The squared distance approach to frequency domain time‐resolved fluorescence analysis

A frequency‐domain (FD) analysis of fluorescence lifetime (FLT) is a unique and rapid method for cellular and intracellular classifications that can serve for medical diagnostics purposes. Nevertheless, its data analysis process demands nonlinear fitting algorithms that may distort the resolution of the FLT data and hence diminish the classification ability of the method. This research suggests a sample classification technique that is unaffected by the analysis process as it is based on the squared distance (D²) between the raw frequency response data (FRD). In addition, it presents the theory behind this technique and its validation in two simulated data sets of six groups with similar widely and closely spaced FLT data as well as in experimental data of 43 samples from bacterial and viral infected and non‐infected patients. In the two simulated tests, the classification accuracy was above 95% for all six groups. In the experimental data, the classification of 41 out of 43 samples matched earlier report and 29 out of 31 agreed with preliminary physician diagnosis. The D² approach has the potential to promote FD‐time resolved fluorescence measurements as a medical diagnostic technique with high specifity and high sensitivity for many of today's conventional diagnostic procedures.      

Utility of the food colorant erythrosine B as an effective green probe for quantitation of the anticancer sunitinib. Application to pharmaceutical formulations and human plasma

Sunitinib is a tyrosine kinase inhibitor used for the treatment of renal cell carcinoma and gastrointestinal stromal tumors. In this study, two spectroscopic methods, spectrofluorometric and spectrophotometric, were utilized to quantify sunitinib in different matrices. In method I, the native fluorescence of erythrosine B was quenched by forming ion-pair complex with increasing quantities of sunitinib. This approach was utilized for measuring sunitinib in its dosage forms and spiked plasma. After excitation at 528 nm, the quenching of fluorescence is linearly related to the concentration across the range of 0.05–0.5 μg mL⁻¹ at 550 nm in Britton–Robinson buffer (pH 4.0), with a correlation value of 0.9999 and a high level of sensitivity with detection limit down to 10 ng mL⁻¹. Method II relies on spectrophotometric measurements of the produced complex at 550 nm across a range of 0.5–10.0 μg mL⁻¹, with good correlation value of 0.9999. This method has a detection limit down to 0.16 μg mL⁻¹. The proposed methodologies were validated according to International Conference on Harmonization (ICH) guidelines with satisfactory results. The stoichiometry of the reaction was determined through the application of Job's method, while the mechanism of quenching was investigated by employing the Stern–Volmer plot. The designated methods were used to estimate sunitinib in its capsules and in spiked human plasma. Additionally, the statistical analysis of the data revealed no substantial differences when compared to previous reported spectroscopic method. Green assessment tools provide further details about the eco-friendly nature of the methods.     

Simultaneous estimation of levodopa and carbidopa by RP‐HPLC using a fluorescence detector: its application to a pharmaceutical dosage form

A reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed and validated for the simultaneous estimation of levodopa and carbidopa in bulk and pharmaceutical formulations. Chromatographic separation was achieved by using a C₁₈ reverse‐phase column and a mixture of an aqueous phase (10 mM potassium dihydrogen phosphate buffer, pH 4.0) and methanol (90:10 v/v) as the mobile phase. Quantitative analysis of levodopa and carbidopa was performed using a fluorescence detector at an excitation wavelength of 280 nm and an emission wavelength of 310 nm. The method was linear between 5 and 500 ng/mL for both levodopa and carbidopa. The detection limits for levodopa and carbidopa were 0.30 and 0.60 ng/mL, respectively, whereas the quantitation limit was 0.80 ng/mL for levodopa and 1.2 ng/mL for carbidopa. The method demonstrated good and consistent recoveries (99.63–100.80% for levodopa and 98.97–100.94% for carbidopa) with low interday and intraday relative standard deviation. The validated method was successfully applied to quantify levodopa and carbidopa simultaneously in a pharmaceutical formulation. The method was found to be precise, sensitive and accurate for the simultaneous determination levodopa and carbidopa in bulk and pharmaceutical formulations. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of methocarbamol and aspirin by RP‐HPLC using fluorescence detection with time programming: its application to pharmaceutical dosage form

