Discrimination Between Drug Abuse and Medical Therapy: Case report of a tranylcypromine overdose-related fatality.

Tranylcypromine is an effective antidepressant from the class of monoamine oxidase inhibitors and is structurally related to amphetamine. However, reports differ regarding the potential metabolism of tranylcypromine to amphetamine and methamphetamine within the human body. We report a 25-year-old woman with severe depression who died due to a fatal tranylcypromine overdose in 2016. She had been prescribed tranylcypromine one day previously and had no history of previous suicide attempts or substance abuse. The body was transferred to a forensic medicine department in Tehran, Iran for the autopsy. A urine sample was positive for tranylcypromine, amphetamine and methamphetamine using gas chromatography/mass spectrometry after derivatisation with heptafluorobutyric acid. As amphetamines were present in the urine sample, it was assumed that the tranylcypromine had been converted to amphetamines metabolically. As such, it is possible that the legitimate use of certain prescription drugs may complicate the interpretation of test results for illegal drugs.

Quantification of antidepressants using gas chromatography-mass spectrometry.

Antidepressants are of great interest to clinical and forensic toxicologists as they are frequently used in suicidal gestures; they can be the source of drug interactions and some have narrow therapeutic indices making the potential for toxicity more likely. There are five categories of antidepressants based on function and/or structure. These are monoamine oxidase inhibitors (MAOI), cyclic antidepressants including tricyclic and tetracyclic compounds (TCA), selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and atypical compounds. This method is designed to detect the presence of antidepressant drugs in blood/serum, urine, and tissue specimens using gas chromatography/mass spectrometry (GC/MS) following liquid-liquid extraction (LLE) and identified by relative retention times and mass spectra.

Stereoselective pharmacokinetics of RS-8359, a selective and reversible MAO-A inhibitor, by species-dependent drug-metabolizing enzymes.

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine selectively and reversibly inhibits monoamine oxidase A (MAO-A). After oral administration of rac-RS-8359 to rats, mice, dogs, monkeys, and humans, plasma concentrations of the (R)-enantiomer were greatly higher than were those of the (S)-enantiomer in all species studied. The AUC((R)) to AUC((S)) ratios were 2.6 in rats, 3.8 in mice, 31 in dogs, and 238 in monkeys, and the (S)-enantiomer was almost negligible in human plasma. After intravenous administration of RS-8359 enantiomers to rats, the pharmacokinetic parameters showed that the (S)-enantiomer had a 2.7-fold greater total clearance (CL(t)) and a 70% shorter half-life (t(1/2)) than those for the (R)-enantiomer but had no difference in distribution volume (V(d)). No significant difference in the intestinal absorption rate was observed. The principal metabolites were the 2-keto form, possibly produced by aldehyde oxidase, the cis-diol form, and the 2-keto-cis-diol form produced by cytochrome P450 in rats, the cis-diol form in mice, RS-8359 glucuronide in dogs, and the 2-keto form in monkeys and humans. Thus, the rapid disappearance of the (S)-enantiomer from the plasma was thought to be due to the rapid metabolism of the (S)-enantiomer by different drug-metabolizing enzymes, depending on species.

Chiral characterization of deprenyl-N-oxide and other deprenyl metabolites by capillary electrophoresis using a dual cyclodextrin system in rat urine.

A chiral capillary electrophoresis method has been developed for the simultaneous separation of the enantiomers of deprenyl and eight of its metabolites, among them the recently described metabolite deprenyl-N-oxide. Although heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) was suitable for the enantioresolution of deprenyl and its dealkylated derivatives, the enantiomers of deprenyl-N-oxide were just partly resolved. Carboxymethyl-beta-cyclodextrin (CMBCD) in as low as 2 mM concentration was capable of the enantiomer separation of all the nine examined compounds, however co-migration of 1R,2S-(-)-norephedrine and 1R,2R-(-)-pseudoephedrine, as well as 1S,2R-(+)-ephedrine and R-(-)-amphetamine was observed. This problem could be overcome by the use of a dual cyclodextrin system containing 4 mM DIMEB in addition to 2 mM CMBCD; simultaneous separation of all the compounds could be achieved. The optimized method was used for the analysis of rat urine samples after 10 days of treatment of animals with either R-(-)- or S-(+)-deprenyl. The stereospecific biotransformation of both deprenyl enantiomers was confirmed, and the stereoselectivity of N-oxide formation was demonstrated.

