Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250 µL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500 ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500 ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500 ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.

BACKGROUND: Short-chain volatile amines (SCVA) are an interesting compound class playing crucial roles in physiological and toxicological human settings. Dimethylamine (DMA), trimethylamine (TMA), diethylamine (DEA), and triethylamine (TEA) were investigated in detail. METHODS: Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) was used for the simultaneous qualitative and quantitative determination of four SCVA in different human body fluids. Four hundred microliters of Li-heparin plasma and urine were analyzed after liberation of volatile amines under heated conditions in an aqueous alkaline and saline environment. Target analytes were separated on a volatile amine column and detected on a Thermo DSQ II mass spectrometer scheduled in single ion monitoring mode. RESULTS: Chromatographic separation of selected SCVA was done within 7.5 minutes. The method was developed and validated with respect to accuracy, precision, recovery and stability. Accuracy and precision criteria were below 12% for all target analytes at low and high levels. The selected extraction procedure provided recoveries of more than 92% from both matrices for TMA, DEA and TEA. The recovery of DMA from Li-heparin plasma was lower but still in the acceptable range (>75%). The newly validated method was successfully applied to plasma and urine samples from healthy volunteers. Detected concentrations of endogenous metabolites DMA and TMA are comparable to already known reference ranges. CONCLUSION: Herein, we describe the successful development and validation of a reliable and broadly applicable HS-GC-MS procedure for the simultaneous and quantitative determination of SCVA in human plasma and urine without relying on derivatization chemistry.

Serum Ethylamine Levels as an Indicator of l-Theanine Consumption and the Risk of Type 2 Diabetes in a General Japanese Population: The Hisayama Study.

OBJECTIVE: This study investigated the association between serum ethylamine levels as an indicator of l-theanine consumption and the development of type 2 diabetes in a Japanese community. RESEARCH DESIGN AND METHODS: A total of 2,253 community-dwelling Japanese individuals aged 40-79 years without diabetes were monitored for 7 years. Serum ethylamine levels were divided into quartiles: ≤0.86, 0.87-2.10, 2.11-5.28, and ≥5.29 ng/mL. Kinetic analysis of serum ethylamine concentrations was performed after ingestion of l-theanine-rich green tea products containing 8 mg of l-theanine by 12 healthy volunteers. RESULTS: During follow-up, 282 subjects developed type 2 diabetes. The age- and sex-adjusted cumulative incidence of type 2 diabetes decreased significantly with elevating levels of serum ethylamine (P for trend = 0.04). This association remained unchanged after adjusting for potential confounding factors. The multivariable-adjusted hazard ratio (HR) for type 2 diabetes was significantly lower in the fourth quartile of serum ethylamine than in the first quartile (HR 0.69, 95% CI 0.49-0.98). This trend of decrease in diabetic risk across serum ethylamine levels was more prominent in middle-aged subjects and in subjects with prediabetes, obesity, or insulin resistance. Kinetic analysis estimated that the minimum concentration at the steady state was >5.90 ng/mL in the case of twice-daily ingestion with an interval of 12 h. CONCLUSIONS: Higher serum ethylamine was significantly associated with lower risk of the development of type 2 diabetes in a general Japanese population. The measurement of serum ethylamine concentration would be a useful biomarker for the objective estimation of l-theanine consumption.

Volatile organic compounds in plasma for the diagnosis of esophageal adenocarcinoma: a pilot study.

BACKGROUND AND AIMS: A noninvasive screening test that can detect esophageal adenocarcinoma (EAC) at an earlier stage could improve the prognosis associated with EAC. The role of plasma volatile organic compounds (VOCs) for the diagnosis of EAC has not been previously studied. METHODS: Plasma samples were collected from subjects with EAC and GERD before endoscopy. Twenty-two preselected VOCs were analyzed with selected ion flow tube mass spectrometry. RESULTS: The headspaces from 39 plasma samples (20 EAC, 19 GERD) were analyzed. The levels of 9 VOCs (acetonitrile, acrylonitrile, carbon disulfide, isoprene, 1-heptene, 3-methylhexane, [E]-2-nonene, hydrogen sulfide, and triethylamine) were significantly altered in EAC patients compared with GERD patients. A multivariable logistic regression analysis was performed to build a model for the prediction of EAC. The model identified patients with EAC with an area under the curve of 0.83 (95% confidence interval, 0.67-0.98). CONCLUSIONS: Plasma VOCs may be useful in diagnosing EAC. Larger studies are needed to confirm our pilot study observations.

Ethylone-Related Deaths: Toxicological Findings.

