Hollow-fiber-supported liquid membrane microextraction of amlodipine and atorvastatin.

A simple, environmentally friendly, and efficient method, based on hollow-fiber-supported liquid membrane microextraction, followed by high-performance liquid chromatography has been developed for the extraction and determination of amlodipine (AML) and atorvastatin (ATO) in water and urine samples. The AML in two-phase hollow-fiber liquid microextraction is extracted from 24.0 mL of the aqueous sample into an organic phase with microliter volume located inside the pores and lumen of a polypropylene hollow fiber as acceptor phase, but the ATO in three-phase hollow-fiber liquid microextraction is extracted from aqueous donor phase to organic phase and then back-extracted to the aqueous acceptor phase, which can be directly injected into the high-performance liquid chromatograph for analysis. The preconcentration factors in a range of 34-135 were obtained under the optimum conditions. The calibration curves were linear (R(2) ≥ 0.990) in the concentration range of 2.0-200 µg/L for AML and 5.0-200 µg/L for ATO. The limits of detection for AML and ATO were 0.5 and 2.0 µg/L, respectively. Tap water and human urine samples were successfully analyzed for the existence of AML and ATO using the proposed methods.

Polytetrafluorethylene film-based liquid-three phase micro extraction coupled with differential pulse voltammetry for the determination of atorvastatin calcium.

In this paper, we describe a new combination method based on polytetrafluorethylene (PTFE) film-based liquid three-phase micro extraction coupled with differential pulse voltammetry (DPV) for the micro extraction and quantification of atorvastatin calcium (ATC) at the ultra-trace level. Different factors affecting the liquid-three phases micro extraction of atorvastatin calcium, including organic solvent, pH of the donor and acceptor phases, concentration of salt, extraction time, stirring rate and electrochemical factors, were investigated, and the optimal extraction conditions were established. The final stable signal was achieved after a 50 min extraction time, which was used for analytical applications. An enrichment factor of 21 was achieved, and the relative standard deviation (RSD) of the method was 4.5% (n = 4). Differential pulse voltammetry exhibited two wide linear dynamic ranges of 20.0-1000.0 pmol L(-1) and 0.001-11.0 µmol L(-1) of ATC. The detection limit was found to be 8.1 pmol L(-1) ATC. Finally, the proposed method was used as a new combination method for the determination of atorvastatin calcium in real samples, such as human urine and plasma.

Voltammetric oxidation and determination of atorvastatin based on the enhancement effect of cetyltrimethyl ammonium bromide at a carbon paste electrode.

An electrochemical method has been described for the voltammetric oxidation and determination of an antihyperlipoproteinemic drug, atorvastatin (ATOR), at a carbon paste electrode (CPE) in the presence of an enhancing agent, cetyltrimethyl ammonium bromide (CTAB) using cyclic and differential pulse voltammetry (DPV). The results indicated that the voltammetric response of ATOR was improved distinctly in the low concentration of CTAB, suggesting that CTAB exhibits noticeable enhancement effect to the determination of ATOR. The dependence of current on pH, concentration and scan rate were investigated to optimize the experimental conditions for the determination of ATOR. The anodic peak was characterized and the process was adsorption-controlled. The number of electrons transferred in the oxidation process was calculated and a plausible oxidation mechanism was proposed. In the range of 0.05-10 µM, the current measured by DPV presents a good linear property as a function of the concentration of ATOR with a detection limit of 4.08 nM with good selectivity and sensitivity. The proposed method was successfully applied to ATOR determination in pharmaceutical samples and urine as a real sample. This method can be employed in clinical analysis, quality control and routine determination of drugs in pharmaceutical formulations.

Discovery of safety biomarkers for atorvastatin in rat urine using mass spectrometry based metabolomics combined with global and targeted approach.

