Syncope without prodromes is associated with excessive plasma release of adenosine at the time of syncope during head-up tilt table test.

BACKGROUND: In syncopal patients without underlying structural disease, we sought to investigate the association of Adenosine Plasma Levels (ADP) with the clinical presentation of neurally mediated syncope (NMS) and the outcomes of Head-Up Tilt Table Test (HUTT) and Adenosine test (ADT). METHODS: We studied 124 patients with different clinical types of NMS, i.e., Vasovagal (VVS, n=58), non-prodromes (NPS, n=18), or situational syncope (SS, n=48), using a standard protocol including HUTT and ADT. During HUTT, ADP was measured in the supine position, at table tilting and in syncope. RESULTS: Baseline ADP did not differ among groups. ADP at syncope were higher in NPS (n=5) compared to VVS (n=20): 0.23 vs. 0.12 µΜ, p=0.03, and SS (n=22): 0.04 µΜ, p=0.02. In NPS, ADP increased from supine to syncope (n=5): 0.15 vs. 0.23 µΜ, p=0.04. In VVS, ADP increased only from supine to tilt position: 0.11 vs. 0.14 µΜ, p=0.02. In SS, ADP did not change during HUTT. In positive vasodepressor HUTT, ADP increased from supine to tilt position (p=0.002) and at syncope (p=0.01). In SS, 20.0% exhibited cardioinhibitory HUTT vs. 6.8% in other forms of syncope (p=0.04). In SS, 22.9% manifested positive ADT vs 6.6% in other types of syncope (p=0.012). CONCLUSION: The subset of NPS patients with positive HUTT, show excessive ADP release at the time of syncope. This may explain the lack of prodromes in this form of syncope. Such observations contribute to the understanding of distinct profiles of clinical forms of syncope and may differentiate the management approach accordingly.

Detection and determination of C12-, C14-, C16-alkyldimethylamines in human blood using gas chromatography-mass spectrometry.

RATIONALE: N,N-Dimethyldodecylamine is produced from lauryl alcohol and dimethylamine. C12-C16 alkyldimethylamines are used as intermediates for the manufacture of amineoxides and quaternary amino compounds. In the present study a gas chromatography-mass spectrometry (GC/MS) method for the determination of C12-C16 alkyldimethylamines in blood was developed and validated. The reason for this study was the detection of the above compounds in the postmortem blood sample of a fatal suicide case. METHODS: Analysis of amines was performed using a gas chromatograph (Agilent Technologies 7890A) with an MS 5975C inrXL, EI/CI MSD with triple-axis detector in selected ion monitoring mode, after liquid-liquid extraction. Four different organic solvents (butyl acetate, ethyl acetate, n-hexane and n-heptane) were used for the optimization of the extraction procedure, resulting in ethyl acetate being the solvent of choice for the extraction procedure. A QuEChERS step was applied (20 mg of MgSO4 , 5 mg of NaCl) to 1 mL of blood and pH was adjusted at 12 (K2 CO3 buffer solution). After the addition of the extraction solvent, samples were vortexed, centrifuged and directly injected into the GC/MS system. RESULTS: In validation, the method was found to be selective and sensitive (limit of detection from 0.3 to 0.5 ng/mL, limit of quantitation from 10.0 to 20.0 ng/mL), whilst validation included recovery, stability, accuracy and precision (relative standard deviation). Validation results were found satisfactory: intra- and interday precision ranged from 0.4% to 2% and from 0.6% to 1.9% respectively, while intra- and interday accuracy ranged from 87% to 109% and from 86% to112.8%. C12-C16 alkyldimethylamines were detected in blood samples at a concentration of 8.39 µg/mL (C12), 3.01 µg/mL (C14) and 0.42 µg/mL (C16). CONCLUSIONS: A rapid, sensitive and reliable method was developed for the determination of C12-C16 alkyldimethylamines in postmortem blood, after optimization of the sample preparation procedure, and finally successfully applied to a real postmortem blood sample from a fatal case involving these compounds.

Application of a hybrid zwitterionic hydrophilic interaction liquid chromatography column in metabolic profiling studies.

