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Biochemical testing in urine is a powerful new tool in the investigation of non-adherence to cardiovascular medications Drug testing using liquid chromatography tandem mass spectrometry (LC-MS-MS) is the mainstay of the laboratory test but may be subject to pre-analytical factors that could impact on test results. The stability of cardiovascular medications in urine is one such factor that has not been fully explored in non-adherence testing and has the potential to result in patients appearing falsely non-adherent to their therapy. The stability of 29 cardiovascular medications in patients' urine samples were assessed at room temperature (RT) and at -80°C using a LC-MS-MS screening method. All drugs and drug metabolites were found to be stable under the storage conditions studied. The findings imply that the medication stability in urine samples does not have any impact on non-adherence results and thus allowing samples to be taken and transported without the need for specialist sample handling procedures. The stability of cardiovascular drugs in urine samples will allow adherence testing to be utilized more widely into routine clinics and research., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Meldonium is a metabolic drug whose inclusion in the 2016 List of Prohibited Substances and Methods followed the analysis of data collected under the 2015 World Anti-Doping Agency Monitoring Program. In the early months of 2016, anti-doping laboratories reported an unusually high number of cases in which urine samples contained high concentrations of meldonium. Consequently, the meldonium excretion period in healthy athletes and the substance's long-term urine and blood (plasma) pharmacokinetics became central questions for the anti-doping community to address, to ensure appropriate assessment of the scientific and medical situation, and also fair treatment of athletes from a result management and legal standpoint. At the present time, data on meldonium pharmacokinetics is limited to a few studies, with no known data available on long-term excretion of high oral doses. The primary objective of this open-label study was to determine long-term urine and plasma pharmacokinetic parameters of meldonium in healthy volunteers. Study design included single and repeated functional load testing and assessment of L-carnitine administration on meldonium excretion and pharmacokinetics. Thirty-two volunteers were equally divided into two groups receiving either 1.0 g or 2.0 g of oral meldonium daily for 3 weeks. The study found meldonium takes several days to attain a steady state in blood and displays an elimination period over several months after cessation of treatment. Moreover, findings demonstrate that the daily dose, periodicity and duration of treatment with meldonium are the most important factors to consider in calculating the substance's elimination and complete body clearance., (© 2018 John Wiley & Sons, Ltd.)

In the last few years, a number of studies were conducted which aimed at understanding the mechanisms of cardiovascular drug, metabolism, and there is still the need to determine the metabolites of cardiac drugs for the purpose of metabolism control. In this study, we employ a direct combination of electrochemical oxidation and mass spectrometric (EC-MS) identification for monitoring the oxidation pathway of ten cardiovascular drugs (metoprolol, propranolol, propafenone, mexiletine, oxprenolol, pirbuterol, pindolol, cicloprolol, acebutolol and atenolol). Oxidation was accomplished in an electrochemical thin-layer cell coupled on-line to electrospray ionization mass spectrometry (EC/ESI-MS). For further characterization of electrochemical products, the approach involving liquid chromatography linked to tandem mass spectrometry was used. Appropriate conditions for oxidation and identification processes with such parameters as the potential value, mobile phase (type and pH) and working electrode were optimized. Optimization was performed with the use of central composite design (CCD). Besides electrochemical oxidation of analytes (phase I of metabolic transformation), addition of glutathione (GSH) for follow-up reactions (phase II conjunction) was also investigated. The electrochemical results were compared to in-vivo experiments by analyzing plasma and urine samples from patients who had been administered selected cardiovascular drugs. These results show that electrochemistry coupled to mass spectrometry turned out to be an analytical tool suitable to procure a feasible analytical base for the envisioned in vivo experiments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An effective dual preconcentration method involving off-line membrane supported liquid-liquid-liquid microextraction (MS-LLLME) and on-line field-amplified sample injection (FASI) was proposed for the extraction of six cardiovascular drugs, including mexiletine, xylocaine, propafenone, propranolol, metoprolol, and carvedilol from aqueous solution prior to CE-UV. In MS-LLLME, the analytes were extracted from 9 mL sample solution into toluene, and then back extracted into a drop of acceptor phase of 10 μL 20 mmol/L acetic acid. After that, the acceptor phase was directly introduced into CE for FASI without any modification. In FASI process, water plug was hydrodynamically injected (50 mbar, 3 s) into the capillary prior to sample injection (+6 kV, 18 s). Six target analytes were separated in less than 10 min at 25°C with a BGE consisting of 70 mmol/L Tris-H3 PO4 (pH 2.2) containing 10% v/v methanol. Under the optimized conditions, LODs obtained by the proposed MS-LLLME-FASI-CE-UV method were in the range of 0.02-0.82 μg/L (based on S/N = 3) with enrichment factors of 546- to 7300-fold for the target analytes. The RSDs of the developed method were in the range of 6.7-12.9% (n = 7). Good linearity (R(2) = 0.9928-0.9997) was obtained in concentration range of 0.1-100 μg/L for mexiletine and propranolol, 0.2-100 μg/L for xylocaine and metoprolol, 0.5-100 μg/L for propafenone and 2.0-100 μg/L for carvedilol, respectively. The developed method was successfully applied for real-time determination of metoprolol in human urine samples within 26 h after uptake., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

