Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD

An HPLC-DAD method for the quantitative analysis of Δ(9)-tetrahydrocannabinol (THC), Δ(9)-tetrahydrocannabinolic acid-A (THCA-A), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. For determination of the THC content of plant material, this method combines quantitation of THCA-A, which is the inactive precursor of THC, and free THC. Plant material was dried, homogenized and extracted with methanol by ultrasonication. Chromatographic separation was achieved with a Waters Alliance 2695 HPLC equipped with a Merck LiChrospher 60 RP-Select B (5μm) precolumn and a Merck LiChroCart 125-4 LiChrospher 60 RP-Select B (5μm) analytical column. Analytes were detected and quantified using a Waters 2996 photo diode array detector. This method has been accepted by the public authorities of Switzerland (Bundesamt für Gesundheit, Federal Office of Public Health), and has been used to analyse 9092 samples since 2000. Since no thermal decarboxylation of THCA-A occurs, the method is highly reproducible for different cannabis materials. Two calibration ranges are used, a lower one for THC, CBN and CBD, and a higher one for THCA-A, due to its dominant presence in fresh plant material. As provider of the Swiss proficiency test, the robustness of this method has been tested over several years, and homogeneity tests even in the low calibration range (1%) show high precision (RSD≤4.3%, except CBD) and accuracy (bias≤4.1%, except CBN).

AN EVALUATION OF THE CONSISTENCY OF IMRT PATIENT-SPECIFIC QA TECHNIQUES

To ensure the integrity of an intensity modulated radiation therapy (IMRT) treatment, each plan must be validated through a measurement-based quality assurance (QA) procedure, known as patient specific IMRT QA. Many methods of measurement and analysis have evolved for this QA. There is not a standard among clinical institutions, and many devices and action levels are used. Since the acceptance criteria determines if the dosimetric tools’ output passes the patient plan, it is important to see how these parameters influence the performance of the QA device. While analyzing the results of IMRT QA, it is important to understand the variability in the measurements. Due to the different form factors of the many QA methods, this reproducibility can be device dependent. These questions of patient-specific IMRT QA reproducibility and performance were investigated across five dosimeter systems: a helical diode array, radiographic film, ion chamber, diode array (AP field-by-field, AP composite, and rotational composite), and an in-house designed multiple ion chamber phantom. The reproducibility was gauged for each device by comparing the coefficients of variation (CV) across six patient plans. The performance of each device was determined by comparing each one’s ability to accurately label a plan as acceptable or unacceptable compared to a gold standard. All methods demonstrated a CV of less than 4%. Film proved to have the highest variability in QA measurement, likely due to the high level of user involvement in the readout and analysis. This is further shown by how the setup contributed more variation than the readout and analysis for all of the methods, except film. When evaluated for ability to correctly label acceptable and unacceptable plans, two distinct performance groups emerged with the helical diode array, AP composite diode array, film, and ion chamber in the better group; and the rotational composite and AP field-by-field diode array in the poorer group. Additionally, optimal threshold cutoffs were determined for each of the dosimetry systems. These findings, combined with practical considerations for factors such as labor and cost, can aid a clinic in its choice of an effective and safe patient-specific IMRT QA implementation.

Particle size spectrum analysis during Maria S. Merian cruise MSM26, spring 2013

A Laser In-Situ Scattering Transmissometer (LISST) was used to collect vertical distribution data of particles from 2.5 to 500 µm in size. The LISST uses a multi-ring detector to measure scattering light of particles from a laser diode. Particles are classified into 32 log-spaced bins and the concentration of each bin is calculated as micro-liters per liter (µl/l). The instrument is rated to a depth of 300 m, and also records temperature and pressure. The sample interval was set to record every second. The LISST was attached to the LOPC frame to conduct casts and allow for particle-size comparisons between the two instruments. The LOPC is rated to a depth of 2000 m, thus a short deployment to a depth of 300 m was first conducted with both instruments. The instruments were then returned to the deck and the LISST removed via a quick release bracket so deep LOPC casts could be continued at a station. Raw LISST size-spectrum data is presented as concentrations for each of the 32 size bins for every second of the cast.

