Urinary excretion profile of methiopropamine in mice following intraperitoneal administration: A liquid chromatography-tandem mass spectrometry investigation.

We have considered the urinary excretion profile of methiopropamine (MPA), a thiophene ring-based structural analog of methamphetamine with similar stimulant effects, with the aim of selecting the most appropriate marker(s) of intake that may be useful in forensic analysis. For this purpose, in vitro studies were preliminarily performed on human liver microsomes for tracing the phase I metabolic pathways of MPA, preselecting the best candidates as potential target analytes, and designing the optimal experimental strategy. In vivo studies were then conducted on mice, after the intraperitoneal administration of a 10-mg/kg dose. Urine samples were collected every 3 h in the first 9 h and, subsequently, from 24 to 36 h, and stored at -80°C until further analysis. The measurements were performed using a targeted procedure based on liquid/liquid extraction followed by liquid chromatography-tandem mass spectrometry analysis. Our results show that in the time interval 0-9 h after administration, MPA was extensively oxidized mainly to nor-MPA, oxo-MPA, and two hydroxylated metabolites (ie, hydroxy-aryl-methiopropamine and hydroxy-alkyl-methiopropamine). All phase I metabolites underwent phase II metabolism, with the formation of nor-hydroxy-methiopropamine only in phase II, confirmed by the results obtained after enzymatic hydrolysis with ß-glucuronidase and arylsulfatase. In the time interval 24-36 h after administration, only unchanged MPA and nor-MPA were detected, suggesting that these two markers are those endowed with the highest diagnostic value. The method was validated for these two principal markers, proving to be fit for anti-doping, toxicological, and forensic analyses.

Colorimetric Urinalysis for On-Site Detection of Metabolic Biomarkers.

Over the past few decades, colorimetric assays have been developed for cost-effective and rapid on-site urinalysis. Most of these assays were employed for detection of biomarkers such as glucose, uric acid, ions, and albumin that are abundant in urine at micromolar to millimolar levels. In contrast, direct assaying of urinary biomarkers such as glycated proteins, low-molecular-weight reactive oxygen species, and nucleic acids that are present at significantly lower levels (nanomolar to picomolar) remain challenging due to the interferences from the urine sample matrix. State-of-the-art assays for detection of trace amounts of urinary biomarkers typically utilize time-consuming and equipment-dependent sample pretreatment or clean-up protocols prior to assaying, which limits their applicability for on-site analysis. Herein, we report a colorimetric assay for on-site detection of trace amount of generic biomarkers in urine without involving tedious sample pretreatment protocols. The detection strategy is based on monitoring the changes in optical properties of poly(3-(4-methyl-3'-thienyloxy)propyltriethylammonium bromide) upon interacting with an aptamer or a peptide nucleic acid in the presence and absence of target biomarkers of relevance for the diagnosis of metabolic complications and diabetes. As a proof of concept, this study demonstrates facile assaying of advanced glycation end products, 8-hydroxy-2'-deoxyguanosine and hepatitis B virus DNA in urine samples at clinically relevant concentrations, with limits of detection of ∼850 pM, ∼650 pM, and ∼ 1 nM, respectively. These analytes represent three distinct classes of biomarkers: (i) glycated proteins, (ii) low-molecular-weight reactive oxygen species, and (iii) nucleic acids. Hence, the proposed methodology is applicable for rapid detection of generic biomarkers in urine, without involving sophisticated equipment and skilled personnel, thereby enabling on-site urinalysis. At the end of the contribution, we discuss the opportunity to translate the homogeneous assay into a paper-based format.

