Solid-Contact Potentiometric Sensors Based on Main-Tailored Bio-Mimics for Trace Detection of Harmine Hallucinogen in Urine Specimens.

All-solid-state potentiometric sensors have attracted great attention over other types of potentiometric sensors due to their outstanding properties such as enhanced portability, simplicity of handling, affordability and flexibility. Herein, a novel solid-contact ion-selective electrode (SC-ISE) based on poly(3,4-ethylenedioxythiophene) (PEDOT) as the ion-to-electron transducer was designed and characterized for rapid detection of harmine. The harmine-sensing membrane was based on the use of synthesized imprinted bio-mimics as a selective material for this recognition. The imprinted receptors were synthesized using acrylamide (AA) and ethylene glycol dimethacrylate (EGDMA) as functional monomer and cross-linker, respectively. The polymerization process was carried out at 70 °C in the presence of dibenzoyl peroxide (DBO) as an initiator. The sensing membrane in addition to the solid-contact layer was applied to a glassy-carbon disc as an electronic conductor. All performance characteristics of the presented electrode in terms of linearity, detection limit, pH range, response time and selectivity were evaluated. The sensor revealed a wide linearity over the range 2.0 × 10-7-1.0 × 10-2 M, with a detection limit of 0.02 µg/mL and a sensitivity slope of 59.2 ± 0.8 mV/hamine concentration decade. A 40 mM Britton-Robinson (BR) buffer solution at pH of 6 was used for all harmine measurements. The electrode showed good selectivity towards harmine over other common interfering ions, and maintained a stable electrochemical response over two weeks. After applying the validation requirements, the proposed method revealed good performance characteristics. Method precision, accuracy, bias, trueness, repeatability, reproducibility, and uncertainty were also evaluated. These analytical capabilities support the fast and direct assessment of harmine in different urine specimens. The analytical results were compared with the standard liquid chromatographic method. The results obtained demonstrated that PEDOT/PSS was a promising solid-contact ion-to-electron transducer material in the development of harmine-ISE. The electrodes manifested enhanced stability and low cost, which provides a wide number of potential applications for pharmaceutical and forensic analysis.

Concentrations of selected heterocyclic aromatic amines among US population aged ≥ 6 years: data from NHANES 2013-2014.

Data from National Health and Nutrition Examination Survey for US population aged ≥ 6 years for 2013-2014 were used to analyze data for four heterocyclic aromatic amines (HCAA), namely 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), harman, and norharman. Data were analyzed separately for children aged 6-11 years (N = 416), adolescents aged 12-19 years (N = 475), adults aged 20-64 years (N = 1913), and seniors aged ≥ 65 years (N = 458). Adult males had lower concentrations of AαC and harman than adult females (1.44 vs. 2.22 pg/mL for AαC, p < 0.01 and 136.8 vs. 163.2 pg/mL for harman, p = 0.04). Racial/ethnic differences were observed in the adjusted concentrations of HCAAs. For adults, adjusted concentrations of HCAAs were lower for non-Hispanic Asians and Hispanics as compared to non-Hispanic blacks and whites. For example for AαC, the adjusted concentrations for non-Hispanic Asians, Hispanics, non-Hispanic blacks and whites were 1.16, 2.00, 2.37, and 2.16 pg/mL respectively. Adjusted concentrations of AαC were found to be lower among nonsmokers as compared to smokers for adolescents (0.34 vs. 1.32 pg/mL, p < 0.01), adults (0.40 vs. 7.91 pg/mL, p < 0.01), and seniors (0.30 vs. 4.29 pg/mL, p < 0.01). For both harman and norharman, adult nonsmokers had lower adjusted concentrations than smokers (125.7 vs. 177.6 pg/mL, p < 0.01 for harman, 296.1 vs. 421.6 pg/mL, p < 001, for norharman). Exposure to environmental tobacco smoke was found to be associated with higher concentrations of AαC among adolescents (p = 0.01) and adults (p = 0.01) and for harman (p = 0.01) and norharman (p = 0.01) among seniors. In conclusion, concentrations of selected HCAAs can be several fold higher among smokers as compared to nonsmokers and gender as well as race/ethnicity also affect the observed concentrations of HCAA.

