Identification and characterization of amiodarone metabolites in rats using UPLC-ESI-QTOFMS-based untargeted metabolomics approach.

Amiodarone is a class III anti-arrhythmic benzofuran derivative extensively utilized in treatment of life-threatening ventricular and supraventricular arrhythmias. However, amiodarone also produces adverse side effects including liver injury due to its metabolites rather than parent drug. The purpose of the present study was to identify metabolites of amiodarone in the plasma and urine of rats administered the drug by using an untargeted metabolomics approach. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and results subjected to multivariate data analysis. A total of 49 amiodarone metabolites were identified and their structures were characterized by tandem mass spectrometry. Amiodarone metabolites are presumed to be generated via five major types of metabolic reactions including N-desethylation, hydroxylation, carboxylation (oxo/hydroxylation), de-iodination, and glucuronidation. Data demonstrated that an untargeted metabolomics approach appeared to be a reliable tool for identifying unknown metabolites in a complex biological matrix.

Metabolite identification studies on amiodarone in in vitro (rat liver microsomes, rat and human liver S9 fractions) and in vivo (rat feces, urine, plasma) matrices by using liquid chromatography with high-resolution mass spectrometry and multiple-stage mass spectrometry: characterization of the diquinone metabolite supposedly responsible for the drug's hepatotoxicity.

RATIONALE: Several mechanisms have been anticipated for the toxicity of amiodarone, such as oxidative stress, lipid peroxidation, phospholipidosis, free radical generation, etc. Amiodarone is structurally similar to benzbromarone, an uricosuric agent, which was withdrawn from European markets due to its idiosyncratic hepatotoxicity. A proposed reason behind the toxicity of benzbromarone was the production of a reactive ortho-diquinone metabolite, which was found to form adducts with glutathione. Therefore, taking a clue that a similar diquinone metabolite of amiodarone may be the reason for its hepatotoxicity, metabolite identification studies were carried out on the drug using liquid chromatography/mass spectrometry (LC/MS) tools. METHODS: The studies involved in vitro (rat liver microsomes, rat liver S9 fraction, human liver S9 fraction) and in vivo (rat feces, urine, plasma) models, wherein the samples were analyzed by employing LC/HRMS, LC/MS(n) and HDE-MS. RESULTS AND CONCLUSIONS: A total of 26 metabolites of amiodarone were detected in the investigated in vitro and in vivo matrices. The suspected ortho-diquinone metabolite was one of them. The formation of the same might be an added reason for the hepatotoxicity shown by the drug.

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry.

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.

Rapid liquid chromatographic assay for the determination of amiodarone and its N-deethyl metabolite in plasma, urine, and bile.

A rapid high-performance liquid chromatographic assay was developed for the determination of amiodarone (1) and its N-deethyl metabolite (desethylamiodarone, 2) in plasma, urine, and bile. Analysis was performed on a C18 reversed-phase column and precolumn using a mobile phase consisting of methanol:water:58% ammonium hydroxide (94:4:2) delivered at a flow rate of 1.5 mL/min. The eluant was monitored at 244 nm. Under these conditions, 1, 2, and the internal standard eluted with retention times of 5.5, 4.6, and 6.8 min, respectively. Samples (100 microL) of plasma were prepared by precipitating the plasma proteins with acetonitrile containing the internal standard and injecting an aliquot of the supernatant directly onto the column. Samples (100 microL) of urine and bile were prepared for injection by acidifying the sample with concentrated HCl and then extracting the mixture with six volumes of 2,2-dimethoxyproprane. The recovery of 1 and 2 from plasma was virtually complete. The recovery from urine and bile was 80-90% for 1 and 60-65% for 2. The limit of sensitivity of both compounds in plasma was 100 ng/mL. For urine and bile, the detection limits were 1 and 5 micrograms/mL, respectively. Over the plasma concentration range of 0.1-10.0 micrograms/mL, the within-day CV ranged from 1 to 10% for 1 and from 1 to 8% for 2. The between-day CV ranged from 2 to 12% and from 1 to 17% for 1 and 2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylamiodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether-acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol-acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C18 column operating in combination with a radial compression separation unit and a methanol-25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay. The limit of sensitivity of the assay was 0.025 micrograms/ml with a precision of +/- 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.

Rapid high-performance liquid chromatographic method for the measurement of amiodarone in blood plasma or serum at the concentrations attained during therapy.

A simple high-performance liquid chromatographic method has been developed for the measurement of the antiarrhythmic drug amiodarone in small (200 microliter) volumes of plasma or serum. After addition of 2 mole/l phosphate solution, pH 4.5 (20 microliter), containing the internal standard, the sample is vortex-mixed with diisopropyl ether (200 microliter) for 30 sec. A portion (100 microliter) of the resulting extract is analysed on a microparticulate (5 micron) silica column using methanol-diethyl ether (85:15) containing perchloric acid (0.02% v/v) as the mobile phase, and the absorption of the column effluent is monitored at 240 nm. No endogenous sources of interference have been observed, and interference from other drugs is minimal. The procedure is rapid, an analysis in duplicate taking less than 15 min to complete. The limit of sensitivity of the assay is 0.05 mg/l, and the concentrations of amiodarone measured in plasma samples from patients under treatment with this compound ranged from 0.15 to 4.5 mg/l.