In order to develop a safety biomarker for atorvastatin, this drug was orally administrated to hyperlipidemic rats, and a metabolomic study was performed. Atorvastatin was given in doses of either 70 mg kg(-1) day(-1) or 250 mg kg(-1) day(-1) for a period of 7 days (n=4 for each group). To evaluate any abnormal effects of the drug, physiological and plasma biochemical parameters were measured and histopathological tests were carried out. Safety biomarkers were derived by comparing these parameters and using both global and targeted metabolic profiling. Global metabolic profiling was performed using liquid chromatography/time of flight/mass spectrometry (LC/TOF/MS) with multivariate data analysis. Several safety biomarker candidates that included various steroids and amino acids were discovered as a result of global metabolic profiling, and they were also confirmed by targeted metabolic profiling using gas chromatography/mass spectrometry (GC/MS) and capillary electrophoresis/mass spectrometry (CE/MS). Serum biochemical and histopathological tests were used to detect abnormal drug reactions in the liver after repeating oral administration of atorvastatin. The metabolic differences between control and the drug-treated groups were compared using PLS-DA score plots. These results were compared with the physiological and plasma biochemical parameters and the results of a histopathological test. Estrone, cortisone, proline, cystine, 3-ureidopropionic acid and histidine were proposed as potential safety biomarkers related with the liver toxicity of atorvastatin. These results indicate that the combined application of global and targeted metabolic profiling could be a useful tool for the discovery of drug safety biomarkers.

ABCG2 polymorphism markedly affects the pharmacokinetics of atorvastatin and rosuvastatin.

The ABCG2 c.421C>A single-nucleotide polymorphism (SNP) was determined in 660 healthy Finnish volunteers, of whom 32 participated in a pharmacokinetic crossover study involving the administration of 20 mg atorvastatin and rosuvastatin. The frequency of the c.421A variant allele was 9.5% (95% confidence interval 8.1-11.3%). Subjects with the c.421AA genotype (n = 4) had a 72% larger mean area under the plasma atorvastatin concentration-time curve from time 0 to infinity (AUC(0-infinity)) than individuals with the c.421CC genotype had (n = 16; P = 0.049). In participants with the c.421AA genotype, the rosuvastatin AUC(0-infinity) was 100% greater than in those with c.421CA (n = 12) and 144% greater than in those with the c.421CC genotype. Also, those with the c.421AA genotype showed peak plasma rosuvastatin concentrations 108% higher than those in the c.421CA genotype group and 131% higher than those in the c.421CC genotype group (P < or = 0.01). In MDCKII-ABCG2 cells, atorvastatin transport was increased in the apical direction as compared with vector control cells (transport ratio 1.9 +/- 0.1 vs. 1.1 +/- 0.1). These results indicate that the ABCG2 polymorphism markedly affects the pharmacokinetics of atorvastatin and, even more so, of rosuvastatin-potentially affecting the efficacy and toxicity of statin therapy.

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.

Glucuronidation of statins in animals and humans: a novel mechanism of statin lactonization.

The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding delta-lactone. By means of an LC-NMR technique, the glucuronide structure was established to be a 1-O-acyl-beta-D-glucuronide conjugate of the statin acid. The formation of statin glucuronide and statin lactone in human liver microsomes exhibited modest intersubject variability (3- to 6-fold; n = 10). Studies with expressed UDP glucuronosyltransferases (UGTs) revealed that both UGT1A1 and UGT1A3 were capable of forming the glucuronide conjugates and the corresponding lactones for all three statins. Kinetic studies of statin glucuronidation and lactonization in liver microsomes revealed marked species differences in intrinsic clearance (CL(int)) values for SVA (but not for AVA or CVA), with the highest CL(int) observed in dogs, followed by rats and humans. Of the statins studied, SVA underwent glucuronidation and lactonization in human liver microsomes, with the lowest CL(int) (0.4 microl/min/mg of protein for SVA versus approximately 3 microl/min/mg of protein for AVA and CVA). Consistent with the present in vitro findings, substantial levels of the glucuronide conjugate (approximately 20% of dose) and the lactone form of SVA [simvastatin (SV); approximately 10% of dose] were detected in bile following i.v. administration of [(14)C]SVA to dogs. The acyl glucuronide conjugate of SVA, upon isolation from an in vitro incubation, underwent spontaneous cyclization to SV. Since the rate of this lactonization was high under conditions of physiological pH, the present results suggest that the statin lactones detected previously in bile and/or plasma following administration of SVA to animals or of AVA or CVA to animals and humans, might originate, at least in part, from the corresponding acyl glucuronide conjugates. Thus, acyl glucuronide formation, which seems to be a common metabolic pathway for the hydroxy acid forms of statins, may play an important, albeit previously unrecognized, role in the conversion of active HMG-CoA reductase inhibitors to their latent delta-lactone forms.

