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The ERETIC (Electronic REference To access In vivo Concentrations)2 method is a new qNMR experimental technique to measure analytes based on the signal of the reference compound without additional hardware equipment. In this study, ERETIC2 method was validated, and we sought to identify whether it would be possible to apply this method to a specific compound analysis of metabolites in plant. The 90° pulse value (P1) and spin-lattice relaxation time (T 1 ) of each compound were measured for ERETIC2. The 9 1 H of 3-(trimethylsilyl) propionic-2,2,3,3-d 4 acid (TSP) was used as a reference peak for ERETIC 2, and then, a suitable solvent and pulse sequence for each compound were selected. Under the NOESY-presat sequence, the relative accuracy error for quantitative analyses of primary metabolites was within the range of 5%, with the exception of glucose, which showed ≥ 55% error due to saturation. It showed excellent results for the quantification of glucose by using a 30° pulse sequence, which did not suppress the water peak. In addition, the quantitative accuracy for secondary metabolites was extremely accurate, with an error ≤5% when considering the purity of the standard sample. The ERETIC2 method showed outstanding linearity, precision, and accuracy.

BACKGROUND AND OBJECTIVEIntoxications 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 SETTINGSThis 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.METHODSFour 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.RESULTSPatients’ 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.CONCLUSIONThe 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.

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ObjectivesThe 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 methodsThis 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.ResultsMean 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.ConclusionThis 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.

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.

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