Pseudo-random single photon counting for space-borne atmospheric sensing applications

The ability to accurately observe the Earth's carbon cycles from space gives scientists an important tool to analyze climate change. Current space-borne Integrated-Path Differential Absorption (IPDA) Iidar concepts have the potential to meet this need. They are mainly based on the pulsed time-offlight principle, in which two high energy pulses of different wavelengths interrogate the atmosphere for its transmission properties and are backscattered by the ground. In this paper, feasibility study results of a Pseudo-Random Single Photon Counting (PRSPC) IPDA lidar are reported. The proposed approach replaces the high energy pulsed source (e.g. a solidstate laser), with a semiconductor laser in CW operation with a similar average power of a few Watts, benefiting from better efficiency and reliability. The auto-correlation property of Pseudo-Random Binary Sequence (PRBS) and temporal shifting of the codes can be utilized to transmit both wavelengths simultaneously, avoiding the beam misalignment problem experienced by pulsed techniques. The envelope signal to noise ratio has been analyzed, and various system parameters have been selected. By restricting the telescopes field-of-view, the dominant noise source of ambient light can be suppressed, and in addition with a low noise single photon counting detector, a retrieval precision of 1.5 ppm over 50 km along-track averaging could be attained. We also describe preliminary experimental results involving a negative feedback Indium Gallium Arsenide (InGaAs) single photon avalanche photodiode and a low power Distributed Feedback laser diode modulated with PRBS driven acoustic optical modulator. The results demonstrate that higher detector saturation count rates will be needed for use in future spacebourne missions but measurement linearity and precision should meet the stringent requirements set out by future Earthobserving missions.

The spatial and temporal representation of a tone on the guinea pig basilar membrane

In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites across the width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25- to 1.40-mm length of BM centered on the CF place.

X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation.

The x-ray structure of carbon monoxide (CO)-ligated myoglobin illuminated during data collection by a laser diode at the wavelength lambda = 690 nm has been determined to a resolution of 1.7 A at T = 36 K. For comparison, we also measured data sets of deoxymyoglobin and CO-ligated myoglobin. In the photon-induced structure the electron density associated with the CO ligand can be described by a tube extending from the iron into the heme pocket over more than 4 A. This density can be interpreted by two discrete positions of the CO molecule. One is close to the heme iron and can be identified to be bound CO. In the second, the CO is dissociated from the heme iron and lies on top of pyrrole ring C. At our experimental conditions the overall structure of myoglobin in the metastable state is close to the structure of a CO-ligated molecule. However, the iron has essentially relaxed into the position of deoxymyoglobin. We compare our results with those of Schlichting el al. [Schlichting, I., Berendzen, J., Phillips, G. N., Jr., & Sweet, R. M. (1994) Nature 317, 808-812], who worked with the myoglobin mutant (D122N) that crystallizes in the space group P6 and Teng et al. [Teng, T. Y., Srajer, V. & Moffat, K. (1994) Nat. Struct. Biol. 1, 701-705], who used native myoglobin crystals of the space group P2(1). Possible reasons for the structural differences are discussed.

Determinação de vitaminas antioxidantes em suplementos e avaliação da rotulagem nutricional

