A multi-analyte LC-MS/MS method for the determination of 57 pharmaceuticals and illicit drugs in plasma, and its application to poisoning cases.

The diagnostic methods in an emergency scenario must be simple, fast, and efficient to provide an effectiveness and efficient treatment, thus reducing the consequences of exposure. Considering the sample analysis, the protein precipitation combined with LC-MS/MS has been shown to be a good strategy for the simultaneous determination of compounds of toxicological interest, such as medicines and drugs of abuse. In this study, a rapid and simple multi-analyte method was developed and validated for the quantification of 57 pharmaceuticals and illicit drugs in plasma samples. Sample pre-treatment consists of protein precipitation of 50 µL of the sample with 240 µL of organic solvent mixture (MeOH:ACN, 3:1, v/v), centrifugation, and injection into the LC-MS/MS, with a chromatographic run time of 7 min. The method was validated considering lower limit of quantification (LLOQ), interferences, linearity, precision, accuracy, dilution integrity, carryover, and matrix effect. The LLOQs ranged from 5 to 20 ng/mL and all analytes were linear (r2>0.99) in the tested concentration ranges. The method proved to be precise and accurate, presenting QC concentrations for all analytes within acceptable limits by the guideline used (CV % ≤20 % and bias ± 20 %). The developed method was successfully applied in 470 plasma samples of real cases of poisoning. A total of 80 % of the samples were positive for at least one substance, with acetaminophen (32.1 %), diazepam (25.1 %), and lidocaine (18.9 %) being the most detected. The most prevalent exposure circumstance among the cases was suicide attempt. The most frequent age groups were young adults between 20 and 29 years old and children under 5 years old. The methodology developed proved to be efficient in the simultaneous determination of 57 substances of toxicological interest, contributing to a correct diagnosis and, consequently, to the most appropriate management and treatment of the intoxicated patient. Furthermore, it is possible to observe the most commonly involved toxic agents in the Rio Grande do Sul, southern Brazil, helping to trace a profile of the poisoning patient, important in toxicovigilance actions.

Molecular Level Sucrose Quantification: A Critical Review.

Sucrose is a primary metabolite in plants, a source of energy, a source of carbon atoms for growth and development, and a regulator of biochemical processes. Most of the traditional analytical chemistry methods for sucrose quantification in plants require sample treatment (with consequent tissue destruction) and complex facilities, that do not allow real-time sucrose quantification at ultra-low concentrations (nM to pM range) under in vivo conditions, limiting our understanding of sucrose roles in plant physiology across different plant tissues and cellular compartments. Some of the above-mentioned problems may be circumvented with the use of bio-compatible ligands for molecular recognition of sucrose. Nevertheless, problems such as the signal-noise ratio, stability, and selectivity are some of the main challenges limiting the use of molecular recognition methods for the in vivo quantification of sucrose. In this review, we provide a critical analysis of the existing analytical chemistry tools, biosensors, and synthetic ligands, for sucrose quantification and discuss the most promising paths to improve upon its limits of detection. Our goal is to highlight the criteria design need for real-time, in vivo, highly sensitive and selective sucrose sensing capabilities to enable further our understanding of living organisms, the development of new plant breeding strategies for increased crop productivity and sustainability, and ultimately to contribute to the overarching need for food security.

Fusarium cerealis causing Fusarium head blight of durum wheat and its associated mycotoxins.

Fusarium Head Blight (FHB) is a very important fungal disease that affects small grain cereals worldwide. This disease not only causes yield loses but also crops contamination with mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV). Species within the Fusarium graminearum species complex have been described as the main causal agents of this disease, however lately there have been few reports of Fusarium cerealis causing the disease in wheat and barley in different parts of the world. This study evaluated the aggressiveness of F. cerealis to durum wheat cultivars and also mycotoxin production in planta. Moreover, the mycotoxin profile of F. cerealis strains was characterized molecularly and chemically. All durum wheat cultivars showed typical FHB symptoms but the disease severity varied among them in levels up to 66%. In addition, seventeen different compounds were detected in the infected heads including DON, NIV and nivalenol-3-ß-d-glucose (NIV3G). NIV was detected in all cultivars and was the most produced mycotoxin with levels ranging from 1.04 to 6.8 mg/kg. On the other hand, the molecular analysis of F. cerealis strains showed that all of them possessed NIV genotype while the chemical assessment showed that the strains were able to produce not only this toxin in vitro but also DON, zearalenone and other twenty-one secondary metabolites. The increasing incidence of F. cerealis and the possible contamination of crops with the mycotoxins that it produces are of great concern for food security and world cereal trade since it has been reported that NIV is more toxic for humans and animals than DON.

