Classification of Angelica species found in various foods using an LC-QTOF/MS-based metabolomics approach.

In Korea, Angelica gigas is commonly known as Danggui. However, two other species on the market, Angelica acutiloba and Angelica sinensis, are also commonly called Danggui. Since the three Angelica species have different biologically active components, thus, different pharmacological activities, clear discrimination between them is needed to prevent their misuse. A. gigas is used not only as a cut or powdered product but also in processed foods, where it is mixed with other ingredients. To discriminate between the three Angelica species, reference samples were analysed as non-targeted using liquid chromatography-quadrupole time of flight/mass spectrometry (LC-QTOF/MS) and a metabolomics approach in which a discrimination model was established by partial least squares-discriminant analysis (PLS-DA). Then, the Angelica species in the processed foods were identified. First, 32 peaks were selected as marker compounds and a discrimination model was created using PLS-DA, and its validation was confirmed. Classification of the Angelica species was undertaken using the YPredPS value, and it was confirmed that all 21 foods examined contained the appropriate Angelica species indicated on the product packaging. Likewise, it was confirmed that all three Angelica species were accurately classified in the samples to which they were added.

Identification of a novel bromoindazole synthetic cannabinoid analogue in seized e-cigarette liquid: ADB-BRINACA.

A wide variety of new synthetic cannabinoids have emerged around the world in recent years, and because of this rapid emergence, the detection and monitoring of this class of abused drugs remain a challenge. In this study, a new cannabimimetic indazole-3-carboxamide derivative, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)- 5-bromo-1 H-indazole-3-carboxamide, was identified from seized e-cigarette liquid samples and newly named as ADB-BRINACA by referring to the names of other known indazole-class synthetic cannabinoids. Structure identification was accomplished based on gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-high resolution quadrupole mass spectrometry (HPLC-QTOF-MS/MS), and nuclear magnetic resonance spectrometry (NMR). The concentration range of ADB-BRINACA in six e-cigarette liquid samples was found to be 2228-4203 mg/L using ERETIC 2, a quantitative NMR method, which is advantageous in the absence of a reference material. As there have been no chemical or pharmaceutical reports on ADB-BRINACA until now, this is the first report presenting a comprehensive analytical characterization of ADB-BRINACA.

Discrimination of three Angelica herbs using LC-QTOF/MS combined with multivariate analysis.

Angelica gigas, a popular medicinal herb in Korea, is locally called Danggui; this name is similarly used for Angelica acutiloba and Angelica sinensis, which are also sold in the retail market. These three herbs have differing therapeutic effects and should be used according to their prescribed purposes. In some retail markets, though, all three herbs are known by the same common name rather than a scientific name and can therefore be confused with each other. In particular, in the case of powdered products, intentional or unintentional wrong sales activity by the seller may occur. In this study, non-targeted analysis was performed using liquid chromatography quadrupole time-of-flight mass spectrometry to discriminate between the three Angelica herbs, and marker compounds were identified by principal component analysis. Principal component analysis was applied to the whole dataset with the variables being sample name, peak name (m/z with retention time), and ion intensity extracted in advance by peak finding, alignment, and filtering. All three herbs were visually and clearly differentiated in the score plot, and the marker compounds that contributed to their discrimination were found in the loading plot through principal component variable grouping (PCVG). Among the marker compounds, coumarins contributed to the classification of A. gigas, and phthalides contributed to the classification of A. sinensis. The three Angelica herbs were well discriminated from each other. Within the three Angelica species investigated, marker compounds can determine the species of even powdered or extracted samples that cannot be visually identified.

Determination of nitrite and nitrate in postmortem whole blood samples of 10 sodium nitrite poisoning cases: The importance of nitrate in determining nitrite poisoning.

