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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. Keywords: Mad honey, Grayanotoxin, LC-MS/MS, Blood, Urine, Pulse, Blood pressure

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 0.1 ng/5 mg hair (diethylnorephedrine, fenfluramine, ketamine, and MA) to 0.5 ng/5 mg hair (amfepramone, MDA, phendimetrazine, and phentermine). The LLOQs were 1 ng/5 mg 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.

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.

A high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the determination of flavonoid glycosides, polymethoxyflavones, and coumarins in herbal medicines of Citrus fruits and peels and Poncirus fruits. The analytes were rutin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, sinensetin, isosinensetin, nobiletin, heptamethoxyflavone, tangeretin, imperatorin, phellopterin, and isoimperatorin, and, together with puerarin (the internal standard), were separated in short run time using a gradient flow of acetonitrile and 0.1% formic acid. Mass spectrometry detection was performed in the multiple reaction monitoring mode. Ion detection was operated alternately between negative and positive ion modes in a single injection. The main fragmentation characteristics of the analytes were investigated to discriminate between isomeric pairs. Validation of the analytical method showed linearity, r > 0.999; accuracy,...

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. Copyright © 2014 John Wiley & Sons, Ltd.

This paper describes a pattern recognition method of Magnoliae flos based on a gas chromatographic/mass spectrometric (GC/MS) analysis of the essential oil components. The botanical drug is mainly comprised of the four magnolia species (M. denudata, M. biondii, M. kobus, and M. liliflora) in Korea, although some other species are also being dealt with the drug. The GC/MS separation of the volatile components, which was extracted by the simultaneous distillation and extraction (SDE), was performed on a carbowax column (supelcowax 10; 30 m{\time}0.25 mm{\time}0.25{\mu}m$) using temperature programming. Variance in the retention times for all peaks of interests was within RSD 2% for repeated analyses (n = 9). Of the 74 essential oil components identified from the magnolia species, approximately 10 major components, which is -pinene, -pinene, sabinene, myrcene, d-limonene, eucarlyptol (1,8-cineol), -terpinene, p-cymene, linalool, -terpineol, were commonly present in the four species. For statistical analysis, the original dataset was reduced to the 13 variables by Fisher criterion and factor analysis (FA). The essential oil patterns were processed by means of the multivariate statistical analysis including hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). All samples were divided into four groups with three principal components by PCA and according to the plant origins by HCA. Thirty-three samples (23 training sets and 10 test samples to be assessed) were correctly classified into the four groups predicted by PCA. This method would provide a practical strategy for assessing the authenticity or quality of the well-known herbal drug, Magnoliae flos.

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.

A rapid, accurate and sensitive liquid chromatography –tandem mass spectrometry method has been developed for the determination of a quaternary nitrogen muscle relaxant, rocuronium, in human blood. The procedure involves protein precipitation with chloroform and trichloroacetic acid, and purification using methanol. The chromatography was performed using a phenyl-hexyl column (150 3 2.0 mm i.d., 3 mm; Phenomenex) with a mobile phase consisting of 5 mM ammonium formate ( pH 3.0) and acetonitrile. Multiple reaction monitoring was used for quantification. The assay was linear over a concentration range of 4–500 ng/mL for rocuronium with R 2 � 0.998. The recoveries for this compound ranged from 96.0 to 109.1%. The intra-day and inter-day precision was less than 10.5% and the accuracy ranged from 106.6 to 114.9%. The validated method was applied to quantify the content of rocuronium in blood and a variety of tissues of a victim suspected of overdose. In conclusion, the method was successfully applied for the analysis of rocuronium in biological samples for forensic toxicology.

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.