Y.E. Park

Advanced analytical method of nereistoxin using mixed-mode cationic exchange solid-phase extraction and GC/MS

Nereistoxin(NTX) was originated from a marine annelid worm Lumbriconereis heteropoda and its analogue pesticides including cartap, bensultap, thiocyclam and thiobensultap have been commonly used in agriculture, because of their low toxicity and high insecticidal activity. However, NTX has been reported about its inhibitory neuro toxicity in human and animal body, by blocking nicotinic acetylcholine receptor and it cause significant neuromuscular toxicity, resulting in respiratory failure. We developed a new method to determine NTX in biological fluid. The method involves mixed-mode cationic exchange based solid phase extraction and gas chromatography/mass spectrometry for final identification and quantitative analysis. The limit of detection and recovery were substantially better than those of other methods using liquid-liquid extraction or headspace solid phase microextraction. The good recoveries (97±14%) in blood samples were obtained and calibration curves over the range 0.05-20 mg/L have R2 values greater than 0.99. The developed method was applied to a fatal case of cartap intoxication of 74 years old woman who ingested cartap hydrochloride for suicide. Cartap and NTX were detected from postmortem specimens and the cause of the death was ruled to be nereistoxin intoxication. The concentrations of NTX were 2.58 mg/L, 3.36 mg/L and 1479.7 mg/L in heart, femoral blood and stomach liquid content, respectively. The heart blood/femoral blood ratio of NTX was 0.76.

Estimation of the synthetic routes of seized methamphetamines using GC-MS and multivariate analysis

One hundred and twenty six seized methamphetamine (MA) samples were analyzed using GC-MS. All the peaks that appeared in the chromatograms were investigated and 61 impurities including n-octacosane (internal standard) were identified. Among them, 37 impurities were already known or newly identified by comparing with commercial library entries and 18 impurities were detected for the first time. To estimate the synthetic routes of MA samples, route specific impurities had to be selected for each method. Two naphthalenes, 1,3-dimethyl-2-phenylnaphthalene and 1-benzyl-3-methylnaphthalene were selected as Nagai route specific impurities and three diasteromers, UK-19.62(58_165_178) I, UK-19.95(58_165_178) II, UK-20.49(58_165_178) III were also selected not only for their high frequency detection only in Nagai samples but also for the high principal component analysis (PCA) correlation values. For the Emde route, N,N-dimethyl-3,4-diphenylhexane-2,5-diamine and N-methyl-1-{4-[2-(methylamino)propyl]phenyl}-1-phenylpropan-2-amine were selected as route specific impurities, and N,N-di(β-phenylisopropyl)amine I (DPIA I), N,N-di(β-phenylisopropyl)amine II (DPIA II), N,N-di(β-phenylisopropyl)methylamine I (DPIMA I) and N,N-di(β-phenylisopropyl)methylamine II (DPIMA II) were selected for the Leuckart route. With these route specific impurities, synthetic routes could be identified for 78 of the 126 samples. The 61 impurities were registered in AMDIS target component library and the GC-MS data were deconvoluted. After AMDIS deconvolution, a matrix file was composed and then multivariate analyses were performed to estimate the synthetic route for unknown samples. The unsupervised methods, hierarchical clustering analysis (HCA) and PCA clustered the samples according to the closeness between samples. Two classification functions were obtained from discriminant analysis (DA) and the synthetic routes of the unknown samples were predicted using these two functions.

Determination of the PDE-5 Inhibitors and Their Analogues by GC-MS and TMS Derivatization

Eighteen of the PDE-5 inhibitors and their analogues were analyzed using GC-EI-MS. Fourteen of them could be identified by simple GC-MS method without derivatization, but hydroxyhongdenafil, hydroxyvardenafil, xanthoanthrafil and mirodenafil could not be identified without derivatization for the high polarity due to the presence of hydroxyl groups. N,O-bis(trime- thylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), widely used trimeth- ylsilyl (TMS) derivatizing reagents, were used to improve the sensitivity of the hydroxylated analogues. And the analytes could be identified by GC-MS after the derivatization.