Sodium nitrite (NaNO2), a widely used food preservative, has become a popular agent in South Korea for use in committing suicide since the mid-2010s because of its easy access. After ingesting sodium nitrite, nitrite ions oxidize hemoglobin to methemoglobin in red blood cells (RBCs), causing methemoglobinemia which can be fatal depending on the severity. As the number of deaths involving sodium nitrite has increased rapidly over the years, we developed a quantitative analysis method for nitrite and its oxidized form, nitrate, using ion chromatography (IC) with a conductivity detector. A simple ultrafiltration method was used for sample preparation because chloride ions which usually interfere with nitrite in most IC methods were completely separable using the developed analytical method. The limit of detection and lower limit of quantitation of nitrite were 0.5 and 1 mg/L, respectively. Nitrite and nitrate showed good linearity in the range of 1-500 mg/L and 5-500 mg/L, respectively. The established method was successfully applied to 10 authentic sodium nitrite poisoning cases, resulting in low nitrite concentrations (32.4 ± 29.5 mg/L in peripheral blood samples and 20.4 ± 18.7 mg/L in heart blood samples) and high nitrate concentrations (298.0 ± 25.6 mg/L in peripheral blood samples and 252.0 ± 41.3 mg/L in heart blood samples). The imbalance between nitrite and nitrate was due to the extensive conversion of nitrite to nitrate in postmortem bloods, which was confirmed by spiking nitrite into blank blood samples. In conclusion, not only the blood concentrations of nitrite but also those of nitrate should be quantified and considered for the determination of sodium nitrite poisoning, especially in postmortem blood samples.

A novel method for cyanide quantification in human whole blood using ion chromatography with amperometric detection and its application to cyanide intoxication cases.

Cyanide is a highly toxic agent that has been frequently used for suicide in South Korea. It is also used in various industrial fields, such as metal plating, in which many accidental cyanide intoxications have occurred. To overcome the disadvantages of conventional cyanide analysis methods, a simple and fast method for the analysis of cyanide in whole blood using ion chromatography (IC) with amperometric detection was developed in this study. Whole blood samples were deproteinized, diluted, and analyzed using an IC-amperometric detection system. The limits of detection and quantitation were 0.1 and 0.2 mg/L, respectively. The method showed good linearity in the range of 0.2 to 50 mg/L with R2  > 0.99. The intra- and inter-assay precision and accuracy values were <10%. The established method was successfully applied to analyze whole blood samples from three cyanide intoxication cases.

Clinical relationship between blood concentration and clinical symptoms in aconitine intoxication.

BACKGROUND: Aconitine is well-known for its potential analgesic, anti-inflammatory, and circulation promoting effects and has been widely used as a folk medicine in South Korea. Owing to its extremely toxic nature and relatively low safety margin, intoxication is sometimes fatal. The toxic compound mainly affects the central nervous system, heart, and muscle, resulting in cardiovascular complications. PURPOSE: To determine the exact relationship between blood concentration of aconitine and clinical manifestation. BASIC PROCEDURES: The National Forensic Service (NFS) was commissioned to assist in a quantitative analysis of highly toxic aconitine and corresponding blood concentrations by analyzing the body fluids of three patients who were suspected of aconitine poisoning. MAIN FINDINGS: Aconitine blood values tested by the NFS showed that patients with a blood concentration below a certain level developed symptoms slowly and showed a high severity of clinical manifestation. There was no correlation between blood concentration and symptoms or ECG results. CONCLUSIONS: In case of suspected aconitine poisoning, an emergency care department should be visited, even with symptomatic improvement, and the patient should be monitored for at least 24 h, depending on the level of recovery and changes in ECG results.

Identification of phytolaccosides in biological samples from pokeweed intoxication patients using liquid chromatography-tandem mass spectrometry.

In Phytolaccaceae family, Phytolacca americana L. (American pokeweed) and P. esculenta Van Houtte (Chinese pokeweed) are the two representative species among the genus. Pokeweeds have triterpenoid saponins as toxic compounds in every part of the plant. The saponins phytolaccoside A, B, D, E, and G were isolated from P. americana, and esculentoside H, J, L, K, M, I, and N were isolated from P. esculenta. Along with saponins, their aglycones (phytolaccagenin, phytolaccagenic acid, esculentic acid and jaligonic acid) were also isolated from P. americana and P. esculenta. Two people who unknowingly ate misidentified pokeweed plant roots were transferred to the emergency room. Urine and gastric content after irrigation were collected from the first patient (patient 1), and blood and urine were collected from the second patient (patient 2). The samples were analyzed to identify toxic substances with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the blood sample, 1.9 ng/mL of esculentoside A and 1.5 ng/mL of esculentoside C were detected, while the concentration of esculentoside B and H were below the LLOQ. In gastric contents and ingested roots, esculentoside A, B, C, and H were identified. Esculentoside A, C, and H were identified in the urine of patient 1, and esculentoside A and C were identified in the urine sample of patient 2. The developed analytical method was validated for parameters such as linearity, limit of detection, precision, accuracy, matrix effect, recovery, and process efficiency, and they showed clear and unbiased results.

