Sensitive spectrofluorimetric determination of tizanidine in pharmaceutical preparations,human plasma and urine through derivatization with dansyl chloride

A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1‐dimethylaminonaphthalene‐5‐sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence–concentration plots were rectilinear over the ranges 50–500 and 20–300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t‐ and F‐tests. Copyright © 2011 John Wiley & Sons, Ltd.     

Optimization of ASE and SPE conditions for the HPLC‐FLD detection of piperazine in chicken tissues and pork

Accelerated solvent extraction (ASE) and solid‐phase extraction (SPE) conditions were optimized by a high‐performance liquid chromatography‐fluorescence detector (HPLC‐FLD) method for the detection of piperazine in chicken tissues and pork. Piperazine residues were determined by precolumn derivatization with trimethylamine and dansyl chloride. Samples were extracted with 2% formic acid in acetonitrile using an ASE apparatus and purified using a Strata‐X‐C SPE column. The monosubstituted product of the reaction of piperazine with dansyl chloride was 1‐dansyl piperazine (1‐DNS‐piperazine). Chromatographic separations were performed on an Athena C₁₈ column (250 × 4.6 mm, id: 5 μm) with gradient elution using ultrapure water and acetonitrile (5:95, V/V) as the mobile phase. The calibration curves showed good linearity over a concentration range of LOQ‐200.0 μg/kg with a coefficient of determination (R²) ≥ .9992. The recoveries and relative standard deviations (RSD values) ranged from 78.49% to 97.56% and 1.19% to 5.32%, respectively, across the limit of quantification (LOQ) and 0.5, 1, and 2.0 times the maximum residue limit (MRL; μg/kg). The limits of detection (LODs) and LOQs were 0.96 to 1.85 μg/kg and 3.20 to 5.50 μg/kg, respectively. The method was successfully applied for the validation of animal products in the laboratory.     

Development and validation of a sensitive spectrofluorimetric method for the determination of amoxapine in human plasma and urine

A selective and sensitive spectrofluorimetric method was developed and validated for the determination of amoxapine in human plasma and urine. The developed method is based on labeling with 5‐dimethylaminonaphthalene‐1‐sulfonyl chloride (dansyl chloride) and monitoring at 397 nm (excitation)/514 nm (emission). The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The calibration curves were linear over a concentration range of 250–2500 and 50–1250 ng/mL for plasma and urine, respectively. The LOD values were calculated to be 13.31 and 13.17 ng/mL for plasma and urine, respectively. The proposed method was applied to study of amoxapine in human plasma and urine. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of three triterpenic acids in dried rosemary using HPLC‐fluorescence detection and 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride derivatization

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride (DIB‐Cl) and detected using HPLC‐fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB‐Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB‐triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 μm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λ_ex) and 490 nm (λ_em) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5–100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin,a novel highly lipophilic camptothecin derivative,in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2±0.5 min and 8.0±0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at −70°C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Utility of the fluorogenic characters of benzofurazan for analysis of tigecycline using spectrometric technique; application to pharmacokinetic study,urine and pharmaceutical formulations

Tigecycline (TIGE) is the newest tetracycline derivative antibiotic with low toxicity, it is used for management of infectious diseases caused by Gram‐positive and Gram‐negative bacteria. Hence, an efficient, selective and sensitive method was developed for analysis of TIGE in commercial formulations, human plasma and urine. The spectrofluorimetric technique based on the reaction of secondary amine moiety in TIGE with 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) in slightly alkaline medium producing a highly fluorescent product measured at 540 nm (λ_ex at 470 nm) after heating for 15 min at 75°C. The proposed strategy was upgraded and approved by ICH rules and bio‐analytical validated using US‐FDA recommendations. A linear relationship between fluorescence intensity and TIGE concentration was observed over the concentration range 40–500 ng mL^?1 with limit of quantification (LOQ) 21.09 ng mL^?1 and limit of detection (LOD) 6.96 ng mL^?1.The ultra‐affectability and high selectivity of the proposed strategy permits analysis of TIGE in dosage form, human plasma and urine samples with good recovery ranged from 97.23% to 98.72% and from 99.36% to 99.80% respectively, without any interfering from matrix components. Also, the developed strategy was used to examine the stability of TIGE in human plasma and applied for pharmacokinetic investigation of TIGE.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

