Atypical retinopathy in patients with nephronophthisis type 1: an uncommon ophthalmological finding.

BACKGROUND: Progressive retinal degeneration without retinal pigmentation has been repeatedly observed in Korean nephronophthisis (NPHP) type 1 patients with a total homozygous deletion of NPHP1. DESIGN: Retrospective case series. PARTICIPANTS: Patients with clinical diagnosis of NPHP and genetic diagnosis of total deletion of NPHP1 (n = 5) were included in this study. METHODS: Patients with clinical diagnosis of NPHP (n = 57) were screened for total deletion of NPHP1 by polymerase chain reaction (PCR) for the 20 exons of NPHP1. The clinical and ophthalmological findings of NPHP type 1 patients were reviewed. Additionally, four exons of MALL, a gene adjacent to NPHP1, were amplified using PCR, and amplification failure was considered a homozygous deletion encompassing the corresponding exons. MAIN OUTCOME MEASURE: Ophthalmological findings in NPHP type 1 patients. RESULTS: Five of 57 patients with clinical diagnosis of NPHP were diagnosed as having NPHP type 1 by genetic analysis. Chronic renal failure was diagnosed in these five patients at 7.9-15.4 years of age. All the patients with NPHP type 1 had progressive decline in visual acuity with various ages of onset (2-17 years). Ophthalmological examinations revealed unexpected findings of retinopathy with large or small flecks, which was compatible with Stargardt-like retinopathy or albipunctatus retinopathy in majority of them (four of five). The genetic study revealed an additional deletion of exon 1 of the adjacent gene MALL. CONCLUSIONS: We report the unexpectedly common retinal involvement of NPHP type 1 with an additional MALL deletion in a Korean cohort.

Atypical hemolytic uremic syndrome: Korean pediatric series.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease with a genetic predisposition. Few studies have evaluated the disease in the Asian population. We studied a Korean pediatric cohort to delineate the clinical characteristics and genotypes. METHODS: A multicenter cohort of 51 Korean children with aHUS was screened for mutations using targeted exome sequencing covering 46 complement related genes. Anti-complement-factor-H autoantibody (anti-CFH) titers were measured. Multiplex ligation-dependent probe amplification assay was performed to detect deletions in the complement factor-H related protein genes (CFHR) in the patients as well as in 100 healthy Korean controls. We grouped the patients according to etiology and compared the clinical features using Mann-Whitney U-test and chi-squared test. RESULTS: Fifteen patients (group A, 29.7%) had anti-CFH, and mutations were detected in 11 (group B, 21.6%), including one with combined mutations. The remaining 25 (group C, 49.0%) were negative for both. The prevalence of anti-CFH was higher than the worldwide level. Group A had a higher onset age than group B, although the difference was not significant. Group B had the worst renal outcome. Gene frequencies of homozygous CFHR1 deletion were 73.3%, 2.7% and 1% in group A, group B + C and the control, respectively. CONCLUSIONS: The incidence of anti-CFH in the present Korean aHUS cohort was high. Clinical outcomes largely conformed to the previous reports. Although the sample size was limited, this cohort provides a reassessment of clinicogenetic features of aHUS in Korean children.

Muscle involvement in Dent disease 2.

BACKGROUND: Dent disease, an X-linked recessive renal tubulopathy, is caused by mutations in either CLCN5 (Dent disease 1) or OCRL (Dent disease 2). OCRL mutations can also cause Lowe syndrome. In some cases it is difficult to differentiate Dent disease 1 and 2 on the basis of clinical features only without genetic tests. Several studies have shown differences in serum levels of muscle enzymes between these diseases. The aim of our study was to test the validity of these findings. METHODS: In total, 23 patients with Dent disease 1 (Group A), five patients with Dent disease 2 (Group B) and 19 patients with Lowe syndrome (Group C) were enrolled in our study. The serum levels of three muscle enzymes [creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST)], were measured. The levels of a hepatic enzyme, alanine aminotransferase (ALT), were also measured as a control. RESULTS: One patient in Group B had muscle hypoplasia of both upper extremities. The serum levels of all three muscle enzymes assayed were higher in Group B or C patients than in Group A patients. Serum ALT levels were normal in all three groups of patients. CONCLUSIONS: The serum levels of muscle enzymes in patients with Dent disease can be used as a biomarker to predict genotypes, even though the patients do not have clinical symptoms of muscle involvement.

Development of a target component extraction method from GC-MS data with an in-house program for metabolite profiling.

