An efficient and label-free LC-MS/MS method for assessing drug's activity at dopamine and serotonin transporters using transporter-transfected HEK293T cells.

BACKGROUND: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. AIMS: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. METHODS: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. RESULTS: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5-160 nM. Amphetamine and cocaine's IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. CONCLUSIONS: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.

Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae.

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 µM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.

Pharmacokinetics of N-ethylpentylone and its effect on increasing levels of dopamine and serotonin in the nucleus accumbens of conscious rats.

N-Ethylpentylone (NEP) is one of the most confiscated synthetic cathinones in the world. However, its pharmacology and pharmacokinetics remain largely unknown. In this study, the pharmacokentics of NEP in rat nucleus accumbens (NAc) was assessed via brain microdialysis after the intraperitoneal (ip) administration of NEP (20 or 50 mg/kg). The concentrations of dopamine (DA) and serotonin (5-HT) and their metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and 5-hydroxyindoleacetic acid (5-HIAA), were simultaneously monitored to elucidate the pharmacological effect of NEP. In addition, the plasma levels of NEP were also assessed. The pharmacokinetics of NEP showed a dose-related pattern, with NEP rapidly passing through the blood-brain barrier and reaching a maximum concentration (Cmax ) at approximately 40-minutes postdose. Approximately 4% of plasma NEP was distributed to the NAc, and considering a homogeneous brain distribution, over 90% of plasma NEP was potentially distributed to the brain. High values of area under curve (AUC) and mean residence time (MRT) of NEP were observed in both the NAc and plasma, indicating large and long-lasting effects. NEP elicited dose-related increases in microdialysate DA and 5-HT and increased the concentration of 3-MT in a dose-related manner. However, the rate of DA converted into 3-MT was unaffected. NEP had a negative effect on the rates of which DA and 5-HT were transformed into DOPAC and 5-HIAA, respectively. In summary, NEP rapidly entered the NAc and showed a long-lasting effect. In addition, DA increased more significantly than 5-HT, indicating a large potential for NEP abuse.

Effects of N-ethylpentylone on locomotor activity and anxiety-like behavior in rats.

N-ethylpentylone (NEP), a new synthetic cathinone, has been rising to be one of the most popular cathinone derivatives in recent years. However, research on NEP is rather limited. In this study, locomotor stimulation and sensitization, as well as anxiety-like behavior induced by NEP were studied in Sprague-Dawley rats, using the open field and elevated plus maze respectively. Rats were administered NEP (5, 20 or 50 mg/kg, intraperitoneal), with saline as the negative control and methamphetamine (5 mg/kg) as a positive control. Acute administration of NEP at all the doses tested significantly promoted locomotor activity, presenting an inverted U-shaped dose-effect curve. The highest activity was observed at the 20 mg/kg dose group, with the average distance traveled 18 times higher than the saline group. Repeated administration of NEP enhanced locomotor activity only at the 5 mg/kg dose group. After a week's withdrawal, re-challenge of NEP failed to induce marked behavioral sensitization. In elevated plus maze experiments, both acute and repeated administration of 20 mg/kg NEP induced anxiolytic-like effects, while no significant alteration was observed in the 5 and 50 mg/kg dose groups. In summary, acute administration of NEP caused significantly enhanced locomotor activity in rats at all the tested doses, while repeated NEP administration enhanced locomotor activity only at a low dose (5 mg/kg), while a high dose (20 mg/kg) of NEP induced anxiolytic-like effects after both acute and repeated administration.

Common-path spectral interferometry for single-shot terahertz electro-optics detection.

We propose a common-path spectral interferometer for single-shot terahertz (THz) electro-optics (EO) detection, where a probe pulse pair with orthogonal polarizations and a relative time delay are generated by simply using a birefringent plate. One of them, as the object, transmits through a THz EO crystal with THz phase modulation, while the other goes through the crystal without any phase imposed by target the THz field as the reference. The co-axial propagation of the pulse pair can effectively reduce the noises due to mechanical vibrations, air turbulences, and temperature fluctuations in the traditional non-common-path spectral interferometers. Our experiments show that, for a given target THz pulse field, the measured THz signals in a single-shot mode have a signal-to-noise ratio (SNR) of 41.2 with our THz common-path spectral interferometer, but 7.91 with a THz Mach-Zehnder spectral interferometer; thus, our design improves the SNR of the THz signal by about 5.2 times.

Ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the simultaneous determination of 12 new antidepressants and 2 antipsychotics in whole blood by gas chromatography-mass spectrometry.

Antidepressant drugs are widely used in the treatment of different psychiatric disorders, as well as in conjunction with antipsychotics for the treatment of major depressive disorder. In this study, a simple and rapid ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) method was developed for the simultaneous determination of 12 new antidepressants (norfluoxetine, fluoxetine, fluvoxamine, agomelatine, mirtazapine, moclobemide, melitracen, N-desmethylmirtazapine, maprotiline, sertraline, citalopram, paroxetine) and 2 antipsychotics (clozapine and haloperidol) in human whole blood by gas chromatography-mass spectrometry (GC-MS). Different parameters affecting the UA-LDS-DLLME were optimized and the optimal conditions were as follows: 100µL of toluene as extraction solvent, extraction pH 12 and 3min of ultrasound stirring. Good linearity (R2≥0.991) was obtained at the concentration range of 15-1500ng/mL for norfluoxetine, fluoxetine, fluvoxamine, melitracen, maprotiline and citalopram, and 5-500ng/mL for agomelatine, mirtazapine, moclobemide, N-desmethylmirtazapine, sertraline, paroxetine, clozapine and haloperidol. The intra-day and inter-day precision were all less than 10%, and accuracy of intra-day and inter-day were in the range of -12.7% to 7.9% and -13.9 to 11.8%, respectively. The extraction recoveries of most analytes were more than 60%. The UA-LDS-DLLME/GC-MS method was demonstrated with acceptable precision, accuracy and good specificity for the simultaneous determination of 12 antidepressants and 2 antipsychotics, and has been successfully applied in a real case.

Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid-phase extraction and liquid chromatography with tandem mass spectrometry.

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 µL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

Two Fatal Intoxications with Cyanohydrins.

Cyanohydrins, also be called cyanoalcohols, are important industrial precursors to carboxylic acids and some amino acids. Acetone cyanohydrin (ACH) and formaldehyde cyanohydrin (glycolonitrile, FCH), which are the typical examples of cyanohydrins, are classified as extremely hazardous substances. As the cyanohydrins can readily decompose, and it is hard to find cyanohydrins in gastric contents and heart blood, the determination study in biological samples can be divided into two parts: the first is the determination of HCN by using a Prussian blue reaction and the HS-GC-MSD after derivatization by chloramine-T. The second is the determination of acetone or formaldehyde. In this part, headspace gas chromatography with flame ionization detector (HS-GC-FID) and solid phase microextraction (SPME)-gas chromatography with mass spectrometric detectors (GC-MSD) had been used. In this report, we reported two fatal intoxication cases of ACH and FCH; one person was killed by his wife by poisoning his food and the other was suicide by poison. Two real cases of ACH and FCH in human blood and gastric contents have been analyzed by using the above-mentioned method. The Prussian blue reaction was positive in the two cases. The peaks of acetone with retention times of 0.998 min appear in specimens of the deceased are consistent with the retention times of pure acetone. The peaks of formaldehyde with a retention time of 1.658 min appear in heart blood of the deceased, and the retention time of formaldehyde of the liquid is 1.674 min, which are consistent with the retention times of pure formaldehyde (1.673 min).

Rapid separation and identification of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap mass spectrometry.

An in vivo study of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap MS is reported for the first time. Two major Strychnos alkaloids compounds including strychnine and brucine were investigated. To obtain optimal extraction efficiency, samples were pretreated by using an SPE plate. The structures of metabolites and their fragment ions were characterized based on the accurate mass and MSn data. Forty-seven metabolites were identified in rat urine, of which 25 were reported for the first time. Four new metabolism pathways were proposed on the basis of the identified metabolites. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied not only in forensic and clinically toxicological relevant cases, but also for the structural characterization of metabolites of other compounds.

[Simultaneous determination of trihexyphenidyl, chlorpromazine and clozapine in blood by GC-MS].

OBJECTIVE: To develop a method to measure trihexyphenidyl, chlorpromazine and clozapine in human blood with GC-MS. METHODS: The specimens were alkalized (pH > 10) and extracted with V (benzene):V(ethyl acetate) = 1:1, and qualitatively analyzed using GC-MS-Full Scan with internal standard SKF525A. The specimens were alkalized (pH > 10) and extracted with V(benzene):V(ethyl acetate) = 1:1, and quantitatively analyzed using GC-MS-SIM with internal standard diazepam-d5. RESULTS: The lowest detection limits of trihexyphenidyl, chlorpromazine and clozapine were 0.3, 0.3 and 0.7 ng/mL (S/N > or = 3) respectively. The calibration curve in 20-10 000 ng/mL showed a good linear distribution. The recovery rate was 79.9% to 85.5%. The RSDs of intraday and interday were less than 5.1%. CONCLUSION: The established method was simple, sensitive and accurate for simultaneous determination of trihexyphenidyl, chlorpromazine and clozapine in human blood, and can be applied in forensic toxicological cases.

[Coumarins from root of Zanthoxylum dimorphophyllum var. spinifolium].

OBJECTIVE: To study the chemical components from dried roots of Zanthoxylum dimorphophyllum var. spinifoliun. METHOD: The chemical components were isolated by low pressure column chromatography and their structures were identified by spectroscopic methods. RESULT: Five compounds were isolated and identified as 6-(2', 3'-dihydroxy-3'-methyl-butyl)-7-hydroxy-5-methoxy-2H-1-benzopyran-2-one (I), 6-(2',3'-dihydroxy-3'-methyl-butyl)-7-methoxy-8-(3'-methyl-but-2'-enyl)-2H-1-benzopyran-2-one (II),6-(2',3'-dihydroxy-3'-methyl-butyl)-7-hydroxy-2H-1-benzopyran-2-one (III), 6-(2', 3'-oxiranyl-3'-methyl-butyl)-7-methoxy-8- (3-methyl-but-2-enyl)-2H-1-benzopyran-2-one (IV), 7-methoxy-8-(3'-methyl-but-2'-enyl)-2H-1-benzopyran-2-one (V). CONCLUSION: These compounds were isolated from the plant for the first time.

[Studies on the coumarins in the root of Zanthoxylum dimorphophyllum].

OBJECTIVE: To study the chemical constituents of the dried roots of Zanthoxylum dimorphophyllumr. spinifolium and to find out the active constituents of the plant. METHOD: Modern chromatography was used to purify chemical constituents, and their structures were identified by various spectral methods. RESULT: Four compounds were isolated and identified as isopimpinellin (I), xanthoxyletin (II), 6-(2', 3'-dihydroxy-3'-methyl-butyl)-7-hydroxy-2H-1-benzopyran-2-one (III), 6-(2'-O-beta-D-glucopyranosyloxy, 3'-dihydroxy-3'-methybutyl)-7-hydroxy-2H-1-benzopyran-2-one (IV). CONCLUSION: All of the above compounds were isolated from the above mentioned plant for the first time.