LC-high resolution (HR)-MS well established in proteomics has become more and more important in bioanalysis of small molecules over the last few years. Its high selectivity and specificity provide best prerequisites for its use in broad screening approaches. Therefore, Orbitrap technology was tested for developing a general metabolite-based LC-HR-MS/MS screening approach for urinalysis of drugs necessary in clinical and forensic toxicology. After simple urine precipitation, the drugs and their metabolites were separated within 10 min and detected by a Q-Exactive mass spectrometer in full scan with positive/negative switching, and subsequent data dependent acquisition (DDA) mode. Identification criteria were the presence of accurate precursor ions, isotopic patterns, five most intense fragment ions, and comparison with full HR-MS/MS library spectra. The current library contains over 1900 parent drugs and 1200 metabolites. The method was validated for typical drug representatives and metabolites concerning recovery, matrix effects, process efficiency, and limits showed acceptable results. The applicability was tested first for cardiovascular drugs, which should be screened for in poisoning cases and for medication adherence of hypertension patients. The novel LC-HR-MS/MS method allowed fast, simple, and robust urine screening, particularly for cardiovascular drugs showing the usefulness of Orbitrap technology for drug testing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A comprehensive study for a sensitivity optimization in MCE with mass spectrometric detection is presented. As a text mixture, we chose a mixture of the cardiac drugs propranolol, bisoprolol, lidocaine, procaine and studied the effect of different chip layouts and experimental parameters with the aim of achieving both high sensitivity in MS detection and adequate chip electrophoretic separation. An important aspect was a comparison of microfluidic layouts containing various sheath-flow channels with that avoiding sheath-flow junctions on-chip. We utilized glass chips with monolithically integrated nanospray emitter tips coupled dead volume-free to an IT mass spectrometer running in fragmentation mode (MS(n) ). With this setup, detection limits down to 0.6 ng/mL for the model compound propranolol were achieved., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Buyang Huanwu decoction (BYHWD), a famous traditional Chinese medicine prescription for the treatment of cerebrovascular diseases, is composed of seven commonly used Chinese herbs--Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen and Pheretima. To determine the main absorptive constituents and the metabolites of BYHWD in vivo, urine samples from Wuzhishan (WZS) miniature pigs orally administered with BYHWD (13.6 g crude drugs/kg) were collected to investigate the characteristic compounds. By comparing the high-performance liquid chromatography of a drug-containing urine sample with that of a drug-free sample, 17 characteristic compounds were isolated from the methanol extract of a drug-containing urine sample by column chromatography. Their structures, including 11 isoflavanoids, 2 pterocarpanoids and 4 isoflavonoids, were identified by spectroscopic means. Of the 17 compounds, 8 (1-8) were new compounds with the following structures: 3S-7,3',4'-trihydroxyisoflavan-3'-O-β-D-glucuronide (1), 3S-7,3',4'-trihydroxyisoflavan-4'-O-β-D-glucuronide (2), 3S-7,2',4'-trihydroxyisoflavan-2'-O-β-D-glucuronide (3), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-2'-O-β-D-glucuronide (4), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-2'-O-β-D-glucuronide-6"-methyl ester (5), 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucuronide-6"-methyl ester (6), 3R-7,2',3'-trihydroxy-4'-methoxyisoflavan-3'-O-β-D-glucuronide-6"-methyl ester (7), and 3S-7,4',5'-trihydroxy-2',3'-dimethoxyisoflavan-5'-O-β-D-glucuronide (8). Based on the possible relationship and metabolic pathways of the 17 compounds in vivo, 3R-7,2'-dihydroxy-3',4'-dimethoxyisoflavan (isomucronulatol, 11), 6aR,11aR-3-hydroxy-9,10-dimethoxypterocarpan (methylnissolin, astrapterocarpan, 13), 7,3'-dihydroxy-4'-methoxyisoflavone (calycosin, 16) and 7-hydroxy-4'-methoxyisoflavone (formononetin, 17) were thought to be the most important absorptive original isoflavonoid constituents of BYHWD in vivo, which underwent reactions of glucuronidation, hydroxylation, demethylation and reduction. The other 13 compounds were deduced to be their metabolites.