Development of an automated data processing method for sample to sample comparison of seized methamphetamines

The information about the sources of supply, trafficking routes, distribution patterns and conspiracy links can be obtained from methamphetamine profiling. The precursor and synthetic method for the clandestine manufacture can be estimated from the analysis of minor impurities contained in methamphetamine. Also, the similarity between samples can be evaluated using the peaks that appear in chromatograms. In South Korea, methamphetamine was the most popular drug but the total seized amount of methamphetamine whole through the country was very small. Therefore, it would be more important to find the links between samples than the other uses of methamphetamine profiling. Many Asian countries including Japan and South Korea have been using the method developed by National Research Institute of Police Science of Japan. The method used gas chromatography-flame ionization detector (GC-FID), DB-5 column and four internal standards. It was developed to increase the amount of impurities and minimize the amount of methamphetamine. After GC-FID analysis, the raw data have to be processed. The data processing steps are very complex and require a lot of time and effort. In this study, Microsoft Visual Basic Application (VBA) modules were developed to handle these data processing steps. This module collected the results from the data into an Excel file and then corrected the retention time shift and response deviation generated from the sample preparation and instruments analysis. The developed modules were tested for their performance using 10 samples from 5 different cases. The processed results were analyzed with Pearson correlation coefficient for similarity assessment and the correlation coefficient of the two samples from the same case was more than 0.99. When the modules were applied to 131 seized methamphetamine samples, four samples from two different cases were found to have the common origin and the chromatograms of the four samples were appeared visually identical. The developed VBA modules could process raw data of GC-FID very quickly and easily. Also, they could assess the similarity between samples by peak pattern recognition using whole peaks without spectral identification of each peak that appeared in the chromatogram. The results collectively suggest that the modules would be useful tools to augment similarity assessment between seized methamphetamine samples.

New metabolites of hongdenafil, homosildenafil and hydroxyhomosildenafil

HighlightsThe new metabolites of hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using LC‐Q TOF‐MS/MS.Biological samples of rats for in vivo study and human liver microsome for in vitro study were determined and compared.Major metabolites were identified at m/z 461.1966 or 439.2455 by piperazine N‐dehydroxyethylation or N‐deethylation.From five to seven metabolites were identified in hongdenafil, homosildenafil and hydroxy homosildenafil treated samples.These new metabolites could be fundamental data for the toxicity study of sildenafil analogues and forensic science fields. ABSTRACT Recently, illegal sildenafil analogues have emerged, causing serious social issues. In spite of the importance of sildenafil analogues, their metabolic profiles or clinical effects have not been reported yet. In this study, new metabolites of illegal sildenafil analogues such as hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using liquid chromatography quadrupole‐time of flight mass spectrometry (LC‐Q‐TOF‐MS) and tandem mass spectrometry (LC‐Q‐TOF‐MS/MS). To prepare metabolic samples, in vitro and in vivo studies were performed. For in vivo metabolites analysis, urine and feces samples of rats treated with sildenafil analogues were analyzed. For in vitro metabolites analysis, human liver microsomes incubated with sildenafil analogues were extracted and analyzed. All metabolites were characterized by LC‐Q‐TOF‐MS and LC‐Q‐TOF‐MS/MS. As a result, five, six, and seven metabolites were determined in hongdenafil, homosildenafil, and hydroxyhomosildenafil treated samples, respectively. These results could be applied to forensic science and other analytical fields. Moreover, these newly identified metabolites could be used as fundamental data to determine the side effect and toxicity of illegal sildenafil analogues.

FRI0080 Effects of IL-6 Receptor Inhibition Therapy on The Serum Levels of IL-33 and IL-6 in Patients with Rheumatoid Arthritis