Detection, quantification, metabolism, and behavioral effects of selegiline in horses.

Selegiline ([R]-[-]N,alpha-dimethyl-N-2- propynylphenethylamine or l-deprenyl), an irreversible inhibitor of monoamine oxidase, is a classic antidyskinetic and antiparkinsonian agent widely used in human medicine both as monotherapy and as an adjunct to levodopa therapy. Selegiline is classified by the Association of Racing Commissioners International (ARCI) as a class 2 agent, and is considered to have high abuse potential in racing horses. A highly sensitive LC/MS/MS quantitative analytical method has been developed for selegiline and its potential metabolites amphetamine and methamphetamine using commercially available deuterated analogs of these compounds as internal standards. After administering 40 mg of selegiline orally to two horses, relatively low (<60 ng/ml) concentrations of parent selegiline, amphetamine, and methamphetamine were recovered in urine samples. However, relatively high urinary concentrations of another selegiline metabolite were found, tentatively identified as N- desmethylselegiline. This metabolite was synthesized and found to be indistinguishable from the new metabolite recovered from horse urine, thereby confirming the chemical identity of the equine metabolite. Additionally, analysis of urine samples from four horses dosed with 50 mg of selegiline confirmed that N-desmethylselegiline is the major urinary metabolite of selegiline in horses. In related behavior studies, p.o. and i.v. administration of 30 mg of selegiline produced no significant changes in either locomotor activities or heart rates.

Urinary excretion of selegiline N-oxide, a new indicator for selegiline administration in man.

1. The metabolism of selegiline (SG) has been studied by investigating the time-course of urinary excretion of SG and its metabolites using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) in combination with solid-phase extraction. 2. The excretion profiles of SG and its four major metabolites, selegiline-N-oxide (SGO), N-desmethylselegiline (DM-SG), methamphetamine (MA) and amphetamine (AP), were investigated in six healthy volunteers after oral administrations of SG hydrochloride in a single dose of 2.5 or 7.5mg, and a repeat twice-daily dose of 5.0 mg day(-1) (for 3 days). 3. The cumulative amount of SGO excreted within approximately the first 8-12h was comparable with MA, and the amount in the first 72 h was 2.0-7.8 times larger (2.8-13.2% of the dose) than that of DM-SG. 4. These results demonstrate that SGO can be used in place of DM-SG, which is known to be a main specific metabolite of SG, as a new indicator for the discrimination of SG use compared with MA abuse.

Quantitative liquid chromatography-mass spectrometry determination of isatin in urine using automated on-line extraction.

Here we describe a simple, fast and sensitive liquid chromatography/mass spectrometry method with automated on-line extraction to quantify isatin, an endogenous monoamine oxidase, and atrial natriuretic peptide inhibitor, in urine. After derivatisation of isatin to isatinoxime with hydroxylamine hydrochloride and zinc sulfate precipitation, samples were loaded on the extraction column, washed and, after activation of the column-switching valve, backflushed onto the analytical column. Using electrospray ionisation, [M+H]+ ions could be detected in the selected ion monitoring mode. The assay was linear from 5 to 5000 ng/ml (r2>0.99) and analytical recovery was >80%. Inter-assay precision for the quality control samples was less than 3% and inter-assay accuracy was within +/- 5%.

Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry.

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (HPLC-ESI-MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C18 cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.x 150 mm) for HPLC separation and ESI-MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile-10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10-20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users' urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.

Determination of enantiomeric metabolites of l-deprenyl, d-methamphetamine, and racemic methamphetamine in urine by capillary electrophoresis: comparison of deprenyl use and methamphetamine use.

