Variability amongst urine toxicology amphetamine readings with concurrent administration of fenofibrate.

OBJECTIVE: The aim of this study was to highlight that concurrent administration of the common lipid-lowering agent fenofibrate may lead to false-positive amphetamine results in often-used immunoassay-based urine drug screens. It also aimed to show that there are significant moral and clinical challenges associated with the interpretation of such results amongst psychiatric inpatients. CONCLUSIONS: It is evident that different pathology laboratories may utilise different commercial urine drug-screen immunoassays in their toxicology analysis, with variability in the test specificities. Despite the relatively high prevalence of substance misuse in the population of psychiatric inpatients, there exists a need for increased vigilance towards the possibility of false-positive amphetamine results owing to likely cross-reactivity of fenofibrate with the test reagents. In cases where there is uncertainty when correlating clinically, or where false positives are suspected, gold-standard urine-sample analysis by mass spectrometry should be considered, particularly when the consequences for patients may include restrictive measures.

Development of a LC-MS/MS method for the estimation of clinofibrate in human urine.

A highly sensitive and rapid liquid chromatography-tandem mass spectrometry method was developed for the determination of clinofibrate in human urine. The analyte and IS were extracted through a simple protein precipitation by the mixture of acetonitrile and 1 mol/L hydrochloric acid (95:5, v/v) and separated on an Inspire C18 (150 mm x 4.6 mm I.D., 5 µm particle size) column using isocratic elution with methanol and water containing 0.1% formic acid and 10 mM ammonium acetate (90:10, v/v). Mass spectrometric detection was performed in electrospray positive ionization MRM mode. The mass transition was m/z 486.3-->175.0 for clinofibrate and m/z 361.1-->233.1 for IS, respectively. The flow rate was 0.6 mL/min and the column oven temperature was set at 35 °C. The total run time was 6.5 min. Good linear relationships were obtained for all analytes over the concentrations ranging of 0.1002-10.02 µg/mL (r2 = 0.9991) and the limit of quantification was 0.1002 µg/mL. The extraction recovery was larger than 87.4% and intra- and inter-batch precision and accuracy with RSD were all less than 6.5%. The total amount of unchanged clinofibrate excreted in urine was less than 0.34%. This method was successfully applied to the pharmacokinetic study of clinofibrate in human urine.

New metabolites of fenofibrate in Sprague-Dawley rats by UPLC-ESI-QTOF-MS-based metabolomics coupled with LC-MS/MS.

Fenofibrate has been widely used for the treatment of dyslipidaemia with a long history. Species differences of its metabolism were reported, but its metabolites in rodent have not been fully investigated. Urine and plasma samples were collected before and after oral dosages of fenofibrate in Sprague-Dawley rats. Urine samples were subjected to ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) analysis, and projection to latent structures discriminant analysis was used for the identification of metabolites. New metabolites in urine and plasma were also studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolism pathway was studied in rat hepatocytes. Synthesized and purchased authentic compounds were used for metabolite identification by LC-MS/MS. Five ever-reported metabolites were identified and another four new ones were found. Among these new metabolites, fenofibric acid taurine and reduced fenofibric acid taurine indicate new phase II conjugation pathway of fenofibrate.

Investigation of electrochemical behavior of lipid lowering agent atorvastatin calcium in aqueous media and its determination from pharmaceutical dosage forms and biological fluids using boron-doped diamond and glassy carbon electrodes.

The electrochemical behavior of atorvastatin calcium at glassy carbon and boron-doped diamond electrodes has been studied using voltammetric techniques. The possible mechanism of oxidation was discussed with model compounds. The dependence of the peak current and potentials on pH, concentration, scan rate and nature of the buffer were investigated for both electrodes. The oxidation of atorvastatin was irreversible and exhibited a diffusion-controlled fashion on the diamond electrode. A linear response was obtained within the range of 9.65 x 10(-7) - 3.86 x 10(-5) M in 0.1 M H(2)SO(4) solution for both electrodes. The detection limits of a standard solution are estimated to be 2.11 x 10(-7) M with differential pulse voltammetry (DPV) and 2.05 x 10(-7)M with square wave voltammetry (SWV) for glassy carbon electrode, and 2.27 x 10(-7) M with DPV and 1.31 x 10(-7)M with SWV for diamond electrodes in 0.1 M H(2)SO(4) solution. The repeatability of the methods was found good for both electrodes. The methods were fully validated and successfully applied to the high-throughput determination of the drug in tablets, human serum and human urine with good recoveries.