Introdução: Diante das mudanças nos hábitos de consumo alimentar da população brasileira, suplementos vitamínicos e alimentos enriquecidos são veículos comumente empregados para atender as necessidades de ingestão de micronutrientes. A diversidade de suplementos vitamínicos comercializados atualmente leva à necessidade de desenvolvimento de métodos analíticos de fácil execução e alta produtividade. Informações confiáveis sobre os teores de vitaminas poderão ser obtidas somente com métodos analíticos validados. Objetivos: Validar metodologias analíticas e avaliar o teor de vitaminas antioxidantes em suplementos adquiridos no comércio do município de São Paulo - Brasil, o efeito do armazenamento nestes compostos e confrontar os valores analisados com os valores declarados na rotulagem. Métodos: As metodologias analíticas para determinação de vitaminas antioxidantes por cromatografia líquida de alta eficiência com detector de arranjo de diodos (CLAE-DAD) e de vitamina C por titulação potenciométrica foram validadas para as matrizes sólidas, oleosas e líquidas de suplementos vitamínicos. A estabilidade das vitaminas foi avaliada a cada 6 meses durante 12 meses de armazenamento e a avaliação da rotulagem foi realizada de acordo com as legislações vigentes no Brasil. Resultados: Para os métodos cromatográficos, os limites de detecção (LDs) e de quantificação (LQs) variaram entre 0,3 e 4,3 µg/mL, e entre 0,5 e 14,0 µg/mL respectivamente. As recuperações dos padrões adicionados nas matrizes variaram entre 92 por cento e 109 por cento e entre 86 por cento e 108 por cento no material de referência. A repetitividade foi calculada pelo desvio padrão relativo (RSD), apresentando valores entre 0,2 por cento e 9,6 por cento . Para a determinação de vitamina C pelo método potenciométrico, o LD e o LQ foram respectivamente 1 mg e 3 mg; a recuperação no material de referência foi de 99,8 por cento e a precisão variou entre 0,4 e 3,9 por cento . Das 57 amostras avaliadas, 59 por cento e 35 por cento apresentaram teores de vitaminas A e E respectivamente, abaixo dos valores declarados no rótulo; por outro lado, 20 por cento das amostras apresentaram teores de vitamina E acima dos valores declarados. Em relação aos teores de vitamina C, 60 por cento das amostras estavam de acordo com os valores declarados. O estudo da estabilidade demonstrou degradação significativa das vitaminas A, E e C em aproximadamente 90 por cento das amostras com 12 meses de armazenamento. Na avaliação da rotulagem dos suplementos vitamínicos, 47 das amostras apresentaram uma ou mais irregularidades. Conclusão: Os métodos propostos se mostraram adequados para análise de diferentes matrizes de suplementos vitamínicos. Os resultados das análises de vitaminas nestes produtos mostraram a necessidade urgente de monitoramento em conjunto com ações de fiscalização, pois verificou-se que a maioria das amostras não atenderam a legislação, principalmente quanto aos teores declarados na informação nutricional da rotulagem. A sobredosagem de vitaminas pode ser necessária para manter os teores declarados durante o armazenamento, porém, a quantidade adicional de vitamina a ser incluída no suplemento deve estar dentro de limites seguros e depende de cada amostra, pois além da matriz, diversos fatores relacionados aos compostos e à embalagem também podem influenciar na estabilidade das vitaminas.

Self-mixing-based demodulation technique for dynamic fiber Bragg grating strain sensors

We report a novel demodulation scheme for the detection of small Bragg wavelength shifts in a fiber Bragg grating strain sensor by exploiting the optical feedback reflected from the grating structure back into a 1310 nm laser diode integrating a photodiode. The dynamic strain generated by a mechanical vibrator is applied transversely to the fiber Bragg grating and the desired longitudinal strain values inferred from the detected sawtooth-like optical feedback signals. Preliminary results demonstrate the feasibility of this demodulation technique for strain measurement which could be further extended to fiber Bragg grating-based sensors for the detection of different measurands in general.

Self-mixing-based demodulation technique for dynamic fiber Bragg grating strain sensors

We report a novel demodulation scheme for the detection of small Bragg wavelength shifts in a fiber Bragg grating strain sensor by exploiting the optical feedback reflected from the grating structure back into a 1310 nm laser diode integrating a photodiode. The dynamic strain generated by a mechanical vibrator is applied transversely to the fiber Bragg grating and the desired longitudinal strain values inferred from the detected sawtooth-like optical feedback signals. Preliminary results demonstrate the feasibility of this demodulation technique for strain measurement which could be further extended to fiber Bragg grating-based sensors for the detection of different measurands in general.

