Neuronal antibody prevalence in children with seizures under 3 years: A prospective national cohort.

OBJECTIVE: To report the prevalence of anti-neuronal antibodies in a prospective whole-nation cohort of children presenting with seizures before their third birthday. METHODS: This was a prospective population-based national cohort study involving all children presenting with new-onset epilepsy or complex febrile seizures before their third birthday over a 3-year period. Patients with previously identified structural, metabolic, or infectious cause for seizures were excluded. Serum samples were obtained at first presentation and tested for 7 neuronal antibodies using live cell-based assays. Clinical data were collected with structured proformas at recruitment and 24 months after presentation. In addition, patients with seizures and clinically suspected autoimmune encephalitis were independently identified by a review of the case records of all children <3 years of age in Scotland who had undergone EEG. RESULTS: Two hundred ninety-eight patients were identified and recruited and underwent autoantibody testing. Antibody positivity was identified in 18 of 298 (6.0%). The antibodies identified were GABA receptor B (n = 8, 2.7%), contactin-associated protein 2 (n = 4, 1.3%), glycine receptor (n = 3, 1.0%), leucine-rich glioma inactivated 1 (n = 2, 0.7%), NMDA receptor (n = 1, 0.3%), and GABA receptor A (n = 1, 0.3%). None of these patients had a clinical picture of autoimmune encephalitis. Seizure classification and clinical phenotype did not correlate with antibody positivity. CONCLUSIONS: Autoimmune encephalitis is very rare in early childhood. However serum neuronal antibodies are identified in 6.4% of children presenting with seizures at <3 years of age. Antibody testing should not be a routine clinical test in early childhood-onset epilepsy because, in the absence of other features of autoimmune encephalitis, antibody positivity is of doubtful clinical significance. Antibody testing should be reserved for patients with additional features of encephalitis.

Incidence and phenotypes of childhood-onset genetic epilepsies: a prospective population-based national cohort.

Epilepsy is common in early childhood. In this age group it is associated with high rates of therapy-resistance, and with cognitive, motor, and behavioural comorbidity. A large number of genes, with wide ranging functions, are implicated in its aetiology, especially in those with therapy-resistant seizures. Identifying the more common single-gene epilepsies will aid in targeting resources, the prioritization of diagnostic testing and development of precision therapy. Previous studies of genetic testing in epilepsy have not been prospective and population-based. Therefore, the population-incidence of common genetic epilepsies remains unknown. The objective of this study was to describe the incidence and phenotypic spectrum of the most common single-gene epilepsies in young children, and to calculate what proportion are amenable to precision therapy. This was a prospective national epidemiological cohort study. All children presenting with epilepsy before 36 months of age were eligible. Children presenting with recurrent prolonged (>10 min) febrile seizures; febrile or afebrile status epilepticus (>30 min); or with clusters of two or more febrile or afebrile seizures within a 24-h period were also eligible. Participants were recruited from all 20 regional paediatric departments and four tertiary children's hospitals in Scotland over a 3-year period. DNA samples were tested on a custom-designed 104-gene epilepsy panel. Detailed clinical information was systematically gathered at initial presentation and during follow-up. Clinical and genetic data were reviewed by a multidisciplinary team of clinicians and genetic scientists. The pathogenic significance of the genetic variants was assessed in accordance with the guidelines of UK Association of Clinical Genetic Science (ACGS). Of the 343 patients who met inclusion criteria, 333 completed genetic testing, and 80/333 (24%) had a diagnostic genetic finding. The overall estimated annual incidence of single-gene epilepsies in this well-defined population was 1 per 2120 live births (47.2/100 000; 95% confidence interval 36.9-57.5). PRRT2 was the most common single-gene epilepsy with an incidence of 1 per 9970 live births (10.0/100 000; 95% confidence interval 5.26-14.8) followed by SCN1A: 1 per 12 200 (8.26/100 000; 95% confidence interval 3.93-12.6); KCNQ2: 1 per 17 000 (5.89/100 000; 95% confidence interval 2.24-9.56) and SLC2A1: 1 per 24 300 (4.13/100 000; 95% confidence interval 1.07-7.19). Presentation before the age of 6 months, and presentation with afebrile focal seizures were significantly associated with genetic diagnosis. Single-gene disorders accounted for a quarter of the seizure disorders in this cohort. Genetic testing is recommended to identify children who may benefit from precision treatment and should be mainstream practice in early childhood onset epilepsy.