Metabolic phenotyping studies using mouse liver extracts as a model, performed on a novel zwitterionic HILIC UHPLC column, which is based on ethylene-bridged hybrid organic/inorganic particles bonded with sulfobetaine groups and packed into column hardware modified with hybrid surface technology are reported. Initially the chromatographic performance was evaluated under different mobile phase conditions using selected metabolite standards. Following optimization of the chromatographic conditions for 88 hydrophilic metabolites both targeted and untargeted profiling analyses were performed on tissue extracts using LC-MS/MS and LC-TOF/MS, respectively. Chromatographic efficiency parameters such as peak resolution, peak shapes, selectivity and precision in retention and peak areas as well as characteristics that are critical for metabolic profiling analysis such as metabolite coverage and retention time distribution were assessed. The hybrid zwitterionic column exhibited efficient chromatographic separations providing analysis of ca 80 hydrophilic metabolites from different chemical classes and polarities. Utilizing a one-dimensional separation both targeted and untargeted profiling provided comprehensive metabolic signatures that enabled the acquisition of the metabolic phenotypes of the tissue extracts.

Headspace gas chromatography-mass spectrometry in the analysis of lavender's essential oil: Optimization by response surface methodology.

A static headspace gas chromatography - mass spectrometry (HS-GC/MS) method was developed and optimized with the aim to be applied in the analysis of lavender essential oil. To obtain a comprehensive profile of the essential oil, the optimum HS-GC/MS method parameters were selected based on a Design of Experiments (DοE) process. Plackett-Burman experimental design was applied by utilizing seven parameters of the HS injection system. Incubation equilibration temperature and time, agitator's vortex speed, post injection dwell time, inlet temperature, split ratio and injection flow rate were screened to select the optimum conditions on the basis of the number and the intensity of the identified compounds. Other parameters, such as sample volume and dilution solvent ratio, were also examined to achieve a comprehensive profile in a chromatographic run of 55 min. With the obtained optimum method, more than 40 volatile compounds were identified in lavender's essential oils from different geographical regions in Greece. The method can be utilized for the quality assessment of lavender's essential oil and provide information on its characteristic aroma and discrimination among species based on the acquired GC-MS profiles.

Liquid chromatography tandem mass spectrometry for the determination of nine insecticides and fungicides in human postmortem blood and urine.

Pesticide poisoning is a common occurrence due to their widespread use, easy access and high toxicity even in small concentrations. The most common poisoning fatalities have been observed due to exposure to organophosphates, carbamates and neonicotinoids, thus development of a method for the rapid determination of these compounds in blood and urine is of great importance for clinical and toxicology laboratories. A simple, fast and reliable method was developed for the determination of 9 pesticides in blood and urine using HPLC-MS/MS instrumentation. In order to find the most suitable sample pretreatment technique, three different sample preparation procedures: SPE, protein precipitation and QuEChERS were compared. The final optimized analytical method was fully validated with the values of parameters such as calibration linearity, accuracy, precision, recovery, matrix effect and stability being acceptable. The method proved reliable, accurate, robust and sensitive and was successfully applied for the quantitation of pesticides in three postmortem cases of pesticides poisoning.

Development and validation of a RPLC-MS/MS method for the quantification of ceramides in human serum.

Ceramides are key-role lipids involved in numerous central cellular processes. A plethora of studies have demonstrated that the levels of ceramides in blood circulation are related to different disease states, such as type 2 diabetes, cardiovascular diseases, ovarian cancer, multiple sclerosis and others. Herein, a RPLC-MS/MS method for the rapid quantification of ceramides Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0) and Cer(d18:1/24:1) in human blood serum was developed and validated. Different sample preparation strategies including SLE, LLE and QuECheRS were tested with the aim to attain effective, accurate and reproducible determination of ceramides in serum samples. Intra and inter-day accuracy were found to be between 80.0-111% and 87.8-106%, respectively, for all ceramides, while intra and inter-day precision were found to vary from 0.05% to 10.2% %RSD and 2.2% to 14.0% %RSD, respectively. The lower limits of quantification were 2.3 ng/mL for Cer(d18:1/16:0) and Cer(d18:1/18:0) and 1.4 ng/mL for Cer(d18:1/24:0) and Cer(d18:1/24:1). The method was evaluated in accordance to bioanalytical method guidelines and was used for the determination of serum ceramides of patients with coronary artery disease to evaluate its utility in clinical analyses.

Quantification of endogenous aminoacids and aminoacid derivatives in urine by hydrophilic interaction liquid chromatography tandem mass spectrometry.

