Study on the interaction between erythrosine B and the cardiac drug amiodarone using fluorescence, scattering, and absorbance spectra and their analytical application.

In an acidic buffered solution, erythrosine B can react with amiodarone to form an association complex, which not only generates great enhancement in resonance Rayleigh scattering (RRS) spectrum of erythrosine B at 346.5 nm but also results in quenching of fluorescence spectra of erythrosine B at λemission = 550.4 nm/λexcitation = 528.5 nm. In addition, the formed erythrosine B-amiodarone complex produces a new absorbance peak at 555 nm. The spectral characteristics of the RRS, absorbance, and fluorescence spectra, as well as the optimum analytical conditions, were studied and investigated. As a result, new spectroscopic methods were developed to determine amiodarone by utilizing erythrosine B as a probe. Moreover, the ICH guidelines were used to validate the developed RRS, photometric, and fluorimetric methods. The enhancements in the absorbance and the RRS intensity and the decrease in the fluorescence intensity of the used probe were proportional to the concentration of amiodarone in ranges of 2.5-20.0, 0.2-2.5, and 0.25-1.75 µg/mL, respectively. Furthermore, limit of detection values were 0.52 ng/mL for the spectrophotometric method, 0.051 µg/mL for the RRS method, and 0.075 µg/mL for the fluorimetric method. Moreover, with good recoveries, the developed spectroscopic procedures were applied to analyze amiodarone in its commercial tablets.

Basic micro-pollutants in sludge from municipal wastewater treatment plants in the Northwest Spain: Occurrence and risk assessment of sludge disposal.

Sludge is one of the most problematic residues generated during wastewater treatment. Herein, we validate a single-step, sensitive procedure for the determination of a selection of 46 basic micro-pollutants, either used as pharmaceuticals or pesticides, in sludge from municipal sewage treatment plants (STPs), using liquid chromatography tandem mass spectrometry as determination technique. The proposed method permitted to achieve accurate recoveries (values from 70% to 120%, for samples spiked at different concentration levels) using solvent-based calibration standards. This feature, combined with limits of quantification lower than 5 ng g-1 (dry weight), allowed the rapid and sensitive quantification of target compounds in freeze-dried sludge samples. Out of 46 investigated pollutants, 33 species showed detection frequencies above 85% in a group of 48 sludge samples, obtained from 45 STPs located in the Northwest of Spain. The assessment of eco-toxicological risks associated to sludge disposal as fertilizer in agriculture and/or forestry, considering average concentrations found in sludge samples, highlighted eight pollutants (sertraline, venlafaxine, N-desethyl amiodarone, amiodarone, norsertraline, trazodone, amitriptyline and ketoconazole) representing an environmental hazard based on ratios between predicted soil levels and non-effect concentrations estimated using the equilibrium partition method.

Quantitation of amiodarone and N-desethylamiodarone in single HepG2 cells by single-cell printing-liquid vortex capture-mass spectrometry.

Quantitative measure of a drug and its associated metabolite(s) with single-cell resolution is often limited by sampling throughput or other compromises that limit broad use. Here, we demonstrate the use of single-cell printing-liquid vortex capture-mass spectrometry (SCP-LVC-MS) to quantitatively measure the intracellular concentrations of amiodarone (AMIO) and its metabolite, N-desethylamiodarone (NDEA), from thousands of single cells across several AMIO incubation concentrations ranging from 0 to 10 µM. Concentrations obtained by SCP-LVC-MS were validated through comparison with average assays and traditional measurement of cells in bulk. Average of SCP-LVC-MS measurements and aggregate vial collection assay the concentrations differed by < 5%. Both AMIO and NDEA had clear log-normal distributions with similar standard deviation of concentrations in the cell population. The mean of both AMIO and NDEA intracellular concentrations were positively correlated with AMIO incubation concentration, increasing from 0.026 to 0.520 and 0.0055 to 0.048 mM for AMIO and NDEA, respectively. The standard deviation of AMIO and NDEA log-normal distribution fits were relatively similar in value across incubation concentrations, 0.15-0.19 log10 (mM), and exhibited a linear trend with respect to each other. The single cell-resolved conversion ratio of AMIO to NDEA increased with decreasing incubation concentration, 7 ± 2%, 18 ± 3%, and 20 ± 7% for 10.0, 1.0, and 0.1 µM AMIO incubation concentrations, respectively. Association with simultaneously measured lipids had several ions with statistically significant difference in intensity but no clear correlations with AMIO intracellular content was observed.

Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Detection of glutathione conjugates of amiodarone and its reactive diquinone metabolites in rat bile using mass spectrometry tools.

RATIONALE: Amiodarone is reported to cause hepato and pulmonary toxicity in humans, which has been envisaged to be due to formation of its reactive metabolites, essentially based on its structural similarity to benzbromarone, a drug withdrawn from the market due to reasons of similar hepatotoxicity. Therefore, the purpose of this study was to detect glutathione conjugates of amiodarone and its reactive diquinone metabolites in rat bile using mass spectrometry tools. METHODS: Wistar rats were dosed orally with an amiodarone suspension and bile was collected via bile duct cannulation followed by solid-phase extraction, protein precipitation and centrifugation. Samples were analysed by liquid chromatography coupled with linear ion trap mass spectrometry using tandem mass and constant neutral loss scan in positive electrospray ionization mode. RESULTS: Glutathione adducts of amiodarone and its reactive diquinone metabolites were identified and characterized with the characteristic neutral loss of 129 Da. Glucuronide conjugates of previously reported stable phase-1 metabolites were also observed. CONCLUSIONS: This study confirmed generation of reactive metabolites of amiodarone for the first time, as was hypothesised earlier by various research groups. Also, the responsible toxicophore was identified to be a benzofuran moiety liable to form reactive diquinone species. However, the results need to be further confirmed in human subjects. Copyright © 2016 John Wiley & Sons, Ltd.

Identification and characterization of stress degradation products of dronedarone hydrochloride employing LC-UV/PDA, LC-MS/TOF and MS(n) studies.

Dronedarone HCl was subjected to forced decomposition conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH guideline Q1A(R2). The drug showed significant degradation under alkaline hydrolytic and alkaline photolytic conditions while it remained stable in neutral, acidic, thermal and oxidative conditions. In total, six degradation products (I-VI) were formed, which could be separated by chromatography on C18 (250 mm × 4.6 mm; 5 µ, Xterra) column using isocratic elution method. Detection wavelength was selected as 288 nm. Multi-stage (MS(n)) and MS/TOF accurate mass studies were carried out to establish the complete fragmentation pathway of the drug which in turn was utilized in characterization of the products. The degradation pathway of the drug leading to generation of products I-VI was postulated and this has not been reported so far.

Validated HPLC and Ultra-HPLC Methods for Determination of Dronedarone and Amiodarone Application for Counterfeit Drug Analysis.

Two simple, accurate, and precise chromatographic methods have been developed and validated for the determination of dronedarone (DRO) HCl and amiodarone (AMI) HCl either alone or in binary mixtures due to the possibility of using AMI as a counterfeit of DRO because of its lower price. First, an RP-HPLC method is described for the simultaneous determination of DRO and AMI. Chromatographic separation was achieved on a BDS Hypersil C18 column (150×4.6 mm, 5 µm). Isocratic elution based on potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (10+90, v/v) at a flow rate of 2 mL/min with UV detection at 254 nm was performed. The second method is RP ultra-HPLC in which the chromatographic separation was achieved on an AcclaimTM RSLC 120 C18 column (100×2.1 mm, 2.2 µm) using isocratic elution with potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (5+95, v/v) at a flow rate of 1 mL/min with UV detection at 254 nm. Linearity, accuracy, and precision of the two methods were found to be acceptable over the concentration ranges of 5-80 µg/mL for both DRO and AMI. The results were statistically compared using one-way analysis of variance. The optimized methods were validated and proved to be specific, robust, precise, and accurate for the QC of the drugs in their pharmaceutical preparations.