Direct solid phase microextraction combined with gas chromatography - Mass spectrometry for the determination of biogenic amines in wine.

A direct method based on immersion solid phase microextraction (DI-SPME) gas chromatography mass-spectrometry (GC-MS) was optimized and validated for the determination of 16 biogenic amines in Polish wines. In the analysis two internal standards were used: 1,7-diaminoheptane and bis-3-aminopropylamine. The method allows for simultaneous extraction and derivatization, providing a simple and fast mode of extraction and enrichment. Different parameters which affect the extraction procedure were studied and optimized including ionic strength (0-25%), fiber materials (PDMS/DVB, PDMS/DVD + OC, Polyacrylate, Carboxen/PDMS and DVB/CAR/PDMS) and timings of the extraction, derivatization and desorption processes. Validation studies confirmed the linearity, sensitivity, precision and accuracy of the method. The method was successfully applied to the analysis of 44 wine samples originating from several regions of Poland and 3 wine samples from other countries. Analysis showed that many of the samples contained all examined biogenic amines. The method, assessed using an Eco-Scale tool with satisfactory results, was found to be green in terms of hazardous chemicals and solvents usage, energy consumption and production of waste. Therefore the proposed method can be safely used in the wine industry for routine analysis of BAs in wine samples with a minimal detrimental impact on human health and the environment.

Determination and long-term stability of twenty-nine cathinones and amphetamine-type stimulants (ATS) in urine using gas chromatography-mass spectrometry.

A method was developed for the screening and quantification of seven amphetamine-type stimulants (ATS) and 22 cathinones, including three metabolites, in urine with Gas Chromatography-Mass Spectrometry. This method allowed the detection and quantification of ATS and cathinones group molecules using one procedure. A study of the stability of the drug mixtures for a period of 201 days in human urine samples under three different conditions has been carried. The ATS and cathinones include amphetamine, methamphetamine, MDA, MDEA, MDMA, PMA, PMMA, cathinone, methcathinone, 3'-position-substituted, ring-substituted, methylenedioxy-substituted, N-alkyl-substituted and pyrrolidinyl-substituted. Twenty drugs out of twenty-nine were validated with a quantitative method. This method can be applied to the nine remaining drugs as a screening method. The linearity of the assay was from 50 to 2000 ng/ml, with limits of detection of 0.5 to 10 ng/ml. In terms of accuracy, between-run and within-run precision were ≤20% for 20 compounds with good selectivity. No carryover was seen, and the recovery was between 80 and 120% for most drugs tested. ATS and pyrrolidinyl-substituted groups were conducted to be stable compounds under all conditions. All compounds tested were stable at -20 °C. Some cathinones were primarily degraded after 21 days at 4 °C. They were detectable but unstable after 201 days at 4 °C. Most cathinones were unstable after a day and completely lost after 28 days at RT.

OC04 - A weaning plan for high flow nasal therapy (HFNT) in bronchiolitis - a nurse-led initiative.

UNLABELLED: Theme: Nursing education, management and leadership. INTRODUCTION: The use of HFNT in bronchiolitis is a new phenomenon in paediatrics, with insufficient evidence on its effectiveness (Beggs et al 2014). This paper reports the findings of a clinical audit which resulted in the development of a nurse-led weaning plan to support infant recovery with the potential to reduce hospital stay. OBJECTIVE: To highlight the effectiveness of a nurse-led weaning plan in a general paediatric unit. METHOD: The data of 32 infants were analysed between October 2014 and March 2015. Further data will be collected between October 2015 and March 2016. Data will then be compared. RESULTS: Initial data demonstrates the average time infants spent on HFNT was 2.6 days compared to five days in previous studies (Bressan et al 2013). DISCUSSION: A nurse-led weaning plan has been developed from data collected. CONCLUSION: The audit demonstrates a clinical need for efficient weaning of infants on HFNT.

Capillary gas chromatography using a γ-cyclodextrin for enantiomeric separation of methylamphetamine, its precursors and chloro intermediates after optimization of the derivatization reaction.

