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Hypoxia-inducible factor (HIF) stabilizer belongs to a novel class of pharmacologically active substances, which are capable of inducing the endogenous erythropoietic system. The transcriptional activator HIF has been shown to significantly increase blood hemoglobin and is well set for the treatment of anemia resulting from chronic kidney disease. This research work reports a comprehensive study of the most popular HIF stabilizer roxadustat and its metabolites in thoroughbred horse urine after oral administration. The plausible structures of the detected metabolites were postulated using liquid chromatography-high-resolution mass spectrometry. Under the experimental condition 13 metabolites (7 phase I, 1 phase II, and 5 conjugates of phase I metabolism) were positively detected (M1-M13). The major phase I metabolites identified were formed by hydroxylation. Dealkylated and hydrolyzed phase I metabolites were also observed in this study. In phase II, a glucuronic acid conjugate of roxadustat was detected as the major metabolite. The sulfonic acid conjugates were observed to be formed from phase I metabolites. The characterized in vivo metabolites can potentially serve as target analytes for doping control analysis; hence, the result is an important tool for assessing its use and abuse in competitive sport., (© 2021 John Wiley & Sons, Ltd.)

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation., (© 2020 John Wiley & Sons, Ltd.)

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes. In addition, hydroxylated IOX-2 and the corresponding glucuronic acid conjugates were identified as major metabolites in a microdose elimination study, contributing to enhanced initial testing and confirmation procedures., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

EMA401, (the S-enantiomer of 5-(benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), also known as Olodanrigan, is an orally active selective angiotensin II type 2 receptor (AT 2 R) antagonist that is in Phase IIb clinical development as a novel analgesic for the relief of chronic pain. The main purpose of the present work was to investigate the disposition of a single 14 C- labeled EMA401 in non-clinical studies. The in vitro metabolism studies of EMA401 were undertaken to understand the hepatic biotransformation pathways in animal species used in toxicology studies and how they compare to human. Furthermore, investigation of EMA401's PK was carried out in vivo in rats. The study demonstrates the rapid absorption and distribution of drug-related material mainly to the tissues associated with absorption and elimination (GI tract, liver, and kidney). EMA401was then readily eliminated metabolically via the bile (95% of dose) predominantly in the form of the direct acylglucuronide (40% of dose), which was further hydrolysed by the intestinal flora to the active parent drug. Other metabolic pathways such as dealkylations and hydroxylation were also involved in the elimination of EMA401 to a lesser extent. EMA401 was metabolically unstable in hepatocytes of all species investigated and the key metabolites produced in the in vitro model were also detected in vivo. Independent of the dosing route, the S-enantiomer EMA401 showed a good in vivo chiral stability. Overall, the present study provides the first full characterization of the disposition of EMA401 in preclinical species., (© 2020 John Wiley & Sons, Ltd.)

Rationale: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites., Methods: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data., Results: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis., Conclusions: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network., (© 2019 John Wiley & Sons, Ltd.)

Background: YM758 monophosphate is a novel If channel inhibitor that has an inhibitory action for If current and shows a strong and specific activity, selectively lowering the heart rate and decreasing oxygen consumption by heart muscle., Objectives: The objectives of the current study were to investigate the in vivo metabolic profiles of YM758 in mice, rats, rabbits, dogs, and monkeys and to elucidate the structures of YM758 metabolites., Methods: Biological samples were analyzed by liquid chromatography hyphenated with a radiometric detection system and liquid chromatography coupled with a mass spectrometer to clarify their metabolic patterns. To elucidate their structures, metabolites were isolated and analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy., Results: Our results from in vivo metabolic profiling in humans and animals indicated there is no significant species difference in the metabolism of YM758, and the metabolic pathways of YM758 are considered to be oxidation, hydration, and demethylation followed by sulfate or glucuronide conjugation.