A sensitive and selective ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed for the fast, simultaneous quantification of three novel cardiac drugs (aliskiren, prasugrel and rivaroxaban) in human urine. Sample preparation was performed with microextraction with packed sorbent (MEPS), which is a miniaturization of solid phase extraction. The optimal conditions for MEPS extraction were obtained using C8 sorbent, small sample volumes and a short time period (about 3min for the entire sample preparation step). Chromatographic separation of the selected compounds was achieved in less than 1.5min on a Zorbax Rapid Resolution High Definition SB-C18 column using isocratic elution with 0.1% formic acid and acetonitrile (70:30, v/v) at a flow rate of 0.8mLmin(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode. The method was fully validated according to the latest recommendations of international guidelines. The lower limit of quantification was 5.0pgmL(-1) for aliskiren and rivaroxaban and 0.5pgmL(-1) for prasugrel. The intra- and inter-day precision was within 7.12% and the accuracy ranged from -7.54% to 4.17%. The mean extraction recoveries of the MEPSC8 methodology were found to be 98.3% for aliskiren, 100.3% for rivaroxaban and 99.9% for prasugrel. This MEPSC8-UHPLC-MS/MS method offers a fast, simple and precise way to determine selected novel cardiac drugs in human urine that could be applied to therapeutic drug monitoring and pharmacokinetic studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

One of the major challenges facing the medicine today is developing new therapies that enhance human health. To help address these challenges the utilization of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. In the last decade various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an increasingly important tool for monitoring drugs and their metabolites. Furthermore, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages since its introduction. This paper provides an overview of current trends in fast chromatography for the analysis of cardiovascular drugs and their metabolites in clinical applications. Current trends in fast liquid chromatographic separations involve monolith technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive complementary method to traditional methodology used for routine applications. The practical aspects of, recent developments in and the present status of fast chromatography for the analysis of biological fluids for therapeutic drug and metabolite monitoring, pharmacokinetic studies and bioequivalence studies are presented., (Copyright © 2013 Elsevier B.V. All rights reserved.)

