Simultaneous determination of 20 drugs of abuse in oral fluid using ultrasound-assisted dispersive liquid-liquid microextraction.

Drugs of abuse and new psychoactive substances (NPS) for recreational purposes are in constant evolution, and their consumption constitutes a significant risk to public health and road safety. The development of an analytical methodology to confirm the intake of illicit drugs in biological fluids is required for an effective control of these substances. An ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was developed for simultaneous determination of 10 synthetic cathinones and 10 illicit drugs in oral fluid easily sampled through non-invasive maneuvers. The UPLC-MS/MS method was coupled to an ultrasound-assisted dispersive liquid-liquid microextraction (US-DLLME), which is a miniaturized and inexpensive technique that uses reduced volumes of solvents and samples. The US-DLLME was optimized by using a 213441//18 asymmetric screening design and a Doehlert design. Sample volume, dispersion and extraction solvent volumes, pH, US time, and amount of sodium chloride were evaluated. The US-DLLME-UPLC-MS/MS method was validated according to international guidelines. Limits of quantitation (LOQs) ranged from 0.25 to 5 ng mL-1, and the linear range spanned from LOQ to 500 ng mL-1 with R2 higher than 0.9907, for most of the target drugs. Precision ranged from 1.7 to 14.8 %RSD. Accuracy, i.e., extraction recovery, ranged from 74 to 129%. The proposed method was successfully applied to the analysis of 15 samples from patients on a drug detoxification program.

Characterization of Pseudomonas syringae pv. actinidiae biovar 3 on kiwifruit in north-west Portugal.

AIMS: Bacterial kiwifruit canker disease, caused by Pseudomonas syringae pv. actinidiae (Psa) was detected in north-west Portugal in 2010, and has since caused significant losses. The objectives of this work were to characterize the Portuguese population(s) of Psa and to define the actual prevalence of Psa biovars in the most productive kiwifruit region in Portugal. METHODS AND RESULTS: Isolates obtained from Actinidia deliciosa orchards were characterized by morphological, biochemical, physiological, fatty acids and molecular tests (PCR, BOX-PCR, duplex-PCR, multiplex-PCR and RFLP), phaseolotoxin, housekeeping and effector genes and pathogenicity. Results established that only Psa biovar 3 is present in the north-west of Portugal, despite phenotypic and genetic variability among the isolates. CONCLUSIONS: This work provides new information on P. syringae pv. actinidiae (Psa) genetic profile in Portugal, indicating for the first time, that two genetically different subpopulations of Psa biovar 3 are present. SIGNIFICANCE AND IMPACT OF THE STUDY: A new subpopulation of Psa biovar 3 was found for the first time in Portugal, contributing to increase knowledge about this population worldwide and to support further understanding of the impact of Psa.

Detection and characterization of Pseudomonas syringae pv. actinidifoliorum in kiwifruit in Spain.

AIMS: Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is currently the major threat to its commercial production worldwide. In 2011, the most virulent type (Psa3) was detected for the first time in Northwest-Spain, in the province of Pontevedra. In 2013 surveys, leaves and flower buds with mild symptoms were observed in Actinidia deliciosa 'Hayward' vines in an orchard at the province of A Coruña, suggesting the presence of P. syringae pv. actinidifoliorum (Psaf). METHODS AND RESULTS: Isolates obtained from such orchard were characterized by morphological, biochemical and physiological tests, fatty acids (FA) profile and molecular tests (PCR, BOX-PCR, duplex PCR, multiplex PCR, real-time PCR, PCR-C, phytotoxins, housekeeping and effector genes). Pathogenicity tests were also carried out on plants and fruits of A. deliciosa 'Hayward' and on different cultivated plants and fruits. Results demonstrated the presence of P. syringae pv. actinidifoliorum in Spain. CONCLUSIONS: The work provides new information on the pathovar P. syringae pv. actinidifoliorum, which has only been found previously in New Zealand, Australia and France. SIGNIFICANCE AND IMPACT OF STUDY: The results are relevant for taxonomy of isolates of P. syringae from kiwifruit, especially those of low virulence not belonging to pathovar actinidiae.

Active biomonitoring with the moss Pseudoscleropodium purum: Comparison between different types of transplants and bulk deposition.