After gas chromatography-mass spectrometry (GC-MS) analysis, data processing, including retention time correction, spectral deconvolution, peak alignment, and normalization prior to statistical analysis, is an important step in metabolomics. Several commercial or free software packages have been introduced for data processing, but most of them are vendor dependent. To design a simple method for Agilent GC/MS data processing, we developed an in-house program, "CompExtractor", using Microsoft Visual Basic. We tailored the macro modules of an Agilent Chemstation and implanted them in the program. To verify the performance of CompExtractor processing, 30 samples from the three species of the genus Papaver were analyzed with Agilent 5973 MSD GC-MS. The results of CompExtractor processing were compared with those of AMDIS-SpectConnect processing by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The two methods showed good classification according to their species in HCA. The PC1+PC2 scores were 54.32-63.62% for AMDIS-SpectConnect and 56.65-85.92% for CompExtractor in PCA. Although the CompExtractor processing method is an Agilent GC-MS-specific application and the target compounds must be selected first, it can extract the target compounds more precisely in the raw data file with batch mode and simultaneously assemble the matrix text file.

Seson, a novel zinc finger protein, controls cilia integrity for the LR patterning during zebrafish embryogenesis.

In zebrafish embryos, bilateral symmetry is broken by asymmetric nodal flow generated in Kupffer's vesicle (KV), the transient cilia-rich organ, analogous to the mouse node. Asymmetric nodal flow induces the asymmetric expression of several genes, which are critical for the determination of correct LR body patterning. seson encoding three consecutive C2H2 zinc finger protein is predominantly expressed in the cilia-rich organs including KV. Inhibition of its function by the injection of a seson-specific MO inhibited the left-side biased expression of spaw, and resulted in randomization of the heart, gut looping and brain laterality. Disruption of the LR patterning in seson morphants appeared to be due to severe cilia defects in KV. Seson function was also required for ciliogenesis in other tissues such as the pronephros and olfactory organs. Collectively, our data suggest that Seson has critical roles in ciliogenesis and LR body axis patterning.

Poria cocos water extract (PCW) protects PC12 neuronal cells from beta-amyloid-induced cell death through antioxidant and antiapoptotic functions.

Beta-amyloid (Abeta)-induced neurotoxicity is considered to be mediated through the formation of reactive oxygen species (ROS). In this study, the protective effects of Poria cocos water extract (PCW) against Abeta1-42-induced cell death were investigated using rat pheochromocytoma (PC12) cells. Exposure of PC12 cells to the Abeta1-42 (20 microM) for 48h resulted in neuronal cell death, whereas pretreatment with PCW at the concentration range of 5-125 microg/ml reduced Abeta1-42-induced cell death. In addition, PC12 cells treated with Abeta1-42 exhibited increased accumulation of intracellular oxidative damages and underwent apoptotic death as determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL staining). However, PCW attenuated Abeta1-42-induced cytotoxicity, apoptotic features, and accumulation of intracellular oxidative damage. Moreover, PCW (5 to 125 microg/ml) decreased expression of apoptotic protein Bax and activity of caspase-3, but enhanced expression of anti-apoptotic protein Bcl-2. These results suggest that PCW may protect cells through suppressing the oxidative stress and the apoptosis induced by Abeta1-42, implying that PCW may be potential natural agents for Alzheimer's diseases.

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair.

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.

Metformin restores leptin sensitivity in high-fat-fed obese rats with leptin resistance.

To evaluate whether metformin enhances leptin sensitivity, we measured leptin sensitivity after 4 weeks of metformin treatment (300 mg/kg daily) in both standard chow and high-fat-fed obese rats. Anorexic and fat-losing responses after intracerebroventricular leptin infusion for 7 days (15 microg daily per rat) in standard chow rats were enhanced by metformin treatment, and these responses to leptin were attenuated in high-fat-fed obese rats compared with age-matched standard chow rats. However, these responses to leptin were corrected by metformin treatment in high-fat-fed obese rats. Moreover, serum concentrations of leptin and insulin were decreased dramatically by leptin in metformin-treated standard chow and high-fat-fed obese rats. The hypothalamic phosphorylated AMP-activated protein kinase level was decreased by lower leptin dose in metformin-treated rats than in untreated rats. In an acute study, metformin treatment also increased the anorexic effect of leptin (5 microg), and this was accompanied by an increased level of phosphorylated signal transducer and activator of transcription 3 in the hypothalamus. These results suggest that metformin enhances leptin sensitivity and corrects leptin resistance in high-fat-fed obese rats and that a combination therapy including metformin and leptin would be helpful in the treatment of obesity.

The study of metabolite-to-parent drug ratios of methamphetamine and methylenedioxymethamphetamine in hair.

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.

Validation of the immunalysis microplate ELISA for the detection of methamphetamine in hair.

