Pharmacokinetics of dronedarone in rat using a newly developed high-performance liquid chromatographic assay method.

A sensitive and selective high-performance liquid chromatographic method for the determination of dronedarone in rat plasma was developed. Dronedarone was extracted using one-step liquid-liquid extraction. The separation of dronedarone was accomplished using a C18 analytical column. The mobile phase was composed of a combination of monobasic potassium phosphate and acetonitrile. The UV detection was at 254 nm for ethopropazine, the internal standard, and after its elution, changed to 290 nm for dronedarone detection. The total analytical run time was 20 min. Mean recovery was >80%; the assay had excellent linear relationships (>0.999) between peak height ratios and plasma concentrations; the lower limit of quantification 25 was ng/mL, based on 100 µL of rat plasma. Accuracy and precision were <18% over the concentration range of 25-500 ng/mL. The assay was applied successfully to the measurement of dronedarone plasma concentrations in rats given the drug orally.

Rational design and synthesis of water-compatible molecularly imprinted polymers for selective solid phase extraction of amiodarone.

Novel water-compatible molecularly imprinted polymers (MIPs) selective for amiodarone (AD) were designed via a new methodology which relies on screening library of non-imprinted polymers (NIPs). The NIP library consisted of eighteen cross-linked co-polymers synthesized from monomers commonly used in molecular imprinting. The binding capacity of each polymer in the library was analyzed in two different solvents. Binding in water was used to assess non-specific (hydrophobic) interactions and binding in an appropriate organic solvent was used to assess specific interactions. A good correlation was found between the screening tests and modeling of monomer-template interactions performed using computational approach. Additionally, analysis of template-monomer interactions was performed using UV-vis spectroscopy. As the result, 4-vinylpyridine (4-VP) was selected as the best monomer for developing MIP for AD. The 4-VP-based polymers demonstrated imprinting factor equal 3.9. The polymers performance in SPE was evaluated using AD and its structural analogues. The recovery of AD was as high as 96% when extracted from spiked phosphate buffer (pH 4.5) solution and 82.1% from spiked serum samples. The developed MIP shown as a material with specific binding to AD, comparing to its structural analogues, 1-(2-diethylaminoethoxy)-2,6-diiodo-4-nitrobenzene and lidocaine, which shown 9.9% and 25.4% of recovery from the buffer solution, correspondingly. We believe that the screening of NIP library could be proposed as an alternative to commonly used computational and combinatorial approaches.

Identification of amiodarone metabolites in human bile by ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Amiodarone is recognized as an effective drug in the treatment of arrhythmias. Previous experiments demonstrated that mono-N-desethylamiodarone (MDEA) was the major circulating metabolite in humans. In addition, dealkylation, hydroxylation, and deamination were minor metabolic pathways. The purpose of this study was to identify the metabolites of amiodarone in the bile obtained from patients with T-tube drainage after oral drug administration. Amiodarone metabolism in vitro was also investigated using human liver microsomes (HLMs) and S9 fraction. Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) revealed 33 metabolites in human bile, including 22 phase I and 11 phase II metabolites. The major metabolites were MDEA (M7) and ω-carboxylate amiodarone (M12). Metabolite M12 was isolated from human bile, and the chemical structure was confirmed using UPLC-Q/TOF MS and ¹H NMR. Moreover, the authentic standards of two hydroxylated metabolites, 2-hydroxylamiodarone and 3'-hydroxylamiodarone, were obtained through microbial transformation. Several novel metabolic pathways of amiodarone in human were proposed, including ω-carboxylation, deiodination, and glucuronidation. The in vitro study demonstrated that incubation of HLMs with amiodarone did not give rise to any carboxyl metabolites. In contrast, M12 and its metabolites were detected in human liver S9 incubation samples, and the production of these metabolites were inhibited almost completely by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, suggesting the involvement of alcohol dehydrogenase in the ω-carboxylation of amiodarone. Overall, UPLC-Q/TOF MS analysis leads to the discovery of several novel amiodarone metabolites in human bile and underscores the importance of bile as an excretion pathway.

Development of a liquid chromatography-mass spectrometry (LC/MS) assay method for the quantification of PSC 833 (Valspodar) in rat plasma.

A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.

Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood. I. Amiodarone and desethylamiodarone.

We describe the use of a new form of solid-phase material, the Empore solid-phase extraction membrane (SPEM), for therapeutic drug monitoring. We evaluated the new extraction procedure with the companion high-performance liquid chromatographic (HPLC) method for the antiarrhythmic drug amiodarone and its metabolite, desethylamiodarone, in patients' serum. Acidified serum (250 microliters) was passed through an octyl (C8) SPEM secured in an MF-1 microfilter unit. Serum proteins and potential interferences were removed with an acetonitrile:water wash, and the retained drugs eluted with HPLC mobile phase. This eluate was injected directly onto the analytical column. Both drugs averaged 85% recovery with a linear response from a lower limit of detection at 0.05 mg/L up to 6 mg/L, and between-run precision coefficients of variation ranging from 3.1 to 6.4% over the concentration range of 0.5-3.0 mg/L. We observed significant advantages of the novel SPEM over conventional liquid-liquid or large-particle size solid-phase sorbents packed in cartridges. Minimal amounts of solvents were required, elution volume was smaller, time-consuming evaporating/concentrating steps that can influence drug stability were avoided, and little throw-away material was generated. Only the small membrane was discarded.