Active biomonitoring with terrestrial mosses can be used to complement traditional air pollution monitoring techniques. Several studies have been carried out to compare the uptake capacity of different types of moss transplants. However, until now the relationship between the uptake of elements in devitalized moss bags and in irrigated transplants has not been explored. In this study, the final concentrations of Cd, Cu, Hg, Pb and Zn were determined in irrigated and devitalized moss transplants in the surroundings of a steelworks. The concentrations were also compared with those of the same elements in the bulk deposition to determine which type of moss transplant yields the closest correlations. Devitalized moss retained higher concentrations of all of the elements (except Hg) than the irrigated moss. Both irrigated and devitalized moss transplants appear to detect the same type of contamination (i.e. particulate matter and dissolved metals rather than gaseous forms) as significant correlations were found for Cu, Hg, Pb and Zn, whereas, neither type of the moss transplant was sensitive enough to detect changes in the soluble fraction load of bulk deposition. Further studies will be needed to a better understanding of the correlation between the concentrations of elements in moss transplants with the particulate fraction of the bulk deposition. This will enable the establishment of a more robust and accurate biomonitoring tool.

Transcaval approach for aortic endoprosthesis insertion. A new anesthetic challenge.

The treatment of acute aortic syndrome has been benefited in recent years from the huge progress in endovascular techniques, compared to classical surgical treatment, by open surgery. Nevertheless, for endovascular treatment to be successful, it is essential for the patient to present adequate vascular access. Those cases with unfavourable vascular anatomy make it necessary to consider open surgery with significant morbidity, or even to reject surgery. A new approach to the abdominal aorta has recently been described as an indication for these patients with impossibility of other vascular access and absolute or relative contraindication to the transthoracic approach. The anesthetic management of the aortic syndrome is well known and, even though there are a variety of options, all of them have proven safety and efficacy. The implementation of new surgical approaches and new possible complications imply a challenge for the anesthesiologist which, for now, has little or none scientific evidence. We present the first case of transcaval aortic endoprosthesis implantation in Spain, its anesthetic implications, and a review of the literature.

Matching times: Trying to improve the correlation between heavy metal levels in mosses and bulk deposition.

The relationship between the concentrations of metals in moss tissues and atmospheric deposition is highly complex, resulting in a general lack of correlations between these two matrices. Here, we tried to improve the significance of the moss-bulk deposition (BD) relationship by eliminating the mismatch between the time that the moss tissue selected for analysis is exposed to atmospheric deposition, and the time during which BD is collected. For this, we analysed the concentrations of Cd, Hg and Pb in new grown tissue of Pseudoscleropodium purum and BD collected monthly, for one year, in 21 sampling sites (SS) under different degrees of pollution. Additionally, we assessed how different moss tissues, including native moss (green parts and new grown tissues of P. purum) and moss transplants of Sphagnum denticulatum, reflect BD to find out which moss tissues provide a better estimate of the atmospheric deposition of heavy metals. First of all, our results showed that eliminating the mismatch between native moss exposure time and BD collection period is not enough to improve their correlation. Environmental variation emerged as the main driver of tissue content variation altering the moss-BD relationship unpredictably. Secondly, native P. purum represents BD values better than devitalized transplants by displaying a greater number of significant correlations with BD. Specifically, green parts of P. purum generally represent better BD than new grown tissues. Overall, we conclude that neither native mosses nor transplants are good estimators of atmospheric heavy metal deposition rates. However, they are good qualitative indicators of the atmospheric deposition, by allowing us to differentiate SS subject to a wide range of pollution levels. Additionally, green parts of P. purum, and likely of other mosses with similar growth forms, should be used in passive biomonitoring studies to make results from different studies comparable.

A fast bioanalytical method based on microextraction by packed sorbent and UPLC-MS/MS for determining new psychoactive substances in oral fluid.

The emergence in recent years of potentially dangerous new psychoactive substances (NPS) that are not under international control has led to the development of multi-analyte procedures for their unequivocal quantification. A fast ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS), in combination with a sample pretreatment based on microextraction by packed sorbent (MEPS), was for the first time used in this work for the simultaneous determination of NPS in oral fluid. This matrix is an effective alternative to typical biological samples for drug control in substitution therapy programs, and also for the prevention and reduction of traffic accidents. The proposed method allowed the separation and quantification of eleven synthetic cathinones, six opiates, scopolamine, cocaine and two metabolites in less than 3.0min by using appropriate isotope-labelled internal standards. The MEPS procedure, which is a miniaturized version of the SPE technique, is completed within 15min. The influence of variables such as the washing solution and eluent volumes, phase type, number of aspirate-dispense cycles and pH was investigated by using a 3441//16 asymmetric screening design and a response surface methodology based on a Doehlert design. The MEPS process performed optimally with a mixed-mode C8/SCX sorbent and a sample pH of 9. The proposed method was validated according to major guidelines and found to span the linear concentration range 0.5-500ngmL-1 (R2 ≥ 0.9903), and to be selective and precise (within- and between-day precision as %RSD were both lower than 13.7%). The accuracy, in terms of analyte extraction recovery, ranged from 75% to 125% for most of the analytes. The MEPS-UPLC-MS/MS method was successfully used to analyse twelve real samples from patients on a drug detoxification programme and proved an effective tool for drug monitoring.