The enantiomeric ratio of methylamphetamine (MAMP) is closely related to the optical activity of precursors and reagents used for the synthesis and this knowledge can provide useful information concerning the origins and synthetic methods used for illicit manufacture. The information can be utilized for regulation of the precursors and investigation of the manufacturing sources but this requires analytical procedures to determine purity of drug substances, impurity profiling and enantiomeric composition. In this study, a gas chromatography (GC) coupled with mass spectrometry (MS) method using a γ-cyclodextrin chiral stationary phase was developed and optimized for the simultaneous enantiomeric separations of MAMP and its common precursors, ephedrine, and pseudoephedrine, as well as its chlorointermediates formed during MAMP synthesis by the Emde method, after derivatization with trifluoroacetic anhydride. The optimization was performed using multivariate statistics (cluster analysis and principal components analysis) in order to select and compare optimal experimental conditions. Under the optimized experimental conditions, the calculated calibration curves showed good linearity range up to 0.1µg/mL for all tested analytes. The limits of detection were in the range of 0.002-0.008µg/mL and the coefficient of variability was between 1.0 and 3.9%. The method has the advantage of achieving excellent precision under repeatability and reproducibility conditions while detection by MS allows for the identity of analytes to be confirmed in a single analysis. The method was therefore applied satisfactory to MAMP analysis.

Development of a robust chromatographic method for the detection of chlorophenols in cork oak forest soils.

A major concern for the cork and wine industry is 'cork taint' which is associated with chloroanisoles, the microbial degradation metabolites of chlorophenols. The use of chlorophenolic compounds as pesticides within cork forests was prohibited in 1993 in the European Union (EU) following the introduction of industry guidance. However, cork produced outside the EU is still thought to be affected and simple, robust methods for chlorophenol analysis are required for wider environmental assessment by industry and local environmental regulators. Soil samples were collected from three common-use forests in Tunisia and from one privately owned forest in Sardinia, providing examples of varied management practice and degree of human intervention. These provided challenge samples for the optimisation of a HPLC-UV detection method. It produced recoveries consistently >75% against a soil CRM (ERM-CC008) for pentachlorophenol. The optimised method, with ultraviolet (diode array) detection is able to separate and quantify 16 different chlorophenols at field concentrations greater than the limits of detection ranging from 6.5 to 191.3 µg/kg (dry weight). Application to a range of field samples demonstrated the absence of widespread contamination in forest soils at sites sampled in Sardinia and Tunisia.

Multi-residue method for the determination of 16 recently used pesticides from various chemical groups in aqueous samples by using DI-SPME coupled with GC-MS.

A simple and solvent-free multi-residue method has been optimized to determine 16 currently used pesticides from different chemical groups in aqueous samples. The extraction of analytes was carried out with direct immersion solid-phase microextraction (DI-SPME) and for the identification and quantitative determination gas chromatography coupled with mass spectrometry (GC-MS) was applied. Two commonly used adsorbent coatings have been applied and compared: 100 µm of polydimethylsiloxane (PDMS) and 85 µm of polyacrylate (PA). The method development parameters of DI-SPME, analyte desorption and GC-MS analysis have been outlined along with the final experimental conditions. When the optimum extraction conditions were applied (extraction time 60 min, 10% (w/v) NaCl solution, 45°C) the limits of detection (LODs) were in the range of 0.015-0.13 µgL(-1) and the relative standard deviations (RSDs) were between 1.9 and 9.6%. The developed analytical method was successfully applied to the analysis of natural water samples from the following sources: river, sea, canal and rain.

Chiral analysis of chloro intermediates of methylamphetamine by one-dimensional and multidimentional NMR and GC/MS.

Impurity profiling and classification of abused drugs using chiral analytical techniques is of particular interest and importance because of the additional information obtained from this approach. When these methods are applied to the synthesis of illicitly used substances, they can supply valuable information about the conditions/chemicals used in the synthesis. We have applied GC and NMR methods to the study of intermediates found in methylamphetamine manufacture with the aim of linking the intermediates to the ephedrine/pseudoephedrine starting materials. Therefore, determination of the stereochemical makeup within samples of forensic interest is important giving further specific information to the analyst. This study investigates the stereochemical course of the Emde synthesis of methylamphetamine with particular focus on intermediate formation via the chlorination of ephedrine and pseudoephedrine enantiomers. The configurations of these chloro-phenethylamines were determined by 1D and 2D NMR analysis, and thereafter, the GC/MS analysis was carried out. We have shown here that chlorination of the ephedrine/pseudoephedrine compounds occurs via inversion (S(N)2) and retention (S(N)i) of configuration around the α carbon and mixture of diastereoisomers (chloroephedrine and chloropseudoephedrine) were formed, with the ratio of the resulting compounds dependent on the precursors used. The preparation and analytical properties of these intermediate standards provide data for laboratories interested in the stereochemical analysis of methylamphetamine intermediates such as forensic/law enforcement, and illustrate the value of using a combination of analytical methodology.