Synthetic cathinones are an emerging class of designer drugs, frequently with deceptive labels and a multitude of analogs to circumvent drug control regulations. Research regarding the pharmacological effects and toxicity of these amphetamine derivatives is scarce, heightening the risk to the public health and safety. The composition of synthetic cathinone products continually changes and laboratories began to notice ethylone-positive products in late 2011. This report presents nine postmortem cases in whom ethylone was identified. Ethylone was isolated using solid-phase extraction and detected by gas chromatography-mass spectrometry. Seven of the cases had measurable concentrations of ethylone in blood, ranging from 38 to 2,572 ng/mL; ethylone was detected in the blood sample of one case with a concentration below the assay limit of quantification (25 ng/mL), and one case did not have detectable ethylone in blood. Besides ethylone, all but one case were also positive for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; seven cases had other drugs quantified in blood, including ethanol, alprazolam, benzoylecgonine, diphenhydramine, morphine and tramadol. In five cases where ethylone was present at blood concentrations >400 ng/mL, no other drugs excluding ethanol, cannabis metabolite and doxylamine (one case) were found. The assay also tested for mephedrone, methylone and three dimethoxyamphetamine analogs; no case was positive for these analytes. The present report documents postmortem blood concentrations of ethylone, a novel synthetic cathinone, along with other concurrently identified substances. The findings provide valuable information for developing analytical assays and evaluating a toxic concentration range of ethylone.

Development, validation and application of an LC-MS/MS bioanalytical method for the quantification of GF449, a novel 5-HT1A agonist, in rat plasma and brain.

We have recently reported a novel class of selective 5-HT1A agonists among which GF449 emerged for its high potency and almost full agonist activity (pKi 5-HT1A = 8.8; pD2 = 9.22, %Emax = 91.6). In order to quantify GF449 in rat plasma and brain, a sensitive LC-MS/MS method was developed and validated. Solid phase extraction (SPE) or a combined protein precipitation SPE permitted an efficient analyte recovery and sample clean-up. Multiple reaction monitoring (MRM) was used to track both GF449 and its internal standard (IS), MM189. GF449 was determined and quantitated to nanomolar concentrations in both plasma and brain matrix (LOQs = 0.0025 nmol/mL). Specificity was ensured using three further MRM qualifier transitions for both analyte and IS. Linearity was found in the range of 0.0025 nmol/mL to 1.00 nmol/mL (R(2) = 0.9965) and from 0.0025 nmol/mL to 50 nmol/mL (R(2) = 0.9999) for plasma and brain respectively. Intraday trueness ranged from 94.0% to 117.5% for brain and from 93.7% to 108.1% for plasma, while precision values were within 3.0% - 6.7% and 2.5% - 9.2% for plasma and brain respectively. The interday trueness of plasma ranged from 89.6% to 107.7% and the precision values (CV%) ranged from 4.6% to 7.5%. Interday trueness and precision (CV%) of the brain ranged from 94.3% to 101.2% and from 1.6% to 11.5% respectively. The method was validated in accordance with the EMEA guidelines and was successfully applied to plasma and brain samples obtained from rats treated with a 10 mg/kg single oral dose of GF449, thus demonstrating its applicability to preclinical pharmacokinetic studies.

[Significance of chromatographic methods coupled with mass spectrometry for identification of drugs for medico-legal purposes exemplified by chlorphenoxamine]. / Znaczenie metod chromatograficznych sprzezonych z spektrometria mas (LC/MS, GC/MS) w identyfikacji leków do celów orzecznictwa sadowo-lekarskiego na przykladzie chlorfenoksyaminy.

This work presented the death of the 17-year old women M.F. The direct cause of death was violent strangulation. Toxicological investigation made possible a wider explanation of the case, since M.F. was under influence of associative action of diphenhydramine and ethanol which could not avoid having an influence on the tragic events accompanying the death. Besides a comprehensive interpretation of the toxicological results in medico--legal aspect large methodic documentation has been presented which proves the usefulness of chromatographic methods coupled with spectrometry for identification of xenobiotics found.

1H NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)3.

1H NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)3 at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M(r) metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz 1H NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO>3)3 was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.

Application of capillary gas chromatography to the study of hydrolysis of the nerve agent VX in rat plasma.

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl)methylphosphonothiolate] as well as its P-S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53 +/- 4% of 374 microM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P-S bond (Km = 2.5 mM and Vmax = 13.3 nmol min-1 ml-1 of rat plasma). The activity was totally inhibited by 1 mM Hg2+ and was also inhibited by metal chelators.

Effect of dietary intake of trimethylamine on human metabolism of the industrial catalyst dimethylethylamine.

