New synthetic bile acid analogue agonists of FXR and TGR5 receptors: Analytical methodologies for the study of their physico-chemical properties, pharmacokinetic activity and metabolism.

This thesis reports an integrated analytical approach for the study of physicochemical and biological properties of new synthetic bile acid (BA) analogues agonists of FXR and TGR5 receptors. Structure-activity data were compared with those previous obtained using the same experimental protocols on synthetic and natural occurring BA. The new synthetic BA analogues are classified in different groups according also to their potency as a FXR and TGR5 agonists: unconjugated and steroid modified BA and side chain modified BA including taurine or glycine conjugates and pseudo-conjugates (sulphonate and sulphate analogues). In order to investigate the relationship between structure and activity the synthetic analogues where admitted to a physicochemical characterization and to a preliminary screening for their pharmacokinetic and metabolism using a bile fistula rat model. Sensitive and accurate analytical methods have been developed for the quali-quantitative analysis of BA in biological fluids and sample used for physicochemical studies. Combined High Performance Liquid Chromatography Electrospray tandem mass spectrometry with efficient chromatographic separation of all studied BA and their metabolites have been optimized and validated. Analytical strategies for the identification of the BA and their minor metabolites have been developed. Taurine and glycine conjugates were identified in MS/MS by monitoring the specific ion transitions in multiple reaction monitoring (MRM) mode while all other metabolites (sulphate, glucuronic acid, dehydroxylated, decarboxylated or oxo) were monitored in a selected-ion reaction (SIR) mode with a negative ESI interface by the following ions. Accurate and precise data where achieved regarding the main physicochemical properties including solubility, detergency, lipophilicity and albumin binding . These studies have shown that minor structural modification greatly affect the pharmacokinetics and metabolism of the new analogues in respect to the natural BA and on turn their site of action, particularly where their receptor are located in the enterohepatic circulation.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

Reactions of atomic and molecular ions with acetone, 1,1,1- trifluoroacetone, and hexafluoroacetone: An investigation of the effects of molecular structure on the dynamics and kinetics of ion-molecule reactions

A selected ion flow tube study of the reactions of a series of gas-phase atomic cations (S⁺, Xe⁺, O⁺, Kr⁺, N⁺, Ar⁺ and Ne⁺) and molecular ions (SF _n⁺ (n = 1-5), CF_n⁺ (n = 1-3), CF₂Cl⁺, H₃O⁺, NO⁺, N ₂O⁺, CO₂⁺, CO⁺, and N₂⁺) spanning a large range of recombination energies (6.3-21.6 eV), with acetone, 1,1,1-trifluoroacetone, and hexafluoroacetone has been undertaken with the objective of exploring the nature of the reaction ion chemistry as the methyl groups in acetone are substituted for CF₃. The reaction rate coefficients and product ion branching ratios for all 66 reactions, measured at 298 K, are reported. The experimental reaction rate coefficients are compared to theoretically calculated collisional values. Several distinct reaction processes were observed among the large number of reactions studied, including charge transfer (non-dissociative and dissociative), abstraction, ion-molecule associations and, in the case of the reactions involving the reagent ion H₃O⁺, proton transfer. 

Rapid continuous chemical analysis of meat chicken shed emissions by SIFT–MS

Assessing and addressing odour impacts from poultry production is extremely difficult and subjective because the odorants involved and their dynamics over time and space are poorly understood. This knowledge gap is due, in part, to the lack of suitable analytical tools for measuring and monitoring odorants in the field. The emergence of Selected Ion Flow Tube – Mass Spectrometry (SIFT–MS) and similar instruments is changing that. These tools can rapidly quantify targeted odorants in ambient air in real time, even at very low concentrations. Such data is essential for developing better odour abatement strategies, assessment methods and odour dispersion models. This project trialled a SIFT–MS to determine its suitability for assessing the odorants in meat chicken shed emissions over time and space. This report details evaluations in New Zealand and Australia to determine the potential of SIFT–MS as a tool for the chicken meat industry, including odour measurement (as a proxy for dynamic olfactometry). The report is specifically targeted at those funding and conducting poultry odour research. It will be of interest to those involved with environmental odour monitoring and assessment in general. The high upfront cost of SIFT–MS will lead to potential users wanting compelling evidence that SIFT–MS will meet their needs before they invest in one.

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.

Anxiety-like Effects Of Meta-chlorophenylpiperazine In Paradoxically Sleep-deprived Mice.