3-n-Butylphthalide (NBP) is a cardiovascular drug currently used for the treatment of cerebral ischemia. The present study aims to investigate the metabolism, pharmacokinetics, and excretion of NBP in humans and identify the enzymes responsible for the formation of major metabolites. NBP underwent extensive metabolism after an oral administration of 200 mg NBP and 23 metabolites were identified in human plasma and urine. Principal metabolic pathways included hydroxylation on alkyl side chain, particularly at 3-, ω-1-, and ω-carbons, and further oxidation and conjugation. Approximately 81.6% of the dose was recovered in urine, mainly as NBP-11-oic acid (M5-2) and glucuronide conjugates of M5-2 and mono-hydroxylated products. 10-Keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-hydroxy-NBP (M3-2), and M5-2 were the major circulating metabolites, wherein the areas under the curve values were 1.6-, 2.9-, 10.3-, and 4.1-fold higher than that of NBP. Reference standards of these four metabolites were obtained through microbial biotransformation by Cunninghamella blakesleana. In vitro phenotyping studies demonstrated that multiple cytochrome P450 (P450) isoforms, especially CYP3A4, 2E1, and 1A2, were involved in the formation of M3-1, M3-2, and 11-hydroxy-NBP. Using M3-2 and 11-hydroxy-NBP as substrates, human subcellular fractions experiments revealed that P450, alcohol dehydrogenase, and aldehyde dehydrogenase catalyzed the generation of M2 and M5-2. Formation of M5-2 was much faster than that of M2, and M5-2 can undergo β-oxidation to yield phthalide-3-acetic acid in rat liver homogenate. Overall, our study demonstrated that NBP was well absorbed and extensively metabolized by multiple enzymes to various metabolites prior to urinary excretion.

Piperphentonamine hydrochloride (PPTA) is a new calcium sensitizer. A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of piperphentonamine and its metabolites M1 and M6 was developed for the first time and applied to a pharmacokinetics study. Protein precipitation was used for pre-treatment of plasma samples, and solid phase extraction method was used for pre-treatment of urine samples. The chromatographic separation was achieved on a C(18) column using gradient elution in this study: A: 1% acetic acid aqueous solution, and B: acetonitrile. The whole analysis lasted for 10.5min and the gradient flow rate was 0.25mL/min constantly. The detection was performed of a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via a positive electrospray ionization source. The results were that the m/z ratios of monitored precursor ions and product ions of PPTA, M1 and M6 were 354.0→191.8, 356.0→148.7 and 358.0→148.7, respectively. From the standard curve, the concentration ranges of both PPTA and M1 in blood and urine samples were 0.1-500ng/mL and 0.1-200ng/mL, respectively; the concentration ranges of M6 in blood sample and urine sample were 0.2-500ng/mL and 0.2-200ng/mL, respectively; and the correlation coefficient of standard curve was r>0.99. A total of 31 healthy Chinese subjects participated in the pharmacokinetic study of single bolus intravenous injection of piperphentonamine hydrochloride. They were divided into three dosage groups and given 0.2, 0.4 and 0.6mg/kg of PPTA. After drug administration, concentrations of PPTA, M1 and M6 in human plasma and urine samples were determined to evaluation the pharmacokinetic characteristics of PPTA and its metabolites M1 and M6., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A sensitive ultra-performance liquid chromatography tandem mass spectrometry method with electrospray ionisation (UHPLC-ESI-MS/MS) was developed for the simultaneous determination of 52 compounds: β-blockers, polyphenols (antioxidants) and their metabolites in mixture of standards and after addition the 52 standard solutions to human urine samples. The analyses of urine samples obtained from patients treated with β-blockers were also carried out. The separation of analytes was performed on a Hypersil GOLD™ column (100 mm × 2.1mm, 1.9 μm) using a gradient elution profile for 10 min and mobile phase consisting of 0.1% formic acid in water and acetonitrile. In these conditions, some of the tested compounds were not separated, but this was compensated by the use of MS/MS detection. The drugs, polyphenols and their metabolites were detected with a tandem mass spectrometer after being ionised positively or negatively (depending on the molecule) using an electrospray ionisation (ESI) source. The MS system was operated in the selected reaction monitoring (SRM) mode, where one quantitation and one confirmation transition was done for each analyte. The quantitative method was validated for selectivity, linearity, low limits of quantitation, accuracy, precision, recovery, matrix effect and analyte stability. The LLOQ varied from 0.01 to 0.40 ng mL(-1) for β-blockers and from 0.05 to 40.0 ng mL(-1) for polyphenols. The linear range was 0.08-1000 ng mL(-1) for the drugs and 0.10-2300 ng mL(-1) for the polyphenols. Intra-day and inter-day precision was less than 8%, and the accuracy ranged from -4.40 to 2.23% for all analytes. The average recoveries for all compounds analysed were better than 90%. The developed method can be successfully used to monitor cardiovascular drugs and their metabolites in urine samples of patients treated with β-blockers and can also be used to study the effect of polyphenols on the metabolism of drugs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris-H(3)PO(4) buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris-H(3)PO(4) buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Objectives: Vascular calcification is a major cause of morbidity and mortality in dialysis patients. Human and animal studies indicate that sodium thiosulfate (STS) may prevent the progression of vascular calcifications. The pharmacokinetics of STS in hemodialysis patients has not been investigated yet., Design, Setting, Participants, & Measurements: STS was given intravenously to 10 hemodialysis patients on- and off-hemodialysis. Additionally, STS was applied to 9 healthy volunteers once intravenously and once orally. Thiosulfate concentrations were measured by using a specific and sensitive HPLC method., Results: In volunteers and patients, mean endogenous thiosulfate baseline concentrations were 5.5 ± 1.82 versus 7.1 ± 2.7 μmol/L. Renal clearance was high in volunteers (1.86 ± 0.45 ml/min per kg) and reflected GFR. Nonrenal clearance was slightly, but not significantly, higher in volunteers (2.25 ± 0.32 ml/min per kg) than in anuric patients (2.04 ± 0.72 ml/min per kg). Hemodialysis clearance of STS was 2.62 ± 1.01 ml/min per kg. On the basis of the nonrenal clearance and the thiosulfate steady-state serum concentrations, a mean endogenous thiosulfate generation rate of 14.6 nmol/min per kg was calculated in patients. After oral application, only 4% of STS was recovered in urine of volunteers, reflecting a low bioavailability of 7.6% (0.8% to 26%)., Conclusions: Given the low and variable bioavailability of oral STS, only intravenous STS should be prescribed today. The biologic relevance of the high hemodialysis clearance for the optimal time point of STS dosing awaits clarification of the mechanisms of action of STS.