[Conjugation reactions of ciprofibrate with human and laboratory animals]. / Konjugationsreaktionen von Ciprofibrat bei Menschen und Versuchstieren.

An open problem of the lipid lowering agent ciprofibrate (rac-2-[4-(2,2-dichlorocyclopropyl)-phenoxy]-2-methylpropanoic acid, CAS 52214-84-3) is its metabolism concerning the conjugation with amino acids and glucuronic acid. It could be solved by syntheses of the needed reference compounds--unknown up to now--and administration of ciprofibrate to volunteers and rats. Unexpectedly the conjugation compounds with amino acids are stable in vitro and in metabolism. There was no evidence for any conjugation reaction with amino acids by investigating samples of urine and faeces. On the contrary the urine of humans contains 90-97% of beta-O-acylglucuronide, whereas rat urine shows only 10% of the calculated amount.

Gemfibrozil and its oxidative metabolites: quantification of aglycones, acyl glucuronides, and covalent adducts in samples from preclinical and clinical kinetic studies.

A gradient reversed-phase HPLC analysis for the direct measurement of gemfibrozil (GEM) and four oxidative metabolites in plasma and urine of humans and in tissue homogenates of rats was developed. The corresponding acyl glucuronides and the covalently bound protein adducts (in protein precipitates) were determined after liberation from the respective conjugates via alkaline hydrolysis. The limits of detection for the covalent adducts in human plasma are: 10 ng ml(-1) (GEM), 20 ng ml(-1) (M1), 0.5 ng ml(-1) (M2, M4), and 5 ng ml(-1) (M3). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy. It has been applied to the analysis of preclinical and clinical studies. Pharmacokinetic profiles of gemfibrozil, its metabolites, and covalent adducts in human plasma and rat tissue homogenates are given.

Isolation and identification of novel metabolites of gemfibrozil in rat urine.

Gemfibrozil (GEM) is a clofibrate analog used to treat moderate to severe hypertriglyceridemias. In lab animals, GEM causes peroxisome proliferation, an effect that has been associated with hepatocarcinogenesis in rats. In humans, hepatobiliary disorders, but not carcinogenesis, have been associated with GEM therapy. In the present study [14C]GEM was administered orally to rats at a dose of 2000 mg/kg. At various time points, radioactivity in urine was analyzed by liquid scintillation spectrometry, high-pressure liquid chromatography, liquid chromatography/mass spectrometryn, gas chromatography/mass spectroscopy, and nuclear magnetic resonance. Nine metabolites of GEM were identified, some that have not been reported previously. Although the majority of metabolites were glucuronidated, some nonglucuronidated metabolites were identified in urine, including a diol metabolite (both ring methyls hydroxylated), and the product of its further metabolism, the acid-alcohol derivative (ortho ring methyl hydroxylated, meta ring methyl completely oxidized to the acid). Hydroxylation of the aromatic ring also was a common pathway for GEM metabolism, leading to the production of two phenolic metabolites, only one of which was detected in the urine in the nonconjugated or free form. Also of interest was the finding that both acyl and ether glucuronides were produced, including both glucuronide forms of the same metabolite (e. g., 1-O-GlcUA, 5'-COOH-GEM, and 5'-COO-GlcUA-GEM); the positions and functionality of the glucuronide conjugates were identified using base hydrolysis or glucuronidase treatment, in combination with liquid chromatography/MSn and nuclear magnetic resonance.

[High-performance liquid chromatographic method for the determination of fenofibric acid and reduced fenofibric acid in human blood, plasma and urine].