Amiodarone hydrochloride: enhancement of solubility and dissolution rate by solid dispersion technique

abstract Amiodarone HCl is an antiarrhythmic agent, which has low aqueous solubility and presents absorption problems. This study aimed to develop inclusion complexes containing hydrophilic carriers PEG 1500, 4000 and 6000 by fusion and kneading methods in order to evaluate the increase in solubility and dissolution rate of amiodarone HCl. The solid dispersion and physical mixtures were characterized by X-ray diffraction, FT-IR spectra, water solubility and dissolution profiles. Both methods and carriers increased the solubility of drug, however PEG 6000 enhanced the drug solubility in solid dispersion better than other carriers. Different media were evaluated for the solubility study, including distilled water, acid buffer pH 1.2, acetate buffer pH 4.5 and phosphate buffer pH 6.8 at 37 ºC. Based on the evaluation of the results obtained in the study phase solubility carriers PEG 4000 and PEG 6000 were selected for the preparation of the physical mixture and solid dispersion. All formulations were prepared at drug-carrier ratios of 1:1 to 1:10(w/w). The results of in vitro release studies indicated that the solid dispersion technique by fusion method in proportion of 1:10 (w/w) increased significantly the dissolution rate of the drug. X-ray diffraction studies showed reduced drug crystallinity in the solid dispersions. FT-IR demonstrated interactions between the drug and polymers.

resumo Cloridrato de amiodarona é um agente antiarrítmico que possui baixa solubilidade aquosa e apresenta problemas de absorção. Este estudo teve como objetivo desenvolver complexos de inclusão contendo carreadores hidrofílicos PEG 1500, 4000 e 6000 através dos métodos de fusão e amassamento para avaliar o aumento da solubilidade e taxa de dissolução do cloridrato de amiodarona. As dispersões sólidas e misturas físicas foram caracterizadas por difração de raios-X, espectroscopia no infravermelho com transformada de Fourier, solubilidade em água e perfis de dissolução. Ambos os métodos e carreadores aumentaram a solubilidade do fármaco, no entanto o PEG 6000 aumentou a solubilidade do fármaco na dispersão sólida mais que os outros carreadores. Diferentes meios foram avaliados para o estudo de solubilidade, incluindo água destilada, tampão ácido pH 1,2, tampão acetato pH 4,5 e tampão fosfato pH 6,8. Com base na avaliação dos resultados obtidos no estudo de solubilidade de fases, os carreadores PEG 4000 e PEG 6000 foram selecionados para a preparação das misturas físicas e dispersões sólidas. Todas as formulações foram preparadas nas razões fármaco-carreador de 1:1 a 1:10 (p/p). Os resultados de liberação in vitro que a técnica de dispersão sólida pelo método de fusão na proporção 1:10 (p/p) aumentou significativamente a taxa de dissolução do fármaco. Estudos de difração de raios-X mostraram redução da cristalinidade do fármaco na dispersão sólida. Análise por espectroscopia no infravermelho mostrou interações entre o fármaco e o carreador.

Synthesis and characterization of new related substances of the antiarrhythmic drug dronedarone hydrochloride.

Two new potential impurities of antiarrhythmic drug substance Dronedarone Hydrochloride together with debutyldronedarone were detected by LC-MS analysis during process development. A successful synthetic strategy for the synthesis of these potential impurities was developed facilitating the access to new impurity reference standards. Their synthesis and characterization are discussed in detail. The availability of these impurity standards allowed cost reduction through the increase of process control.

Determination of the cardiac drug amiodarone and its N-desethyl metabolite in sludge samples.

For the first time, a procedure for the simultaneous determination of the iodinated drug amiodarone and its major metabolite, N-desethylamiodarone, in sludge from urban sewage treatment plants (STPs) is proposed. Matrix solid-phase dispersion (MSPD) followed by on-line cationic exchange clean-up, in modular configuration, was used as sample preparation technique. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, was employed for the selective determination of target compounds. The optimized procedure provided exhaustive recoveries with little effect of the sample matrix in the efficiency of electrospray ionization (ESI). The overall recoveries of the method ranged between 95 and 111%, for samples spiked at different concentration levels. The achieved limits of quantification (LOQs) remained below 10ngg(-1) for both compounds, and the linear response range extended up to 2500ngg(-1). Amiodarone and N-desethylamiodarone were ubiquitous in sludge samples, from different STPs located in the Northwest of Spain, with maximum concentrations above 300ngg(-1) referred to the freeze-dried matrix. They were also present in stabilized sludge (mixed with lime and thermally dehydrated), which is mostly disposed in agriculture fields as fertilizer. Furthermore, mono-iodinated analogues of amiodarone and N-desethylamiodarone were also tentatively identified in some samples from their accurate MS and MS/MS spectra.