Dexrazoxane is approved as a cardioprotective agent for use in female patients with breast cancer who are receiving doxorubicin. The effect of renal insufficiency on elimination is not known. The pharmacokinetics of dexrazoxane 150 mg/m(2), given as a 15-minute constant-rate intravenous infusion, were assessed in 24 men and women with varying degrees of renal function in a single-dose, open-label, parallel-group study. Blood and urine samples were measured by a validated liquid chromatography/mass spectrometry assay. Dexrazoxane pharmacokinetic parameters were derived by standard noncompartmental methods. The effect of kidney function and effect of body surface area on the pharmacokinetics of dexrazoxane were analyzed using linear and nonlinear regression in the SPSS statistical program. Dexrazoxane clearance is decreased in subjects with kidney dysfunction. Compared with normal subjects (creatinine clearance [CL(CR)] >80 mL/min), mean area under the concentration curve from time 0 to infinity (AUC(0-inf)) was 2-fold greater in subjects with moderate (CL(CR) 30-50 mL/min) to severe (CL(CR) <30 mL/min) renal dysfunction. Modeling demonstrated that equivalent exposure (AUC(0-inf)) could be achieved if dosing were reduced by 50% in subjects with CL(CR) less than 40 mL/min compared with control subjects (CL(CR) >80 mL/min). Modeling study results suggested that equivalent exposure could be achieved if dosing was halved in subjects with CL(CR) less than 40 mL/min compared with controls.

This paper describes the quantitation of acyl-glucuronide metabolites (M26 and M5) of a cardiovascular-drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base-treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base-hydrolysis of an acyl-glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether-glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet-chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

(-)-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.

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Recent data indicate that bradykinin participates in the regulation of neonatal glomerular function and also acts as a growth regulator during renal development. The aim of the present study was to investigate the involvement of bradykinin in the maturation of renal function. Bradykinin beta2-receptors of newborn rabbits were inhibited for 4 days by Hoe 140. The animals were treated with 300 microg/kg s.c. Hoe 140 (group Hoe, n = 8) or 0.9% NaCl (group control, n = 8) twice daily. Clearance studies were performed in anesthetized rabbits at the age of 8-9 days. Bradykinin receptor blockade did not impair kidney growth, as demonstrated by similar kidney weights in the two groups, nor did it influence blood pressure. Renal blood flow was higher, while renal vascular resistance and filtration fraction were lower in Hoe 140-treated rabbits. No difference in glomerular filtration rate was observed. The unexpectedly higher renal perfusion observed in group Hoe cannot be explained by the blockade of the known vasodilator and trophic effect of bradykinin. Our results indicate that in intact kallikrein-kinin system is necessary for the normal functional development of the kidney.