In this study, a very reliable HPLC method was developed for the determination of fenofibric acid and reduced fenofibric acid in the biological samples described as follows. After addition of the internal standard solution and 0.5 M HCl to the biological sample, fenofibric acid, reduced fenofibric acid and the internal standard were extracted with a mixed solvent of n-hexane and ethyl acetate (90:10) from the mixture. The acids were back-extracted from the organic phase with 0.1 M Na2HPO4 and then re-extracted from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5) after addition of 0.5 M HCl. The organic phase was evaporated to dryness under the vacuum. The residue was dissolved in MeOH and diluted with distilled water. An aliquot of the resulting solution was injected on the HPLC. High reproducibility was observed in this HPLC method (C.V.% less than 4%). Moreover it was confirmed that the conjugates in the urine could be hydrolyzed by incubation at 37 degrees C for 18 h after addition of 400 IU of beta-glucuronidase.

Effect of clofibrate on the chiral inversion of ibuprofen in healthy volunteers.

OBJECTIVES: To determine the influence of the hypolipidemic drug clofibrate on the stereoselective metabolism of ibuprofen in humans. METHODS: Healthy male subjects (n = 12) ingested a dose of 400 mg pseudoracemic ibuprofen (200 mg R-ibuprofen, 160 mg S-ibuprofen, and 40 mg 13C-S-ibuprofen) on two occasions after either pretreatment with clofibrate (2 gm/day over 1 week) or no pretreatment in a randomized order. RESULTS: When subjects were pretreated with clofibrate, clearances of R-ibuprofen and 13C-S-ibuprofen increased significantly from 55.0 and 66.4 ml/min to 186.2 and 106.7 ml/min (p < 0.01), respectively. This increase was similarly reflected in the clearance by inversion of R-ibuprofen (control, 36.0 ml/min; treated, 118.8 ml/min; p < 0.01), as well as in the clearance by noninversion (control, 19.0 ml/min; treated, 67.4 ml/min; p < 0.01). Unbound clearance values significantly increased for R-ibuprofen (control, 19.5 L/min; treated, 38.7 L/min) but not for 13C-S-ibuprofen (11.8 versus 10.6 L/min, respectively). The fractional inversion of ibuprofen calculated from the urinary metabolite data was increased after clofibrate pretreatment (clofibrate group, 66.4%; control, 53.5%; p < 0.01). However, this was not evident when fractional inversion was calculated from the plasma concentration-time data for the unmetabolized drug. CONCLUSIONS: Clofibrate altered the stereoselective disposition of ibuprofen in healthy volunteers by increased formation of R-ibuprofenoyl-coenzyme A rather than by an effect on oxidative metabolism of ibuprofen. This interaction has potential therapeutic implications.

[The pharmacokinetics of hypolipemic agents. 10. The dehalogenation of the hypolipemic agent ciprofibrate]. / Zur Pharmakokinetik von Lipidsenkern, 10. Mitt.: Zweiter Beitrag zur Frage der Dehalogenierung des Lipidsenkers Ciprofibrat.

rac-2 described as a metabolite of rac-1 was synthesized in four steps starting with rac-3. Partial dehalogenation occurs with LiAlH4. A new structure assignment of the resulting stereoisomers resulted from NMR spectroscopy. After oral administration of rac-1 in multiple dose studies to volunteers, rac-2 could not be detected within the limitations of sensitivity of HPLC (UV-detector) in plasma or in urine.

In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers.

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (+/-)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 microM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid beta-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (-)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 x 10(-4) and 0.177 x 10(-4) mol/mol protein. Multiple dosing increased irreversible binding 3- to 4-fold.

Hydroxypropylcyclodextrins in parenteral use. II: Effects on transport and disposition of lipids in rabbit and humans.