A new simple, rapid and sensitive reversed‐phase liquid chromatographic method was developed and validated for the simultaneous determination of methocarbamol (MET) and aspirin (ASP) in their combined dosage form. The separation of these compounds was achieved within 6.0 min on a CLC Shim‐pack C₈ column (250 × 4.6 mm, 5 µm particle size) using isocratic mobile phase consisting of acetonitrile and 0.02 M dihydrogenphosphate buffer (30:70, v/v) at pH = 5.0. The analysis was performed at a flow rate of 1.0 mL/min with fluorescence detection at 277/313 nm for MET and 298/410 nm for ASP using real‐time programming. The selectivity, linearity of calibration, accuracy, inter‐ and intra‐day precision and recovery were examined as parts of the method validation. The concentration–response relationship was linear over concentration ranges of 0.02‐0.20 and 0.02‐0.40 µg/mL for MET and ASP, respectively, with a limit of detection of 6 and 32 ng/mL for MET and ASP, respectively. The proposed method was successfully applied for the analysis of both MET and ASP in prepared tablets with average recoveries of 99.88 ± 0.65% for MET and 100.44 ± 0.78% for ASP. The results were favourably compared to those obtained by a reference method. Copyright © 2012 John Wiley & Sons, Ltd.     

Validated sensitive spectrofluorimetric method for determination of antihistaminic drug azelastine HCl in pure form and in pharmaceutical dosage forms: application to stability study

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of azelastine HCl (AZL) in either its pure state or pharmaceutical dosage form. The proposed method was based on measuring the native fluorescence of the studied drug in 0.2 M H₂SO₄ at λ_em = 364 nm after excitation at λ_ex = 275 nm. Different experimental parameters were studied and optimized carefully to obtain the highest fluorescence intensity. The proposed method showed a linear dependence of the fluorescence intensity on drug concentration over a concentration range of 10–250 ng/mL, with a limit of detection of 1.52 ng/mL and limit of quantitation of 4.61 ng/mL. Moreover, the method was successfully applied to pharmaceutical preparations, with percent recovery values (± SD) of 99.96 (± 0.4) and 100.1 (± 0.52) for nasal spray and eye drops, respectively. The results were in good agreement with those obtained by the comparison method, as revealed by Student's t‐test and the variance ratio F‐test. The method was extended to study the stability of AZL under stress conditions, where the drug was exposed to neutral, acidic, alkaline, oxidative and photolytic degradation according to International Conference on Harmonization (ICH) guidelines. Copyright © 2016 John Wiley & Sons, Ltd.     

A validated HPLC method for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) with fluorescence detection in raw material and pill form: application to an in vitro dissolution test and a content uniformity test

A simple, sensitive and rapid HPLC method with fluorescence detection for the determination of dimethyl‐4,4′‐dimethoxy‐5,6,5′,6′‐dimethylene dioxybiphenyl‐2,2′‐dicarboxylate (DDB) in the raw material and pill form was developed. Liquid chromatography was performed on a C₁₈ column (250 × 4.6 mm i.d., 5 µm particle size), the mobile phase consisted of methanol and 0.05 M sodium dihydrogen phosphate buffer (80 : 20, v/v), and the apparent pH of the mobile phase was adjusted to 3. The fluorescence detector was operated at excitation/emission wavelengths of 275/400 nm. The proposed method allows the determination of DDB within concentration range 0.1–1.5 µg/mL with a limit of detection of 0.032 µg/mL, a limit of quantification of 0.097 µg/mL and a correlation coefficient of 0.9997. The proposed method has been successfully applied for the analysis of DDB in its pills with a percentage recovery of 98.45 ± 0.32. The method was fully validated according to ICH guidelines. Moreover, the high sensitivity of the method permits its use in an in vitro dissolution test for DDB under simulated intestinal conditions. In addition, the proposed method was extended to a content uniformity test according to USP guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Isoquinoline-based intrinsic fluorescence assessment of erythropoiesis-stimulating agent,Roxadustat (FG-4592), in tablets: applications to content uniformity and human plasma evaluation