Metabolism and disposition of N,N-dimethyltryptamine and harmala alkaloids after oral administration of ayahuasca.

Ayahuasca is an Amazonian psychotropic plant tea obtained from Banisteriopsis caapi, which contains ß-carboline alkaloids, chiefly harmine, harmaline and tetrahydroharmine. The tea usually incorporates the leaves of Psychotria viridis or Diplopterys cabrerana, which are rich in N,N-dimethyltryptamine (DMT), a psychedelic 5-HT(2A/1A/2C) agonist. The ß-carbolines reversibly inhibit monoamine-oxidase (MAO), effectively preventing oxidative deamination of the orally labile DMT and allowing its absorption and access to the central nervous system. Despite increased use of the tea worldwide, the metabolism and excretion of DMT and the ß-carbolines has not been studied systematically in humans following ingestion of ayahuasca. In the present work, we used an analytical method involving high performance liquid chromatography (HPLC)/electrospray ionization (ESI)/selected reaction monitoring (SRM)/tandem mass spectrometry(MS/MS) to characterize the metabolism and disposition of ayahuasca alkaloids in humans. Twenty-four-hour urine samples were obtained from 10 healthy male volunteers following administration of an oral dose of encapsulated freeze-dried ayahuasca (1.0 mg DMT/kg body weight). Results showed that less than 1% of the administered DMT dose was excreted unchanged. Around 50% was recovered as indole-3-acetic acid but also as DMT-N-oxide (10%) and other MAO-independent compounds. Recovery of DMT plus metabolites reached 68%. Harmol, harmalol, and tetrahydroharmol conjugates were abundant in urine. However, recoveries of each harmala alkaloid plus its O-demethylated metabolite varied greatly between 9 and 65%. The present results show the existence in humans of alternative metabolic routes for DMT other than biotransformation by MAO. Also that O-demethylation plus conjugation is an important but probably not the only metabolic route for the harmala alkaloids in humans.

Screening of non-polar heterocyclic amines in urine by microextraction in packed sorbent-fluorimetric detection and confirmation by capillary liquid chromatography.

A rapid and simple procedure for the direct screening of urine samples is described. The method involves microextraction in a packed sorbent (MEPS) that is on-line coupled to a capillary liquid chromatograph with fluorimetric detection. The overall arrangement works as a screening/confirmatory system for monitoring non-polar heterocyclic aromatic amines (HAAs) in urine samples. This configuration allows the selective retention of HAAs from urine on a C(18) MEPS cartridge integrated in the needle of a micro-well plate autosampler. Retained HAAs were eluted with methanol/water (90:10, v/v) and directly injected into the fluorimetric detector. This screening method provides a yes/no binary response that may require confirmation. The samples for which the concentration of HAAs was close to or above the established threshold limit (30 ng mL(-1)) were subjected to capillary liquid chromatography (CLC) for confirmation purposes. A mobile phase of acetonitrile and triethylamine (25 mM) at pH 2.5, through a gradient of composition at a flow rate of 20 µL min(-1), resulted in good separations between the analytes in less than 11 min. This confirmation method allowed the determination of the analytes in the 10-100 ng mL(-1) range for harmane and norharmane and from 20 to 200 ng mL(-1) for 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b] indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido-[4,3-b] indole (Trp-P-2), 2-amino-9H-pyrido-[2,3-b] indole (AαC) and 2-amino-3-methyl-9H-pyrido-[2,3-b] indole (MeAαC), with relative standard deviation (RSD) values between 2.12% and 3.73%, and limits of detection between 1.6 and 5.6 ng mL(-1) for all the HAAs.