C60-SIMS imaging of nanoparticles within mammalian cells.

To achieve successful drug delivery via nanoparticles the interactions between the nanoparticle and the chemistry of the surrounding biological environment is of central importance. A thorough understanding of these interactions is necessary in order to better elucidate information regarding drug pathways and mechanisms of action in treatment protocols. As such, it is important to identify the location of the nanoparticle, the state of its functionalization, as well as any changes in the cellular environment. The use of cluster secondary ion mass spectrometry (SIMS) using C60 (+) primary ions makes simultaneous acquisition of this information possible. Here, SIMS has been successfully used to chemically image gold nanoparticles (AuNPs) within a model, single cell system involving macrophage-like RAW 264.7 cells. The macrophage-like properties of this cell line make it extremely well-suited for cell-uptake studies. Both AuNPs and two pharmaceutical compounds, amiodarone and elacridar, were successfully imaged within a cellular system using cluster SIMS. To verify that SIMS can also be used to detect functionalization and nanoparticles simultaneously, fluorophore-functionalized AuNPs were studied as a model system. The fluorescent characteristics of these functionalized nanoparticles enabled the visual confirmation of the presence and location of the particles within the cell.

Systematic evaluation of plasma phospholipids for reliable and precise determination of dronedarone and desbutyldronedarone by LC-MS/MS.

BACKGROUND: The objective of the present work was to minimize or eliminate the matrix effect due to plasma phospholipids as observed during sample preparation for accurate determination of dronedarone and its active metabolite, desbutyldronedarone by LC-ESI-MS/MS. RESULTS: The extraction recovery and matrix factors ranged from 93.27 to 95.14% and 0.99 to 1.02, respectively, for both the analytes. A linear concentration range of 0.10 to 150 ng/ml was established for both the analytes. The analytes were efficiently resolved (R(s) 2.37) on Kromasil(®) (AkzoNobel, Bohus, Sweden) C18 column within 3.0 min. The assay reproducibility was determined by reanalysis of 72 incurred samples with % change within ± 10%. CONCLUSION: The optimized solid-phase extraction provided cleaner extracts with reduced matrix effect from plasma phospholipids compared with protein precipitation and liquid-liquid extraction.

Quality by design (QbD) based development and validation of an HPLC method for amiodarone hydrochloride and its impurities in the drug substance.

The USP monograph describes an HPLC method for seven impurities in the amiodarone drug substance using a L1 column, 4.6mm×150mm, 5µm packing (PF listed ODS2 GL-Science, Inertsil column) at 30°C with detection at 240nm. The standard contains 0.01mg/mL of amiodarone, and USP specified impurities D and E with a resolution requirement of NLT 3.5 between peaks D and E. Impurities in a 5mg/mL sample are quantitated against the standard. Impurity A peak elutes just before peak D. We observed two problems with the method; the column lot-to-lot variability resulted in unresolved A, D, and E peaks, and peak D in the sample preparation eluted much later than that in the standard solution. Therefore, optimization experiments were conducted on the USP method following the QbD approach with Fusion AE™ software (S-Matrix Corporation). The resulting optimized conditions were within the allowable changes per USP 〈621〉. Lot-to-lot variability was negligible with the Atlantis T3 (Waters Corporation) L1 column. Peak D retention time remained constant from standard to sample. The optimized method was validated in terms of accuracy, precision, linearity, range, LOQ/LOD, specificity, robustness, equivalency to the USP method, and solution stability. The QbD based development helped in generating a design space and operating space with knowledge of all method performance characteristics and limitations and successful method robustness within the operating space.