Stabilizing the labile factor HIF (hypoxia inducible factor) for therapeutic use has led to the development of various molecules by pharmaceutical companies. These HIF stabilizers show promising erythropoiesis stimulating capacities and are of great interest for patients with chronical kidney disease and anemia. Amongst them FG-4592 from FibroGen is now under phase 3 of clinical studies. While this drug is still under investigation, a parallel market already allows to buy this product, which could be tempting for some athletes willing to increase their performances. To avoid such a use for doping purpose, WADA has listed HIF stabilizers and FG-4592 in particular as prohibited substances since 2011 and some anti-doping laboratories have developed a technique of identification of FG-4592 in urine. Here, we described the first case ever identified by an anti-doping laboratory of an athlete using FG-4592. Detection and confirmation in urinary samples was performed by LC-MS/MS. In addition, potential indirect markers erythropoietin (EPO) and hematological parameters followed in the Athlete Biological Passport (ABP) were analyzed during and after the period of use but showed no profound alterations. Only ABPS (abnormal blood profile score) reached (but did not exceed) the upper limit proposed by the ABP adaptive model just after the period of use of FG-4592. Altogether this case sends a warning for anti-doping laboratories which now must strengthen surveillance on HIF stabilizers and develop sensitive methods of detection for this new class of drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The new analytical method for the determination of palonosetron in human plasma and urine has been developed based on liquid chromatography-mass spectrometry. The method utilized tramadol as the internal standard (IS). Separation was carried out on a Zorbax Eclipse TC-C(18) column using methanol-1mM ammonium formate in water (containing 0.1% formic acid, v/v, pH=2.8) as mobile phase for gradient elution. Detection is carried out by multiple reaction monitoring (MRM) on 3200 Qtrap mass spectrometry. The method has a chromatographic run time of 5.5 min and is linear within the concentration range 0.01-5.00 ng/mL for plasma and 0.10-30.00 ng/mL for urine both with a LOD of 0.003 ng/mL. Intra- and inter-day RSD of the concentration was 3.66-6.60%, 1.29-7.71% for plasma and 2.39-5.76%, 2.06-7.13% for urine. The relative error (RE) was -4.58% to 3.26% for plasma and -1.47% to 2.53% for urine. The recovery rates of palonosetron and IS both for plasma and urine were more than 90%. Palonosetron was stable under all the conditions tested. The method was successfully used to analyze palonosetron in human plasma and urine over a period of 168 h after intravenously pumping a single dose of 0.25mg to volunteers. No significant differences were found between the pharmacokinetic parameters and urine accumulated excretory rate for male and female volunteers (P>0.05). A two-compartment model was obtained after administrations. Palonosetron was eliminated at a slow rate in volunteers. The mean urine accumulated excretory rate was 25.97 ± 12.87%. Inter-individual differences could not be neglected due to the high coefficient of variety in several pharmacokinetic parameters and the urine accumulated excretion., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Hypoxia-inducible factor (HIF) stabilizers increase blood haemoglobin levels after oral administration and their use in sports was recently banned by the World Anti-Doping Agency. For the support of analytical assay development, the metabolic fate of two model HIF stabilizers, based on the isoquinoline-3-carboxamide scaffold of the lead drug candidate FG-2216, was assessed by in vitro methods. The analytes were identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative ionization mode using an API 4000 Qtrap as well as an exactive high resolution-high accuracy MS. The model HIF stabilizer N-[(1-chloro-4-hydroxy-7-isopropoxy-isoquinolin-3-carbonyl)-amino]-acetic acid (1), was converted into 3 major phase I metabolites by hydroxylation, dealkylation, and dehydrogenation. The structures of the hydroxylated and the dealkylated metabolites were confirmed by LC-coupled nuclear magnetic resonance spectroscopy. Moreover, glucuronic acid conjugates of the active drug and one of the dealkylated phase I metabolite were identified. Hydroxylation of model compound 2 (N-[(1-chloro-4-hydroxy-isoquinolin-3-carbonyl)-amino]-acetic acid) yielded two metabolites, regioisomeric to the dealkylated product of 1. Mass spectral data of compounds 1 and 2, as well as a structure-related analogue were included into a multi-target analytical assay based on direct injection and LC-MS/MS analysis of human urine. The method was validated for quantitative purposes. In an approach of preventive doping research, more comprehensive screening methods applying precursor ion (m/z 166) and neutral loss (-10 Da) scans were developed, allowing for the detection of unknown metabolites and structurally analogous HIF stabilizers emerging from ongoing lead structure developments., (Copyright © 2011 John Wiley & Sons, Ltd.)

The fluorescence spectra of berberine, palmatine, jatrorrhizine, and coptisine in ionic liquids were studied and found to increase significantly in ionic liquids, with [C(8)MIM][PF(6)] having the greatest increase. Further studies showed that these drugs could be extracted from an aqueous solution by [C(8)MIM][PF(6)] using the temperature-assisted ionic liquid dispersive liquid phase microextraction method. The enrichment factors were 81.8-82.3, and the extraction recovery was 98.5%, 98.1%, 98.3%, and 98.8% for berberine, palmatine, jatrorrhizine, and coptisine, respectively. Based on the [C(8)MIM][PF(6)] preconcentration, separation, and sensitized fluorescence for these drugs, a new selective and sensitive method for the determination of concentration of these four drugs in aqueous samples was presented. At optimum conditions, the linear relationship was obtained in the ranges of 0.8-130 ng mL(-1), 0.9-160 ng mL(-1), 0.7-140 ng mL(-1), and 0.6-110 ng mL(-1), respectively. The proposed method was successfully applied for the determination of the drugs in pharmaceutical preparations, urine, and plasma samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Palonosetron is used for the prevention of chemotherapy-induced nausea and vomiting. However, quantification of this drug in human urine has been rare., Results: A one-step dilution method for the analysis of palonosetron in human urine using LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines. The method uses 200 µl of urine and covers a working range from 2.5-1000 ng/ml with a LLOQ of 2.5 ng/ml., Conclusion: This new LC-MS/MS assay is sensitive and specific despite using an external standard method. It is suitable for clinical studies of palonosetron.

(-)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758) is a novel inhibitor of the "funny" If current channel (If channel) that is expressed in the sinus node of heart and is being developed as a treatment for stable angina and atrial fibrillation. Its metabolites were identified in human urine, plasma, and feces by radio-high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analyses after oral administration of [(14)C]YM758. 6,7-Dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124), (5R)-5-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]piperidin-2-one (YM-385459), 2-{[(3R)-1-{2-[(4-fluorobenzoyl)amino]ethyl}piperidin-3-yl]carbonyl}-7-methoxy-1,2,3,4-tetrahydroisonolin-6-yl beta-D-glucopyranosiduronic acid (AS2036329), and the unchanged drug were detected as major constituents in both urine and plasma, whereas N-(4-fluorobenzoyl)glycine (YM-385461) was detected in plasma, but not in urine. The renal and hepatic uptake transporters for these metabolites were investigated by assessing their inhibitory effect on uptake activity in human (h) organic cation transporter (OCT) 1-3/rat (r) Oct1-3, human organic anion transporter (OAT) 1/rOat1, hOAT3/rOat3, and organic anion-transporting protein 1B1/1B3-expressing HEK293 cells. IC(50) values of YM-252124 for 1-methyl-4-phenylpyridinium uptake via hOCT2 and rOct2 were 93.9 and 1700 microM, respectively, suggesting that this metabolite is secreted into urine via hOCT2/rOct2 and that the large difference in the inhibitory potentials between hOCT2 and rOct2 explains the species difference in the urinary excretion ratio of the radioactivity. The renal secretion of YM-385461, one derivative of p-aminohippuric acid, via hOAT1/rOat1, and hepatic uptake of YM-252124 via hOCT1/rOct1 was also expected. This kind of study was useful in investigating the relationship between the urinary/hepatic elimination and the transport activity for metabolites.