Tunable master-oscillator power-amplifier based on chirped quantum-dot structures

A broadly tunable master-oscillator power-amplifier (MOPA) picosecond optical pulse source is demonstrated, consisting of an external cavity passively mode-locked laser diode with a tapered semiconductor amplifier. By employing chirped quantum-dot structures on both the oscillator's gain chip and amplifier, a wide tunability range between 1187 and 1283 nm is achieved. Under mode-locked operation, the highest output peak power of 4.39 W is achieved from the MOPA, corresponding to a peak power spectral density of 31.4 dBm/nm. © 1989-2012 IEEE.

Avaliação da atividade anti-inflamatória do extrato aquoso, frações e compostos identificados nos frutos de Hancornia Speciosa Gomes (Apocynaceae)

Hancornia speciosa Gomes (Apocynaceae), popularly known as ‘mangabeira’, has been used in folk medicine to treat inflammatory disorders, hypertension, dermatitis, diabetes, liver diseases and stomach disorders. Regarding the Hancornia speciosa fruits, the ethnobotany indicates its use especially for treating inflammation and tuberculosis. However, no study has been done so far to prove such biological activities. The objective was evaluation anti-inflammatory activity from the fruits of Hancornia speciosa Gomes (mangabeira). Aqueous extract was prepared by decoction, subsequently submitted the liquid-liquid fractionation. The secondary metabolites were identified by high performance liquid chromatography coupled with detector diode array (HPLC-DAD) and liquid chromatography diode array detector coupled with mass spectrometry (LC-DAD-MS). The anti-inflammatory properties of the aqueous extract, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions of the fruits from H. speciosa, as well as rutin and chlorogenic acid were investigated using in vitro and in vivo models. In vivo tests comprised the xylene-induced ear edema that was measured the formation of edema, carrageenan-induced peritonitis was evaluated the total leukocytes at 4h and zymosan-induced air pouch was measured the total leukocytes and differential cell count at 6, 24 and 48 hours, whereas in vitro tests were evaluated levels of cytokines IL-1β, IL-6, IL-12 and TNF-α using ELISA obtained of carrageenan-induced peritonitis model. The results showed the presence of rutin and chlorogenic acid were detected in the aqueous extract from H. speciosa fruits by HPLC-DAD and LC-DAD-ME. Furthermore, the aqueous extracts and fractions, as well as rutin and chlorogenic acid significantly inhibited the xilol-induced ear edema and reduced cell migration in the animal models such as carrageenan-induced peritonitis and zymosan-induced air pouch. In addition, reduced levels of cytokines IL-1β, IL-6, IL-12 and TNF-α were observed. This is the first study that demonstrated the anti-inflammatory effect of aqueous extract from Hancornia speciosa fruits against different inflammatory agents in animal models, suggesting that their bioactive molecules, especially rutin and chlorogenic acid contributing, at least in part, to the anti-inflammatory effect of aqueous extract. These findings support the widespread use of Hancornia speciosa in popular medicine and demonstrate that this aqueous extract has therapeutic potential for the development of a herbal drugs with anti-inflammatory properties.

Microphytobenthos composition in Heron Reef, Australia, by High Performance Liquid Chromatography (HPLC) (July 2012)

We quantified pigment biomarkers by high performance liquid chromatography (HPLC) to obtain a broad taxonomic classification of microphytobenthos (MPB) (i.e. identification of dominant taxa). Three replicate sediment cores were collected at 0, 50 and 100 m along transects 5-9 in Heron Reef lagoon (n=15) (Fig. 1). Transects 1-4 could not be processed because the means to have the samples analysed by HPLC were not available at the time of field data collection. Cores were stored frozen and scrapes taken from the top of each one and placed in cryovials immersed in dry ice. Samples were sent to the laboratory (CSIRO Marine and Atmospheric Research, Hobart, Australia) where pigments were extracted with 100% acetone during fifteen hours at 4°C after vortex mixing (30 seconds) and sonication (15 minutes). Samples were then centrifuged and filtered prior to the analysis of pigment composition with a Waters - Alliance HPLC system equipped with a photo-diode array detector. Pigments were separated using a Zorbax Eclipse XDB-C8 stainless steel 150 mm x 4.6 mm ID column with 3.5 µm particle size (Agilent Technologies) and a binary gradient system with an elevated column temperature following a modified version of the Van Heukelem and Thomas (2001) method. The separated pigments were detected at 436 nm and identified against standard spectra using Waters Empower software. Standards for HPLC system calibration were obtained from Sigma (USA) and DHI (Denmark).