In the present study, a first validated and green spectrofluorimetric approach for its assessment and evaluation in different matrices was investigated. After using an excitation wavelength of 345 nm, Roxadustat (ROX) demonstrates a highly native fluorescence at an emission of 410 nm. The influences of experimental factors such as pH, diluting solvents, and different organized media were tested, and the most appropriate solvent choice was ethanol. It was confirmed that there was a linear relationship between the concentration of ROX and the relative fluorescence intensity in the range 60.0–1000.0 ng ml^−1, with the limit of detection and limit of quantitation, respectively, being 17.0 and 53.0 ng ml⁻¹. The mean recoveries % [±standard deviation (SD), n = 5] for pharmaceutical preparations were 100.11% ± 2.24%, whereas for plasma samples, they were 100.08 ± 1.08% (±SD, n = 5). The results obtained after the application of four greenness criteria, Analytical Eco-Scale metric, NEMI, GAPI, and AGREE metric, confirmed its eco-friendliness. In addition, the whiteness meter (RGB12) confirmed its level of sustainability. The International Council for Harmonisation (ICH) criteria were used to verify the developed method through the study in both spiked plasma samples and content uniformity evaluation. An appropriate standard for various applications in industry and quality control laboratories was developed.     

Application of liquid chromatographic method with fluorescence detection for the determination of triclabendazole in tablets and biological fluids

A simple and rapid liquid chromatographic method was developed and validated for the determination of triclabendazole with high accuracy and precision within 6 min. Good chromatographic separation was achieved using a CLC Shim‐pack C₈ (250 × 4.6 mm, 5 µm particle size) using the mobile phase containing a mixture of 0.02 m phosphate buffer and methanol with a ratio of (20 : 80 v/v) at pH 4.0 was pumped at a flow rate of 1.2 mL/min with fluorescence detection for the first time at 338 nm after excitation at 298 nm. Losartan potassium was used as an internal standard. The method showed good linearity in the ranges of 0.05–2.0 µg/mL with limits of detection and quantification of 14.1 and 42.6 ng/mL, respectively. The suggested method was successfully applied for the analysis of triclabendazole in tablets. The high sensitivity of the method enabled the determination of the studied drug in spiked human plasma with mean percentage of recoveries of 99.79 ± 5.09. Statistical evaluation of the data was performed according to ICH Guidelines. Copyright © 2013 John Wiley & Sons, Ltd.     

The fluorescence quenching of Ru(bipy)32+: an application for the determination of bilirubin in biological samples

A simple, sensitive and efficient fluorescence method has been established for the quantitative analysis of bilirubin. The fluorometric determination method was based on the kinetic quenching of ruthenium(II) fluorescence. The quenching effect may be due to the complexation reaction of bilirubin with ruthenium(II). Therefore, the effects of ruthenium concentrations and different surfactants have been studied. Under the optimized experimental parameters, the fluorescence intensity decreased proportionally with the bilirubin concentration and linearity was established in the range of 3.3 × 10⁻⁷ to 3.0 × 10⁻⁴ M bilirubin. The detection limit calculated from the calibration graph was found to be 5.2 × 10⁻⁸ M. The relative standard deviation (RSD) of 10 consecutive measurements of 8.0 × 10⁻⁶ M bilirubin was 3.0%, while the recoveries of bilirubin in both human serum and urine samples were obtained in the range 94.0–99.5%. The interference study shows that the developed fluorescence based technique is fast, easy to carry out and shows negligible interference. The developed technique was successfully applied for the analysis of bilirubin in human urine and serum samples. All the experimental results and quality parameters confirmed the sensitivity and reproducibility of the proposed technique for bilirubin determination in human urine and serum samples .     