In silico, in vitro and in vivo metabolite identification of brexpiprazole using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

RATIONALE: Brexpiprazole is a novel serotonin-dopamine activity modulator approved by the USFDA in July 2015 for the treatment of schizophrenia and as an adjunctive therapy with other antidepressants for major depressive disorder in adults. However, limited numbers of metabolites are reported in the literature for brexpiprazole. Our prime intent behind this study is to revisit metabolite profiling of brexpiprazole and to identify and characterize all possible in vitro and in vivo metabolites. METHODS: Firstly, the site of metabolism for brexpiprazole was predicted by a Xenosite web predictor model. Secondly, in vitro metabolite profiling was performed by incubating the drug individually with rat liver microsomes, human liver microsomes and rat S9 fraction at 37°C for 1 h in incubator shaker. Finally, for in vivo metabolite identification, a 50 mg kg-1 dose of brexpiprazole was administered to male Sprague-Dawley rats and the presence of various metabolites was confirmed in rat plasma, urine and feces. RESULTS: The predicted atomic site of metabolism was obtained as a color gradient by the Xenosite web predictor tool and, from this study, probable metabolites were listed. In total, 14 phase I and 2 phase II metabolites were identified and characterized in the in vitro and in vivo matrices using ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC/QTOF-MS/MS). The majority of metabolites were found in the sample incubated with human liver microsomes and in rat urine, while in the other matrices only a few metabolites were detected. CONCLUSIONS: All the 16 metabolites were identified and characterized using UHPLC/QTOF-MS/MS. The study revealed that brexpiprazole is metabolized via hydroxylation, glucuronidation, S-oxidation, N-oxidation, dioxidation, oxidative deamination, N-dealkylation, etc.

A further insight into the metabolic profile of the nuclear receptor Rev-erb agonist, SR9009.

The reactions involved in the metabolic pathways of SR9009 were characterized by liquid chromatography-mass spectrometry (LC-MS) to identify the most appropriate marker(s) of use. The effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009 were also evaluated. In vitro approaches were based on the use of human liver microsomes and cytochrome P450 isoforms. Sample preparation included an enzymatic hydrolysis (performed only for the phase II investigation) followed by liquid-liquid extraction. The chromatographic separation was carried out using a reverse-phase column; detection was performed by either a triple-quadrupole or a time-of-flight system in positive electrospray ionization and different acquisition modes. In the presence of human liver microsomes, SR9009 was biotransformed to 13 metabolites by CYP3A4, CYP3A5, CYP2C19, and CYP2D6 isoenzymes. The reactions included hydroxylation, de-alkylation, oxidation, and combinations thereof, the de-alkylated metabolites being the most abundant. Once formed the mentioned metabolites underwent glucuronidation. Concerning the effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009, our observation have shown the following: (a) No significant alterations were measured between female and male, (b) significant differences were registered using either the CYP2D6 or CYP2C19 allelic variants, and finally (c) significant alterations were registered in the presence of ketoconazole, miconazole, fluoxetine, nefazodone and paroxetine; moderate variation were instead registered with fluconazole, itraconazole, gestodene, and levonorgestrel. This observation put in evidence the importance to take into account both genetic polymorphism and drug-drug interaction to select the most appropriate marker(s) of use in doping analysis.

Determination of a PDE4 inhibitor Hemay005 in human plasma and urine by UPLC-MS/MS and its application to a PK study.

AIM: Hemay005 is a novel small-molecule inhibitor of phosphodiesterase-4 developed for the treatment of psoriasis. Measurement of Hemay005 in biological samples is critical for evaluation of its pharmacokinetics in clinical studies. Methodology & results: Plasma and urine samples were extracted and then chromatographed on an Acquity UPLC HSS T3 column with a gradient elution. Detection was performed on a Xevo TQ-S tandem mass spectrometer using negative ESI. CONCLUSION: For the first time, a sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the quantitative determination of Hemay005 in human plasma and urine, and it was successfully applied to evaluate the pharmacokinetics of Hemay005 in healthy subjects in a first-in-human study.

Acute Methiopropamine Intoxication After "Synthacaine" Consumption.

Use of methiopropamine (MPA), a synthetic metamfetamine analog, has been detected since 2011 in Europe, but there is limited information on its acute toxicity. A 30-year-old man was admitted to the emergency department in a confused state, with paranoid delusion, auditory and visual hallucinatory experiences, and incoherent speech following the use of "synthacaine" (a slang term derived from "synthetic" and "cocaine"). Toxicological screening for pharmaceuticals and drugs of abuse by liquid chromatography-diode-array detector, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS-MS) detected MPA, which was subsequently quantified by a specific LC-MS-MS method. Of note, 13 h after presentation to the emergency department, the plasma concentration of MPA was 14 ng/mL. This case report confirms the toxicity of MPA and the need for toxicological analysis to confirm the substance actually ingested by users of new psychoactive substances.