The biotransformation of the antiplasmodial naphthylisoquinoline alkaloid dioncophylline A by rat liver microsomes and its pharmacokinetics in male rats were studied. Incubation of dioncophylline A with rat liver microsomes resulted in the formation of the major metabolite 5'-O-demethyldioncophylline A, and a second minor metabolite, corresponding to the mass of an as yet unknown 4-hydroxydioncophylline A. Kinetic constants of the formation of 5'-O-demethyldioncophylline A were Km = 32 nmol and Vmax = 20 pmol min-1 mg-1). Administration of dioncophylline A at a dose of 6.67 mg kg-1 body weight to rats intravenously and orally (n = 4 per group) resulted in peak plasma levels of 0.84 and 0.11 microg ml-1, respectively. Levels of metabolites were below the limit of quantitation (LOQ). The following pharmacokinetic parameters of dioncophylline A were determined: oral bioavailability of 25%, plasma half-life of 2.5 h and partition volume of 8 l kg-1 body weight. Concentrations of dioncophylline A metabolites in all plasma and urine samples were below the limit of detection (LOD) and recovery of dioncophylline A in urine was very low, suggesting distribution into lipid rich tissues.

Trabectedin (Yondelis, formerly ET-743) is an anti-cancer drug currently undergoing phase II development. Despite extensive pharmacokinetic studies, the human disposition and excretory pathways of trabectedin remain largely unknown. Our objective was to determine the mass balance of trabectedin in humans. To this aim, we intravenously administered [(14)C]trabectedin to 8 cancer patients, followed by collection of whole blood, urine and faeces samples. A 24-h infusion was administered to 2 patients, whereas the other 6 patients received a 3-h infusion. Levels of total radioactivity and unchanged trabectedin were determined and used for calculation of pharmacokinetic parameters. No schedule dependency of pharmacokinetic parameters was observed apart from C(max). Plasma and whole blood concentrations of [(14)C]trabectedin related radioactivity were comparable. Only 8% of the plasma exposure to [(14)C]trabectedin related compounds is accounted for by trabectedin, indicating the importance of metabolism in trabectedin elimination. Trabectedin displays a large volume of distribution (+/-1700 L), relative to total radioactivity (+/-220 L). [(14)C]trabectedin related radioactivity is mainly excreted in the faeces (mean: 55.5% of the dose). Urinary excretion accounts for 5.9% of the dose on average resulting in a mean overall recovery of 61.4% (3-h administration schedule). The excretion of unchanged trabectedin is very low both in faeces and in urine (< 1% of dose). In conclusion, trabectedin is extensively metabolised and principally excreted in the faeces.

Endogenously synthesised norsalsolinol derivatives are elevated in Parkinson's disease (PD) and have been considered potentially useful biological markers of the disease. However, little is known about the impact of dopaminergic drugs on the formation of these compounds. We prospectively examined the urine concentrations of norsalsolinol, N-methyl-norsalsolinol and salsolinol in 47 PD patients and 14 control subjects. Patients and control subjects were re-examined after approximately 1 year to assess long term changes. Norsalsolinol derivatives were low in controls and untreated patients with early PD. Increased urine concentrations of norsalsolinol derivatives were significantly associated with levodopa treatment. They were elevated more markedly in the urine of patients treated with high (>600 mg daily) doses of levodopa compared with patients receiving medium (300-600 mg) or low (<300 mg) doses of the drug. There was no correlation with disease parameters such as the severity of motor disability or deficits in the cognitive performance. In the patient group, the concentrations of all three norsalsolinol derivatives declined over the period of investigation, however, they still remained elevated compared with the control group. We conclude that systemic levels of norsalsolinol derivatives in treated patients with PD are likely to derive from the metabolism of levodopa and cannot be regarded as intrinsic markers of the disease. The limited ability of norsalsolinol derivatives to pass the blood-brain barrier prevents an intracerebral accumulation of these possibly harmful compounds, which are biochemically similar to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

A liquid chromatography-electrospray-mass spectrometry technique for the screening and determination of five non-depolarizing neuromuscular blocking agents (NDBAs), atracurium and its product of degradation/metabolite laudanosine, rocuronium, pancuronium, vecuronium, and mivacurium has been developed using ambenonium as the internal standard (I.S.). Samples were acidified upon reception by adding 20 micro L 0.5M H(2)SO(4) to 500 micro L of biofluid. Sample preparation consisted of simple blood purification and/or protein precipitation using 1 mL I.S. in acetonitrile. Chromatographic separation was carried out on an X-TERRA trade mark column along with a gradient of acetonitrile in 2mM ammonium formate (pH 3). Detection was carried out in the positive selected ion monitoring mode, targeting one quantitation ion and one confirmation ion per compound. The limit of quantitation was 2.5 micro g/L for mivacurium and laudanosine, 5 micro g/L for rocuronium and pancuronium and 10 micro g/L for atracurium and vecuronium in serum (i.e., in the range of, or less than, therapeutic levels). The technique was found to be linear between the respective LOQs and 2000 micro g/L, with correlation coefficients higher than 0.999 in all matrices. Intra- and interday precision and accuracy in serum fulfilled the international criteria. This method was employed for the investigation of a case of suicide by infusion of drugs. Laudanosine, the metabolite or degradation product of both atracurium and cisatracurium, and rocuronium were found in urine and whole blood, at supratherapeutic concentrations in the latter (rocuronium: 1.53 and 2.18 mg/L, laudanosine: 8.86 and 0.31 mg/L, respectively), and even therapeutic concentrations would have been lethal in the absence of respiratory assistance.