How Long are Residual Newborn Screening Specimens Useful for Retesting when Stored in Suboptimal and Uncontrolled Conditions of Temperature and Humidity?

Abstract Residual DBS specimens from newborns diagnosed with Phenylketonuria, Congenital Hypothyroidism, Cystic Fibrosis, Congenital Adrenal Hyperplasia and Galactosemia collected within 1995-2018, stored in cardboard boxes at ambient temperature in uncontrolled conditions, were retested for phenylalanine (Phe), thyrotropin (TSH), immunoreactive trypsinogen (IRT), total galactose (TGal) and 17-hydroxyprogesterone (17OHP), to demonstrate how long are they stable in these conditions and useful to reconfirm a previous abnormal result. Recovery percentage at retesting and qualitative interpretation regarding the current cutoff were evaluated. Phe, TSH and IRT recoveries showed decreasing trends along time. Phe recovery was 64 % after 2-years storage; TSH decayed rapidly recovering 47.3 % at 1-year, while IRT showed recoveries of 60 % at 1-year. Although 17OHP recovery presented a wide variation of results, a decaying trend was also found. Results suggest 17OHP is more stable than TSH and IRT, as supported by recoveries > 71 % when stored ≤ 2-years. TGal recovery presented an erratic behavior, so that it was not possible to estimate expected concentrations as a function of storage time. TGal recoveries above 100 % were found in UDP-galactose-4-epimerase and galactose-1-phosphate uridyltransferase deficiencies, evidencing possible galactose liberation from other sources. These results make a very valuable contribution for programs storing residual DBS in uncontrolled conditions.

Urinary biomarkers in lupus nephritis.

Systemic lupus erythematosus (SLE) is the prototypical autoimmune disease that can affect any organ of the body. Multiple mechanisms may contribute to the pathophysiology of systemic lupus, including failure to remove apoptotic bodies, hyperactivity of self-reactive B and T lymphocytes, abnormal exposure to autoantigens, and increased levels of B-cell stimulatory cytokines. The involvement of the kidney, called lupus nephritis (LN), during the course of the disease affects between 30% and 60% of adult SLE patients, and up to 70% of children. LN is an immune-mediated glomerulonephritis that is a common and serious finding in patients with SLE. Nowadays, renal biopsy is considered the gold standard for classifying LN, besides its degree of activity or chronicity. Nevertheless, renal biopsy lacks the ability to predict which patients will respond to immunosuppressive therapy and is a costly and risky procedure that is not practical in the monitoring of LN because serial repetitions would be necessary. Consequently, many serum and urinary biomarkers have been studied in SLE patients for the complementary study of LN, existing conventional biomarkers like proteinuria, protein/creatinine ratio in spot urine, 24 â€‹h urine proteinuria, creatinine clearance, among others and non-conventional biomarkers, like Monocyte chemoattractant protein-1 (MCP-1), have been correlated with the histological findings of the different types of LN. In this article, we review the advances in lupus nephritis urinary biomarkers. Such markers ideally should be capable of predicting early sub-clinical flares and could be used to follow response to therapy. In addition, some of these markers have been found to be involved in the pathogenesis of lupus nephritis.

A fast and simple approach for the quantification of 40 illicit drugs, medicines, and pesticides in blood and urine samples by UHPLC-MS/MS.

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL-1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology.

Development of a simple HPLC-DAD multi-analyte procedure and its application in cases evaluated by the Poison Control Center of São Paulo, Brazil.

This work describes a simple approach to overcome challenges in emergency toxicological analysis, using liquid-liquid extraction and high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD). A rapid procedure has been developed, for the extraction and detection of 19 analytes from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants and drugs of abuse. These substances are relevant in the context of emergency toxicology in Brazil. The method has been validated according to international guidelines by establishing parameters such as lower limit of quantification, sensitivity, linearity, accuracy and precision. The intra and inter-day precision values, at the lowest concentration levels, have always been less than 20% considering its relative standard deviation. As for accuracy values, these have also been satisfactory (above 81.3%). This method was successfully applied in 201 blood samples from patients with suspected poisoning of the Poison Control Center of São Paulo (PCC-SP), Brazil. Finally, the developed method has shown to be relevant for emergency toxicology due to its high sensitivity and it could be also very useful in both fields of clinical and forensic toxicology.