In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant.

BACKGROUND: Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. MATERIALS & METHODS: A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive™ Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites. RESULTS/CONCLUSION: Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.

A Fatal Case of Isolated Methiopropamine (1-(Thiophen-2-yl)-2-Methylaminopropane) Toxicity: A Case Report.

Methiopropamine (1-(thiophen-2-yl)-2-methylaminopropane) is a synthetic methamphetamine analogue and is classified as a novel psychoactive substance. The use of novel psychoactive substance has been increasing substantially for recreational purpose in recent years. Methiopropamine was first detected in 2011 in Finland and was later detected in the United Kingdom. It can be purchased on the Internet and is currently poorly regulated. Reported adverse effects of methiopropamine use are mostly anecdotal user reports on Internet forums, and there are limited data on its pharmacodynamics and toxicity in the literature. Death as a direct result from methiopropamine toxicity has not been reported in Australia. We report here the first case of death caused by recreational use of methiopropamine in Australia. This same incident highlights the first ever death from isolated methiopropamine use. Being an analogue of methamphetamine, we hypothesize that the mechanism of death caused by methiopropamine would not be dissimilar to methamphetamine.

Application of an ultra-performance liquid chromatography method with tandem mass spectrometry to pharmacokinetics, tissue distribution and excretion in the study of DAT-230, a novel tubulin-binding agent candidate, in rats.

A rapid, sensitive and high-throughput ultra-performance liquid chromatography method with tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of DAT-230 in rat plasma, urine, feces and tissues (heart, liver, spleen, lung, kidney, stomach, intestine and brain). The biological samples were prepared by protein precipitation, and separation was achieved on an ACQUITY™ UPLC BEH C18 column (50 mm × 2.1mm, 1.7 µm) with a mobile phase that consisted of methanol - 0.2% formic acid water (80:20, v/v) at a flow rate of 0.2 mL/min. The MS/MS ion transitions were monitored at m/z 372.17 → 357.17 for DAT-230 and m/z 406.08 → 345.16 for COH-203 (internal standard, IS). The calibration curve was linear in the range of 0.1-5200 ng/mL for all biological matrices (r(2) ≥ 0.996), and it had the same value for the lower limit of quantification. The validated method was successfully applied to the pharmacokinetics, tissue distribution and excretion study after intravenous administration of a 5mg/kg dose of DAT-230 to healthy Sprague-Dawley rats. The mean pharmacokinetic parameters of t1/2 and AUC0-12 were (1.1 ± 0.4)h and (861.0 ± 281.2) ng h/mL, respectively. Tissue distribution results indicated that DAT-230 exhibited rapid distribution and high liver, kidney, spleen, stomach and intestine uptake; these organs were indicated as the major target organs of the drug. In total, 5.3% of the administered DAT-230 was excreted in an unconverted form in urine, feces and bile, which implies that DAT-230 was excreted primarily in the form of metabolites.

Metabolism and excretion of canagliflozin in mice, rats, dogs, and humans.

Canagliflozin is an oral antihyperglycemic agent used for the treatment of type 2 diabetes mellitus. It blocks the reabsorption of glucose in the proximal renal tubule by inhibiting the sodium-glucose cotransporter 2. This article describes the in vivo biotransformation and disposition of canagliflozin after a single oral dose of [(14)C]canagliflozin to intact and bile duct-cannulated (BDC) mice and rats and to intact dogs and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in both animals and humans. In BDC mice and rats, most radioactivity was excreted in bile. The extent of radioactivity excreted in urine as a percentage of the administered [(14)C]canagliflozin dose was 1.2%-7.6% in animals and approximately 33% in humans. The primary pathways contributing to the metabolic clearance of canagliflozin were oxidation in animals and direct glucuronidation of canagliflozin in humans. Unchanged canagliflozin was the major component in systemic circulation in all species. In human plasma, two pharmacologically inactive O-glucuronide conjugates of canagliflozin, M5 and M7, represented 19% and 14% of total drug-related exposure and were considered major human metabolites. Plasma concentrations of M5 and M7 in mice and rats from repeated dose safety studies were lower than those in humans given canagliflozin at the maximum recommended dose of 300 mg. However, biliary metabolite profiling in rodents indicated that mouse and rat livers had significant exposure to M5 and M7. Pharmacologic inactivity and high water solubility of M5 and M7 support glucuronidation of canagliflozin as a safe detoxification pathway.