The enantiomeric analysis of urine collected from rats administered l-deprenyl, d-methamphetamine (MA), or dl-MA and from healthy male volunteers who ingested l-deprenyl by capillary electrophoresis (CE) using carboxy methylated-beta-cyclodextrin (CMCD) as a chiral selector was investigated to compare the metabolic pattern of l-deprenyl with the metabolism of d- or dl-MA. Urine from illegal drug abusers was also analyzed for the comparison of therapeutic drug (l-deprenyl) use with illicit drug (d-MA) use. MA enantiomers (l-, d-), amphetamine (AM) enantiomers (l-, d-), l-deprenyl, and desmethylselegiline (DMS) enantiomers (l-, d-) were simultaneously separated and detected with clear resolution. L-deprenyl and its metabolites, l-MA, l-AM, and l-DMS, were detected in rat urine sample collected up to 24 h after oral administration of l-deprenyl (10 mg/kg), and the urinary l-AM/l-MA ratio was 2.45 +/- 0.55. This AM/MA ratio was significantly higher than the ratios obtained from rats administered with d-MA (5 mg/kg) and dl-MA (10 mg/kg). The d-AM/d-MA ratio was 0.98 +/- 0.25 for the d-MA treatment, and the d-AM/d-MA and l-AM/l-MA ratios were 0.72 +/- 0.24 and 0.71 +/- 0.21, respectively, for the dl-MA treatment. Analysis of human urine revealed that, unlike in rat urine, the MA content was much greater than the AM content, resulting in the AM/MA ratios being far lower in cases of healthy adult men treated with l-deprenyl (10 mg) and MA abusers. The AM/MA ratio from l-deprenyl users (0.33 +/- 0.03) was significantly higher than the ratio from MA abusers (0.20 +/- 0.12). Results indicate that although metabolic patterns of the drugs in rat and humans may be different, the AM/MA ratio from l-deprenyl use is significantly higher than the ratio from MA use in both rat and human urine. This ratio, however, cannot give conclusive proof of deprenyl or MA use in humans. The simultaneous chiral separation for all the metabolites of l-deprenyl and MA by CE analysis used in this study could provide rapid and simple discrimination between therapeutic drug use and illegal drug abuse.

Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake.

Biological effects of dietary isoflavones, such as daidzein and genistein are of interest in preventive medicine. We estimated the dietary intake of isoflavones from dietary records and compared the values with the plasma concentrations and urinary excretions in Japanese middle-aged women. The dietary intake of daidzein and genistein was 64.6 and 111.6 mumol /day/capita (16.4 and 30.1 mg/day/capita), respectively. The isoflavones intake was mostly attributable to tofu, natto and miso. The median of plasma daidzein and genistein concentration was 72.46 and 206.09 nmol/L, respectively. The median of urinary excretion was 20.54 mumol /day for daidzein, 10.79 for genistein, 15.74 for equol and 1.64 for O-desmethylangolensin (O-DMA). Equol and O-DMA were excreted by 50% and 84% of all participants, respectively. Equol metabolizers were significantly lower the plasma and urinary daidzein and urinary O-DMA. The dietary intake of daidzein and genistein after the adjustment for total energy intake was significantly correlated with the urinary excretion (r = 0.365 for daidzein and r = 0.346 for genistein) and plasma concentration (r = 0.335 for daidzein and r = 0.429 for genistein). The plasma concentration of isoflavones was also significantly correlated with the urinary excretion. We conclude that in epidemiological studies measurements of plasma concentration or urinary excretion of these isoflavones are useful biomarkers of dietary intake and important for studies on their relation to human health.

Urinary excretion of isoflavonoids and the risk of breast cancer.

Isoflavonoids are a group of biologically active phytochemicals that humans are exposed to mainly through soy food intake. Because of the similar chemical structure of these compounds and estradiol, it has been hypothesized that isoflavonoids may be related to the risk of breast cancer. Overnight urine samples from 60 incident breast cancer cases and their individually matched controls were assayed for urinary excretion rates of five major isoflavonoids (daidzein, genistein, glycitein, equol, and O-desmethylangolensin) and total phenols. These subjects were from a large population-based case-control study conducted in Shanghai, and urine samples from breast cancer cases were collected before any cancer therapy to minimize the potential influence of the disease and its sequelae on study results. Urinary excretion of total phenols and all individual isoflavonoids, particularly glycitein, was substantially lower in breast cancer cases than controls. For total isoflavonoids, the mean excretion was 13.95 nmol/mg creatinine (SD, 20.76 nmol/mg creatinine) for cases and 19.52 nmol/mg creatinine (SD, 25.36 nmol/mg creatinine) for controls (P for difference = 0.04). The case-control difference was more evident when median levels of these compounds were compared, with the median excretion of all major isoflavonoids being 50-65% lower in cases than in controls. Individuals in the highest tertile of daidzein, glycitein, and total isoflavonoids had about half the cancer risk of those in the lowest tertile. The adjusted odds ratio for breast cancer was 0.14 (95% confidence interval, 0.02-0.88) for women whose urinary excretion of both phenol and total isoflavonoids was in the upper 50% compared with those in the lower 50%. The results from this study support the hypothesis that a high intake of soy foods may reduce the risk of breast cancer.