The systemic hemodynamic and renal responses to conivaptan hydrochloride (YM087; 4'-(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine -6-carbonyl)-2-phenylbenzanilide monohydrochloride), a vasopressin V1A and V2 receptor antagonist, were determined in pentobarbital-anesthetized dogs after 2 to 3 weeks of rapid right ventricular pacing. Congestive heart failure, characterized by decreases in first derivative of left ventricular pressure (left ventricular d P/dt(max)) and cardiac output, and increases in left ventricular end-diastolic pressure and total peripheral vascular resistance, was induced by chronic rapid right ventricular pacing at 260-280 beats/min. Intravenous administration of conivaptan (0.1 mg/kg) significantly increased left ventricular dP/dt(max) and cardiac output and significantly decreased left ventricular end-diastolic pressure and total peripheral vascular resistance. Conivaptan also increased urine flow and reduced urine osmolality by markedly increasing free water clearance. These results indicate that conivaptan produced hemodynamic improvement and marked aquaresis in dogs with congestive heart failure. Therefore, conivaptan may find clinical use in treating patients with congestive heart failure.

A sensitive and selective high-performance liquid chromatographic method with native detection of fluorescence was developed and validated for the quantitation of ivabradine and its N-demethylated metabolite in plasma (rat, dog, human) and human urine. The procedure involves the use of an analogue as internal standard, solid-phase extraction on cyano cartridges, separation on a Nova-Pak C8 column and fluorescence detection. Calibration curves are linear in the concentration ranges from 0.5 to 100 ng/ml in plasma and 2.0 to 500 ng/ml in urine with a limit of quantitation set at 0.5 and 2.0 ng/ml in plasma and urine, respectively. The analysis of plasma and urine samples (spiked with the analytes at low, medium and high concentrations of the calibration range) demonstrates that both analytes can be measured with precision and accuracy within acceptable limits. Quality controls spiked with analyte concentrations up to 10000 ng/ml can also be analysed with excellent precision and accuracy after dilution of the samples. The parent drug and its metabolite are stable in plasma and urine after short-term storage (24 h at room temperature and after three freeze-thaw cycles) as well as after long-term storage at -20 degrees C (at least 6 months in animal plasma and 12 months in human plasma and urine). The method has been used to quantify both compounds in plasma and urine samples from clinical and non-clinical studies with ivabradine.

A sensitive and selective ion-pair reversed-phase HPLC method has been developed for the quantitative determination of KC 12291 and its major metabolite, KC 13194, in rat plasma and urine. An Ultrasphere ODS column constructed by using a mobile phase of 1 mM 1-octanesulfonic acid containing acetonitrile-0.1 M triethylamine phosphate buffer, pH 2.2 (29:71, v/v in plasma and 27:73, v/v in urine), an internal standard and a fluorescent detector (excitation 265 nm, emission 370 nm) were used for the separation and the quantitative determination, respectively. The plasma samples were made alkaline and both compounds were cleaned up by the use of liquid-liquid extraction. The limit of quantification was 10 ng/ml for KC 12291 in plasma and urine and for KC 13194 and 100 ng/ml in plasma, respectively. The assay has been validated with respect to system suitability, accuracy, precision, recovery, stability and ruggedness. All validated parameters were found to be within the necessary limits.

Background: Drugs affecting the cardiovascular and hematopoietic systems are frequently involved in poisoning. As a continuation of our previously published study about the concentrations of drugs of abuse, we have compiled published data about these drugs and subjected them to selection and unification on the basis of conservative criteria and our own experience., Results: A compilation of the concentrations of 90 drugs affecting heart, circulation, blood or hematopoietic organs, in whole blood, serum/plasma, and urine, corresponding to therapeutic, toxic or lethal concentrations is given. Although the interpretation of the concentrations is a complex and difficult problem, the presented table can be helpful in interpretation from the actual concentrations of this group of drugs encountered in clinical, toxicological and forensic cases.