Determination of free and total sulfur(IV) compounds in coconut water using high-resolution continuum source molecular absorption spectrometry in gas phase.

This work proposes a method for the determination of free and total sulfur(IV) compounds in coconut water samples, using the high-resolution continuum source molecular absorption spectrometry. It is based on the measurement of the absorbance signal of the SO2 gas generate, which is resultant of the addition of hydrochloric acid solution on the sample containing the sulfating agent. The sulfite bound to the organic compounds is released by the addition of sodium hydroxide solution, before the generation of the SO2 gas. The optimization step was performed using multivariate methodology involving volume, concentration and flow rate of hydrochloric acid. This method was established by the sum of the absorbances obtained in the three lines of molecular absorption of the SO2 gas. This strategy allowed a procedure for the determination of sulfite with limits of detection and quantification of 0.36 and 1.21mgL-1 (for a sample volume of 10mL) and precision expressed as relative standard deviation of 5.4% and 6.4% for a coconut water sample containing 38.13 and 54.58mgL-1 of free and total sulfite, respectively. The method was applied for analyzing five coconut water samples from Salvador city, Brazil. The average contents varied from 13.0 to 55.4mgL-1 for free sulfite and from 24.7 to 66.9mgL-1 for total sulfur(IV) compounds. The samples were also analyzed employing the Ripper´s procedure, which is a reference method for the quantification of this additive. A statistical test at 95% confidence level demonstrated that there is no significant difference between the results obtained by the two methods.

Solid-phase extractions in flow analysis

ABSTRACT Coupling solid-phase extraction (SPE) to flow systems has promoted a synergistic development. Whereas SPE mechanization leads to improved precision and higher sample throughput, as well as diminishes systematic errors and contamination risks, analyte concentration and separation from the sample matrix provides a remarkable impact on detectability and selectivity in flow analysis. Historical aspects, main cornerstones, tips for system design, and recent applications are critically reviewed, in the context of analyte(s) separation/concentration, sample clean-up, and release of sorbed chemical species involving both packed (e.g. mini-columns, cartridges, and disks) or fluidized (e.g. beads and magnetic materials) particles. Novel (bio)sorbents, selective synthetic materials, and stationary phases for low-pressure chromatography are also discussed. Moreover, the feasibility of SPE for sample treatment before chromatographic separation, as well as the exploitation of direct measurements on the solid phase (optosensing) are emphasized.

Aplicação da análise multivariada para o mapeamento dos casos de intoxicações agudas atendidos no Centro de Controle de Intoxicações da cidade de São Paulo / Application of multivariate analysis for the mapping of acute intoxication cases attended in Poison Control Center of the São Paulo city

A Toxicologia tem desempenhado um importante papel na identificação de efeitos nocivos à população, gerando subsídios para a tomada de decisões na prática clínica, auxiliando desta forma no bom prognóstico de pacientes intoxicados. De acordo com a Pan American Health Organization, o sucesso em qualquer intervenção em saúde somente pode ser obtido com a criação e manutenção de um banco de dados confiável, que seja capaz de predizer as diversas particularidades das intoxicações, como a população-alvo e suas suscetibilidades. Assim, recomenda-se que médicos, especialistas, legisladores e administradores em saúde adotem em sua rotina uma coleta de dados sistêmica e integrada para o mapeamento e caracterização das intoxicações. Neste cenário, técnicas de análises multivariadas poderiam ser empregadas para evidenciar possíveis intercorrelações; entretanto, seu uso ainda não é comum na toxicologia clínica. Neste trabalho foram identificados e quantificados agentes exógenos em amostras biológicas (sangue e urina) provenientes do Centro de Controle de Intoxicações da cidade de São Paulo, correlacionando os dados obtidos dessas análises com o perfil clínico e prognóstico dos pacientes. Fármacos benzodiazepínicos, antidepressivos, anticonvulsivantes, paracetamol, drogas de abuso e praguicidas foram selecionados de acordo com a incidência reportada por esse centro no período de 2013 a 2014. Do total de amostras analisadas (n = 320), 192 foram positivas para alguma substância, sendo 101 positivas para etanol e 131 positivas para as demais substâncias. Os dados obtidos foram submetidos à análise de correspondência múltipla e análise hierárquica de cluster. A partir da análise multivariada foi possível agrupar os indivíduos em 3 clusters, o que correspondeu a 66,5% do total de informações. No primeiro eixo houve a separação dos pacientes do gênero feminino, que se intoxicaram ou foram expostos a medicamentos e drogas de abuso, por tentativa de suicídio, dos pacientes do gênero masculino, de idade entre 30 a 39 anos, que se intoxicaram com drogas de abuso. No segundo eixo fatorial foram agrupados os pacientes que se intoxicaram com etanol isoladamente, juntamente com pacientes que se intoxicaram com diazepam. Este trabalho contribuiu para o mapeamento dos casos de intoxicação atendidos pelo CCI-SP e foi um estudo inicial para a criação de um banco de dados que poderá ser alimentado constantemente e assim, oferecer ao sistema de toxicovigilância uma base para políticas educativas