Development and application of an analytical approach to assess an antibody's potential for disulfide reduction

Monoclonal antibody (mAb) interchain disulfide bond reduction has been observed in a recent large-scale clinical manufacturing operation. A massive reduction/precipitation at post-clarification steps has occurred. This note presents the development of a novel analytical approach to identify the “potential reduction”—a unique approach to predict the propensity of a monomeric-profiled mAb to be reduced in the post-harvest stage, such as harvest clarification and/or purification steps. The core of this new approach includes comparing the non-reducing capillary electrophoresis profiles of pre- and post-vacuum treated mAb in harvest cell culture fluid (HCCF). Using this approach, the potential reductions of two in-house mAbs in the unclarified and clarified cell culture harvest were assessed.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Preparation of aminated core‐shell fluorescent nanoparticles and their application to the synchronous fluorescence determination of γ‐globulin

Amino‐modified silica nanoparticles (FSNPs) doped with fluorescein isothiocyanate (FITC) were synthesized by using an aqueous core of reverse‐micelle microemulsion as the nanoreactor in an easy one‐pot method. Due to the FITC conjugating with (3‐aminopropyl)triethoxysilane (APTS), the nanoparticles prevent the FITC from leaching from the silica matrix when immersed in aqueous solution. SEM, FTIR, fluorescence lifetime, a photobleaching experiment and synchronous fluorescence spectra were used to characterize the FSNPs. The synchronous fluorescence signal of FSNPs was enhanced when trace amounts of γ‐globulin (γ‐G) were added. Under the optimal experimental conditions, the enhanced fluorescence intensity (ΔF) was linear with the concentration of γ‐G (c) in the range 0.3–4.8 µg/mL, with a detection limit of 0.04 µg/mL. The proposed method is simple, sensitive for the determination of trace amounts of γ‐G and used to determine the content of γ‐G in synthetic samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

优化的绿色荧光蛋白在超嗜热嗜酸古菌冰岛硫化叶菌中的表达与应用

【目的】开发可用于在极端嗜热嗜酸模式泉古菌冰岛硫化叶菌(Sulfolobus islandicus)中进行高效表达的eCGP123(enhanced consensus green protein variant 123)荧光蛋白,并用作S.islandicus的细胞内蛋白定位工具。【方法】绿色荧光蛋白突变体eCGP123具有极高的热稳定性、耐酸性和可逆的荧光特性等。本研究主要对eCGP123的基因根据S.islandicus密码子偏好性进行优化与合成,在大肠杆菌(Escherichia coli)中表达并研究其蛋白性质;通过在eCGP123的C末端分别融合具有不同细胞内定位的蛋白(包括E.coli来源的Fts Z和S.islandicus来源的Ups E、PCNA1和SiRe_1200等),构建eCGP123及其融合蛋白的表达菌株,用激光共聚焦显微镜分析eCGP123及其融合蛋白在E.coli和S.islandicus活细胞中的亚细胞定位。【结果】我们确认了在E.coli中表达并纯化密码子优化后的e CGP123具有与野生型绿色荧光蛋白相同的吸光值和较高的热稳定性。细胞学分析显示细胞分裂相关蛋白FtsZ和Si Re_1200分别主要定位于E.coli和S.islandicus分裂细胞的中间;鞭毛组分蛋白Ups E呈点状均匀分布,可能定位于细胞膜上;DNA复制滑动夹亚基PCNA1呈区域性点状分布,暗示了DNA复制区域的位置。蛋白的亚细胞定位与预期结果基本吻合。【结论】绿色荧光蛋白e CGP123可以作为报告蛋白,应用于S.islandicus细胞的蛋白定位分析中,可作为该模式菌株中功能基因研究的重要工具,但需要进一步优化条件。     

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.     