Background Several pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, IL-8 and IL-15 are known to be critical in synovial inflammatory process in RA and successful results have been obtained in RA treatment with targeting pro-inflammatory cytokines including TNF-α, IL-1 and IL-6. Objectives This study sought to investigate the role of IL-33 and IL-6 in RA patients receiving IL-6 receptor inhibition therapy. Methods We analyzed the association of the IL-33 and IL-6 level with disease activity and serologic features in 83 patients with RA. We also measured the serum level of IL-33 and IL-6 before and after the administration of tocilizumab for 24 weeks in 40 patients. Results Serum IL-33 level showed significant correlation with RF (rho =0.660, p<0.001) but did not correlate with DAS28, ESR, hsCRP or RA duration. IL-6 level was significantly correlated with hsCRP (rho =0.482, p<0.001) but not correlate with DAS28, ESR, RF or RA duration. There was no correlation between serum IL-6 and IL-33 levels. Serum IL-33 level significantly decreased after 24 weeks of IL-6 receptor inhibition in patients with RA (p<0.001). When comparing subgroups according to ACR20 response, serum IL-33 levels were significantly decreased after 24 weeks of IL-6 receptor inhibition therapy in ACR20 responders (p<0.001) but not in the non-responders (p=0.084). Baseline IL-33 levels were not significantly different between the two subgroups (p=0.765). Serum IL-6 levels were not significantly changed after 24 weeks of IL-6 receptor inhibition therapy (median 7.1 to 8.9 pg/mL, p=0.503). Changes of IL-6 levels were insignificant both in ACR20 responders and non-responders after 24 weeks of IL-6 receptor inhibition therapy. Baseline IL-6 levels were not different between ACR20 responders and non-responders. Conclusions The use of IL-6 receptor inhibitor decreased the serum level of IL-33 and this effect seems to be led by the responder group. IL-33 could be a useful indicator to monitor the response in IL-6 receptor inhibition therapy. Disclosure of Interest None declared

G.P.260

Nemaline myopathy (NM) is a clinically heterogeneous congenital myopathy characterized by the presence of nemaline rods in the skeletal muscle fibers. We investigated the clinical variation and pathological features in Korean patients with NM. Thirteen patients were diagnosed through muscle biopsy. Six patients had the typical congenital type, which showed the motor development delay and hypotonia at birth. Six patients showed the mild childhood type with the gait disturbance. One patient had the intermediate congenital type, who needed mechanical ventilation. Regarding the distribution of weakness, five patients presented distal dominant weakness across the clinical type. High arched palates and feet deformity were observed. Equino varus deformity was identified in two patients. But, we could not find the cardiac muscle involvement. Typical nemaline rods were recognized on light microscopy with muscle biopsy from 11 patients. However, we could identify the nemaline filamentous aggregation in electro microscope (EM) from 2 patients. One of them showed a mitochondrial abnormality in EM. Type 1 fiber predominance was in all patients. The causative mutation form 8 patients was investigated, we found a missense heterozygous mutation in exon 1 of TPM3 gene (c.32T>A, p.Met11Lys). Seven patients showed negative results in ACTA1 gene. The thirteen patients in this study did not share much common feature except the dysmorphic appearance. The clinical diversity was prominent with the distribution of muscle weakness and the severity of respiratory dysfunction. This study showed the phenotypic heterogeneity of NM is not only with clinical features but also with pathological findings in our patient pool. Wider application of whole exome sequencing may help overcome the phenotypic and genotypic diversity in the diagnosis of NM.

G.P.267

Nebulin is a giant sarcomeric protein spanning whole length of thin filament, and its coding gene (NEB) mostly causes autosomal recessive nemaline myopathy. The NEB mutations may also cause distal nebulin myopathy with initial presentation of foot drop, and recent reports added another muscle disease of core-rod myopathy with double presentation of nemaline rods and cores. In this study, two patients with nemaline rods in muscle pathology were recruited, one of whom (patient 1) was the proband of a symptomatic sibling. Patient 1 initially presented with foot drop and has been a slow runner since childhood. His fourth elder brother also complained of foot drop and gait disturbance. Patient 2 had gait disturbance since age 5, and his ankle dorsiflexors were the most weak among all muscles. All the patients were still ambulant and never complained of respiratory restriction. Muscle CT scans revealed atrophy of anterior tibial muscles in both of patients. Muscle pathology additionally showed core lesions and mitochondrial abnormalities, as well as nemaline rods. Nemaline myopathy-causing genes were first excluded and then, whole exome sequencing and followed targeted sequencing were detected three novel mutations in NEB gene: one missense, one single bp deletion and del/ins mutations. One missense mutation was shared by all of them. This study represents the disease associated with novel NEB mutations marked by the presence of additional pathological features, as well as nemaline rods. Although mixed pathology has been already reported in core-rod myopathy with NEB mutations, muscle pathology in these patients is more characteristic, and clinical manifestation is much milder compared with the previous ones. This report suggests the expanded clinical and pathological spectrum of nebulin-associated myopathy with new genetic and pathologic features. Further, next genome sequencing might be helpful for searching mutations in big, huge-sized genes, such as NEB.