Influence of hepatic impairment on the pharmacokinetics of the dopamine agonist rotigotine.

The transdermally applied dopamine receptor agonist rotigotine is extensively metabolized in the liver. An open-label, parallel-group study was conducted to evaluate the effects of moderate hepatic impairment on the pharmacokinetics, safety and tolerability of rotigotine. Eight subjects with normal hepatic function and nine with moderate hepatic impairment (Child-Pugh class B) received one rotigotine transdermal patch (providing a dose of 2 mg/24 h) daily for 3 days with a 24-h patch-on period. Blood and urine samples were collected to evaluate pharmacokinetic parameters characterizing drug bioavailability and elimination. Primary variables included plasma and urine concentrations of unconjugated rotigotine (active parent compound) and total rotigotine (unconjugated rotigotine plus sulfate and glucuronide conjugates) under steady-state (SS) conditions. For unconjugated rotigotine, point estimates for the ratios of AUC(0-24)SS and C max,SS between the two groups (normal vs. impaired hepatic function) were near 1: AUC(0-24)SS, 0.90 (90 % CI 0.59, 1.38) and C max,SS, 0.94 (90 % CI 0.66, 1.35); t max,SS and t 1/2 were lower in subjects with hepatic impairment, while renal clearance was unaffected and overall clearance was higher. For total rotigotine, C max,SS was higher in subjects with hepatic impairment compared with those with normal hepatic function (P = 0.0239, ANOVA). A tendency to reduced non-renal clearance was observed in subjects with hepatic impairment, consistent with their higher plasma concentrations of total rotigotine. Thus, moderate hepatic impairment did not influence the pharmacokinetics of unconjugated rotigotine under steady-state conditions suggesting that dose adjustment will not be required for patients with mild or moderate hepatic insufficiency. In addition, the rotigotine patch was well tolerated in subjects with moderate hepatic impairment.

Asparagus, urinary odor, and 1,2-dithiolane-4-carboxylic acid.

The derivatives of 1,2-dithiolane are scattered widely throughout the natural world where they are generally exploited for their biocidal properties. One of these, known as asparagusic acid (1,2-dithiolane-4-carboxylic acid), is present in the frequently consumed vegetable, asparagus, and though apparently innocuous toxicologically, it may be responsible for the distinctive urine odor produced after consuming this food. This review collects together, for the first time, historical observations associating asparagus ingestion with this unique odor-producing phenomenon and collates data implicating a 1,2-dithiolane structure as the major chemical precursor responsible.

2-methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques.

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling "research chemicals." The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors' well-established gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MS(n)) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography-high-resolution linear ion trap mass spectrometry (LC-HR-MS(n)) and the phase II metabolites by LC-HR-MS(n). The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user's dose, 2-MPA and its metabolites were identified in rat urine using the authors' GC-MS and the LC-MS(n) screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.

Gas chromatographic-mass spectrometry method for the detection of busulphan and its metabolites in plasma and urine.