Hollow fibre-supported liquid membrane extraction and LC-MS/MS detection for the analysis of heterocyclic amines in urine samples.

Heterocyclic amines (HCAs) are potent mutagens/carcinogens to which humans are frequently exposed through the consumption of cooked meat and fish food. The effect of normal intake of HCAs and their role in the aetiology of human cancer is unknown. To some extent, limitations of the existing analytical methods in monitoring the low levels of HCAs in biological samples have hindered obtaining conclusive results. In this study, a method for the analysis of HCAs in human urine has been studied to detect HCAs and metabolites at levels resulting from consumption of food cooked at ordinary conditions. The analytical method consisted of extraction and clean-up by the novel technique liquid-phase microextraction combined with LC-MS/MS. The effect of pH during the extraction and hydrolysis step was examined. High sensitivity was achieved when the extraction was performed in raw urine adjusted to pH 5.5, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine being detected from 2 pg/g urine, levels comparable with a normal exposure. Good reproducibility and repeatability was obtained for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, below 9% using isotopic dilution. The performance of the method on 9H-pyrido[3,4-b]indole, 2-amino-1-methyl-6-(4'-hydroxyphenyl)imidazo[4,5-b]pyridine and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine was also studied.

Excretion of beta-carbolines harman and norharman in 24-hour urine of chronic alcoholics during withdrawal and controlled abstinence.

Animal experiments suggest that endogenous substances that could result from the interaction between neurotransmitters (dopamine and indoleamines) and ethanol and its metabolite acetaldehyde might be involved in the pathogenesis and maintenance of alcohol dependence. Therefore, aromatic beta-carbolines (norharman and harman) were investigated repeatedly in 24-hr urine of 13 male severe alcoholics without any psychiatric comorbidity during a controlled inpatient abstention program of up to 8 weeks. Harman excretion was approximately 2-fold above levels in control subjects, with a steady decline after 3 weeks of abstinence and lower levels in patients with a longer duration of alcohol dependence. Severity of withdrawal symptoms and actual feelings of anxiety/depression were negatively associated with urinary harman excretion. Positive associations could be established with daily ethanol consumption the month before admission and the score on the scale "reward dependence" according to Cloninger's Tridimensional Personality Questionnaire. Moreover, patients without alcohol-dependent first-degree relatives and higher "reward dependence" exhibited an increased excretion of harman. Therefore, harman levels might characterize a distinct subgroup of alcoholic patients, who in part resemble the so-called type l alcoholics of Cloninger. However, this awaits further study in a larger number of individuals. In contrast, norharman excretion was elevated up to 6-fold, compared with nonalcoholics over 6 to 8 weeks of controlled abstention. No correlations to demographic or clinical variables could be observed. Therefore, increased norharman levels might be proposed as a "residual marker" or a trait variable. Whether the observed changes are specific markers of at least certain aspects of alcoholism or dependence remain to be elucidated.

Quantitation of human plasma levels of the anticancer agent carboxyamidotriazole by high-performance liquid chromatography.

The predominant cause of death of cancer patients is growth and metastasis of their tumors. By targeting signal transduction pathways as sites of therapeutic intervention, we have identified a novel anticancer drug carboxyamidotriazole (CAI). A straight-forward and reliable method of detection and quantitation of human CAI plasma levels using solid-phase organic extraction followed by isocratic reversed-phase chromatography is now reported. This assay detected CAI over the concentration range 0.04-10.0 micrograms/ml, which brackets the range shown to be physiologically and biochemically effective. Linearity was demonstrated by linear regression analysis of calibration curves (r2 = 0.999). Equivalence of recovery of extracted versus non-extracted CAI over a broad concentration range was demonstrated (r2 = 0.998, coefficients of variability < 10%). The method was applied to quantitate CAI plasma levels from patients now entered on the Phase I clinical trial underway at the National Cancer Institute.

Biotransformation of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid to harmalan, tetrahydroharman and harman in rats.