The liver is the major site of drug metabolism in the body. However, many drugs undergo metabolism in extrahepatic sites and in the gut wall and lumen. In this study, the distribution and activity of reductases in rat that reduced potential cytostatic oracin to its principal metabolite 11-dihydrooracin (DHO) were investigated. The extension and stereospecificity of oracin reduction to DHO were tested in microsomal and cytosolic fractions from the liver, kidney, heart, lung and wall of small intestine, caecum and large intestine. Intestinal bacterial reduction of oracin was studied as well. The amount of DHO enantiomers was measured by HPLC with Chiralcel OD-R as chiral column. Reductive biotransformation of oracin was mostly stereospecific for (+)-DHO, but the enantiomeric ratio differed significantly among individual tissues and subcellular fractions (from 56% (+)-DHO in heart microsomes to 92% (+)-DHO in liver cytosol). Stereospecificity for (-)-DHO (60%) was observed in bacterial oracin reduction in the lumen of small intestine, caecum and large intestine. Shift of the (+)-DHO/(-)-DHO enantiomeric ratio from 90:10 (in liver subcellular fractions) to 60:40 (in-vivo) clearly demonstrated the importance of the contribution of extrahepatic metabolism to the total biotransformation of oracin to DHO.

Eschscholtzia californica preparations are in use as phytopharmaceuticals and as herbal drugs. Studies are described on the metabolism and the toxicological analysis of the Eschscholtzia californica alkaloids californine and protopine in rat urine using gas chromatography-mass spectrometry. The identified metabolites indicated that californine is extensively metabolized by N-demethylation and/or single or double demethylenation with consecutive catechol-O-methylation of one of the hydroxy groups. Protopine, however, only undergoes extensive demethylenation of the 2,3-methylenedioxy group followed by catechol-O-methylation. All phenolic hydroxy metabolites were found to be partly conjugated. The authors' systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of californine and protopine in rat urine after a dose which should correspond to that of drug users. Therefore, use of Eschscholtzia californica preparations should also be detectable in human urine by the authors' systematic toxicological analysis procedure.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new isoquinolinedione derivative, 7-anilino-5,8-isoquinolinedione (IQO4), in rat plasma, urine, blood and tissue homogenates using diazepam as an internal standard. A 2 volume of acetonitrile was added to deproteinize the biological sample. A 50 microl aliquot of the supernatant was injected onto a C(18) reversed-phase column. The mobile phase, 0.05 M acetate buffer (pH 3):acetonitrile:methanol (40:40:20, v/v/v), was run at a flow rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 298 nm. The retention times for IQO4 and the internal standard were approximately 5 and 7 min, respectively. The detection limits of IQO4 in rat plasma, urine and tissue homogenates (including blood) were 0.05, 0.1 and 0.1 microg/ml, respectively. The coefficients of variation of the assay were below 9.4% for rat plasma, urine and tissue homogenates. No interferences from endogenous substances were found., (Copyright 2002 Elsevier Science B.V.)

Quinapril is an antihypertensive drug commonly used in the treatment of hypertension and congestive heart failure. In this work, a capillary zone electrophoresis system is optimized for the analysis of quinapril and its active metabolite quinaprilat in urine, as well as for the determination of the drug and its combination with hydrochlorothiazide in pharmaceuticals. The separation takes place in a fused-silica capillary. The running electrolyte consists of a 60 mM borate buffer solution, pH 9.5. The analysis of urine samples requires a previous extraction step using C8 solid-phase cartridges. Under the optimum experimental conditions, the separation of the two analytes and the internal standard takes less than 5 min. The detection limits obtained (75 and 95 ng/mL for quinapril and quinaprilat, respectively) allow the application of the electrophoretic method to the determination of the drug and its metabolite in urine samples obtained from four patients treated with quinapril.

Ecteinascidin 743 (ET-743) is a potent anti-tumoral agent of a marine origin. It is currently being tested in phase II clinical trials using a 3-weekly 24-h i.v. infusion of 1500 microg/m(2) and 3-h infusions of 1650 microg/m(2). Knowledge of the metabolism of ET-743 is, however, still scarce. In the present study, a qualitative chromatographic discovery of metabolites of ET-743 in man is reported. ET-743 and its demethylated analog ET-729 were incubated at 37 degrees C in the presence of enzyme systems, pooled human microsomes, pooled human plasma and uridine 5'-diphosphoglucuronyltransferase, respectively, in appropriate media. Reaction products were investigated chromatographically using photodiode array and ion spray-mass spectrometric detection (LC-MS). The main reaction products in microsomal incubations of ET-743 resulted from a remarkable breakdown of the molecule. In plasma the drugs were deacetylated, and the transferase did actually yield a glucuronide of both ET-743 and ET-729. In contrast, screening of urine, plasma and bile, collected from patients treated with ET-743 at the highest dose levels, using a sensitive LC-MS assay, did not result in detection of ET-729 and metabolites which were generated in vitro. The urinary excretion of ET-743 in man was lower than 0.7% of the administered dose for a 24-h infusion.