Busulphan is an alkylating agent used as conditioning regimen prior to stem cell transplantation. Busulphan is metabolized in the liver and four major metabolites have been identified. The first metabolite is tetrahydrothiophene which is oxidized to tetrahydrothiophene 1-oxide, then sulfolane and finally 3-hydroxy sulfolane. Despite the low molecular weight and wide polarity range of busulphan and its four metabolites, the use of a fused silica non-polar column significantly enhanced the automated gas chromatography-mass spectrometry of their detection in one simple method. The limit of quantification was 0.5µM for busulphan and all its metabolites except 3-OH sulfolane, which was 1.25µM. This method was validated for all the compounds in both human plasma and urine. Lower limits of quantifications (LLOQs) were run in pentaplicate per compound and all results were within 20% of the nominal values. The recovery was determined by comparing the peak area of two quality control (QC) samples, before and after extraction in plasma and urine, in triplicate. Acceptable precision and accuracy have been obtained; at least 3 standard curves have been run for each compound using three different QCs covering the calibration curve in triplicate. The QC values were within 15% (SD) of the nominal values. Selectivity and sensitivity of all compounds have been measured. Compounds were stable up to 50 days after extraction in -20°C and 48h at RT. Moreover, the compounds were stable for three cycles of freezing and thawing. The method was applied in a clinical case where the patient received high dose busulphan; all the compounds have been detected, identified and quantified both in plasma and urine.

Novel methods for assessing oral direct factor Xa and thrombin inhibitors: use of point-of-care testing and urine samples.

Rivaroxaban and dabigatran are new oral anticoagulants (NOACs) that inhibit directly factor Xa and thrombin, respectively. These NOACs effectively prevent thromboembolic complications using fixed doses without the need for dose adjustment according to laboratory results. About 60% of rivaroxaban is cleared from circulation by glomerular filtration, 30% of which is excreted as active drug. About 80% of dabigatran is excreted into urine as active compound. Accordingly, both NOACs can be determined in urine by means of chromatographic methods. Only a few laboratories are able to perform such methods, and results are not available within short time frames. New methods have to be developed to obtain results within minutes and possibly as point-of-care (POC) techniques. This testing may be useful for special patient populations such as those with acute deterioration of renal function due to any disease, before surgical interventions, during unexpected bleeding or thrombotic episodes while on therapy with NOACs, the oldest and youngest populations, pregnancy, suspicion of overdose and intoxication, and to determine adherence to therapy. Here we describe results of a POC qualitative assay using urine samples from patients on treatment with dabigatran and rivaroxaban.

Combination of docking, molecular dynamics and quantum mechanical calculations for metabolism prediction of 3,4-methylenedioxybenzoyl-2-thienylhydrazone.

In modern drug discovery process, ADME/Tox properties should be determined as early as possible in the test cascade to allow a timely assessment of their property profiles. To help medicinal chemists in designing new compounds with improved pharmacokinetics, the knowledge of the soft spot position or the site of metabolism (SOM) is needed. In silico methods based on docking, molecular dynamics and quantum chemical calculations can bring us closer to understand drug metabolism and predict drug-drug interactions. We report herein on a combined methodology to explore the site of metabolism prediction of a new cardioactive drug prototype, LASSBio-294 (1), using MetaPrint2D to predict the most likely metabolites, combined with structure-based tools using docking, molecular dynamics and quantum mechanical calculations to predict the binding of the substrate to CYP2C9 enzyme, to estimate the binding free energy and to study the energy profiles for the oxidation of (1). Additionally, the computational study was correlated with a metabolic fingerprint profiling using LC-MS analysis. The results obtained using the computational methods gave valuable information about the probable metabolites of (1) (qualitatively) and also about the important interactions of this lead compound with the amino acid residues of the active site of CYP2C9. Moreover, using a combination of different levels of theory sheds light on the understanding of (1) metabolism by CYP2C9 and its mechanisms. The metabolic fingerprint profiling of (1) has shown that the metabolites founded in highest concentration in different species were metabolites M1, M2 and M3, whereas M8 was found to be a minor metabolite. Therefore, our computational study allowed a qualitative prediction for the metabolism of (1). The approach presented here has afforded new opportunities to improve metabolite identification strategies, mediated by not only CYP2C9 but also other CYP450 family enzymes.

Stimulating luteinizing hormone.

Low molecular weight luteinizing hormone (LMWLH) receptor agonists could be of interest as a potential doping substance for athletes. These orally active compounds induce the production of endogenous hormones such as testosterone in a similar way to LH. A method for the detection of these compounds needs to be direct as their effect--the excess production of endogenous hormones--cannot be proven by analysis techniques which test for endogenous hormones. In order to detect a broad range of potential LMWLH receptor agonists, a precursor ion monitoring liquid chromatography tandem mass spectrometry method was developed. The method was tested against a selection of urine samples to ascertain potential problems with background analytes interfering with the compounds of interests. Selected compounds were extracted with an established methodology from urine to determine suitability of implementing into general screening methodologies. The two available LMWLH receptor agonists could be detected at concentrations of 100 ng/ml in urine samples. This establishes a basic precursor ion monitoring method suitable for screening purposes for the detection of LMWLH receptor agonists in urine samples.