Serial measurement of glyphosate blood concentration in a glyphosate potassium herbicide-intoxicated patient: A case report.

INTRODUCTION: This report describes changes in blood and urine concentrations of glyphosate potassium over time and their correlations with clinical symptoms in a patient with acute glyphosate potassium poisoning. CASE REPORT: A 67-year-old man visited the emergency center after ingesting 250 mL of a glyphosate potassium-based herbicide 5 h before. He was alert but presented with nausea, vomiting, and bradyarrhythmia with atrial fibrillation (tall T waves). Laboratory findings revealed a serum potassium level of 6.52 mEq/L. After treatment with an injection of calcium gluconate, insulin with glucose, bicarbonate, and an enema with polystyrene sulfonate, the patient's serum potassium level normalized and the bradyarrhythmia converted to a normal sinus rhythm. During admission, the blood and urine concentration of glyphosate and urine aminomethylphosphonic acid (AMPA, a glyphosate metabolite) was measured at regular time intervals. The patient's glyphosate blood concentration on admission was 11.48 mg/L, and it had decreased rapidly by 16 h and maintained about 1mgl/L by 70 h after admission. Urine glyphosate and AMPA levels had also decreased rapidly by 6 h after admission. DISCUSSION: Glyphosate potassium poisoning causes hyperkalemia. Blood concentrations of glyphosate were decreased rapidly by 16 h after admission, and urine concentrations were also decreased by 6 h after admission.

Identification of trichothecene-type mycotoxins in toxic mushroom Podostroma cornu-damae and biological specimens from a fatal case by LC-QTOF/MS.

In some autopsy cases, there are unknown natural toxins that are suspected to cause serious damage to the person. However, without reference materials, it is almost impossible to identify the suspicious natural toxins by GC-MS or LC-MS. In this case, a man drank mushroom -liquor with a meal at his home. Seven hours later, he was transported to the emergency room, and 12hours later, he died. In the ingested mushroom-infused-liquor, there were pieces of mushroom that were estimated to be Podostroma cornu-damae (Hypocreaceae) based on their morphological characteristics. To identify the species, chemical component analysis was conducted using LC-QTOF-MS/MS. Monoisotopic mass, fragment ions, and isotope distributions were obtained from the LC-QTOF-MS/MS analysis. In addition, fragment ions and structure matching were tested for target compound confirmation. In this analysis, several toxic trichothecene-type mycotoxins were identified including roridin D, roridin E, roridin Q, satratoxin G, satratoxin H, satratoxin H 12'-acetate, satratoxin H 13'-acetate, satratoxin H 12',13'-diacetate, and verrucarol. At autopsy, heart blood, peripheral blood, and the stomach contents were collected, and only satratoxin H was detected in these samples. This is the first finding of a trichothecene-type mycotoxin in a human biological sample from an expected case of P. cornu-damae intoxication. We demonstrated that LC-QTOF-MS/MS analysis was an effective method for mushroom intoxication cases in the absence of reference materials. Additionally, the experience, knowledge, and analytical methods we obtained in this study will be great assets for solving other cases of possible natural toxin intoxication.

Grayanotoxin levels in blood, urine and honey and their association with clinical status in patients with mad honey intoxication.

OBJECTIVES: The purpose of this study was to investigate whether there is an association between grayanotoxin levels in urine and blood of patients with mad honey intoxication and in the honey consumed, and the resulting clinical picture. The pilot data acquired from this study was analysed in National Forensic Service, Daejeon Institute, South Korea and first results were published as a preliminary study. PATIENTS AND METHODS: This descriptive study was conducted at a university hospital emergency department in Turkey. 25 cases diagnosed with mad honey intoxication were obtained the study. Samples of mad honey consumed by patients were obtained. Blood and urine specimens were collected at presentation to the emergency department. GTX 1 and GTX 3 levels from patients' blood, urine and honey consumed were investigated simultaneously using the LC-MS/MS system. RESULTS: Mean GTX 1 concentration in blood was 4.82 ng/mL and mean GTX 3 level 6.56 ng/mL. Mean GTX concentration in urine was 0.036 µg/mL and mean GTX 3 level 0.391 µg/mL. Mean GTX I concentration in honeys consumed was 8.73 µg/gr and mean GTX 3 level 27.60 µg/gr. CONCLUSION: This descriptive study is show grayanotoxin levels in body fluids of patients with mad honey intoxication. No association was determined between grayanotoxin levels in blood and clinical data.