Quinapril is an antihypertensive drug that belongs to the family of angiotensin-converting enzyme inhibitors. It is metabolized to quinaprilat, which is the compound that is really responsible for the therapeutic action. In this study, a rapid and simple liquid chromatographic method with photometric detection is described and applied to the determination of quinapril and quinaprilat in urine. The cleanup procedure for the urine samples consists of a solid-liquid extraction using C8 cartridges. Under these conditions, both compounds and the internal standard (enalapril maleate) are separated in less than 9 min. Recoveries for quinapril and quinaprilat are greater than 80%. The method is sensitive enough (detection limit of 60 ng/mL for quinapril and 50 ng/mL for quinaprilat) to be applied for the determination of quinapril and quinaprilat in urine samples obtained from four hypertensive patients after the intake of a therapeutic dose.

The present study was undertaken to investigate whether the consumption of green tea and black tea influences the excretion of mutagens and promutagens in rats treated orally with the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Rats were maintained on aqueous extracts (2.5%, w/v) of green tea, black tea or decaffeinated black tea as their sole drinking liquid. After 4 weeks, the animals received, by gastric intubation, a single dose of IQ (5 mg/kg), and urine was collected for 48 h. Direct and indirect mutagenicity, in the presence of an activation system derived from Aroclor 1254-treated rats, was determined in the urine samples using the Ames mutagenicity assay. Consumption of green tea and black tea, but not of decaffeinated black tea, markedly decreased the urinary excretion of mutagens and promutagens. In a further study, supplementation of decaffeinated black tea with caffeine suppressed the excretion of mutagens and promutagens in the urine of rats pretreated with IQ. It is concluded that both green tea and black tea modulate the bioactivation and metabolism of IQ, and that caffeine is largely responsible for this effect., (Copyright 1999 Elsevier Science B.V.)

In vitro and in vivo experiments to investigate possible stereospecific aspects of oracin reduction in relation to rat gender have been conducted. Incubation of oracin with rat microsomes, cytosol, and hepatocytes in the presence of various coenzymes and under aerobic or anaerobic conditions provided evidence for sex differences in the formation of 11-dihydrooracin (DHO) enantiomers. The greatest sex differences were seen in hepatocytes where females showed higher stereospecificity of the reductases than males. While female biotransformation enzymes preferentially generated approximately 82% of (+)-DHO, male enzymes gave only rise to 63% of (+)-DHO. Males displayed higher stereospecificity than females in the microsomal fraction. However, in the cytosolic fraction females exhibited higher stereospecificity than males. Similarly, in in vivo studies, the ratio of (+)- and (-)-DHO in faeces and urine gave no indication of the significant differences between the male and female rat. Enzyme stereospecificity has been defined as preferential formation of the (+)- or (-)-stereoisomer of 11-DHO by the respective enzyme. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as the chiral stationary phase with excellent resolution and stability.

Purpose: To compare the pharmacokinetic behaviour of doxacurium in patients undergoing normothermic or hypothermic cardiopulmonary bypass (CPB) for coronary artery bypass graft surgery., Methods: Twenty patients in two equal groups were studied. Anaesthesia was induced with sufentanil and midazolam after a standard premedication. Doxacurium was administered at 3 x ED95 (80 micrograms.kg-1), and anaesthesia was maintained with 0.5 microgram.kg-1 hr-1 sufentanil, 0.05 mg.kg-1 midazolam and isoflurane 0.5-1%. Systemic temperature for patients in the normothermic and hypothermic groups was maintained at 33-36 C and 26-30 C respectively. Timed blood and urine samples were collected and pharmacokinetic parameters were estimated using a non-compartmental approach., Results: For the normothermic and hypothermic groups, terminal elimination half-life (t1/2 beta) was 100.1 +/- 28 and 183.8 +/- 60 min (P < 0.05) respectively, elimination half-life during the CPB phase (T1/2 CPB) 114.5 +/- 10 and 183.8 +/- 60 min (P < 0.05), mean residence time 108.8 +/- 25 and 164.8 +/- 34 min (P < 0.05) and apparent volume of distribution at steady state 0.20 +/- 0.03 and 0.26 +/- 0.04 L.kg-1 (P < 0.05). Compared with the hypothermic group, the normothermic group had a higher rate of renal clearance (1.40 +/- 0.4 vs 0.93 +/- 0.3 ml.min-1.kg-1; P < 0.05) and a higher value for renal clearance as a percentage of the total clearance (76.2 +/- 10 vs 58.3 +/- 20%)., Conclusion: The elimination rate of doxacurium during normothermic CPB is faster than that in hypothermic CPB.

The tetrahydroisoquinoline alkaloids salsolinol and norsalsolinol were found in human urine samples in concentrations ranging from 0.1 to 29.5 ng/ml. Great interindividual variation was found in urine levels of these alkaloids in a collection of chronic alcoholics and in a group of nonalcoholics. Thus, levels of the individual alkaloids are insufficient markers for distinguishing between alcoholics and nonalcoholics. However, by using the concentration ratio of norsalsolinol and salsolinol, the so-called dopamine-aldehyde adduct ratio (DAAR), significant differences between alcoholics (median 1.3) and nonalcoholics (median 0.6) were detected. This concentration ratio could serve as a marker for the processor state of the dopaminergic system.