High-performance liquid chromatographic determination of seratrodast and its metabolites in human serum and urine.

A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of seratrodast, a new antiasthmatic drug, and its metabolites (M-I to M-III) in human serum and urine. The method for serum and urine with and without enzymatic hydrolysis using beta-glucuronidase involved liquid-liquid extraction and chemical oxidation with iron(III) chloride. The compounds in the extract were analyzed using HPLC with UV detection at 266 nm. The detection limits of seratrodast, M-I, M-II and M-III in serum and urine were 5-10 and 5-20 ng/ml, respectively, and those of deconjugated compounds in urine were 10-50 ng/ml. The method was applicable for human serum and urine from clinical trials.

Influence of carbon chain length on the hepatic effects of perfluorinated fatty acids. A 19F- and 31P-NMR investigation.

Using nuclear magnetic resonance (NMR) spectroscopy, we investigated the importance of carbon chain length with regard to the hepatic effects associated with perfluoro-n-carboxylic acids. Male F-344 rats were administered a single intraperitoneal dose of either perfluoro-n-heptanoic acid (C7-PFA), perfluoro-n-nonanoic acid (C9-PFA), or perfluoro-n-undecanoic acid (C11-PFA). Data from previous studies involving perfluoro-n-octanoic acid (C8-PFA) and perfluoro-n-decanoic acid (C10-PFA) are included for comparison. Food consumption/body weight was monitored daily for all groups. C9- and C11-PFA treatment yields a prolonged hypophagic response while C7-PFA shows a more acute response. Fluorine-19 NMR spectra of urine and bile samples show no evidence of fluorometabolites and suggest that the distribution of perfluorocarbons into urine or bile is dependent upon carbon chain length. The aqueous solubility of C7-PFA appears to facilitate rapid urinary excretion, similar to that observed for C8-PFA. The relative hydrophobicity of C9- and C11-PFA appears to favor biliary enterohepatic recirculation, yielding a more protracted toxicity, similar to C10-PFA. Phosphorus-31 NMR studies of liver in vivo and liver extracts show that perfluorocarbons of > or = C9 carbons produce a significant increase in liver phosphocholine concentration. These data are discussed with regard to the impact of these chemicals on hepatic phospholipid metabolism. Hepatic peroxisomal fatty acyl CoA-oxidase activity (FAO) was measured to determine if C7-, C9-, and C11-PFA are peroxisome proliferators. Data indicate that the induction of peroxisomal enzyme activity by perfluorocarbons requires a chain length greater than seven carbons. In general, these results demonstrate the significance of carbon chain length in the hepatotoxic response and provide clues toward understanding the processes involved in the biological activities associated with exposure to these compounds.

Conventional and enantioselective determination of a new blood glucose-lowering agent in biological fluids using liquid-liquid extraction and high-performance liquid chromatography.

Two analytical methods are described for the determination of 2-(4-tert.-butylphenoxy)-7-(4-chlorophenyl)heptanoic acid sodium salt (I) in animal models (beagle dog and rat). Method 1 is conventional reversed-phase high-performance liquid chromatography on an octadecylsilane column with an eluent of acetonitrile-0.02 M potassium buffer (pH 3) (65:35, v/v). Method 2 is used for the enantioselective determination of I. This method uses a chiral column (Chiralcel OJ) with an eluent of n-hexane-2-propanol (95:5, v/v) containing 3 ml/l trifluoracetic acid. The analytical procedure has a recovery of more than 90%; within-run precision of less than 5.1%, and between-run precision of less than 4.3%.

Pharmacokinetic and pharmacodynamic profiles of vapiprost, a selective, long-lasting thromboxane receptor antagonist, after single and multiple oral administration to healthy volunteers.

