes; nevertheless, obtaining accurate growth rate data is still challenging due to a number of factors that prevail in crystal growth. In industrial crystallization, crystals are generally grown from multi-componentand multi-particle solutions under complicated hydrodynamic conditions; thus, it is crucial to increase the general understanding of the growth kinetics in these systems. The aim of this work is to develop a model of the crystal growth rate from solution. An extensive literature review of crystal growth focuses on themodelling of growth kinetics and thermodynamics, and new measuring techniques that have been introduced in the field of crystallization. The growth of a singlecrystal is investigated in binary and ternary systems. The binary system consists of potassium dihydrogen phosphate (KDP, crystallizing solute) and water (solvent), and the ternary system includes KDP, water and an organic admixture. The studied admixtures, urea, ethanol and 1-propanol, are employed at relatively highconcentrations (of up to 5.0 molal). The influence of the admixtures on the solution thermodynamics is studied using the Pitzer activity coefficient model. Theprediction method of the ternary solubility in the studied systems is introduced and verified. The growth rate of the KDP (101) face in the studied systems aremeasured in the growth cell as a function of supersaturation, the admixture concentration, the solution velocity over a crystal and temperature. In addition, the surface morphology of the KDP (101) face is studied using ex situ atomic force microscopy (AFM). The crystal growth rate in the ternary systems is modelled on the basis of the two-step growth model that contains the Maxwell-Stefan (MS) equations and a surface-reaction model. This model is used together with measuredcrystal growth rate data to develop a new method for the evaluation of the model parameters. The validation of the model is justified with experiments. The crystal growth rate in an imperfectly mixed suspension crystallizer is investigatedusing computational fluid dynamics (CFD). A solid-liquid suspension flow that includes multi-sized particles is described by the multi-fluid model as well as by a standard k-epsilon turbulence model and an interface momentum transfer model. The local crystal growth rate is determined from calculated flow information in a diffusion-controlled crystal growth regime. The calculated results are evaluated experimentally.

Prediction of individual automobile RBNS claim reserves in the context of Solvency II

Automobile bodily injury (BI) claims remain unsettled for a long time after the accident. The estimation of an accurate reserve for Reported But Not Settled (RBNS) claims is therefore vital for insurers. In accordance with the recommendation included in the Solvency II project (CEIOPS, 2007) a statistical model is here implemented for RBNS reserve estimation. Lognormality on empirical compensation cost data is observed for different levels of BI severity. The individual claim provision is estimated by allocating the expected mean compensation for the predicted severity of the victim’s injury, for which the upper bound is also computed. The BI severity is predicted by means of a heteroscedastic multiple choice model, because empirical evidence has found that the variability in the latent severity of injured individuals travelling by car is not constant. It is shown that this methodology can improve the accuracy of RBNS reserve estimation at all stages, as compared to the subjective assessment that has traditionally been made by practitioners.

Commodity Price Driven Risk Management from Viewpoint of High Grade Steel Alloys

The study of price risk management concerning high grade steel alloys and their components was conducted. This study was focused in metal commodities, of which nickel, chrome and molybdenum were in a central role. Also possible hedging instruments and strategies for referred metals were studied. In the literature part main themes are price formation of Ni, Cr and Mo, the functioning of metal exchanges and main hedging instruments for metal commodities. This section also covers how micro and macro variables may affect metal prices from the viewpoint of short as well as longer time period. The experimental part consists of three sections. In the first part, multiple regression model with seven explanatory variables was constructed to describe price behavior of nickel. Results were compared after this with information created with comparable simple regression model. Additionally, long time mean price reversion of nickel was studied. In the second part, theoretical price of CF8M alloy was studied by using nickel, ferro-chrome and ferro-molybdenum as explanatory variables. In the last section, cross hedging possibilities for illiquid FeCr -metal was studied with five LME futures. Also this section covers new information concerning possible forthcoming molybdenum future contracts as well. The results of this study confirm, that linear regression models which are based on the assumption of market rationality, are not able to reliably describe price development of metals at issue. Models fulfilling assumptions for linear regression may though include useful information of statistical significant variables which have effect on metal prices. According to the experimental part, short futures were found to incorporate the most accurate information concerning the price movements in the future. However, not even 3M futures were able to predict turning point in the market before the faced slump. Cross hedging seemed to be very doubtful risk management strategy for illiquid metals, because correlations coefficients were found to be very sensitive for the chosen time span.

A sensitive and robust lc-ms/ms method with monolithic column and electrospray ionization for the quantitation of efavirenz in human plasma: application to a bioequivalence study

An LC-MS/MS method has been developed for the determination of efavirenz (EFZ) in human plasma using hydrochlorothiazide as internal standard (I.S.). An ESI negative mode with multiple reaction-monitoring was used monitoring the transitions m/z 313.88→69.24 (EFZ) and 296.02→204.76 (I.S.). Samples were extracted using liquid-liquid extraction. The total run time was 2.0 min. The separation was achieved with HPLC-RP using a monolithic column. The assay was linear in the concentration range of 100 - 5000 ng mL-1. The mean recovery was 83%. Intra- and inter-day precision were < 9.5% and < 8.9%, respectively and accuracy was in the range ± 8.33%. The method was successfully applied to a bioequivalence study.

Validação de método multirresíduo para determinação de pesticidas em alimentos empregando QuEChERS E UPLC-MS/MS

This paper presents a practical and rapid method which was validated for simultaneous quantification and confirmation of 29 pesticides in fruits and vegetables using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted following the method known as QuEChERS. Using the developed chromatographic conditions, the pesticides can be separated in less than 9 min. Two multiple reaction monitoring (MRM) assays were used for each pesticide. Four representative matrices (lettuce, tomato, apple and grapes) were selected to investigate the effect in recoveries and precision. Typical recoveries ranged from 70-120%, with relative standard deviation (RSDs) lower than 20%.

Determination of hydrochlorothiazide in pharmaceutical formulations by diffuse reflectance spectroscopy

This paper describes a method for quantitative spot test analysis of hydrochlorothiazide using diffuse reflectance spectroscopy. The reflectance measurements were performed analyzing the colored compound (l = 585 nm) produced from the reaction between hydrochlorothiazide and p-dimethylaminocinnamaldehyde (PDAC) in acid medium. This reaction occurred on filter paper after heating to 80ºC for 8 minutes. Factorial designs allowed varying multiple reaction factors simultaneously in order to obtain the best reaction conditions. These factors included heating temperature, heating time, acid volume and PDAC volume. The linearity was studied in the range of 3.36x10-2 to 1.01x10-1 mol L-1 with a correlation coefficient of 0.998. The limit of detection was estimated to be 1.32x10² mol L-1. Commercial samples were analyzed using the proposed method and the results were favorably compared with those of the United States Pharmacopeia method, showing that quantitative spot test analysis by diffuse reflectance could be successfully used to determine hydrochlorothiazide in medicines.