Hydroxypropyl ethers of cyclodextrins, after parenteral administration, come into contact with lipids in tissues and in circulation and form water-soluble inclusion complexes with these lipids. A single intravenous administration of hydroxypropyl-beta-cyclodextrin to a hereditary hyperlipidemic Watanabe rabbit slightly and temporarily decreased the level of total cholesterol in serum. Single injections of hydroxypropyl-alpha-cyclodextrin and of the corresponding gamma-homologue, both of which are less potent solubilizers of cholesterol, had lesser effects. Repeated administration of hydroxypropyl-beta-cyclodextrin to rabbits led to a gradual increase in total cholesterol in circulation and eventually to a slight relief of atherosclerotic lesions in the thoracic aorta. The only untoward effects of repeated treatments (total doses of up to 40 g/kg) were vacuoles in cells of proximal convoluted tubules in the kidneys. Repeated administration also strongly increased cholesterol in urine, probably because of excretion of the soluble cholesterol-hydroxypropyl-beta-cyclodextrin complex. Proteins in urine increased significantly, whereas triglycerides increased only moderately after repeated administrations. Intravenous infusion of hydroxypropyl-beta-cyclodextrin into a patient with hypervitaminosis A led to a release of liver-stored retinoids into serum in quantities much higher than those that could be directly solubilized by hydroxypropyl-beta-cyclodextrin. Levels of total cholesterol in the circulation of this patient decreased during the infusion. Thus, hydroxypropylcyclodextrins may serve as artificial lipid carriers in the circulation, and because the exchanges that involve inclusion complexation occur very quickly, the presence of hydroxypropylcyclodextrins in organisms may catalytically augment the establishment of equilibria in lipid distribution.

Urinary glucuronide excretion of fenofibric and clofibric acid glucuronides in man. Is it polymorphic?

The possible polymorphism of the glucuronidation reaction in man has been investigated using two hypolipidaemic compounds, fenofibrate and clofibrate, as the test probes. The formation of fenofibryl and clofibryl glucuronides was identified by their susceptibility to hydrolyses by beta-glucuronidase. The urinary excretion of the glucuronides was measured in 72 healthy volunteers after a single dose of fenofibrate, and in 104 subjects given a single dose of clofibric acid. Fenofibrate was excreted at a lower rate than clofibrate, since 13.94% and 26.55% of the doses of fenofibrate and clofibrate respectively, were recovered in urine in 8 h. Correlation analysis indicated that sex and body mass index significantly influenced the formation of fenofibryl glucuronide, whereas age and oral contraceptives affected the excretion of clofibryl acid glucuronide. The 8-hour urinary excretion patterns of clofibryl glucuronide and of clofibric acid presented a Gaussian distribution, whereas those of fenofibryl glucuronide and fenofibric acid showed 2 populations. When the metabolic ratio free fenofibric acid/glucuronide was considered, 84.7% of subjects presented the ratio 0.147, and 15.3% had the 3-fold higher ratio of 0.421. The study has shown, in the human population studied, that the glucuronidation of fenofibric acid but not that of clofibric acid may present a polymorphism.

The influence of renal insufficiency and haemodialysis on the kinetics of ciprofibrate.

1. The kinetics of the hypolipidaemic drug, ciprofibrate, were studied after a single oral dose (100 mg) in subjects with normal renal function (n = 6), patients with mild (n = 6) and severe (n = 6) renal insufficiency as well as in haemodialysed patients (n = 5). 2. Under fasting conditions, ciprofibrate, was absorbed rapidly in subjects with normal renal function, and its apparent elimination half-life was approximately 81 h. Both renal clearance (0.15 ml min-1) and cumulative renal excretion (less than 7% of the administered dose) were low. 3. Mild renal insufficiency did not alter the pharmacokinetics of ciprofibrate, but severe renal impairment significantly reduced both its renal clearance and cumulative urinary excretion and increased the apparent elimination half-life. 4. A 5 h haemodialysis session did not lower the plasma concentrations of ciprofibrate. 5. It is concluded that, from a pharmacokinetic point of view, a reduction in the dosage of ciprofibrate should be considered in patients with a glomerular filtration rate below 30 ml min-1/1.73 m2.

Disposition of the hypolipidaemic agent, 2,3-dihydrophthalazine-1,4-dione, in Sprague Dawley rats.