Simultaneous determination of bentazone and its metabolites in postmortem whole blood using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method with solid-phase extraction (SPE) was developed and validated for the detection and quantitation of bentazone and its two hydroxylated metabolites, 6-hydroxybentazone and 8-hydroxybentazone, in postmortem blood. Sample cleanup was performed using a hydrophilic-lipophilic balanced (HLB) SPE cartridge and then separated on a C18 LC column using a gradient elution of 0.1% formic acid in distilled water and 0.1% formic acid in methanol. The identification of bentazone and its hydroxylated metabolites was performed using tandem mass spectrometry with electrospray ionization in negative ion mode with selective reaction monitoring. The retention times of bentazone, 6-hydroxybentazone, 8-hydroxybentazone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA, internal standard) appeared separately in the chromatogram. The matrix effect, recovery, and process efficiency of bentazone were 75.3%, 103.6% and 77.9%, respectively. In addition, good accuracy (88.2-110.5%), precision (0.5-7.5%, bias), and linearity (5-500ng/mL) were obtained with this method. The limit of detection (LOD) of bentazone, 6-hydroxybentazone, and 8-hydroxybentazone were 0.05, 0.5, and 0.5ng/mL, respectively. The method developed herein was applied to authentic samples from three fatal cases from 2016 for the determination of the corresponding bentazone and its metabolites levels. The concentration ranges of bentazone, 6-hydroxybentazone, and 8-hydroxybentazone in the heart blood from the three victims were 46.0-91.8, 4.2-6.2, and 0.2-0.6µg/mL, respectively.

Development of an analytical method for simultaneous detection of psychotropic phenylalkylamines in hair by LC-MS/MS with a multi-mode reversed-phase column using pH gradient elution.

Phenylalkylamine derivatives, such as methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), phentermine, fenfluramine, phendimetrazine, amfepramone, and ketamine, are widely abused recreational or anorectic drugs in Korea, and their abuse has become a serious social problem. Hair is a useful specimen to prove chronic use and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently become a more popular tool for hair analysis due to sensitivity and simplicity in sample preparation. In order to overcome limitations of standard reversed-phase column to separate low molecular weight amines, we adopted a multi-mode reversed-phase column, Scherzo SS-C18, which was composed of strong ionic ligands and C18 ligands, and used pH gradient elution to separate seven psychotropic phenylalkylamines and their metabolites. The essential validation parameters including selectivity, LOD, LLOQ, linearity, intra- and inter-assay precision and accuracy, recovery, and the matrix effect were satisfactory. The LODs ranged from 0.1ng/5mg hair (diethylnorephedrine, fenfluramine, ketamine, and MA) to 0.5ng/5mg hair (amfepramone, MDA, phendimetrazine, and phentermine). The LLOQs were 1ng/5mg hair for all analytes. The developed method was successfully applied to determination of phenylalkylamines in authentic hair samples analyzed previously by a routine gas chromatography/mass spectrometry (GC-MS) method. A good correlation was observed between the two methods, with a slope near one.

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.

Examination using LC-MS/MS determination of grayanotoxin levels in blood, urine, and honey consumed by patients presenting to the emergency department with mad honey intoxication and relations with clinical data: a preliminary study.

BACKGROUND AND OBJECTIVE: Intoxications related to "mad honey" are frequently encountered in the Black Sea region of Turkey. Intoxication is established on the basis of whether honey was consumed when history was taken at presentation. The search for a simple and reliable method for showing the grayanotoxins (GTXs) in mad honey in body fluids and in honey consumed by patients is still at the research stage. The purpose of this preliminary study was to investigate GTX levels in blood, urine, and honey consumed by patients with mad honey intoxication and to determine whether there is an association with clinical status. DESIGN AND SETTINGS: This descrptive study was conducted at the department of Emergency Medicine of Karadeniz Technical University Medical Faculty in Turkey. Mad honey, blood, and urine samples were obtained from patients between September 2013 and October 2014. METHODS: Four cases presenting the Department of Emergency Medicine and diagnosed with mad honey intoxication were included in the study. GTX levels in blood, urine, and honey consumed by patients were determined using liquid chromatography-tandem mass spectrometry. RESULTS: Patients' mean blood GTX I level was 30.62 ng/mL, GTX III level 4.917 ng/mL, urine GTX I level 0.447 mg/mL, and GTX III level 1.998 mg/mL. The mean GTX I level in the honey samples consumed was 4.683 mg/g and GTX III level 8.423 mg/g. CONCLUSION: The present study is unique in representing the first time that GTXs have been determined in human body fluids. There is now an urgent need for a large series of studies to provide statistical evidence whether there is a relationship between levels of toxins in human body fluids and clinical picture.