Analysis of drugs of abuse in human plasma using microextraction by packed sorbents and ultra-high-performance liquid chromatography.

A miniaturized and simple method based on digitally programmed microextraction by packed sorbent (eVol®-MEPS) coupled to ultra-performance liquid chromatography (UPLC) has been developed for quantitative determination of three synthetic cathinones and seven conventional drugs of abuse and metabolites. The influence of several extraction parameters, such as washing and elution solvents were tested. In addition important variables affecting MEPS performance, namely sample volume, sorbent drying time, washing solvent volume, elution volume, number of extraction cycles, sorbent phase and pH, were evaluated using an asymmetrical screening design. The optimal experimental conditions involved 300µL of plasma, loading 10×100µL of sample through a C8/SCX sorbent in a MEPS syringe placed in the semi-automatic eVol® system, washing using 150µL H2O:MeOH (90:10, v/v), drying for 0.5min and elution using 200µL dichloromethane:2-propanol:ammonium hydroxide (78:20:2, v/v/v). The drugs separation was achieved using an ACQUITY BEH Shield RP18 column (2.1mm×100mm×1.7µm) in 3min. Under optimized conditions the proposed method was validated in terms of selectivity, linearity, limits of detection (LOD) and quantitation (LOQ), precision and matrix effect, using standard addition calibration. The combination of MEPS and UPLC provides a method for the primary screening of the analytes in 18min with excellent recoveries at three concentration levels, ranging between 80 and 104% (relative standard deviation <11%). The developed methodology has been successfully applied to plasma samples from polydrug abusers.

[Rupture of left main bronchus due to a left double-lumen tube in patient with a history of radiotherapy]. / Rotura de bronquio principal izquierdo por tubo de doble luz en paciente con antecedentes de radioterapia.

Airway injury caused by double-lumen tubes is a rare but potentially serious complication. We describe the case of a patient who had a bronchial rupture during one-lung ventilation with left double-lumen tube, complicated with a secondary cardiac arrest. She had a full recovery without sequelae. Underlying causes of the patient were a history of radiotherapy, and a possible overinflation of bronchial cuff, that it could contribute to the development of this complication. The possible airway injury should be considered by all practitioners who employ double-lumen tubes for the care of their patients.

Water supply regulates structure, productivity, and water use efficiency of Acacia koa forest in Hawaii.

We studied changes in stand structure, productivity, canopy development, growth efficiency, and intrinsic water use efficiency (WUE=photosynthesis/stomatal conductance) of the native tree koa (Acacia koa) across a gradient of decreasing rainfall (2600-700 mm) with increasing elevation (700-2000 m) on the island of Hawaii. The stands were located on organic soils on either smooth (pahoehoe) or rough (aa) lava flows. In the greenhouse, we also examined growth and WUE responses to different water regimes of koa seedlings grown from seeds collected in the study area. We tested the hypotheses that (1) stand basal area, aboveground net primary productivity (ANPP), leaf area index (LAI), and growth per unit leaf area decreased with decreasing rainfall, (2) WUE increased with decreasing rainfall or water supply, and (3) WUE responses were caused by stomatal limitation rather than by nutrient limitations to photosynthesis. The carbon isotope composition of phyllode tissues (δ13C) was examined as an integrated measure of WUE. Basal area and LAI of koa stands on both pahoehoe and aa lava flows, and ANPP on aa lava flows decreased with elevation. Basal area, LAI, and ANPP of koa in mixed stands with the exotic tropical ash (Fraxinus udhei) were lower compared to single-species koa stands at similar elevations. Along the gradient, phyllode δ13C (and therefore WUE) increased with elevation from -30.2 to -26.8‰. Koa in mixed stands exhibited higher (less negative) δ13C than in single-species stands suggesting that koa and tropical ash competed for water. In the greenhouse, we observed the same trend observed in the field, as phyllode δ13C increased from -27.7 to -24‰ as water supply decreased. Instantaneous gas exchange measurements in the greenhouse showed an inverse correlation of both maximum (morning) photosynthesis (A) and conductance (g) with δ13C values and, also, a good agreement between instantaneous (A/g) and integrated measures of WUE. Phyllode δ13C was not correlated with foliar concentrations of N or other nutrients in either the field or the greenhouse, indicating that differences in δ13C were caused by stomatal limitation rather than by nutrient-related changes in photosynthetic capacity. This study provided evidence that long-term structural and growth adjustments as well as changes in WUE are important mechanisms of koa response to water limitation.