Aminoacids and their derivatives are key biologically important metabolites and reliable, rapid and accurate, quantification for these analytes in urine remains an important analytical challenge. Here a fast and reliable HILIC-tandem MS method is presented for application in clinical or nutritional studies. The developed method was validated according to existing guidelines adapted for endogenous analytes. The validation strategy provided evidence of linearity, LOD and LOQ, accuracy, precision, matrix effect and recovery. The surrogate matrix approach was applied for calibration proving satisfactory accuracy and precision based on standard criteria over the working concentration ranges. Intra and inter day accuracy was found to range between 0.8 and 20% for the LQC (low QC) and between 0.05 and 15 % for MQC (medium QC) and HQC (high QC). Inter and intraday precision were found to be between 3 and 20 % for the LQC and between 1 and 15% for the MQC and HQC. The stability of the analytes, in both surrogate and pooled urine QC samples, was found to be within 15% over a short period at 4 °C or after a up to 3 freeze-thaw cycles. The uncertainty of the method was also assessed to provide increased confidence for the acquired measurements. The method was successfully applied to a subset of human urine samples involved in a study of amino acids dietary uptake. This method may provide a valuable tool for many applications or studies where amino acid metabolic signatures in the excreted urine are under investigation.

Risk factors for fatal drowning in a Greek region: a retrospective case-control study.

BACKGROUND: Fatal drowning is one of the leading causes of unintentional injury mortality worldwide and a persistent public health concern in Greece. While several pathologic and sociodemographic contributing factors have been previously identified, these have not been extensively investigated in conjunction with the effects of psychoactive substances. METHODS: A retrospective case-control study of drowning deaths was conducted in the Greek regions of Northern Greece and Thessaly during a 10-year period. A regression model was constructed examining differences in detected substances, autopsy findings and sociodemographic characteristics between 240 victims of unintentional fatal submersion and 480 victims of other causes of sudden or violent death. RESULTS: The majority of victims were males (69.4%) and foreign nationality was associated with increased odds of drowning. Cardiomegaly and coronary bypass grafts were significantly more likely to have been recorded among drowning victims, while the frequency of other circulatory system disorders was also elevated. Several of these findings were potential arrhythmogenic substrates which could adversely interact with the diving reflex. Selective serotonin reuptake inhibitors (SSRIs) were the most commonly detected pharmacological group (9.0%), and along with tramadol, there was an increased likelihood of exposure to them. These drugs have been previously associated with QT prolongation and other adverse effects which may contribute to fatal outcomes in a seawater environment. In contrast, there was a decreased risk of exposure to dependence-inducing drugs and paracetamol. CONCLUSIONS: Male sex, older age, foreign nationality and cardiovascular disease predisposed individuals to an elevated risk of fatal submersion. SSRI antidepressants and tramadol may contribute to this outcome.

A UHPLC-MS-MS Method for the Determination of 84 Drugs of Abuse and Pharmaceuticals in Blood.

The analysis of blood samples for forensic or clinical intoxication cases is a daily routine in an analytical laboratory. The list of 'suspect' drugs of abuse and pharmaceuticals that should be ideally screened is large, so multi-targeted methods for comprehensive detection and quantification are a useful tool in the hands of a toxicologist. In this study, the development of an ultra-high performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method is described for the detection and quantification of 84 drugs and pharmaceuticals in postmortem blood. The target compounds comprise pharmaceutical drugs (antipsychotics, antidepressants, etc.), some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines, benzodiazepines and new psychoactive substances. Sample pretreatment was studied applying a modified Mini-QuEChERS single step, and the best results were obtained after adding a mixture of 20 mg MgSO4, 5 mg K2CO3 and 5 mg NaCl together with 600 µL of cold acetonitrile in 200 µL of sample. After centrifugation, the supernatant was collected for direct injection. LC-MS analysis took place on a C18 column with a gradient elution over 17 min. The method was found to be selective and sensitive, offering limits of detection ranging from 0.01 to 9.07 ng/mL. Validation included evaluation of limit of quantification, recovery, carryover, matrix effect, accuracy and precision of the method. The method performed satisfactorily in relation to established bioanalytical criteria and was therefore applied to the analysis of blood obtained postmortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification. The method showed good linearity for all analytes within a concentration range from 0.25 to 2 µg/mL for mephedrone, from 0.02 to 0.16 µg/mL for 3,4-MDPV and AH-7921 and from 0.005 to 0.04 µg/mL for JWH-018 and AM-2201. LOD ranged from 0.002 µg/mL (JWH-018 and AM-2201 in blood and urine), to 0.08 µg/mL (mephedrone in urine). LOQ in blood ranged from 0.005 µg/mL for JWH-018 and AM-2201 to 0.25 µg/mL for mephedrone. Accuracy was within acceptable limits with % bias ranging from +20% to -17.98% for intra-assay study and from +18.87% to -11.16% for inter-assay study. Precision was found to be between 2.60% and 17.17% (CV%) for intra-assay study and from 6.03% to 13.72% (CV%) for inter-assay study. An intra laboratory comparison provided proof of the method robustness. The developed method can be used for the reliable and fast quantification of five NPS in blood and the detection of six NPS in urine within the practice of a clinical or forensic toxicology laboratory.