Toxicology has played an important role in the identification of harmful effects to the population, generating subsidies for decision making in clinical practice, helping in this way the good prognosis of acutely intoxicated patients. According to the Pan American Health Organization, success in any health intervention can only be achieved by creating and maintaining a reliable database that is capable of predicting the various characteristics of intoxications, such as the target population and their susceptibilities. Thus, it is recommended that doctors, specialists, legislators and health administrators adopt in their routine a systemic and integrated data collection for the mapping and characterization of intoxications. In this scenario, multivariate analysis techniques could be used to evidence possible intercorrelations; however, its use is not yet common in clinical toxicology. In this work, were identified and quantified exogenous agents in biological samples (blood and urine) from the Poison Control Center São Paulo city, correlating the data obtained from these analyzes with the clinical and prognostic profile of the patients. Benzodiazepines, antidepressants, anticonvulsants, acetaminophen, drugs of abuse and pesticides were selected according to the incidence reported by this center in the period from 2013 to 2014. Of the total number of samples analyzed (n = 320), 192 samples have shown to be positive for some of the analytes, from these 100 were positive for ethanol and 131 positive for other substances. The data were submitted to multiple correspondence analysis and hierarchical cluster analysis. From the multivariate analysis it was possible to group the individuals into 3 clusters, which corresponded to 66.5% of the total information. In the first axis, the patients were separated from the female gender, who were intoxicated or were exposed to drugs and suicide attempt of the male patients, aged between 30 and 39 years, who became intoxicated with drugs of abuse. In the second factorial axis were grouped the patients who were intoxicated with ethanol, together with patients who became intoxicated with diazepam. This work contributed to the mapping of intoxication cases attended by the CCI-SP and was a initial study for the creation of a database that could be fed constantly and thus provide the toxicovigilance system with a basis for educational policies

Practical aspects in gas chromatography-mass spectrometry for the analysis of pesticide residues in exotic fruits.

The most relevant parameters of a multimode inlet were optimized to increase the injection volume up to 25 µL using solvent vent mode in order to improve the sensitivity of the gas chromatography-mass spectrometry system. Consequently, the implementation of a concurrent backflushing was necessary to largely prevent the expected loss of performance derived from such matrix load out of a general-purpose extraction (EN-15622-QuEChERS). Additionally, four mixtures of compounds used as analyte protectants were tested using spiked physalis to enhance the quality of signals. The chosen mixture remarkably improved sensitivity and yield better peak shapes, significantly more than others also tested. The analysis of pesticide residues in exotic fruits using instruments of limited selectivity is challenging since these complex matrices usually give notably dirty extracts. This scheme included an instrumental optimization and the addition of selected compounds that enabled to selectively reach limits of quantitation of 0.01 mg kg(-1) for most analytes.

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics.

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR-based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross-reactions with the predator and related species genomes. Here, we use PCR-free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h(-1) respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h(-1) , respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1-2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.

Analytical applications of microbial fuel cells. Part II: Toxicity, microbial activity and quantification, single analyte detection and other uses.

Microbial fuel cells were rediscovered twenty years ago and now are a very active research area. The reasons behind this new activity are the relatively recent discovery of electrogenic or electroactive bacteria and the vision of two important practical applications, as wastewater treatment coupled with clean energy production and power supply systems for isolated low-power sensor devices. Although some analytical applications of MFCs were proposed earlier (as biochemical oxygen demand sensing) only lately a myriad of new uses of this technology are being presented by research groups around the world, which combine both biological-microbiological and electroanalytical expertises. This is the second part of a review of MFC applications in the area of analytical sciences. In Part I a general introduction to biological-based analytical methods including bioassays, biosensors, MFCs design, operating principles, as well as, perhaps the main and earlier presented application, the use as a BOD sensor was reviewed. In Part II, other proposed uses are presented and discussed. As other microbially based analytical systems, MFCs are satisfactory systems to measure and integrate complex parameters that are difficult or impossible to measure otherwise, such as water toxicity (where the toxic effect to aquatic organisms needed to be integrated). We explore here the methods proposed to measure toxicity, microbial metabolism, and, being of special interest to space exploration, life sensors. Also, some methods with higher specificity, proposed to detect a single analyte, are presented. Different possibilities to increase selectivity and sensitivity, by using molecular biology or other modern techniques are also discussed here.