1-Methyl-1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid (1-carboxytetrahydroharman, 1-CTHH) has been detected in the brain of rats following intracerebroventricular injection of tryptamine and pyruvic acid. We now report the metabolism of this compound. Following intraperitoneal injection of 1-CTHH into rats, harmalan was found to be the major metabolite besides tetrahydroharman (THH) and harman. A high concentration of THH was measured in the lung while most of harman was found in the urine. Harmalan and THH could be detected in the brain in low concentrations. The products were separated following extraction from tissues by high-performance liquid chromatography (HPLC) on a reversed phase C18-DB column. The identity of the metabolites was confirmed by mass spectrometry (MS) analysis. The results demonstrate the role of 1-CTHH as a precursor of the biologically active compounds harmalan, THH and harman.

Ethanol induces an increase of harman in the brain and urine of rats.

Harman occurs in rat brain, with the highest concentration in the cerebellum and the lowest in the striatum. 2 g/kg ethanol were ineffective with respect to the concentration of harman in the brain whereas 5 g/kg ethanol caused a time-dependent increase in the cerebral cortex as well as the cerebellum. A toxic dose (8 g/kg) of ethanol elicited no change of harman in the brain 3 h following the application. The rise in the harman concentration in the brain did not correlate with the increase of acetaldehyde in the blood after treatment with ethanol suggesting that several mechanisms are involved in the changes of the levels of harman. In subchronic experiments rats were treated with ethanol over a period of 5 or 6 days. Harman increased in the brain whereby the effect seemed to be more pronounced in the cerebellum than in the cerebral cortex. The concentration tended to increase over time and reached control levels again during withdrawal. The time course of the excretion of harman into the urine was similar to that of the brain in that it increased continuously during the period of ethanol treatment and reached control levels again during withdrawal.

Is there a correlation between the concentration of beta-carbolines and their pharmacodynamic effects?

Two sensitive and specific methods are presented for the determination of 6-OH-tetrahydronorharmane (6-OH-THN, 6-OH-THBC) and harmane (1-Me-BC). The concentration of 6-OH-THN in blood platelets from men was found 5.19 +/- 0.57 ng (mean +/- SEM) per 10(9) platelets, of acute schizophrenic patients 2.66 +/- 0.38 ng per 10(9) platelets, p less than 0.02, and in rats 6 - 13 ng x 10(9) platelets. The concentration of harmane in the urine of rats was measured 9.72 +/- 1.56 ng per 16 h. A load with ethanol caused an increased excretion of the beta-carboline in some rats. In pharmacological experiments substantial evidence was detected for a correlation between the (3H)-flunitrazepam displacing potency and the conflict-augmenting effects of beta-carbolines. Furthermore, a good correlation was found between the results of binding experiments and the antagonism of the beta-carbolines with respect to the activating effect of low doses of diazepam. No such correlation exists for the sedative effect.

Biliary and urinary excretion of drug conjugates: effect of diuresis and choleresis on excretion of harmol sulphate and harmol glucuronide in the rat.

1. Harmol (7-hydroxy-1-methyl-9H-pyrido [3,4b] indole) is converted to harmol sulphate and harmol glucuronide in the rat in vivo. Harmol sulphate is excreted mainly in urine, while harmol glucuronide is excreted about equally in bile and urine, when harmol is given intravenously. 2. In rats with ligated kidneys, only choleresis produced by glycodehydrocholate and dehydrocholate caused enhanced biliary excretion of harmol sulphate; harmol glucuronide was unaffected. Several other bile salts had no effect. 3. Mannitol diuresis markedly increased urinary excretion of harmol sulphate, and decreased its biliary excretion. Harmol glucuronide was much less affected. 4. Nafenopin pretreatment increased liver weight and bile flow, and enhanced biliary excretion of harmol sulphate at the expense of its urinary excretion. 5. For harmol sulphate, urine and bile are compensatory pathways of elimination that can be influenced by urine and bile flow changes through diuresis and choleresis.