Simultaneous chiral separation of methylamphetamine and common precursors using gas chromatography/mass spectrometry.

Methylamphetamine, ephedrine, and pseudoephedrine were derivatized using trifluoroacetic anhydride and enantiomers of each were analyzed using gas chromatography coupled to mass spectrometry (GC/MS) fitted with a γ-cyclodextrin (Chiraldex™ G-PN) chiral column. A temperature-programmed method was developed and optimized and the results compared with those obtained using a previously published isothermal GC method applied to GC/MS analysis. Trifluoroacetylated 3-(trifluoromethyl)phenethylamine hydrochloride was used as an internal standard, and mass fragmentation patterns are proposed for all derivatives analyzed. Qualitative validation of the optimized chromatographic conditions was completed in accordance with the guidelines published by the United Nations Office on Drugs and Crime (UNODC). Under conditions of repeatability and reproducibility, the method gave relative retention times with a relative standard deviation of less than 0.02% for all six analytes of interest. This surpasses the UNODC's acceptance criteria of 2% for validation of qualitative precision. Ephedrine and pseudoephedrine are common precursors in the clandestine manufacture of methylamphetamine. Seizures of illicit methylamphetamine therefore often contain mixtures of these optically active compounds. The simultaneous enantioseparation of these compounds to produce a profile would provide valuable information to law enforcement agencies regarding the provenance of a methylamphetamine seizure.

Chiral gas chromatography as a tool for investigations into illicitly manufactured methylamphetamine.

The aim of this study was to develop a chiral gas chromatographic method for the separation of compounds likely to be found in the EMDE synthesis of methylamphetamine, a heavily abused stimulant drug. Here we describe the separation of the enantiomers of ephedrine, pseudoephedrine, chlorinated intermediates and methylamphetamine using fluorinated acid anhydrides as chemical derivatization reagents prior to gas chromatographic analysis on a 2,3-di-O-methyl-6-t-butyl silyl-ß-cyclodextrin stationary phase (CHIRALDEX™ B-DM). Separation of the enantiomers of pseudoephedrine, methylamphetamine and chloro-intermediates was achieved using PFPA derivatization, and enantiomers of ephedrine using TFAA derivatization, in run times of less than 40 minutes. The use of HFBA as a derivatization reagent for this set of analytes is also discussed.

The environmental behaviour of polychlorinated phenols and its relevance to cork forest ecosystems: a review.

Pentachlorophenol (PCP) has been used as a herbicide, biocide and preservative worldwide since the 1930s and as a result, extensive and prolonged contamination exists. The environmental impact increases when its many degradation products are taken into consideration. A number of chloroanisols and their related chlorophenols have been found in cork slabs collected from Portuguese oak tree forests before stopper manufacturing, and contamination by PCP and polychlorinated anisole (PCA) has been detected in Canadian forests. It is suggested that the use of polychlorinated phenols, in particular PCP, is thought to be a cause of the cork taint problem in wine, a major socio-economic impact not only for industry but on sensitive and highly biodiverse ecosystems. It also highlights particular issues relating to the regional regulation of potentially toxic chemicals and global economics world wide. To fully understand the impact of contamination sources, the mechanisms responsible for the fate and transport of PCP and its degradation products and assessment of their environmental behaviour is required. This review looks at the current state of knowledge of soil sorption, fate and bioavailability and identifies the challenges of degradation product identification and the contradictory evidence from field and laboratory observations. The need for a systematic evaluation of PCP contamination in relation to cork forest ecosystems and transfer of PCP between trophic levels is emphasised by discrepancies in bioaccumulation and toxicity. This is essential to enable long term management of not only transboundary contaminants, but also the sustainable management of socially and economically important forest ecosystems.