Growth, biomass allocation and photosynthesis of invasive and native Hawaiian rainforest species.

Growth, biomass allocation, and photosynthetic characteristics of seedlings of five invasive non-indigenous and four native species grown under different light regimes were studied to help explain the success of invasive species in Hawaiian rainforests. Plants were grown under three greenhouse light levels representative of those found in the center and edge of gaps and in the understory of Hawaiian rainforests, and under an additional treatment with unaltered shade. Relative growth rates (RGRs) of invasive species grown in sun and partial shade were significantly higher than those for native species, averaging 0.25 and 0.17 g g-1 week-1, respectively, while native species averaged only 0.09 and 0.06 g g-1 week-1, respectively. The RGR of invasive species under the shade treatment was 40% higher than that of native species. Leaf area ratios (LARs) of sun and partial-shade-grown invasive and native species were similar but the LAR of invasive species in the shade was, on average, 20% higher than that of native species. There were no differences between invasive and native species in biomass allocation to shoots and roots, or in leaf mass per area across light environments. Light-saturated photosynthetic rates (Pmax) were higher for invasive species than for native species in all light treatments. Pmax of invasive species grown in the sun treatment, for example, ranged from 5.5 to 11.9 µmol m-2 s-1 as compared with 3.0-4.5 µmol m-2 s-1 for native species grown under similar light conditions. The slope of the linear relationship between Pmax and dark respiration was steeper for invasive than for native species, indicating that invasive species assimilate more CO2 at a lower respiratory cost than native species. These results suggest that the invasive species may have higher growth rates than the native species as a consequence of higher photosynthetic capacities under sun and partial shade, lower dark respiration under all light treatments, and higher LARs when growing under shade conditions. Overall, invasive species appear to be better suited than native species to capturing and utilizing light resources, particularly in high-light environments such as those characterized by relatively high levels of disturbance.

Photosynthesis, carbohydrate storage and survival of a native and an introduced tree species in relation to light and defoliation.

Fraxinus uhdei (Wenz.) Lingelsh (tropical ash), a species introduced to Hawaii from Mexico, invades forests of the endemic tree Acacia koa A.Gray (koa). We examined physiological and morphological characteristics of koa and tropical ash to explore possible mechanisms that may facilitate invasion of koa forests by tropical ash. Seedlings of both species were grown in a greenhouse in three light treatments: 100% photosynthetic photon flux (PPF); 18% PPF; and 2% PPF inside the greenhouse. Light compensation point, maximum CO2 assimilation rate and dark respiration rate of seedlings differed significantly among light treatments, but were similar between species. A defoliation experiment indicated that tropical ash was better able to survive defoliation than koa, especially under high-light conditions. Tropical ash seedlings allocated more carbon (C) and nitrogen (N) to storage per unit PPF than koa seedlings. Total nonstructural carbohydrates were positively correlated with plant survival in both species. The patterns of C and N allocation associated with tropical ash seedlings favor their survival in high light, under intense herbivory and on sites where N availability is seasonal or highly variable. Variation in carbohydrate storage between koa and tropical ash greatly exceeded variation in photosynthetic performance at the leaf level.

Aquatic toxicity of two Corexit dispersants.

The oil spill dispersants, Corexit 9500 and Corexit 9527 have low to moderate toxicity to most aquatic species in laboratory tests. Toxicity estimates are significantly affected by test variables such as species, lifestage, exposure duration, and temperature. Aquatic toxicity data generated from spiked, declining exposures (107 min half-life) are more reflective of actual dispersant use conditions. Decisions to use oil spill response chemicals should not be based solely on aquatic toxicity. Factors to consider include product effectiveness, toxicity of dispersed oil, species/habitats requiring priority protection, and recovery potential of sensitive habitats and populations. An environmental risk assessment approach is recommended where dispersant toxicity data generated under environmentally relevant exposures are compared to estimated environmental concentrations of dispersants.