The object of this study was to validate the Immunalysis Methamphetamine Microplate ELISA for detecting methamphetamine in hair. Twenty-nine scalp hair samples were obtained as routine cases submitted to the National Institute of Scientific Investigation in Seoul by the police. The hair samples were washed with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. The samples were screened using the Immunalysis Methamphetamine Microplate ELISA and confirmed using gas chromatography-mass spectrometry (GC-MS). Twenty-eight hair samples were screened and confirmed as positive for methamphetamine. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h at 60 degrees C. For GC-MS, the samples were extracted for 20 h in methanol containing 1% hydrochloric acid. The methanol/acid solution was evaporated to dryness and the resulting residue was derivatized with trifluoroacetic anhydride. Methamphetamine and amphetamine were detected using selective ion monitoring (SIM) mode. The Immunalysis Methamphetamine Microplate ELISA demonstrated a sensitivity and specificity of 97% and 100%, respectively, using a cut-off concentration of 0.5 ng/mg d-methamphetamine. The ELISA kit showed 63% cross-reactivity with d,l-methamphetamine and did not cross-react to any significant extent with the licit l-methamphetamine isomer. The intra- and interassay precisions were 2.5% and 3.7%, respectively.

Patterns of drugs & poisons in southern area of South Korea in 2014.

The southern area of South Korea consists of three parts; Busan, Ulsan and Gyeongsangnam-do. Busan Institute of National Forensic Service (NFS) performed about 50,000 cases throughout the southern area in 2014, occupying over 15% of total cases covered by NFS. In this study, patterns of drugs and poisons in the southern area of South Korea were investigated. The investigation was carried out by the laboratory information management system of NFS between January and December of 2014. As results, a total of 606 autopsy cases were performed by Busan Institute of NFS in 2014. Among them, 15 cases were determined as drug intoxication or poisons as the cause of death, taking up 2.5% of total cases: 5 cases of intoxication by drugs, 5 by agricultural pesticides, 3 by illicit drugs, and 1 each by detergents and chemical substances. A total of 108 drugs in postmortem bloods were detected from the autopsy cases, and the top 5 drugs were chlorpheniramine, tramadol, diazepam, zolpidem and lidocaine. Meanwhile, a total of 1,728 cases were submitted for illicit drug testing in 2014. Among them, hair was the most common type of specimens, and the rate of positive detection of methamphetamine from the hair, urine, and seized materials in the southern area was over 50% in all cases, indicating that this is the most commonly abused drug in South Korea. A total of 12 types of novel psychoactive substances (NPSs) were detected in the southern area in 2014; 10 were identified as synthetic cannabinoids and 2 as alkyl nitrites.

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(ß-phenylisopropyl)amine I (DPIA I), N,N-di(ß-phenylisopropyl)amine II (DPIA II), N,N-di(ß-phenylisopropyl)methylamine I (DPIMA I) and N,N-di(ß-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.

Leptin-like effects of MTII are augmented in MSG-obese rats.

To evaluate whether MTII, a melanocortin receptor 3/4 agonist, is working in hypophagic and hypothermogenic obese model, we measured food intake, body weight, oxygen consumption, and fat mass following intracerebroventricular (i.c.v.) infusion of MTII in monosodium glutamate (MSG)-induced obese rats. MTII, or artificial cerebrospinal fluid (aCSF), was infused into i.c.v. with an osmotic minipump for 1 week. MSG-obese rats were induced by neonatal injection of MSG. Five-month-old MSG rats were characterized by hypophagia, lower oxygen consumption, hyperleptinemia, and obesity compared to age-matched control rats. The infusion of MTII decreased their food intake, visceral fat, and body weight in MSG-obese rats compared with aCSF-infused rats. The oxygen consumption was increased by MTII treatment in MSG-obese rats compared with aCSF as well as pair fed (PF) rats. Interestingly, these leptin-like effects of MTII were greater in MSG-obese rats than in controls, which might be related to the increased expression of melanocortin receptor 4 (MC4R) in the hypothalamus of MSG-obese rats. Our results suggested that both anorexic and thermogenic mechanisms were activated by MTII in the MSG-obese rats and contributed to the decrease in body weight and fat mass. Moreover, there was a sensitization to MTII caused by upregulation of the melanocortin receptor in the MSG-obese rats.

Correlation of methamphetamine results and concentrations between head, axillary, and pubic hair.

This study was designed to compare the qualitative results and concentrations of methamphetamine (MA) and its metabolite amphetamine (AP) in head hair and hair collected from different parts of the body (axillae and pubis). Hair from subjects (N = 14) suspected MA users was simultaneously collected. Hair preparation involved washing step, fine cutting, overnight extraction, derivatization by the trifluoroacetic anhydride, and gas chromatography/mass spectrometry (GC/MS) using selective ion monitoring. In this study, we found a good correlation of the qualitative results for MA between head hair and hair on other parts of the body, but there were some differences in concentrations of MA and AP. Namely, the concentrations of MA and AP were higher in axillary and pubic hair than in head hair.

The prevalence of MDMA/MDA in both hair and urine in drug users.