The disposition of [14C]2,3-dihydrophthalazine-1,4-dione, a potent hypolipidaemic agent, has been determined after both intravenous and oral administration. Both the routes of administration afforded multi-exponential disposition with an estimated t1/2 of approximately 75 h. After oral administration, the drug was observed to be absorbed rapidly from the intestine and distributed quickly to all tissues of the body. A large quantity of the 14C-radioactivity was found in the skin and carcass. Approximately 35% of the administered radioactivity was excreted in urine after oral administration and 11% in the faeces. Approximately 66% of the radioactivity excreted in urine was the parent drug. There was evidence of an additional metabolite which accounted for 28% of the urinary radioactive excretion. The parent drug has little serum protein binding, is highly water soluble, and is probably taken up by cells by passive diffusion.

Metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-oxazole-4-acetate, a new hypolipidemic agent, in the rat, rabbit, and dog. Glucuronidation of carboxyl group and cleavage of furan ring.

Metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-oxazole-4-acetate (TA-1801), a new hypolipidemic agent, was studied in the rat, rabbit, and dog. Animals were given a single oral dose of 50 mg/kg TA-1801 labeled with 14C. The first metabolic reaction for TA-1801 was hydrolysis of the ester linkage. The resulting metabolite M1 was found to undergo further biotransformations, i.e. glucuronidation at the carboxyl group and ring cleavage of the furan group. These metabolic pathways were observed in all the species examined, although species differences were seen in the amount of metabolites.

Metabolism of the hypolipidemic agent tiadenol in man and in the rat.

The metabolism of the hypolipidemic agent 1,10-bis(hydroxyethylthio)decane (tiadenol, Eulip) has been studied in vivo in man and in the rat and in vitro in the rat. Following oral administration, in both species tiadenol was completely absorbed, extensively metabolized by the liver and more than 95% of the dose was eliminated in this form via kidneys within 48 h. Insignificant was the excretion of the unchanged drug in urine (approximately 1%) as well as that of its metabolites in the feces. 8 metabolites were isolated from human or rat urine and their structures were elucidated by means of electron impact, field desorption and positive and negative fast atom bombardment mass spectrometry. Both in man and in the rat the main metabolic pathway was the oxidation of the thioether sulfur, followed by oxidation or conjugation of the primary alcohol group(s). The urinary excretion of S-oxidized metabolites and sulfoxidized carboxylic metabolites accounted for 75% of the dose and that of S-oxidized conjugated metabolites for 20%. Rat in vitro studies showed that hepatic microsomal cytochrome P-450-dependent monooxygenase catalyzes the S-oxidative pathway, which governs the in vivo elimination of the drug in both species. Thus cytochrome P-450 is the key enzyme in the hepatic detoxification of tiadenol.

Pharmacokinetics and distribution of a fluoresceinated glycosaminoglycan, sulodexide, in rats. Part I: Pharmacokinetics in rats.

Fluorescein-labelled sulodexide pharmacokinetics and urinary excretion were studied in the rat after intravenous administration. The compound showed a pharmacokinetic pattern similar to that of heparin and the plasmatic levels were linearly correlated with anti-factor Xa activity and logarithmically with lipoprotein lipase activation. The compound is well eliminated by urinary route; the urinary recovery was 50% in 24 h and 67% 48 h after administration. The results obtained indicate that fluorescein labelling of glycosaminoglycans may be a valid tool for pharmacokinetics and distribution studies in the laboratory animals.

[Metabolism of beclobrate in rat and man]. / Untersuchungen zur Metabolisierung von Beclobrat bei Ratte und Mensch.

The major pathway of biotransformation of beclobrate [(2-[4-[(4-chlorophenyl)methyl]phenoxy]-2-methylbutyric acid ethyl ester] is the ester cleavage to beclobrinic acid (M1), which is eliminated as glucuronide. Subsequent metabolic attack is occurring via oxidation of the methylene bridge to the carbinol (M2) as well as to the benzophenone (M3). The p-chloro substituted phenyl ring is oxidated via a postulated arene oxide to the 2'- and 3'-phenol metabolites (M5 and M6) and to the trans-2',3'-dihydrodiol (M4). Except M4, all metabolites are eliminated exclusively as glucuronides.