[Trends in the prevalence of tuberculosis among six-year-olds in Albacete (Spain)]. / Evolución de la prevalencia de la infección tuberculosa en la población escolar de 6 años en Albacete.

OBJECTIVES: To analyze the prevalence and evolution of tuberculous infection among 6-year-olds in Albacete and its relation to socioeconomic status of the family. METHOD: A tuberculin test (2UT RT-23 Tween 80) was given to 6-year-old school children in and around Albacete in 1992 and 1999. We calculated the prevalence of tuberculosis and the relation to family socioeconomic level as assessed by the parents' level of education of the area of residence. RESULTS: The tuberculin test was given to 2,783 children, 1,532 in 1992 (8% bacille-Calmette-Guerin-vaccinated) and 1,251 in 1999. The prevalence of tuberculosis infection among non-vaccinated children was 0.78% in 1992 (3.37% among vaccinated children, p = 0.012) and 0.72% in 1999. No significant differences between the two screenings were found except for the vaccinated and non-vaccinated children. The annual decline was 1.1% and the annual decrease in risk of tuberculosis infection was 0.12%. No significant differences related to level of parental education or area of residence were detected. CONCLUSIONS: The prevalence of tuberculosis infection among 6-year-old school children in Albacete is low, although there is a very slight non-significant downward trend, which may be biased by the inclusion of bacille-Calmette-Guerin-vaccinated children. No differences related to family socioeconomic level were found.

[Necrotizing fasciitis as complication of percutaneous endoscopic gastrostomy]. / Fascitis necrotizante como complicación de gastrostomía endoscópica percutánea.

Necrotizing fasciitis (NF) is a rare clinical entity. We describe a case associated with a percutaneous endoscopic gastrotomy (PEG).

[Utility of the clinical history, physical examination and radiography in the localization of bleeding in patients with hemoptysis]. / Utilidad de la historia clínica, la exploración física y la radiografía en la localización del sangrado de los pacientes con hemoptisis.

OBJECTIVE: We examined the medical history, physical examination and chest radiography utility to accurately identifying the site of pulmonary bleeding in patients with hemoptysis. METHODS: We prospectively reviewed and compared the suspected site of bleeding obtained with the medical history, physical examination and chest radiography (right or left) in 466 patients with hemoptysis after the confirmation with a bronchoscopy, computed chest tomography (CT) or bronchial arteriography, and separately analysing the more common etiologies and the volume of bleeding. RESULTS: Age 62.6 years (DS 14), 85% males, 80% smokers with a volume of bleeding of 42.5 ml/day (DS 86) and > or = 100 ml/day in 13.5%. Medical history localized the site of bleeding in 1-13% (p < 0.0001), clinical responses in 8-29% (p < 0.0001), physical examination in 13-47.5% (p < 0.0001) and chest radiography in 14.5-88% (p = 0.04), with a more frequent accurately location findings (p < 0.01) that gradually increased as the previous results with the lung carcinomas and decreased with bronchiectasis or chronic bronchitis. When the volume of bleeding was > or = 100 ml/day, clinical responses utility improved (p = 0.04) as when it was < 100 ml/day with the radiography (p = 0.0001). Specificity, sensitivity and predictive values were variable and better with the radiography than with the medical history or physical examination. CONCLUSIONS: We concluded that chest radiography was most useful than the medical history or physical examination to localize the site of bleeding in patients with hemoptysis. Almost all of the findings that suggests the site of bleeding were accurate and they increased with the radiography or decreased with the physical examination and specially with the medical history in patients with bronchiectasis or chronic bronchitis.

[Neurological complications associated with ultrasound-guided interscalene and supraclavicular block in elective surgery of the shoulder and arm. Prospective observational study in a university hospital]. / Complicaciones neurológicas asociadas a bloqueos interescalénico y supraclavicular guiados con ecografía en cirugía electiva de hombro y extremidad superior. Estudio observacional prospectivo en un hospital universitario.