A randomized, four-way cross-over design was used to assess the disposition of the cardioprotective agent, dexrazoxane, in four male beagle dogs following single I.V. administration of 10, 25, 50, and 100 mg kg-1 doses. Parent drug was quantified in plasma and urine with a validated high-pressure liquid chromatographic-electrochemical assay. A two-compartment open model adequately described the dexrazoxane plasma concentration versus time data. The terminal half-life ranged between 1.1 and 1.3 h and the apparent steady-state distribution volume was 0.67 L kg-1. The systemic clearance (CL) ranged from 10.3 to 11.5 mL min-1 kg-1, while estimates of renal clearance approximated the glomerular filtration rate (GFR approximately 3.2-4.9 mL min-1 kg-1). Over the dose range evaluated, CL was dose independent (ANOVA, p = 0.33), while concentration at the end of infusion (Cend) and the area under the concentration versus time curve (AUC) were directly proportional to the dose (r > 0.999). The blood cell to plasma partitioning ratio was approximately 0.517 and drug was essentially unbound to plasma proteins (fu approximately 0.95). Dexrazoxane appeared to be subject to low organ extraction, since the hepatic and renal drug extraction ratios were on the order of 0.228 +/- 0.054 and 0.184 +/- 0.024, respectively. These results suggest a relatively small drug distribution space (approximately equal to total-body water) and low tissue and plasma protein binding. In light of the low plasma protein binding and extraction ratio exhibited by dexrazoxane, metabolic capacity and renal function would appear to be the predominant variables affecting the CL of this drug. The constancy of the half-life, CL, and VSS with increasing dose indicates dose-independent disposition for dexrazoxane. Thus a linear increase in the systemic exposure can be predicted over this dose range.

The separation and determination of amiloride, metoprolol, deacetylmetipranolol, labetalol and furosemide in human serum and urine by capillary isotachophoresis were investigated. Amiloride and beta-blockers were separated by cationic isotachophoresis in the electrolyte system sodium morpholinoethanesulfonate buffer (pH 5.5) (cL = 10 mM)-glutamic acid. Furosemide was separated using the anionic electrolyte system histidine hydrochloride buffer (pH 6.2) (cL = 10 mM)-morpholinopropanesulfonic acid. Endogenous and the possible exogenous compounds were almost totally removed from serum and urine by solid-phase extraction using a Separon SGX C18 cartridge. The recovery of compounds varied from 98.2 to 103.2%. The linearity range for the compounds was 50-1000 ng/ml. The relative standard deviations varied from 0.1 to 5.6%. The overall limits of determination ranged from 32 to 46 ng/ml of urine and from 39 to 46 ng/ml of serum, depending of the type of drugs.

Enantiomers of disopyramide (DP) and its mono-N-dealkylated metabolite (MND) were determined in human plasma and urine by enantioselective high-performance liquid chromatography using a chiral stationary-phase column. This method was precise and sensitive: the mean recoveries from plasma at a concentration of 0.5 microgram/ml were 101.1% for (+)-DP, 98.0% for (-)-DP, 94.4% for (+)-MND and 82.9% for (-)-MND; the within- and between-day coefficients of variation at the same concentration were 4.4 and 3.3% for (+)-DP, 4.7 and 4.1% for (-)-DP, 6.5 and 4.1% for (+)-MND and 7.8 and 2.4% for (-)-MND for plasma; the lower detection limits were 40 ng/ml for (+)-DP, 80 ng/ml for (-)-DP, 100 ng/ml for (-)-MND and 200 ng/ml for (+)-MND, for 0.5 ml of plasma and 0.2 ml of urine. The ultrafiltration technique was used for determination of the unbound concentration of DP enantiomers in plasma. A preliminary study of the determination of DP and MND enantiomers in plasma and urine samples from a healthy subject given racemic DP demonstrated the clinical applicability of the present method for therapeutic monitoring and pharmacokinetic studies.