A high-performance liquid chromatographic method was developed for the determination of a new proton pump inhibitor, YH1885 (I), in human plasma and urine, and rat blood and tissue homogenate using fenticonazole as an internal standard. The sample preparation was simple: a 2.5 volume of acetonitrile was added to the biological sample to deproteinize it. A 50-microliter aliquot of the supernatant was injected onto a C8 reversed-phase column. The mobile phase employed was methanol-0.005 M tetrabutylammonium dihydrogenphosphate (77:23, v/v), and it was run at a flow-rate of 1.0 ml/min. The column effluent was monitored using an ultraviolet detector at 270 nm. The retention times for I and the internal standard were 9.0 and 10.3 min, respectively. The detection limits for I in human plasma and urine, and in rat tissue homogenate (including blood) were 50, 100 and 100 ng/ml, respectively. The coefficients of variation of the assay (within- day and between-day) were generally low (below 8.84%) for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.

Cisatracurium, one of 10 isomers of atracurium, undergoes pH and temperature-dependent Hofmann elimination in plasma and tissues. The clearance of cisatracurium due to Hofmann elimination and organ elimination was estimated by applying a nontraditional two-compartment pharmacokinetic model with elimination occurring from both compartments to plasma cisatracurium concentration-time data from 31 healthy adult surgical patients with normal renal and hepatic function. The elimination rate constant from the central compartment, intercompartmental rate constants, and the volume of the central compartment were obtained from the model fit. The elimination rate constant from the peripheral compartment could not be independently estimated in vivo and was therefore fixed to the rate of degradation of cisatracurium in human plasma (pH 7.4 and 37 degrees C) and held constant in the model. Total body clearance, Hofmann clearance, organ clearance, and the volume of distribution at steady-state were derived from the model parameter estimates. Renal clearance was calculated from cisatracurium urinary excretion data from 12 of the 31 patients. Clearance values (mean +/- SD) were 5.20 +/- 0.86, 4.00 +/- 1.04, 1.20 +/- 0.71, and 0.85 +/- 0.32 mL.min-1.kg-1 for total body clearance, Hofmann clearance, organ clearance, and renal clearance, respectively. Hofmann clearance accounted for 77% of total body clearance. Organ clearance was 23% of total body clearance. Renal clearance, a component of organ clearance, was 16% of total body clearance. The organ-independent nature of the elimination of cisatracurium was characterized by a relationship between steady-state volume of distribution and total body clearance. The half-life is an independent variable and is not dependent on the total body clearance nor the steady-state volume of distribution. Hofmann elimination is the predominant pathway for cisatracurium elimination in humans.

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-beta-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-beta-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-beta-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-beta-carboline, 6-methyl-1,2,3,4-tetrahydro-beta-carboline, and 6-methoxy-1,2,3,4-tetrahydro-beta-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.

Oracine (I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine (II) and a phenolic product (III). Two further metabolites (IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.

We could quantify the tetrahydroisoquinoline derivative salsolinol in urine of patients with Parkinson's disease and normal control subjects by means of high performance liquid chromatography and electrochemical detection. Urine levels of salsolinol were positively related to the homovanillic acid/3-O-methyl-dopa ratio in the cerebrospinal fluid that reflects dopamine metabolism. In the patient group with visual hallucinations, mean salsolinol level was significantly increased to almost the 3-fold of those found in patients without hallucinations. Since the daily L-dopa doses of both patient groups were nearly identical this result is not due to different L-dopa medications. Additionally, either high values of the main serotonin metabolite, 5-hydroxyindole acetic acid (HIAA) or the L-dopa/3-O-methyl-dopa ratio were found in cerebrospinal fluid of patients with hallucinations. The enhanced salsolinol levels in patients with visual hallucinations seem to be due to an overloaded dopaminergic pathway with an imbalance between dopaminergic and serotonergic systems. Thus, salsolinol appears as a predictor for hallucinosis in Parkinson's disease.

Tetrahydroisoquinolines (TIQs) might be formed endogenously and can act centrally to promote a mechanism governing alcohol drinking behaviour. The possibility that biosynthesis occurs through a stereospecific enzymatic reaction is considered. Several TIQs were transformed into diastereomers by a two-step derivation with N-methyl-N-trimethylsilyltrifluoracetamide and R-(-)-2-phenylbutyrylic acid and were analyzed by gas chromatography-mass spectrometry (GC-MS). High resolution of the TIQ enantiomers was achieved. This method was applied to the quantification of the enantiomers of salsolinol (SAL) in urine and plasma of healthy humans. Deuterated SAL was used as the internal standard. SAL was extracted from biological material using phenyl-boronic phase cartridges and transformed into diastereomers. The sensitivity and specificity of the assay permit the determination of the enantiomeric composition of SAL in plasma and urine. The limit of quantification was found to be 100 pg/ml for each enantiomer. The described method has the advantage that optimal resolution of the SAL enantiomers without peak overlapping between analyte and other compounds can be achieved. Contrary to other findings, our GC-MS studies have demonstrated that endogenously formed SAL is racemic in plasma as well as in urine of healthy subjects.

The tubular uptake and esterolysis of quinapril and quinaprilat were studied in male Sprague-Dawley rats using an in vivo micropuncture technique. [3H]Quinapril or [3H]quinaprilat was injected with [14C]inulin into either proximal or distal segments of the renal tubules, and urine was collected over 30 min. Urine and perfusate were assayed for [14C]-inulin using dual label spectrometry. [3H]Quinapril and [3H]quinaprilat concentrations were determined in urine and perfusate using a reversed-phase HPLC procedure with radiochemical detection, coupled to liquid scintillation spectrometry. These studies demonstrated that quinapril could access the esterase enzyme from tubular fluid and be metabolized to quinaprilat in both proximal and to a lesser extent distal segments of the kidney tubule. Quinapril, but not quinaprilat, was extensively reabsorbed. Its reabsorption along the proximal tubule and/or the loop of Henle could account for as much as 45-50% of the available dose of quinapril. Further, the urinary recovery of quinapril and quinaprilat (after dosing quinapril into proximal segments) was urine flow rate dependent.