Development of a liquid chromatography-tandem mass spectrometry method for the determination of Grayanotoxins in rat blood and its application to toxicokinetic study.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.

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.

Genetic and chemical components analysis of Papaver setigerum naturalized in Korea.

Of the 110 species of genus Papaver, only Papaver somniferum and P. setigerum are controlled poppies in Korea. All poppy samples share similar morphology therefore it is important to check if they contain controlled substances such as morphine and codeine for forensic purpose. Since the alkaloid content of Papaver plants varies according to their growing stage, chemical components analysis alone is not enough to identify exact species. In 2010, hundreds of poppy plants suspected to be P. somniferum were found in Jeju Island, South Korea. They had a slightly different but overall similar appearance to P. somniferum. Using GC-MS analysis, codeine, rhoeadine, papaverine, protopine, noscapine, setigeridine and trace amounts of morphine were detected in these samples. Although their chemical components were different from what has been described in literatures for P. setigerum, they could be assumed to be P. setigerum based on their morphological features and GC-MS results. Also, chromosome numbers using their seeds showed 2n=44 and the numbers were in accordance with those of P. setigerum. Nucleotide substitution or insertion/deletion of ITS (internal transcribed spacer), 18S rRNA (ribosomal RNA), rbcL (large subunit of ribulose 1,5-bisphosphate carboxylase), trnL-trnF IGS (intergenic spacer), trnL intron and psbA-trnH were assessed as universal genetic markers for P. setigerum. Also, genetic analysis using six target genes involved in the biosynthesis of benzylisoquinoline alkaloids, including TYDC (tyrosine/dopa decarboxylase), SAT (salutaridinol-7-O-acetyltransferase), BBE (berberine bridge enzyme), COR (codeinone reductase), CYP80B1 ((S)-N-methylcoclaurine 3'-hydroxylase) and NCS (norcoclaurine synthase) were tested as Papaver-specific genetic markers by the existence of their PCR products. From the results, the sequences of the 6 universal genetic markers and 6 Papaver-specific genetic markers for P. setigerum were identified and then Genbank accession numbers of them were registered in NCBI. Also, the trnL intron and psbA-trnH nucleic acid sequences of the 7 Papaver species were identified and registered.

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.

Determination and validation of tetrodotoxin in human whole blood using hydrophilic interaction liquid chromatography-tandem mass spectroscopy and its application.

A sensitive analytical method was developed for the quantitative determination of tetrodotoxin (TTX), a powerful sodium channel blocker, in human postmortem whole blood. The sample mixture was cleaned up using cation exchange SPE catridge after protein precipitation by methanol and then separated on a PC-HILIC (phosphorylcholine hydrophilic interaction liquid chromatography) column (150 mm × 2.0mm i.d., 5 µm) using a isocratic elution of 1% acetic acid and acetonitrile. The identification of TTX was performed on tandem mass spectrometry with electrospray ionization interface in positive ion mode. The retention time of voglibose (internal standard) and TTX was 5.1 and 6.0 min, respectively. TTX and internal standard (voglibose) were monitored and quantitated using the ion transitions: the respective precursor to product ion combinations, m/z 320/302 for TTX and m/z 268/92 for voglibose in the multiple reaction monitoring (MRM) mode. The recovery of TTX and voglibose was 61.4% and 62.8%, respectively and the good accuracy (97.7-103.9%), linearity (2-1200 ng/mL) and reproducibility were shown in this method. The limit of detection and limit of quantification were 0.32 ng/mL and 1.08 ng/mL, respectively. This method was applied in the case of three fishermen who were poisoned (including one death) by unknown fish on their boat in October 2010. In this case, the levels of TTX were 27.2, 30.0 and 29.7 ng/mL in heart blood, peripheral blood and serum of a victim, were 3.1 and 12.1 ng/mL in peripheral blood and 3.9 and 12.8 ng/mL in serum of two survivors, respectively.