Influence of serum levels of vitamins A, D, and E as well as vitamin D receptor polymorphisms on micronucleus frequencies and other biomarkers of genotoxicity in workers exposed to formaldehyde

Background/Aim: Formaldehyde is classified as carcinogenic to humans, making it a major concern, particularly in occupational settings. Fat-soluble vitamins, such as vitamins A, D, and E, are documented as antigenotoxic and antimutagenic and also correlate with the cell antioxidant potential. This study investigates the influence of these vitamins on genotoxicity biomarkers of formaldehyde-exposed hospital workers. Methods: The target population were hospital workers exposed to formaldehyde (n = 55). Controls were nonexposed individuals (n = 80). The most used genotoxicity biomarkers were the cytokinesis-block micronucleus assay for lymphocytes and the micronucleus test for exfoliated buccal cells. Vitamins A and E were determined by high-performance liquid chromatography with a diode array detector (HPLC-DAD) and vitamin D receptor (VDR) polymorphisms by real-time PCR. Results: Significant correlations were found between genotoxicity biomarkers and between vitamins A and E in controls. Multiple regression showed that vitamin A was significantly associated with a higher mean of nucleoplasmic bridges (p < 0.001), and vitamin E was significantly associated with a decreased frequency of nuclear buds (p = 0.045) in the exposed group. No effect of vitamin D was observed. The VDRBsmI TT genotype carriers presented higher means of all the genotoxicity biomarkers; however, we found no significant associations. Conclusions: The study suggests that vitamin levels may modulate direct signs of genotoxicity.

Design of experiments to optimized the determination of TDIs in agglomerated cork stoppers

Isocyanates are included into a class with an extreme commercial importance because their use in the manufacture of polyurethanes. Polyurethanes are used in several applications such as adhesives, coatings, foams, thermoplastics resins, printing inks, foundry moulds and rubbers. Agglomerated cork stoppers are currently used for still wines, semi-sparkle and gaseous wines, beer and cider. Methylene diphenyl diisocyanate (MDI) is presently the isocyanate used in the production of polyurethane based adhesive in use due to its lowest toxicity comparing with toluene diisocyanate (TDI) previously employed. However, free monomeric TDI or MDI, depending on the based polyurethane, can migrate from agglomerated cork stoppers to beverages therefore it needs to be under control. The presence of these compounds are usually investigated by HPLC with Fluorescence or UV-Vis detector depending on the derivatising agent. Ultra Performance Liquid Chromatography with Diode Array Detector (UPLC-DAD) method is replacing HPLC. The objective of this study is to determine which method is better to analyze isocyanates from agglomerated cork stoppers, essentially TDI to quantify its monomer. A Design of Experiments (DOE) with three factors, column temperature, flow and solvent, at two levels was done. Eight experiments with three replications and two repetitions were developed. Through an ANOVA the significance of the factors was evaluated and the best level’s factors were selected. As the TDI has two isomers and in this method these two isomers were not always separated an ANOVA with results of resolution between peaks was performed. The Design of Experiments reveals to be a suitable statistical tool to determine the best conditions to quantified free isocyanates from agglomerated cork stoppers to real foodstuff. The best level’s factors to maximize area was column temperature at 30ºC, flow to 0,3 mL/min and solvent 0,1% Ammonium Acetate, to maximize resolution was the same except the solvent that was 0,01% Ammonium Acetate.