Hg2+‐selective ratiometric and colorimetric probe based on dansyl–rhodamine and its staining function in cell imaging

A new ratiometric probe composed of a dansyl–rhodamine dyad for the detection of Hg²⁺ via fluorescence resonance energy transfer was designed and synthesized. Rhodamine, dansyl chloride, and hydrazide were selected as the acceptor, donor, and reaction site, respectively. It displayed high selectivity and sensitivity to Hg²⁺ with obvious colour change and fluorescence change due to Hg²⁺‐assisted hydrolysis of rhodamine hydrazide. A good linear relationship ranging from 0 to 16 μM and 0–28 μM for the Hg²⁺ concentration was found based on absorbance and fluorescence assay, respectively. Detection limits of absorbance and fluorescence for Hg²⁺ were calculated to be 1.22 μM and 9.10 μM, respectively.     

Phenylisothiocyanate and dansyl chloride precolumn derivatizations for the high-performance liquid chromatography analysis of the antitumoral agent ES-285 in dog plasma

Chromophor and fluorophor addition reactions involving phenylisothiocyanate (PITC) and dansyl chloride (DC) were optimized to adapt two high-performance liquid chromatography (HPLC) procedures designed for the accurate determination of the novel antitumoral agent ES-285 in Beagle dog plasma. ES-285 was reacted with PITC at 60 degrees C for 15 min in the presence of triethylamine. The dansyl derivative was obtained by reaction of ES-285 with dansyl chloride in a basic medium at 80 degrees C for 20 min. Both reactions also worked for ES-299, a compound structurally related to ES-285 which was used as internal standard. The treatment of ES-285 and ES-299 spiked plasma samples with a basic phosphate buffer and MeOH permitted the extraction of the drug from the matrix. The purification of the analytes was carried out by solid-phase extraction followed by precolumn derivatization with PITC and DC. The phenylisothiocyanate adducts were analyzed by isocratic HPLC with UV detection at 254 nm. The ES-285 and ES-299 derivatives were eluted from a C(18) column at approximately 6.9 and approximately 8.1 min, respectively. The eluent ACN-water (95:5, v/v) was delivered to the column at a flow-rate of 1 ml/min and the analysis was completed in 15 min. The dansyl derivatives were analysed by a two-HPLC column system with fluorescence detection and gradient elution. The column temperature was maintained at 40 degrees C. The analysis lasted 55 min with the elution of ES-285 and ES-299 derivatives at approximately 35.2 and approximately 37.9 min, respectively. The PITC- and DC-based procedures were characterized by limits of quantification of 20 and 15 ng/ml, respectively. Both procedures were validated according to the ICH and FDA guidelines. They were selective for ES-285 and provided accurate, precise and reproducible results. ES-299 was shown to be a suitable internal standard. The HPLC procedure involving derivatization with DC was more sensitive and permitted to process plasma sample volumes as low as 100 microl. On the other hand, the PITC-based procedure characterised by a quite similar LOQ permitted a higher throughput but implied the processing of a 500-microl plasma volume.     

Derivatization and high-performance liquid chromatographic analysis of pentaazapentacosane pentahydrochloride

A rapid high-performance liquid chromatographic method for determination of the dansyl derivative of pentaazapentacosane (PAPC) pentahydrochloride has been developed. The chromatographic system uses a reversed-phase C₈ column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20-fold dansyl chloride excess. The yield of dansyl PAPC increased 10-fold as the reaction pH was changed from 9.5 to 10.5. Under derivatization conditions of pH 8.5–11.0 and 1–30-fold excess dansyl chloride only perdansyl PAPC was found.     