This report describes the change of salsolinol (SAL) levels in the urine of healthy subjects after ethanol ingestion. A new rapid method for SAL extraction from urine sample and a GC-MS assay were used to study the urinary SAL excretion in a group of adult males (n = 14) and females (n = 13) with and without ingestion of ethanol. The results of this study show that the urinary SAL output of all subjects is significantly influenced by the intake of ethanol. A decrease in the SAL level was found in the urine of most of the volunteers (n = 17), whereas only eight subjects showed an increase in the SAL level after administration of ethanol.

An isolated perfused rat kidney model was used to probe the renal disposition of quinapril and quinaprilat after separate administration of each drug species. Control studies were performed with drug-free perfusate (n = 8) and perfusate containing quinapril (n = 9) or quinaprilat (n = 7) at initial drug concentrations of 1000 ng/ml (including corresponding tracer levels of tritiated drug). Physiologic parameters were within the normal range of values for this technique and were stable for the duration of each experiment. Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase HPLC procedure with radiochemical detection, coupled to liquid scintillation spectrometry. Perfusate protein binding was determined using an ultrafiltration method at 37 degrees C. The total renal clearance of quinapril (CLr) was calculated as Dose/AUC(0-infinity), and is represented by the sum of its urinary and metabolic clearances. The urinary clearances (CLe) of quinapril and quinaprilat were calculated as urinary excretion rate divided by midpoint perfusate concentration for each respective species. Of the total renal clearance for quinapril (CLr = 4.49 ml/min), less than 0.1% was cleared as unchanged drug (CLe = 0.004 ml/min); over 99% of the drug was cleared as quinaprilat formed in the kidney. The clearance ratio of quinapril [CR = CLr/(fu.GFR)] was 41.0, a value representing extensive tubular secretion into the renal cells. Following quinaprilat administration, the clearance ratio of metabolite [CR = CLe/(fu.GFR)] was 3.85, indicating a net secretion process for renal elimination.

A new method for the analysis of tetrahydroisoquinolines (TIQ) from biological materials is described. The method combines extraction and acylation of the TIQs from urine in a one-step procedure followed by analysis by gas chromatography/mass spectrometry. By this extractive derivatization method, a decrease of the extraction time, to speed up sample preparation in order to minimize oxidative side reaction, has been achieved. The detection limits for the analysis of salsolinol and salsolin in urine were 10 fmol/ml sample volume. Tetrahydropapaveroline could only be detected in spiked urine samples with a detection limit of 1 pmol/ml urine. The method is not only restricted to urine samples, but can also be applied to a variety of tissue types, including blood components.

The pharmacokinetics of the 1R cis-1'R cis-isomer of atracurium (51W89) and its metabolite, laudanosine, were studied in 11 healthy patients with normal renal function and in 12 patients with chronic renal failure undergoing regular dialysis. A bolus dose of 51W89 (0.1 mg/kg) was given, and the plasma concentration was measured at regular intervals for 480 min. The elimination half-life of 51W89 was significantly longer in renal failure patients than in healthy controls (38.9 min vs 30.6 min; P < 0.05). The plasma laudanosine levels were lower than those reported after an equipotent dose of atracurium besylate. 51W89 may have a prolonged effect in renal failure patients.

CGP 28,014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28,014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28,014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28,014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28,014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.

A gas chromatographic-mass spectrometric method has been developed for the identification of 1,2,3,4-tetrahydroisoquinoline and six metabolites extracted from urine in the picogram range. The derivatization procedure for the substances, formed by reaction of formaldehyde with biogenic amines, employs propionic anhydride and can take place in aqueous medium. In this way artificial formation of these compounds via condensation of biogenic amines with aldehydes or alpha-keto acids during the work-up procedure is eliminated. The procedure results in hydrophobic compounds, which are quantitatively extractable by liquid-liquid extraction with organic solvents. Further clean-up was performed by solid-phase extraction on C18 sample preparation columns.

In 3,4-methylenedioxyamphetamine (MDA)-treated rat brain and urine, 3-methyl-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (3Me6,7MDTIQ) was detected as a new metabolite. The content of 3Me6,7MDTIQ in (R)-MDA-treated rat was significantly higher than that in (S)-MDA-treated brain and urine. This result suggests that this metabolism of MDA contains stereoselective pathways. The pharmacological effects of 3Me6,7MDTIQ on behavior differed from those of MDA. The ambulation score of 3Me6,7MDTIQ was significantly decreased compared to the control, and the rearing score of 3Me6,7MDTIQ was moderately decreased compared to the control in an open-field test.

In a case involving a fatal shooting, toxicology tests on blood and urine demonstrated the presence of cocaine metabolites and a large amount of an unidentified compound. This compound was subsequently identified by mass spectral, gas chromatographic, and thin layer chromatographic tests as laudanosine, a metabolite of the skeletal muscle relaxant atracurium which was administered during emergency surgery. Identification was confirmed by comparison with commercially available standards. Because of the difficulty associated with isolating and chromatographing highly water-soluble compounds, recognition of this artifact is a useful tool in identifying these cases.