Qualitative GC-MS assessment of TCP and TAMORF elimination in rats.

Nerve agents are highly toxic organophosphorus (OP) compounds. They inhibit acetylcholinesterase (AChE), an enzyme that hydrolyses acetycholine (ACh) in the nervous system. Pathophysiological changes caused by OP poisonings are primarily the consequence of surplus ACh on cholinergic receptors and in the central nervous system. Standard treatment of OP poisoning includes combined administration of carbamates, atropine, oximes and anticonvulsants. In order to improve therapy, new compounds have been synthesised and tested. Tenocyclidine (TCP) and its adamantane derivative 1-[2-(2-thienyl)-2-adamantyl] morpholine (TAMORF) have shown interesting properties against soman poisoning. In this study, we developed a qualitative GC-MS method to measure elimination of TCP and TAMORF through rat urine in order to learn more about the mechanisms through which TCP protects an organism from OP poisoning and to determine the duration of this protective effect. GC-MS showed that six hours after treatment with TCP, rat urine contained only its metabolite 1-thienylcyclohexene, while urine of rats treated with TAMORF contained both TAMORF and its metabolites.

Absorption, disposition, metabolic fate, and elimination of the dopamine agonist rotigotine in man: administration by intravenous infusion or transdermal delivery.

The dopamine agonist rotigotine was developed for the treatment of Parkinson's disease and restless legs syndrome. Disposition, metabolism, elimination, and absolute bioavailability of rotigotine were determined in six healthy male subjects by using two different forms of administration in a randomized sequence with a crossover design. Treatment A (continuous infusion) consisted of a single radiolabeled 12-h intravenous infusion of 1.2 mg of rotigotine (0.6 mg of [(14)C] and 0.6 mg of unlabeled rotigotine, 3.7 MBq) solution. Treatment B (transdermal application) consisted of a single 10-cm(2) patch containing 4.5 mg of unlabeled rotigotine with a patch-on period of 24 h. During the 12 h-infusion, total radioactivity concentration rapidly increased within 2 h; there was a slight additional increase toward the end of infusion. Plasma concentrations of total radioactivity declined by 75% within 12 h after completion of infusion. More than 94% of the radioactivity was excreted 216 h after the start of infusion, 71% by the kidneys and 23% by feces. Renal elimination of the parent compound was <1%. Systemically absorbed rotigotine was rapidly metabolized. The major rotigotine biotransformation pathway was conjugation of the parent compound, mainly by sulfation; a second pathway was the formation of phase 1 metabolites (N-desalkylation) with subsequent conjugation. Plasma concentration-time profiles of unchanged rotigotine during and after infusion and during and after patch administration were comparable. Absolute bioavailability of transdermally applied rotigotine was 37%.

Rapid HPLC method for the simultaneous monitoring of duloxetine, venlaflaxine, fluoxetine and paroxetine in biofluids.

A simple and rapid HPLC method is developed for the determination of two serotonin-norepinephrine-reuptake inhibitors (duloxetine and venlaflaxine) and two selective serotonin-reuptake inhibitors (fluoxetine and paroxetine) in human biofluids. Separation was performed on an Inertsil ODS-3 column (250 x 4.0 mm, 5 µm) with acetonitrile-ammonium acetate (0.05 M, 41:59 v/v) at 235 nm, within 7 min. SPE on Oasis(®) HLB cartridges was applied for the isolation of analytes from biofluids. The developed methodology was validated in terms of sensitivity, linearity, accuracy, precision, stability and selectivity. Relative standard deviation was less than 10.4%. Limit of detection was 0.2-0.6 ng/µl in blood plasma and 0.1-0.8 ng/µl in urine. The method was successfully applied to biofluids from a patient under duloxetine treatment.