Metabolic Phenotyping Study of Mouse Brains Following Acute or Chronic Exposures to Ethanol.

The chronic and acute effect of ethanol administration on the metabolic phenotype of mouse brain was studied in a C57BL/6 mouse model of ethanol abuse using both untargeted and targeted ultra performance liquid chromatography-tandem mass spectrometry. Two experiments based on either chronic (8 week) exposure to ethanol of both male and female mice or acute exposure of male mice for 11 days, plus 2 oral gavage doses of 25% ethanol, were undertaken. Marked differences were found in amino acids, nucleotides, nucleosides, and related metabolites as well as a number of different lipids. Using untargeted metabolite profiling, acute ethanol exposure found significant decreases in several metabolites including nucleosides, fatty acids, glycerophosphocholine, and a number of phospholipids, while chronic exposure resulted in increases in several amino acids with notable decreases in adenosine, acetylcarnitine, and galactosylceramides. Similarly, targeted metabolite analysis, focusing on the hydrophilic fraction of the brain tissue extract, identified significant decreases in the metabolism of amino acids and derivatives, as well as purine degradation especially after chronic exposure to ethanol.

Study of Fecal and Urinary Metabolite Perturbations Induced by Chronic Ethanol Treatment in Mice by UHPLC-MS/MS Targeted Profiling.

Alcoholic liver disease (ALD) as a consequence of ethanol chronic consumption could lead to hepatic cirrhosis that is linked to high morbidity and mortality. Disease diagnosis is still very challenging and usually clear findings are obtained in the later stage of ALD. The profound effect of ethanol on metabolism can be depicted using metabolomics; thus, the discovery of novel biomarkers could shed light on the initiation and the progression of the ALD, serving diagnostic purposes. In the present study, Hydrophilic Interaction Liquid Chromatography tandem Mass Spectrometry HILIC-MS/MS based metabolomics analyisis of urine and fecal samples of C57BL/6 mice of both sexes at two sampling time points was performed, monitoring the effect of eight-week ethanol consumption. The altered hepatic metabolism caused by ethanol consumption induces extensive biochemical perturbations and changes in gut microbiota population on a great scale. Fecal samples were proven to be a suitable specimen for studying ALD since it was more vulnerable to the metabolic changes in comparison to urine samples. The metabolome of male mice was affected on a greater scale than the female metabolome due to ethanol exposure. Precursor small molecules of essential pathways of energy production responded to ethanol exposure. A meaningful correlation between the two studied specimens demonstrated the impact of ethanol in endogenous and symbiome metabolism.

Metabolic Profiling: Status, Challenges, and Perspective.

Metabolic profiling has advanced greatly in the past decade and evolved from the status of a research topic of a small number of highly specialized laboratories to the status of a major field applied by several hundreds of laboratories, numerous national centers, and core facilities. The present chapter provides our view on the status of the remaining challenges and a perspective of this fascinating research area.

Quality Control and Validation Issues in LC-MS Metabolomics.

Global metabolic profiling (untargeted metabolomics) of different and complex biological matrices aims to implement an holistic, hypothesis-free analysis of (potentially) all the metabolites present in the analyzed sample. However, such an approach, although it has been the focus of great interest over the past few years, still faces many limitations and challenges, particularly with regard to the validation and the quality of the obtained results. The present protocol describes a quality control (QC) procedure for monitoring the precision of the analytical process involving untargeted metabolic phenotyping of urine and plasma/serum. The described/suggested methodology can be applied to different biological matrices, such as biological biofluids, cell, and tissue extracts.

HILIC-MS/MS Multi-Targeted Method for Metabolomics Applications.