The effect of temperature, pH, light, drug concentration and aeration on the stability of aqueous solutions of GR63178A, a novel anticancer compound, has been investigated. The effect of light upon the stability of GR63178A in a clinical infusion system and in biological fluids, has also been investigated. A high performance liquid chromatographic assay was used to separate the parent compound from a number of degradation products. Data presented here demonstrate that GR63178A is stable in aqueous solutions at normal ambient temperatures in neutral or alkaline pH ranges. The compound is unstable when exposed to light (t1/2 3.2-233 min) and shows an inverse relationship between concentration and rate of degradation. The drug is also unstable under acidic conditions, but shows a more limited spectrum of degradation products than those arising from photolysis.

The daily urinary excretion of salsolinol, 1,2-dehydrosalsolinol, and norsalsolinol, as free, glucuronide, and sulfate, has been measured in parkinsonian patients and age-matched controls in an attempt to examine whether the determination of dopamine-derived alkaloids in urine may be used as a marker of the decrease in brain dopamine levels associated with the disease. In contrast with a preliminary study where the daily urinary excretion of total salsolinol was significantly higher in young controls than in parkinsonians, in the present study no difference was found between parkinsonian patients and controls concerning salsolinol and norsalsolinol excretion. However, the urinary excretion of total 1,2-dehydrosalsolinol was significantly higher in the control group, owing to a statistically significant increase in its excretion as sulfate in this group. Further studies appear to be necessary to establish whether 1,2-dehydrosalsolinol, salsolinol, and/or any other dopamine-derived alkaloid may serve for the detection of subjects with dysfunctions of the dopaminergic system.

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane-dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography-electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a single-to-noise ratio of greater than 3 and greater than 5, respectively. The proposed method is applicable to pharmacokinetic studies.

A highly specific and sensitive method for the simultaneous determination of quinapril and its active metabolite, quinaprilat, in human plasma and urine by gas chromatography-negative-ion chemical ionization mass spectrometry is described. Quinapril and quinaprilat were extracted from human plasma and urine by using a Bond-Elut C18 cartridge. The plasma or urine extract was treated with pentafluorobenzyl bromide followed by trifluoroacetic anhydride to convert quinapril and quinaprilat into their pentafluorobenzyl-trifluoroacetyl derivatives, which were analysed by a selected-ion monitoring method using deuterium-labelled internal standards. The limits of quantitation for both quinapril and quinaprilat were 0.05 ng/ml in plasma and 0.5 ng/ml in urine. The proposed method is applicable to pharmacokinetic and clinical pharmacological studies with satisfactory reliability.

Salsolinol was measured in urine of alcoholics after one through four and after 21 days of withdrawal, in nonalcoholic controls with and without ingestion of alcohol and in young children. HPLC equipped with fluorescence detection was employed. There were no significant differences in salsolinol excretion rate between alcoholics and controls one day after alcohol deprivation. A significant difference in salsolinol output in urine of alcoholics between the first and the twenty-first day of withdrawal was found, but salsolinol output expressed as creatinine-related output did not change. There was no trend of urinary salsolinol excretion rate by alcoholics during the first 4 days of abstinence. The results indicate that the urinary salsolinol excretion rate and the creatinine-related salsolinol output are insufficient to distinguish between alcoholics and nonalcoholics.

We have measured the steady state urinary clearances of atracurium, given by constant infusion, and laudanosine in eight patients undergoing artificial ventilation; all had normal renal function (mean creatinine clearance 81 ml min-1). Mean (SD) urinary clearance of atracurium was 0.55 (0.5) ml kg-1 min-1; that of laudanosine was 0.33 (0.2) ml kg-1 min-1. Simultaneous plasma clearances were 7.1 (1.4) ml kg-1 min-1 and 3.8 (1.5) ml kg-1 min-1, respectively. Notional haemofiltration clearances of the two substances were measured also in seven critically ill patients with renal and respiratory failure undergoing continuous venovenous haemofiltration. Mean (SD) clearances of atracurium and laudanosine in the haemofiltrate fluid were 0.11 (0.06) ml kg-1 min-1 and 0.09 (0.02) ml kg-1 min-1, respectively whilst plasma clearances were atracurium 6.7 (1.8) ml kg-1 min-1 and laudanosine 4.5 (1.8) ml kg-1 min-1. There were no significant differences between the plasma clearances of the drugs in the two groups, despite the difference in severity of sickness. Urinary clearance rates of atracurium and laudanosine were approximately 8 and 9% of that in the plasma, but the haemofiltration clearance of both substances was only 2%.

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic-mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [2H4]salsolinol from dopamine and [2H4]acetaldehyde via a Pictet-Spengler condensation is described; [2H4]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.

The analysis of the urine from 6 chronic alcoholics showed that (R)- and (S)-salsolinol were detectable in 4 subjects, the R enantiomer was only found in one and that both enantiomers were under the limit of detection in another one. 1,2-Dehydrosalsolinol was present in the urine of all of them. There was no correlation between the presence of alcohol in blood upon admission to the hospital and that of either salsolinol enantiomer in urine In a previous study (Strolin Benedetti et al., 1989 b), both salsolinol enantiomers were found in the urine of 3 out of 6 healthy subjects, possibly in relation with regular intake of alcoholic beverages. The content in (R)- and (S)-salsolinol was determined in the same 3 subjects after deprivation of alcohol for 24 h. Under these conditions, only (R)-salsolinol was detected in urine and this also after ingestion of 50 g of alcohol (500 ml Chianti). The possible involvement of the non-physiological enantiomer of salsolinol in alcohol addiction deserves further study.