Chlorophenylpiperazines (CPP) are psychotropic drugs used in nightclub parties and are frequently used in a state of sleep deprivation, a condition which can potentiate the effects of psychoactive drugs. This study aimed to investigate the effects of sleep deprivation and sleep rebound (RB) on anxiety-like measures in mCPP-treated mice using the open field test. We first optimized our procedure by performing dose-effect curves and examining different pretreatment times in naïve male Swiss mice. Subsequently, a separate cohort of mice underwent paradoxical sleep deprivation (PSD) for 24 or 48h. In the last experiment, immediately after the 24h-PSD period, mice received an injection of saline or mCPP, but their general activity was quantified in the open field only after the RB period (24 or 48h). The dose of 5mgmL(-1) of mCPP was the most effective at decreasing rearing behavior, with peak effects 15min after injection. PSD decreased locomotion and rearing behaviors, thereby inhibiting a further impairment induced by mCPP. Plasma concentrations of mCPP were significantly higher in PSD 48h animals compared to the non-PSD control group. Twenty-four hours of RB combined with mCPP administration produced a slight reduction in locomotion. Our results show that mCPP was able to significantly change the behavior of naïve, PSD, and RB mice. When combined with sleep deprivation, there was a higher availability of drug in plasma levels. Taken together, our results suggest that sleep loss can enhance the behavioral effects of the potent psychoactive drug, mCPP, even after a period of rebound sleep.

Quantification of free mycophenolic acid by high-performance liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 mul) using ultrafiltration, The analytes were extracted from the ultrafiltrate (200 mul) using C-18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 mug/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 mug/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

More about the role of 2,6-dichlorophenol in tick courtship: identification and olfactometer bioassay in Amblyomma cajennense and Rhipicephalus sanguineus

This study aimed to identify 2,6-dichlorophenol (2,6-DCP) in Amblyomma cajennense and to evaluate its role in A. cajennense and Rhipicephalus sanguineus courtship. Hexanic extract from attractive females was purified by solid phase extraction and the phenol was identified by the single ion monitoring method using GC/MS. In an olfactometer, the responses of A. cajennense and R. sanguineus males to females, control rubber septa or rubber septa impregnated with 2,6-DCP at 50, 500, and 5000 ng, respectively, were studied. 2,6-DCP was identified in A. cajennense extract and the males oriented themselves toward the concentration of 500 ng. These septa and the females were recognized as copula partners. The septa treated with 2,6-DCP did not attract and were not even recognized by the R. sanguineus males, whereas the females were recognized. Due to the presence of 2,6-DCP in A. cajennense and the results of biological bioassays, it was concluded that this compound acts as an attractant and mounting sex pheromone in this tick, but it does not play any role in R. sanguineus courtship.

Use of accuracy profile for the validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method: quantification of ethyl glucuronide in hair

Introduction: Ethylglucuronide (EtG) is a direct and specific metabolite of ethanol. Its determination in hair is of increasing interest for detecting and monitoring alcohol abuse. The quantification of EtG in hair requires analytical methods showing highest sensitivity and specificity. We present a fully validated method based on gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS). The method was validated using French Society of Pharmaceutical Sciences and Techniques (SFSTP) guidelines which are based on the determination of the total measurement error and accuracy profiles. Methods: Washed and powdered hair is extracted in water using an ultrasonic incubation. After purification by Oasis MAX solid phase extraction, the derivatized EtG is detected and quantified by GC-NCI-MS/MS method in the selected reaction monitoring mode. The transitions m/z 347 / 163 and m/z 347 / 119 were used for the quantification and identification of EtG. Four quality controls (QC) prepared with hair samples taken post mortem from 2 subjects with a known history of alcoholism were used. A proficiency test with 7 participating laboratories was first run to validate the EtG concentration of each QC sample. Considering the results of this test, these samples were then used as internal controls for validation of the method. Results: The mean EtG concentrations measured in the 4 QC were 259.4, 130.4, 40.8, and 8.4 pg/mg hair. Method validation has shown linearity between 8.4 and 259.4 pg/mg hair (r2 > 0.999). The lower limit of quantification was set up at 8.4 pg/mg. Repeatability and intermediate precision were found less than 13.2% for all concentrations tested. Conclusion: The method proved to be suitable for routine analysis of EtG in hair. GC-NCI-MS/MS method was then successfully applied to the analysis of EtG in hair samples collected from different alcohol consumers.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.