OBJECTIVES: The aim was to study the effect of trimethylamine (TMA) on the metabolism of the industrial catalyst dimethylethylamine (DMEA) to ascertain whether biological monitoring of industrial exposure to DMEA is compromised and excretion of the malodorous DMEA in sweat and urine is increased by dietary intake of TMA. METHODS: DMEA (0/25 mg) and TMA (0/300/600 mg) were given simultaneously once weekly for six weeks to five healthy volunteers. Plasma was collected before and one hour after the doses, and urine 0-2, 2-4, 4-6, 6-8, and 8-24 hours after the doses. Specimens were analysed by gas chromatography with a nitrogen sensitive detector. RESULTS: Both amines were readily absorbed from the gastrointestinal tract and excreted in urine within 24 hours (DMEA 80%; TMA 86%). Oral intake of TMA increased the DMEA content of plasma and urine dose dependently, although there were large individual differences. Plasma and urinary TMA concentrations also increased, but not dose dependently. Moreover, the findings suggested the formation of endogenous TMA, little dealkylation of DMEA and TMA, and considerable first-pass metabolism. CONCLUSIONS: Although intake of TMA reduced N-oxygenation of DMEA and TMA, total urinary DMEA values (aggregate of DMEA and its oxide DMEAO excretion) were unaffected. Thus, monitoring occupational exposure to DMEA by analysis of biological specimens is not confounded by dietary intake of TMA, provided that total urinary DMEA is monitored. Although the increased urinary and hidrotic excretion of DMEA may contribute to body odour problems, they were primarily due to TMA excretion, which is much the greater.

Liquid membrane work-up of blood plasma samples applied to gas chromatographic determination of aliphatic amines.

A technique for sample work-up and enrichment using a supported liquid membrane in an automated flow system, connected to a gas chromatograph, was used for the determination of aliphatic amines in human blood plasma. The amines studied were N,N-dimethylethylamine, triethylamine, N-methylmorpholine, cyclohexylamine and N,N-dimethylcyclohexylamine. An efficient clean-up of the complex plasma matrix was achieved, resulting in identical blank chromatograms for plasma samples and aqueous solutions. Different parameters influencing the efficiency and selectivity of the extraction procedure were experimentally studied and theoretically explained. The detection limit depends on the extraction flow-rate and the available sample volume. With 1 ml of sample and a flow-rate giving an extraction time of 16 min, the detection limit was ca. 5 ppb (5 micrograms/l); with 4 ml of sample and a lower flow-rate, sub-ppb detection limits could be reached in ca. 3 h. Linear calibration curves up to 500 ppb were obtained. Blood plasma samples from volunteers exposed to N,N-dimethylethylamine in air were analysed, and the results compared favourably with independent measurements by another method.

Dimethylethylamine in mould core manufacturing: exposure, metabolism, and biological monitoring.

The exposure and metabolism of dimethylethylamine (DMEA) was studied in 12 mould core makers in four different foundries using the Ashland cold box technique. The mean time weighted average (TWA) full work shift DMEA exposure concentration was 3.7 mg/m3. Inhaled DMEA was excreted into urine as the original amine and as its metabolite dimethylethylamine-N-oxide (DMEAO). This metabolite made up a median of 87 (range 18-93) % of the sum of DMEA and DMEAO concentrations excreted into the urine. Occupational exposure did not significantly increase the urinary excretion of dimethylamine or methylethylamine. The data indicate half lives after the end of exposure for DMEA in urine of 1.5 hours and DMEAO of three hours. The postshift summed concentration of DMEA and DMEAO in plasma and urine is a good indicator of the TWA concentration in air during the workday, and might thus be used for biological monitoring. An air concentration of 10 mg/m3 corresponds to a urinary excretion of the summed amount of DMEA and DMEAO of 135 mmol/mol creatinine.

Experimental study on the metabolism of dimethylethylamine in man.

Dimethylethylamine (DMEA) is an aliphatic tertiary amine, which is used as a catalyst in the mould core manufacturing. During 8 h, four healthy volunteers were exposed to four different DMEA air concentrations (10, 20, 40 and 50 mg/m3; 20 mg/m3, two subjects only). DMEA was biotransformed into dimethylethylamine N-oxide (DMEAO). On average, DMEAO, accounted for 90% of the combined amount of DMEA and DMEAO excreted into the urine. The half-lives of DMEA and DMEAO in plasma were 1.3 and 3.0 h, respectively. The urinary excretion of DMEA and DMEAO followed a two-phase pattern. The half-lives in the first phase were 1.5 h for DMEA and 2.5 h for DMEAO. In the second phase, which started about 9 h after the end of exposure, half-lives of 7 h for DMEA and 8 h for DMEAO were recorded. The combined concentration of DMEA and DMEAO, in both plasma and urine, showed an excellent correlation with the air concentration of DMEA. Thus, both urinary excretion and plasma concentration can be used for biological monitoring of exposure to DMEA. An 8-h exposure to 10 mg DMEA/m3 corresponds to a postexposure plasma concentration and 2-h postexposure urinary excretion of 4.9 mumol/l and 75 mmol/mol creatinine, respectively.