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time-profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet-rich plasma (PRP) induced by a stable TXA2 receptor agonist U-46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U-46619 ex vivo were simultaneously examined and compared. In the single-dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one-compartment open model with a first-order absorption. The area under plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives (t1/2) remained approximately constant within the range of 0.99-1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U-46619 and the secondary aggregation by ADP were inhibited; that induced by U-46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time-profile of inhibiting whole blood platelet aggregation that was induced by U-46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary treatment eliminates succinylacetone from the urine of a patient with tyrosinaemia type 1.

Over an 18-month period serial observations of plasma tyrosine, methionine and urinary tyrosine metabolites were made and compared with urinary succinylacetone excretion in an infant with tyrosinaemia type 1 treated by diet alone. Despite broadly similar profiles there were significant temporal and quantitative differences between each of these metabolic parameters. Only when plasma tyrosine was kept in the low-normal range by strict phenylalanine restriction (10-15 mg phenylalanine/kg body weight) was detectable succinylacetone consistently eliminated from the urine. Urinary succinylacetone is the only measure of metabolite accumulation immediately proximal to the enzyme defect and its routine measurement will allow more effective control of dietary treatment.

Disposition of pravastatin sodium, a tissue-selective HMG-CoA reductase inhibitor, in healthy subjects.

Pravastatin sodium, a competitive inhibitor of HMG-CoA reductase, is a new orally effective hypocholesterolaemic agent. In a two-way crossover study, eight healthy male subjects each received an intravenous and an oral dose of [14C]-pravastatin sodium. The oral absorption of [14C] activity from pravastatin sodium was about 34% and the oral bioavailability was about 18%, suggesting first-pass metabolism of pravastatin. After the intravenous dose, the recovery of radioactivity averaged 60% and 34% in urine and faeces, respectively. Corresponding values were 20% (urine) and 71% (faeces) for the oral dose. The estimated average plasma elimination half-life of pravastatin was 0.8 and 1.8 h for the intravenous and oral routes, respectively. The average values for total and renal clearances were 13.5 and 6.3 ml min-1 kg-1, respectively, and the steady-state volume of distribution averaged 0.51 kg-1. These results suggest that both kidney and liver are important sites of elimination for pravastatin.

Hereditary tyrosinemia. Formation of succinylacetone-amino acid adducts.

Succinylacetone (SA) (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumaryl acetoacetate hydrolase activity. Patients with this disease are associated with a number of abnormalities, including aminoaciduria, proteinuria, liver failure, commonly hepatoma, and decreased GSH concentration in the liver. In the course of our studies of tyrosinemia, we found that the urine of patients with this disorder contains material(s) that absorbs light at 315 nm. We investigated the nature of the 315 nm material in detail. SA was found to react with amino acids and protein nonenzymatically, to form stable adducts at physiological temperature and pH. All SA adducts with amino acids and/or proteins exhibited an absorption peak at 315 nm. Although all amino acids reacted with SA, the most reactive amino acid was lysine (Lys), followed, in order, by glycine, methionine, phenylalanine, serine, alanine, and glutamine. SA-adducts were unstable at pH below 6, while they were made considerably more stable after reduction with NaBH4, suggesting that SA forms an adduct via Schiff base formation. High-performance liquid chromatography (HPLC) analysis of urines from patients with tyrosinemia revealed the existence of SA-glycine, SA-methionine, SA-tyrosine, and SA-phenylalanine. After digestion of urines with proteinase K, three more HPLC peaks appeared, which all corresponded to SA-Lys adducts. TLC analysis of SA-Lys showed that SA-Lys could form as many as seven different adducts. No SA-adduct peaks were observed in HPLC in urines from normal subjects, patients with other forms of aminoaciduria, or patients with the nephrotic syndrome. In addition to amino acids and proteins, SA reacted with reduced glutathione (GSH) and formed a stable adduct. These findings suggest that SA adduct formation with amino acids, GSH, and proteins is a significant process occurring in tyrosinemia, and may account for certain of the pathologic findings in this hereditary disorder.