Los valores preyy pospandriales de alt, urea y creatinina no cambian significativamette / Pre and ostpandrrial values of alt, urea and creatinine do not change signifcaantly

Introducción: Para el laboratorio clínico es importante tener una muestra que cumpla con los requerimientos para el análisis, por tal razón el laboratorio de química clínica exige a los pacientes ayuno de 8 horas para practicarse el análisis de urea, ALT y creatinina ya que el metabolismo de los alimentos puede verse reflejado en cambios en el suero y causar interferencia en la lectura de estos analitos. El objetivo de este trabajo es revisar qué tanta interferencia se produce cuando se lee la muestra 2 horas después del desayuno. Materiales y Métodos: Para el presente estudio se tomaron muestras de sangre total pre y posprandiales para el análisis de ALT, urea y creatinina en suero y establecer las posibles interferencias que podrían causar la glucosa, el colesterol y los triglicéridos en dicho análisis. Para el análisis de varianza se utilizó el test de ANOVA, y para determinar el tipo de interferencia y su significancia se utilizó el interferograma. Resultados: El test de ANOVA no mostró diferencias significativas en los resultados, y el interferograma no arrojó diferencias significativas para ninguno de los analitos de interés en este estudio. Discusión: El análisis de ALT, urea y creatinina en condiciones posprandiales sufre interferencia positiva en algunos de los pacientes a causa de la glucosa, el colesterol y los triglicéridos contenidos en la muestra, dicha interferencia no resulta significativa, lo que sugiere que no es necesario acudir en ayunas a la toma de la muestra cuando se toma 2 horas siempre posterior al desayuno con ayuno previo de 8 horas.

Introduction: For the clinical laboratory is important to have a sample that meets the requirements for analysis, reason why the clinical chemistry laboratory demands its patients to fast 8 hours in order to take the urea, creatinine and ALT analyses since the metabolism of food may be reflected in the serum changes and may cause interference in the reading of these analytes. The purpose of this work is to review how much interference occurs when the sample is read 2 hours after breakfast. Materials and Methods: Total, pre and postprandial blood samples for ALT, urea and serum creatinine analysis were taken for this study in order to establish the possible interferences glucose, cholesterol and triglycerides may cause in the analysis. The ANOVA test was used for the variance analysis and interferogram was used in order to determine the type of interference and its significance. Results: ANOVA test showed no significant differences in the results and interferogram did not show significant differences for any of the analytes of interest in this study. Discussion: The ALT, urea and creatinine analysis in fed postpandrial conditions suffer positive interference in some patients due to glucose, cholesterol and triglycerides contained in the sample. This interference is not significant which suggests that it is not necessary to fast when the sample is going to be taken when it can be taken 2 hours after breakfast with a previous 8 hour fast.

Chemometric quality inspection control of pyrantel pamoate, febantel and praziquantel in veterinary tablets by mid infrared spectroscopy.

This paper describes the development and validation of a new multivariate calibration method based on diffuse reflectance mid infrared spectroscopy for direct and simultaneous determination of three veterinary pharmaceutical drugs, pyrantel pamoate, praziquantel and febantel, in commercial tablets. The best synergy interval partial least squares (siPLS) model was obtained by selecting three spectral regions, 3715-3150, 2865-2583, and 2298-1733 cm(-1), preprocessed by first derivative and Savitzky-Golay smoothing followed by mean centering. This model was built with five latent variables and provided root mean square errors of prediction (RMSEP) equal or lower than 0.69 mg per 100 mg of powder for the three analytes. The method was validated according the appropriate regulations through the estimate of figures of merit, such as trueness, precision, linearity, analytical sensitivity, bias and residual prediction deviation (RPD). Then, it was applied to three different veterinary pharmaceutical formulations found in the Brazilian market, in a situation of multi-product calibration, since the excipient composition of these commercial products, which was not known a priori, was modeled by an experimental design that scanned the likely content range of the possible constituents. The results were verified with high performance liquid chromatography with diode array detection (HPLC-DAD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and were in agreement with the predicted values at 95% confidence level. The developed method presented the advantages of being simple, rapid, solvent free, and about ten times faster than the HPLC ones.