Absolute bioavailability and pharmacokinetics of medifoxamine in healthy humans.

Medifoxamine is a new monoamine re-uptake inhibiting antidepressant drug. Twelve volunteers received 100 mg by i.v. infusion over 15 min and 500 mg by mouth fasting. The treatments were given 1 week apart in a randomised cross-over design. Venous blood samples (10 ml) were taken at intervals for 24 h for h.p.l.c. measurement of serum medifoxamine. A biexponential decline of serum medifoxamine concentration was observed after intravenous administration in all subjects and similar terminal elimination half-lives were observed following both routes, indicating that oral absorption is not rate-limiting. The absolute bioavailability of oral medifoxamine was 21%.

Experimental study on the metabolism of triethylamine in man.

Five healthy volunteers were exposed by inhalation to triethylamine (TEA; four or eight hours at about 10, 20, 35, and 50 mg/m3), a compound widely used as a curing agent in polyurethane systems. Analysis of plasma and urine showed that an average of 24% of the TEA was biotransformed into triethylamine-N-oxide (TEAO) but with a wide interindividual variation (15-36%). The TEA and TEAO were quantitatively eliminated in the urine. The plasma and urinary concentrations of TEA and TEAO decreased rapidly after the end of exposure (average half time of TEA was 3.2 h). There was an excellent association between air levels of TEA and the urinary concentrations in samples obtained within two hours of the end of exposure. Thus the urinary level of TEA taken in this period is useful as a biological monitoring of exposure. An air concentration of 10 mg/m3 corresponds to an average urinary concentration of about 40 mmol/mol creatinine (at sedentary work).

[Experimental analysis of embichin interaction with human serum albumin]. / Eksperimental'nyi analiz vazimodeistviia émbikhina s syvorotochnym al'buminom cheloveka.

Study of embichin binding to human serum albumin in vitro by gel chromatography and fluorescence has shown that embichin is bound to human serum albumin with an association constant of 3.3 x 10(3) M-1. Interaction of embichin and albumin, that is reversible in the initial stages, transforms to irreversible on long-term contact of the protein with the drug. Experiments with cell culture indicate that the interaction with albumin potentiates the cytotoxic action of embichin.

High-pressure liquid chromatographic determination of 2-aminoethyl hydrogen sulfate in plasma and urine.

A sensitive and specific method for the analysis of 2-aminoethyl hydrogen sulfate in biological fluids by high-pressure liquid chromatography is described. Aliquots from deproteinized plasma or from acidified urine were subjected to ion-exchange clean-up by eluting with water through two columns packed with Dowex 1-X4 (Cl- form) and Dowex 50W-X8 (H+ form) resins. Recoveries in the eluates were consistent and quantitative (95-100%). Effluents from such treatment were chromatographed on a Durrum cation-exchange column with 0.05 M citrate buffer containing 0.15 N Na+ (pH 2.6). Detection was accomplished with an automated fluorescamine detection system. Using 2 ml of plasma or urine, 2-aminoethyl hydrogen sulfate could be assayed at concentrations of 0.3 mug/ml. Linear responses were observed up to at least 133 mug/ml. The method is both accurate and reproducible with a relative standard deviation of about +/- 4.0%.

Pharmacokinetics and metabolism of lidocaine in patients with renal failure.

The kinetics of distribution and elimination of lidocaine and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), were studied in 4 uremic patients on chronic hemodialysis. Each patient received a loading dose of 75 mg of lidocaine, followed by a 30 mug/kh/min lidocaine infusion. No toxic side effects from lidocaine were seen during the study. Average values for lidocaine steady-state plasma levels (2.3 mug/ml) clearance (12.3 ml/min/kg), terminal half-life (148 min), and total volume of distribution (1.9 L/kg) were found, and are similar to those values reported for normal subjects MFGX and after lidocaine infusion averaged 1/5-2/3 of the corresponding lidocaine level, as in nonuremic subjects, and plateaued by 6-8 hr. GX levels did not reach plateau by 12 hr and remained relatively unchanged after infusion. It is concluded that lidocaine infusion in uremic patients is safe, with no abnormal cumulation of lidocaine or MEGX. GX levels, however, may increase progressively, even after 12 hr.