OBJECTIVES: The incidence of postoperative neurological symptoms after performing interscalene block varies between 4 and 16%. The majority of cases are resolved spontaneously within a year, but some patients have their symptoms permanently. Our objective was to assess the incidence of postoperative neurological symptoms after performing the ultrasound-assisted interscalene and supraclavicular anaesthetic blocks. MATERIAL AND METHODS: A prospective and observational study was conducted on consecutive patients who had undergone upper extremity surgery with an interscalene or supraclavicular block as an isolated technique, or as a complement to general anaesthesia. Seven days after the intervention, a telephone interview was conducted that focused on the detection of neurological symptoms in the operated limb. Further serial interviews were conducted on patients with symptoms (after the first, the third and the sixth month, and one year after surgery) until resolution of symptoms. Neurological evaluation was offered to those patients with persistent symptoms after one year. RESULTS: A total of 121 patients were included, on whom 96 interscalene blocks and 22 supraclavicular blocks were performed. Postoperative neurological symptoms were detected in 9.9% (95% CI, 5-15%) of patients during the first week. No significant differences were observed between interscalene (9%) and supraclavicular block (14%). After 3 months the symptoms persisted in 9 patients (7.4%), with symptoms remaining in 4 patients (3.3%) after 1.5 years. Electromyogram was performed on 3 patients who tested positive for nerve damage. CONCLUSIONS: A high incidence of postoperative neurological symptoms was observed, and a worrying percentage of permanence of them. There were no significant differences in incidence according to the type of block, or any features of the patient or the anaesthesia technique that were associated with the incidence of these symptoms, except a marginal relationship with age. These complications must be clearly explained to the patients before performing these blocks.

Distinguishing metal bioconcentration from particulate matter in moss tissue: testing methods of removing particles attached to the moss surface.

Accurate differentiation of the proportion of bioconcentrated metals (i.e. incorporated into cells) and the proportion that is not bioconcentrated (i.e. adsorbed at the surface) would lead to a better understanding of the uptake processes and would represent an advance in the use of mosses as biomonitors. Traditionally the methods used to remove contaminants that are not bioconcentrated were to wash the plant material with water or to apply the sequential elution technique, but nowadays both options are considered inaccurate for these purposes. The remaining possibilities are to clean the moss samples with a nitrogen jet or by power ultrasound. Samples of terrestrial moss Pseudoscleropodium purum (Hewd.) Fleisch. were collected from five sampling stations. Different nitrogen jet cleaning procedures and ultrasound cleaning procedures were applied to the mosses. To determine whether any of the treatments altered the membrane integrity of the moss samples, the concentrations of K were determined. The shoots were observed under a scanning electron microscope, and the size and number of particles were determined. Nitrogen jet cleaning was determined to be unacceptable because it damaged the phyllids and/or altered the membrane permeability and did not eliminate the particles from the moss surface. Moreover, ultrasound cleaning treatment should also discarded because of the loss of extracellular metals that are transferred to the water in which the moss is cleaned.

Liquid chromatography coupled to ion trap-tandem mass spectrometry to evaluate juvenile hormone III levels in bee hemolymph from Nosema spp. infected colonies.

It has been described a fast, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure juvenile hormone III (JH III), which was used to study of the effects of Nosema spp. infection on JH III levels in bee hemolymph. Honey bee hemolymph was extracted by centrifugation and mixed with a solution of phenylthiourea in methanol. This mixture was then centrifuged and the supernatant removed and evaporated to dryness. The residue was reconstituted in methanol containing the internal standard (methoprene) and injected onto an LC-MS/MS (ion-trap) system coupled to electrospray ionization (ESI) in positive mode. Chromatography was performed on a Synergi Hydro-RP column (4 µm, 30 mm × 4.60 mm i.d.) using a mobile phase of 20 mM ammonium formate and methanol in binary gradient elution mode. The method was fully validated and it was found to be selective, linear from 15 to 14,562 pg/µL, precise and accurate, with %RSD values below 5%. The limits of detection and quantification were: LOD, 6 pg/µL; LOQ, 15 pg/µL. Finally, the proposed LC-MS/MS method was used to analyze JH III levels in the hemolymph of worker honey bees (Apis mellifera iberiensis) experimentally infected with different Nosema spp. (Nosema apis, Spanish and Dutch Nosema ceranae strains). The highest concentrations of JH III were detected in hemolymph from bees infected with Spanish N. ceranae.

Moss bag biomonitoring: a methodological review.

Although the moss bag technique has been used for active biomonitoring for the past 40years, there is still no standardized protocol that enables application of the technique as a tool to monitor air quality. The aim of this review paper is to evaluate the degree of standardization of each of the variables that must be considered in applying the technique (i.e. the variables associated with preparation of the moss and moss bags, exposure of the bags, and post-exposure treatment). For this purpose, 112 scientific papers that report the methods used in applying the moss bag technique were consulted. Finally, on the basis of the conclusions reached, we propose a protocol that will enable each of these variables to be investigated separately, with the final aim of developing a standardized methodology.