[Effects of isatin, an endogenous MAO inhibitor, on dopamine (DA) and acetylcholine (ACh) concentrations in rats].

Isatin (indole-2,3-dione), an endogenous inhibitor of monoamine oxidase (MAO), has several physiological properties for stress and anxiety. We previously identified isatin in the brain of stroke-prone spontaneously hypertensive rats (SHRSP) using gas-chromatography mass spectrometry. This study elucidated the effects of isatin on the ACh and DA levels of brain tissues in rats. Furthermore, we evaluated the effect of isatin on DA levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus. Striatal ACh and DA levels significantly increased at 2 hours after isatin (50-200 mg/kg, i.p.) administration. Perfused through a microdialysis probe, isatin (10(-6)-10(-4) M) also produced a significant and concentration-dependent increase in the ACh and DA concentrations in the perfusate from the rat striatum. Furthermore, urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. Isatin (100 mg/kg, i.p.) significantly increased striatal DA levels in a rat model of Parkinson's disease. These results suggest that urinary isatin may become a diagnostic marker for the clinical severity of Parkinson's disease and that endogenous isatin, a new biological modulator, may play a role in the regulation of the brain levels of ACh by increasing the level of DA under stress.

Daidzein conjugates are more bioavailable than genistein conjugates in rats.

This study compared the bioavailability of conjugates of the soy isoflavones genistein and daidzein in rats. Rats were given a single oral dose of a soy extract that provided 74 micromol genistein and 77 micromol daidzein/kg body wt (as conjugates). Plasma samples were obtained from treated and untreated rats; urine and fecal samples were obtained before and after treatment. Isoflavones, equol (the main end product of bacterial degradation of daidzein), and 4-ethyl phenol (the main end product from genistein) were measured by HPLC. The plasma daidzein concentration was maximal at 2 h (9.5 +/- 0.71 micromol/L) and was almost double that of genistein (P = 0.009). Between 2 and 15 h, the plasma daidzein concentration declined by 32%, but the concentration of genistein changed little. At 15 h, the concentrations of daidzein and genistein were not significantly different. Urinary excretion of daidzein over the 48-h postdose period was 17.4 +/- 1.2% of the dose, but only 11.9 +/- 1.1% of the genistein dose was excreted in urine. Equol excretion was 5.0 +/- 1.5% of the daidzein dose, but 41.9 +/- 5.0% of the genistein dose was excreted as 4-ethyl phenol. Fecal daidzein accounted for 2.3 +/- 0.5% and fecal genistein for 3.4 +/- 0.4% of the respective doses. It is concluded that conjugates of daidzein are more bioavailable than those of genistein, probably because of the greater resistance of the former to degradation by gut bacteria.

Gas-chromatographic study on the stereoselectivity of deprenyl metabolism.

The metabolism and urinary elimination of both (-)-deprenyl and (+)-deprenyl have been studied. Gas-chromatographic analysis with mass specific detection indicated that the metabolism of (-)-deprenyl results in a large excess of methamphetamine compared to amphetamine, while the metabolism of (+)-deprenyl gave nearly equal amounts of amphetamine and methamphetamine. A novel deprenyl metabolite, phenylacetone, was also identified in our studies.

Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans.