Application of a white and green spectrofluorimetric approach for facile quantification of amlodipine,a hypotensive drug,in batch materials,dosage forms,and biological fluids; content homogeneity testing

The suggested study adheres to a particular protocol to ensure that the process is environmentally friendly and sustainable. It is worth mentioning that several tools have been adopted as prospective measures of the method greenness. Fortunately, the established analytical method is identified as white by the white analytical chemistry (WAC) concept, which uses the red/ green/blue color scheme (RGB 12 tool) to combine ecological and functional factors for the first time in studying of the cited drug. Amlodipine (AMD), a cardiovascular treating agent, belongs to the dihydropyridine class of oral calcium channel-blocking agents. This article presents a novel, simple, green, one-pot-processed, fast, and ultrasensitive fluorimetric approach for monitoring and assessment of AMD using molecular-size-dependent fluorescence augmentation of the light scattering-driven signal of eosin, a biological stain at a wavelength of 415 nm. This enhancement was directly proportional to the size of the produced complex. The linearity range was from 30 to 900 ng mL⁻¹, with corresponding sensitivity limits (detection and quantitation levels) of 9.2 and 28 ng mL⁻¹, respectively. The planned approach was also successfully used to track AMD content in bulk, dosage forms, and bio-fluids (human plasma and urine). The developed method's eco-friendliness was established by different eco-rating metric tools.     

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein‐protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero‐FRET and homo‐FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A new meta-ANOVA approach for synthesizing information under signal-heterogeneity setting with application to nuclear magnetic resonance spectroscopic data

A new synthesizing statistical methodology is proposed to resolve issues of signal-heterogeneity in data sets collected through high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy. This signal-heterogeneity is typically caused by subjective operations for processing spectral profiles and measuring peak areas, non-homogeneous biological phases of experimental subjects, and variations of systems in multi-center. All these causes are likely to simultaneously impact signals of metabolic changes and their precision in a nonlinear fashion. As a combined effect, signal-heterogeneity chiefly manifests through non-homomorphic patterns of standardized treatment mean deviations spanning all experiments, and makes most remedial statistical models with linearity structure invalid. By avoiding a huge and very complex model, we develop a simple meta-ANOVA approach to synthesize many one-way-layout ANOVA analyses from individual experiments. A scale-invariant F-ratio statistic is taken as the summarizing sufficient statistic of a non-centrality parameter that supposedly captures the information about metabolic change from each experiment. Then a joint-likelihood function of a common non-centrality is constructed as the basis for maximum likelihood estimation and Chi-square likelihood ratio testing for statistical inference. We apply the meta-ANOVA to detect metabolic changes of three metabolites identified through pattern recognition on NMR spectral profiles obtained from muscle and liver tissues. We also detect effect differences among different treatments via meta-ANOVA multiple comparison.     

Study of characterization and application on the binding between 5-iodouridine with HSA by spectroscopic and modeling

The binding of 5-iodouridine with human serum albumin was investigated under the simulative physiological conditions. The fluorescence spectra in combination with UV absorption and modeling method were used in the present work. A strong fluorescence quenching reaction of 5-iodouridine to HSA was observed and the quenching mechanism was suggested as static quenching procedure. The binding constants (K) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated. It showed that the hydrophobic interaction was a predominant intermolecular force in order to stabilize the complex, which was in agreement with the result of modeling study. The binding distance between 5-iodouridine and HSA was calculated on the basis of the theory of Föster energy transfer. The effects of other ions on the binding constants were also discussed. Synchronous fluorescence spectroscopy (SFS) technique were successfully applied to determine protein in the biological samples.     

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...     

Terbium‐sensitized fluorescence method for the determination of deferasirox in biological fluids and tablet formulation

A novel, rapid and sensitive spectroflurimetric method was developed and validated for the determination of deferasirox in urine, serum and tablet samples based on sensitization of terbium fluorescence. The excitation and emission wavelengths were 328 and 545 nm, respectively. The optimum conditions for the determination of deferasirox were investigated considering the effects of various parameters. The method was quantitatively evaluated in terms of linearity, recovery, reproducibility and limit of detection. Under the optimal conditions, the fluorescence intensities were linear with the concentration of deferasirox in the range of 5 × 10^?9 to 5×10^?6 mol L^?1, with a detection limit of 1.5 × 10^?9 mol L^?1 and a relative standard deviation of 1.1–2.3%. Linearity, reproducibility, recovery and limit of detection made the method suitable for determination of deferasirox in urine, serum and tablets samples. Copyright © 2010 John Wiley & Sons, Ltd.