Metabolomics aims at the identification and quantification of key-end point metabolites, basically polar, in order to study changes in biochemical activities in response to pathophysiological stimuli or genetic modifications. Targeted profiling assays have enjoyed a growing popularity during the last years with LC-MS/MS as a powerful tool for development of such (semi-) quantitative methods for a large number of metabolites. Here we describe a method for absolute quantification of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids, and amines with a hydrophilic interaction liquid chromatography (HILIC) system comprised of ultra (high) performance liquid chromatography (UHPLC) with detection on a triple-quadrupole mass spectrometer operating in both positive and negative electrospray ionization modes.

Rat Fecal Metabolomics-Based Analysis.

Fecal metabolomics-based analysis indisputably constitutes a very useful tool for elucidating the biochemistry of digestion and absorption of the gastrointestinal system. Fecal samples represent the most suitable, non-invasive, specimen for the study of the symbiotic relationship between the host and the intestinal microbiota.It is well established that the balance of the intestinal microbiota changes in response to some stimuli, physiological such as gender, age, diet, exercise and pathological such as gastrointestinal and hepatic disease. Fecal samples have been analyzed using the most widespread analytical techniques, namely, NMR spectroscopy, GC-MS, and LC-MS/MS. Rat fecal sample is a frequently used and particularly useful substrate for metabolomics-based studies in related fields. The complexity and diversity of the nature of fecal samples require careful and skillful handling for the effective quantitative extraction of the metabolites while avoiding their deterioration. Parameters such as the fecal sample weight to extraction solvent volume, the nature and the pH value of the extraction solvent, and the homogenization process are some important factors for the optimal extraction of samples, in order to obtain high-quality metabolic fingerprints, using either untargeted or targeted metabolomics.

Impact of Exercise and Aging on Rat Urine and Blood Metabolome. An LC-MS Based Metabolomics Longitudinal Study.

Aging is an inevitable condition leading to health deterioration and death. Regular physical exercise can moderate the metabolic phenotype changes of aging. However, only a small number of metabolomics-based studies provide data on the effect of exercise along with aging. Here, urine and whole blood samples from Wistar rats were analyzed in a longitudinal study to explore metabolic alterations due to exercise and aging. The study comprised three different programs of exercises, including a life-long protocol which started at the age of 5 months and ended at the age of 21 months. An acute exercise session was also evaluated. Urine and whole blood samples were collected at different time points and were analyzed by LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry). Based on their metabolic profiles, samples from trained and sedentary rats were differentiated. The impact on the metabolome was found to depend on the length of exercise period with acute exercise also showing significant changes. Metabolic alterations due to aging were equally pronounced in sedentary and trained rats in both urine and blood analyzed samples.

Sample Preparation Strategies for the Effective Quantitation of Hydrophilic Metabolites in Serum by Multi-Targeted HILIC-MS/MS.

The effect of endogenous interferences of serum in multi-targeted metabolite profiling HILIC-MS/MS analysis was investigated by studying different sample preparation procedures. A modified QuEChERS dispersive SPE protocol, a HybridSPE protocol, and a combination of liquid extraction with protein precipitation were compared to a simple protein precipitation. Evaluation of extraction efficiency and sample clean-up was performed for all methods. SPE sorbent materials tested were found to retain hydrophilic analytes together with endogenous interferences, thus additional elution steps were needed. Liquid extraction was not shown to minimise matrix effects. In general, it was observed that a balance should be reached in terms of recovery, efficient clean-up, and sample treatment time when a wide range of metabolites are analysed. A quick step for removing phospholipids prior to the determination of hydrophilic endogenous metabolites is required, however, based on the results from the applied methods, further studies are needed to achieve high recoveries for all metabolites.

QSRR Modeling for Metabolite Standards Analyzed by Two Different Chromatographic Columns Using Multiple Linear Regression.

Modified quantitative structure retention relationships (QSRRs) are proposed and applied to describe two retention data sets: A set of 94 metabolites studied by a hydrophilic interaction chromatography system under organic content gradient conditions and a set of tryptophan and its major metabolites analyzed by a reversed-phase chromatographic system under isocratic as well as pH and/or simultaneous pH and organic content gradient conditions. According to the proposed modification, an additional descriptor is added to a conventional QSRR expression, which is the analyte retention time, tR(R), measured under the same elution conditions, but in a second chromatographic column considered as a reference one. The 94 metabolites were studied on an Amide column using a Bare Silica column as a reference. For the second dataset, a Kinetex EVO C18 and a Gemini-NX column were used, where each of them was served as a reference column of the other. We found in all cases a significant improvement of the performance of the QSRR models when the descriptor tR(R) was considered.