The aim of this study was to determine the pharmacokinetics and urinary excretion patterns of the soy isoflavones daidzein and genistein in humans. Six healthy men with a mean age of 37 y and a mean body mass index (in kg/m2) of 24 consumed a soybean flour-based meal on two occasions approximately 6 d apart. Blood samples and total urine were collected at intervals for the measurement of daidzein and genistein with HPLC. Isoflavone concentrations rose slowly and reached maximum values of 3.14 +/- 0.36 micromol/L at 7.42 +/- 0.74 h for daidzein and 4.09 +/- 0.94 micromol/L at 8.42 +/- 0.69 h for genistein. Elimination half-lives were 4.7 +/- 1.1 and 5.7 +/- 1.3 h for daidzein and genistein, respectively. The slow increase in plasma concentrations is consistent with the facilitation of absorption by hydrolysis in the small and large intestines of the glycosidic forms of the isoflavones present in soybean-containing foods to their corresponding aglycones. The rate of urinary excretion of daidzein was greater than that of genistein throughout the postmeal period, with mean recoveries of 62 +/- 6% and 22 +/- 4% (P < 0.001) for daidzein and genistein, respectively. However, the ratio of the areas under the plasma concentration versus time curves for genistein and daidzein was equal to the ratio of the concentrations of the respective isoflavones in the soy meal. It is concluded that the bioavailabilities of daidzein and genistein are similar, not withstanding the difference in urinary excretion.

Regional and molecular separation of the four bioactivities of 'tribulin'.

The endocoid tribulin has four known bioactivities: monoamine oxidase A and B inhibitory activities (MAO-AI and MAO-BI) and peripheral and central benzodiazepine receptor binding inhibitory activities (PBR-I and CBR-I). Analysis of the four bioactivities in rat tissue reveals regional dissociation. Most notably liver was particularly rich in PBR-I yet contained no detectable MAO-AI. In addition we have succeeded in separating the four activities from human urine. MAO-AI and MAO-BI have greater retention on charcoal than PBR-I and CBR-I when eluted step-wise with aqueous methanol. MAO-AI can be separated from MAO-BI and in addition PBR-I can be separated from CBR-I by step aqueous methanol elution from Amberlite XAD-4. Hence we present two lines of evidence that, contrary to the original view, tribulin is composed of a number of distinct molecular components.

Metabolism of selegiline in humans. Identification, excretion, and stereochemistry of urine metabolites.

Nine urinary metabolites of selegiline hydrochloride [N-methyl-N-propargyl [2-phenyl-1-methyl)ethylammonium chloride], a monoamine oxidase inhibitor, after administration to humans were identified. Their identifies were confirmed by comparison of the spectra from GC/MS of peaks with those of authentic compounds. The following metabolites and unchanged drug (selegiline) were detected in urine: (R)-desmethylselegiline, (R)-methamphetamine, (R)-amphetamine, (1S,2R)-norephedrine, (1R,2R)-norpseudoephedrine, (1S,2R)-ephedrine, (1R,2R)-pseudoephedrine, (R)-p-hydroxyamphetamine, and (R)-p-hydroxymethamphetamine. The metabolites excreted 2 days after administration of 2.5-10 mg of selegiline hydrochloride amounted to 44-58% of the dose. Selegiline was metabolized by three distinct pathways: N-dealkylation, beta-carbon hydroxylation, and ring-hydroxylation. The major metabolite was (R)-methamphetamine. During metabolism, no racemic transformation occurred and beta-carbon hydroxylation showed apparently product stereoselectivity.

Determination of isatin in urine and plasma by high-performance liquid chromatography.

A method for the detection and determination of isatin (indole-2,3-dione) in urine and plasma by high-performance liquid chromatography has been developed. It consists of a two-step purification using two different columns with UV detection. With this method, we have reconfirmed that isatin is present in human urine. We have also demonstrated that isatin is present in human plasma and that the isatin levels in spot urine samples reflect the plasma isatin levels. In the present report we describe a rapid and sensitive means of determining urine and plasma isatin for laboratories equipped with a high-performance liquid chromatography system.

Urinary excretion of deprenyl metabolites.

(+)-Deprenyl metabolites in rat's urine, such as nordeprenyl. methamphetamine amphetamine and p-hydroxy. methamphetamine were identified by HPLC-MS. After oral administration of 10 mg of pure (-)- and (+)-deprenyl to human volunteers, their urine was analyzed by gas chromatography. The concentration of methamphetamine was found to be overwhelming in the case of the (-)-isomer, while amphetamine and methamphetamine were excreted in equal amounts when (+)-deprenyl was administered. The metabolic processes of deprenyl resulted in metabolites possessing different lipophilicity, as it has been shown by planar displacement chromatography.