The prevalence and age distribution of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in hair samples by gas chromatography/mass spectrometry (GC/MS) were studied. The recoveries obtained from hair were 97% and 99% for MDMA and MDA, respectively. The inter- and intra-assay precision and accuracy were determined. Out of 791 hair samples, 44 (5.6 %) contained MDMA and/or MDA. Out of these 44 subjects, urinalyses from 35 were negative for both MDMA and MDA, while only 9 were positive. We also evaluated concentrations of MDMA and MDA, and the metabolite-to-parent drug ratios. This study showed that the abuse of MDMA or MDA was found principally among young adults and male abusers. We found the epidemiology of ecstasy users in Korea between March 2002 and April 2003.

Relationship between methamphetamine use history and segmental hair analysis findings of MA users.

The aim of this study was to investigate the relationship between methamphetamine (MA) use history and segmental hair analysis (1 and 3cm sections) and whole hair analysis results in Korean MA users in rehabilitation programs. Hair samples were collected from 26 Korean MA users. Eleven of the 26 subjects used cannabis with MA and two used cocaine, opiates, and MDMA with MA. Self-reported single dose of MA from the 26 subjects ranged from 0.03 to 0.5g/one time. Concentrations of MA and its metabolite amphetamine (AP) in hair were determined by gas chromatography mass spectrometry (GC/MS) after derivatization. The method used was well validated. Qualitative analysis from all 1cm sections (n=154) revealed a good correlation between positive or negative results for MA in hair and self-reported MA use (69.48%, n=107). In detail, MA results were positive in 66 hair specimens of MA users who reported administering MA, and MA results were negative in 41 hair specimens of MA users who denied MA administration in the corresponding month. Test results were false-negative in 10.39% (n=16) of hair specimens and false-positive in 20.13% (n=31) of hair specimens. In false positive cases, it is considered that after MA cessation it continued to be accumulated in hair still, while in false negative cases, self-reported histories showed a small amount of MA use or MA use 5-7 months previously. In terms of quantitative analysis, the concentrations of MA in 1 and 3cm long hair segments and in whole hair samples ranged from 1.03 to 184.98 (mean 22.01), 2.26 to 89.33 (mean 18.71), and 0.91 to 124.49 (mean 15.24)ng/mg, respectively. Ten subjects showed a good correlation between MA use and MA concentration in hair. Correlation coefficient (r) of 7 among 10 subjects ranged from 0.71 to 0.98 (mean 0.85). Four subjects showed a low correlation between MA use and MA concentration in hair. Correlation coefficient (r) of 4 subjects ranged from 0.36 to 0.55. Eleven subjects showed a poor correlation between MA use and MA concentration in hair. Correlation between MA use and MA concentration in hair of remaining one subject could not be determined or calculated. In this study, the correlation between accurate MA use histories obtained by psychiatrists and well-trained counselors and MA concentrations in hair was shown. This report provides objective scientific findings that should considerably aid the interpretation of forensic results and of the results of trials related to MA use.

Analysis of benzodiazepines and their metabolites using DBS cards and LC-MS/MS.

Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC-MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50µL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC-MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30µL of whole blood was spotted on FTA DMPK card C and dried for 2h in a desiccator. A 6-mm disk was punched and vortexed for 1min in a centrifuge tube with 300µL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100µL mobile phase of LC-MS/MS and 5µL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters - LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) - were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem).

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.

Quantitative determination of 11-nor-9-carboxy-tetrahydrocannabinol in hair by column switching LC-ESI-MS(3).

Hair analysis has been regarded as an alternative method to urine analysis in forensic and criminal cases. Cannabis (marijuana) is one of the most widely used drugs in the world and it has been controlled in South Korea since 1976. Identification of 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair can be an important proof of cannabis use because it can exclude the possibility of passive cannabis smoke exposure. In this study, we described a quantitative method of THCCOOH in hair using simple liquid-liquid extraction (LLE), selective column switching liquid chromatography with electrospray ionization (ESI)-MS(3). For the column switching system three columns (precolumn, trap column and analytical column) were used. Valve switch from the precolumn to the trap column was set from 3.0 to 4.0 min because THCCOOH appeared around 3.5 min with this precolumn. After 4.0 min the valve was switched to the original position and the analytes in the trap column were eluted onto the analytical column. Resolution occurred in this column and eluted into the ESI-MS(3) system. The internal standard was THCCOOH-d3. We used ESI-negative-MS(3) transition of ions at m/z 343 to 299 to 245 (343/299/245) and m/z 346 to 302 to 248 (346/302/248) for quantification of THCCOOH and THCCOOH-d3, respectively. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection (LOD) was 0.05 pg/mg and the limit of quantification (LOQ) was 0.10 pg/mg. The range of concentration of THCCOOH from 98 authentic human hair was 0.13-15.75 pg/mg. This method was successfully applied in the analysis of authentic human hair samples.

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.