Breast milk concentrations of amiodarone, desethylamiodarone, and bisoprolol following short-term drug exposure: two case reports.

Two cases of mothers given postpartum short-term administration of amiodarone, with and without bisoprolol, are described along with determinations of amiodarone and (±)-bisoprolol in the breast milk. In one mother given a cumulative total of amiodarone of 8 g over 1 week, concentrations 11 days after the drug had been stopped were initially deemed sufficient to pose a risk to an infant. Over the next 5 days the concentrations steadily dropped with amiodarone and desethylamiodarone concentrations being found to be at a level comprising minimal risk to the infant. Bisoprolol was not found in the expressed breast milk. In the second case the mother was given a single 150 mg dose of amiodarone and breast milk concentrations were measured on postpartum days 4 and 5. Breast milk amiodarone concentrations were very low and of little concern clinically had the mother breast fed her baby. The risk to the baby of ingesting breast milk after amiodarone administration postpartum depends on the duration of amiodarone exposure, with a single dose posing minimal risk. Bisoprolol does not appear to accumulate to any great extent in breast milk.

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.

Stability-indicating UPLC method for determining related substances and degradants in dronedarone.

A simple, sensitive and reproducible method was developed on ultra-performance liquid chromatography coupled with photodiode array detection for the quantitative determination of dronedarone hydrochloride (DRO) in drug substance and pharmaceutical dosage forms. The method is applicable for the quantification of related substances and assays of drug substances. Chromatographic separation was achieved on Acquity UPLC BEH C8 100 mm, 2.1 mm and 1.7 µm columns, using gradient elution within a short run time of 10.0 min. The eluted compounds were monitored at 288 nm, the flow rate was 0.5 mL/min and the column oven temperature was maintained at 40°C. The resolution of DRO and 11 impurities (potentials and by-products) was greater than 2.0 for all pairs of components. The high correlation coefficient value (>0.9995) indicates the clear correlations between the concentrations of investigated compound and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by the relative standard deviation, were less than 2.5%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method, expressed as relative error, was satisfactory. No interference was observed from concomitant substances normally added to the tablets. DRO was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. DRO was found to degrade significantly in acid and base stress conditions and to remain stable in thermal, photolytic degradation, oxidative and hydrolytic conditions. The degradation products were well resolved from primary peak and its impurities, proving that the method is stability indicating. The developed method was validated as per International Conference on Harmonization guidelines with respect to specificity, limit of detection, limit of quantification, linearity, accuracy, precision, solution stability and robustness. This method is also suitable for the determination of DRO drug substance and pharmaceutical dosage forms.

A rapid HPLC method for the simultaneous determination of amiodarone and its major metabolite in rat plasma and tissues: a useful tool for pharmacokinetic studies.

A rapid and sensitive high-performance liquid chromatography (HPLC) method was developed and validated in rat plasma and tissue (heart, liver, kidney and lung) homogenates for the determination of amiodarone and its primary metabolite (desethylamiodarone), using tamoxifen as internal standard. Chromatographic separation was achieved within less than 5 min on a LiChroCART Purospher Star C18 column (55 × 4 mm, 3 µm). The mobile phase, consisting of phosphate buffer (50 mM) with 0.1% formic acid (pH 3.1)-methanol-acetonitrile (45:5:50, v/v/v), was pumped isocratically at a flow rate of 1.2 mL/min. The detection was conducted at 254 nm for all compounds. Calibration curves were linear (r(2) ≥ 0.995) in the range of 0.1-15 µg/mL for amiodarone and desethylamiodarone. The limits of quantification were established at 0.1 µg/mL for both analytes. The overall data of precision and accuracy were in accordance with international guidelines for bioanalytical method validation. Amiodarone and desethylamiodarone were extracted from rat matrices by a liquid-liquid extraction procedure and the mean recovery ranged from 59.9 to 97.6%. This novel HPLC method allows the fast and reliable determination of amiodarone and desethylamiodarone from several rat matrices (plasma, liver, kidneys, lungs and heart) and was successfully applied in a preliminary pharmacokinetic study.