Persistent succinylacetone excretion after liver transplantation in a patient with hereditary tyrosinaemia type I.

A liver transplant was performed on a 4-year-old female in liver failure caused by hereditary tyrosinaemia, with hepatocellular carcinoma following a negative evaluation for metastases. However, serum alpha-fetoprotein levels never returned to normal after the surgery. Urinary succinylacetone (SA) was detected in her urine prior to transplantation despite strict adherence to a low-tyrosine diet. Other patients with severe liver disease awaiting liver transplantation do not excrete SA in the urine. She continued to excrete SA during the postoperative period despite normal liver functions. Oral tyrosine loading resulted in significant elevation of SA excretion. Possible explanations for this observation and clinical and therapeutic relevance are discussed.

Determination of urinary succinylacetone by capillary gas chromatography.

The average analytical recovery of succinylacetone added to urine and separated by capillary gas chromatography was 69% for solvent extraction and 72% for anion exchange separation. Treating succinylacetone with hydroxylamine hydrochloride at a pH of less than 5 caused formation of a derivative separated by capillary gas chromatography into two isomers: 3-methyl-5- isoxazole propionate and 5-methyl-3- isoxazole propionate as their trimethylsilyl derivatives (molecular weight 227). In a pH greater than or equal to 5, succinylacetone dioxime was formed and separated into 3 isomers as their trimethylsilyl derivatives (molecular weight 404). Succinylacetone dioxime was converted to 3(5)-methyl-(3)5- isoxazole propionate whenever the pH of the solution was dropped to less than 5. Mass spectra of both derivatives are shown. This study demonstrates that capillary gas chromatography is suitable for use in urinary succinylacetone determination.

Urinary excretion of deuterated metabolites in patients with tyrosinemia type I after oral loading with deuterated L-tyrosine.

1. The metabolic fate of orally given deuterated L-tyrosine, 50 mg/kg body weight, was investigated in seven patients with tyrosinemia type I in order to obtain evidence that the primary defect is at the level of fumarylacetoacetase. 2. The absence of fumarylacetoacetase could be proved in liver biopsy specimens obtained from four patients. 3. All patients excreted deuterated succinylacetoacetate and deuterated succinylacetone was detected in six out of seven. The total amount of these compounds was rather low; maximal 8.3% of the dose. The peak of the excretion occurred 3-6 h after loading, indicating an endogenous formation of the metabolites. 4. All patients excreted deuterated 4-hydroxyphenyl acids, probably reflecting secondary 4-hydroxyphenylpyruvate dioxygenase deficiency connected with liver damage. 5. No evidence for other secondary routes of tyrosine metabolism was found.

Identification of 4,6-dioxoheptanoic acid (succinylacetone), 3,5-dioxooctanedioic acid (succinylacetoacetate) and 4-Oxo-6-hydroxyheptanoic acid in the urine from patients with hereditary tyrosinemia.

In the urine from patients with hereditary tyrosinemia, three characteristic compounds have been found. They have been identified as 4,6-dioxoheptanoic acid (succinylacetone), 3,5-dioxooctanedioic acid (succinyl-acetoacetate) and 4-oxo-6-hydroxyheptanoic acid. The identities have been established by mass spectrometry of several derivatives, and by comparison with a synthetic sample of 4,6-dioxoheptanoic acid.

Urinary excretion of succinylacetone and delta-aminolevulinic acid in patients with hereditary tyrosinemia.

Succinylacetone was excreted in the urine from four patients, with hereditary tyrosinemia i.e., two patients with the severe infantile type with fatal outcome and two patients with less severe juvenile form. In the urine from two patients with neonatal transient tyrosinemia and from normal individuals succinylacetone was not detectable. The urinary excretion of delta-aminolevulinic acid was also increased in all patients with hereditary tyrosinemia compared to patients with neonatal transient tyrosinemia and to normal individuals. The results presented support the hypothesis of a deficiency of fumarylacetoacetase in hereditary tyrosinemia. Furthermore an analytical method for the quantitative determination of succinylacetone in urine using GC-MS is described.