SAT0059 Baseline Interleukin-17A PREDICT Treatment Response to TOCILIZUMAB and Disease-Modifying Antirheumatic Drugs Therapy

Background Tocilizumab (TCZ) has been developed and investigated in several clinical trials for efficacy and adverse events in RA patients. But it remains to be investigated which biomarkers have early predictive values. Objectives To investigate predictive cytokines of TCZ therapy in rheumatoid arthritis (RA) patients with an inadequate response to disease-modifying antirheumatic drugs (DMARDs). Methods We collected sera as part of CWP-TCZ301, a 24-week, randomized, double–blinded trial of TCZ in RA patients with an inadequate response to DMARDs. Serum levels of cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-17A, IL-21 and IL-23 were determined by luminex multiplex analysis at baseline and after treatment (4, 12 and 24 weeks). IL-6 and soluble IL-6 receptor (sIL-6R) were measured by ELISA. Therapeutic response was evaluated by American College of Rheumatology 20% improvement (ACR20) in the TCZ group (n=47) and placebo group (n=48) after 24 weeks. Results Early withdrawal patients from the study were excluded in the evaluation. In TCZ group (n=40), 29 patients were ACR20 responders and 11 patients were non-responders after 24 weeks of treatment. Baseline serum levels of IL-17A were significantly lower in responders than in non-responders (p <0.05). However IL-17A level did not significantly change during TCZ treatment irrespective of ACR20 response. Levels of IL-21 and IL-23 were not significantly different at baseline in responders and non-responders. However, they were significantly decreased at 12 and 24 weeks in responders (all p <0.005), but not in the non-responders. In the placebo group (n=43), 8 patients were ACR20 responders and 35 patients were non-responders after 24 weeks of treatment. Baseline serum levels of IL-17A and IL-21 in ACR20 responders were significantly lower than in non-responders (p<0.001 and p=0.001, respectively). But they did not significantly change during DMARDs treatment, irrespective of ACR20 response. Multivariable logistic regression analysis showed that lower baseline IL-17A patients had increased the odds ratio of being a responder after TCZ (OR 7.364) as well as placebo (OR 9.333) treatment. Figure 1. ROC curve analyses for ACR responders after TCZ (A) and DMARDSs (B) therapy. Conclusions Baseline serum level of IL-17A could be used to predict response of TCZ and DMARDs therapy in RA patients. References Zhou L, Ivanov, II, Spolski R, Min R, Shenderov K, Egawa T, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 2007;8(9):967-74. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5095

P.12.9 Electrophysiological characterization of novel CLCN1 mutations found in Korean patients with myotonia congenita

Myotonia congenita is the most common non-dystrophic channelopathy of the skeletal muscle. It is characterized by clinical myotonia on voluntary contraction, accompanied by warm-up alleviation with repeated movements. Mutations of the chloride channel gene (CLCN1) are responsible for the disease. Both autosomal dominant and recessive inheritance patterns are reported, while recessive cases are more frequent. We analyzed 38 Korean patients with myotonia congenita. Twelve mutations were found and 8 of them were novel. Patch clamp recording revealed significant difference between mutated genes and WT in current density, current–voltage curve, and open probability. Noteworthy of them were 2 peculiar mutations, R47W and A298T, found together as compound heterozygotes in 6 patients. To our best knowledge, they have not been reported to cause any disease phenotype and R47W is even listed in dbSNP as minor allele. R47W is coded from exon 1 and located at N-terminal; A298T is from exon 8, the hotspot of CLCN1 mutation and reside in domain V. By electrophysiologic techniques we could successfully show the pathogenicity of CLCN1 mutations in Korean patients with myotonia congenita. Mild electrophysiological abberation of R47W may explain the paucity of its homozygous patients despite its relatively high allele frequency. However, compound heterozygosity of R47W with A298T disclosed typical electrophysiologic abnormality in myotonia congenita. Further study on heterodimeric interaction of chloride channel protein is warranted to better understand its function and abnormalities.