Development of a novel capillary electrophoresis chemiluminescence system for amino acid analysis

In the present study, a novel peroxyoxalate CE–CL system was developed to achieve high signal stability and sensitivity based on a design of a new interface including a new mixing mode and a new grounding electrode mode. Amino acids fluorescently tagged with dansyl chloride and naphthalene‐2,3‐dicarboxaldehyde(NDA) were used for the study. Experiment results show this new system is quite effective to separate and detect amine acid with high stability and resolute. The detection limits were 1.1 nmol/L for dansyl‐leucine (Leu) and 2.0 nmol/L for dansyl‐aspartic acid (Asp). The relative standard deviations of peak height and migration time were in the ranges of 2.3–3.8% and 1.2–1.5%, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals

Orciprenaline sulphate (ORP) is a direct‐acting sympathomimetic with mainly beta‐adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0–400.0 ng/ml and 10.0–240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra‐sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4–106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

Quantification of nortriptyline in plasma by HPLC and fluorescence detection

A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C₁₈) chromatography with fluorescence detection. The developed method required only 100 μl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5–5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.     

Development of a micelle‐enhanced high‐throughput fluorometric method for determination of niclosamide using a microplate reader

The present paper describes the development and validation of a simple and sensitive micelle‐enhanced high‐throughput fluorometric method for the determination of niclosamide (NIC) in 96‐microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λ_em 444 nm after excitation at λ_ex 275 nm. Tween‐80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100–150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r²≥ 0.9997) over the concentration ranges of 1–5 and 0.5–5 μg/ml with lower detection limits of 0.01 and 0.008 μg/ml and lower quantification limits of 0.04 and 0.03 μg/ml on using Tween‐80 and or CMC, respectively. The developed high‐throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

High-performance liquid chromatographic analysis of mibefradil in dog plasma and urine

The objective of the study was to develop a sensitive and specific assay for studying the pharmacokinetics of a novel calcium antagonist, a benzimidazolyl-substituted tetraline derivative, mibefradil (I) in the dog. The assay involves liquid-liquid extraction of a biological sample, reversed-phase HPLC separation and fluorescence detection (λ_ex = 270 nm and λ_em = 300 nm) of a sample components. Each sample was eluted with a mobile phase pumping at a flow-rate of 2 ml/min. The mobile phase composition was a mixture of acetonitrile and aqueous solution (38:62, v/v). The aqueous solution contains 0.0393 M KH₂PO₄ and 0.0082 M Na-pentanesulphonic acid. The retention times were 10.7 min for I, and 12.2 min for internal standard Ro 40–6792. Calibration curves with concentrations of I ranging from 10 to 500 ng/ml were linear (r² > 0.99). The detection limit for I was 0.5 ng/ml when 0.5 ml of plasma or urine was used. Intra- and inter-day accuracy and precision were within 10%. The assay was successfully applied to the pharmacokinetic studies of I in dogs.     

Second‐derivative synchronous spectrofluorimetric assay of dapagliflozin: Application to stability study and pharmaceutical preparation

A highly accurate, simple and sensitive spectrofluorimetric analytical method for dapagliflozin (DGF) quantitation was developed. The proposed method was successively applied to DGF analysis in both its pure and pharmaceutical dosage forms. This method was developed to investigate DGF stability in its degradation products, as laid out in International Council for Harmonisation (ICH) rules. Kinetics of alkaline degradation of DGF was also calculated. The half‐life time (t_1/2) of the reaction was 75.32 min. An alkaline degradation pathway was described. The present study involved measurement of the second‐derivative synchronous fluorescence intensity of DGF at Δλ = 30 nm. Peak amplitude was measured at 322 nm. Linear range of the calibration curve was 0.1–1.0 μg ml^?1. Lower detection and quantitation limits were 0.023 and 0.071 μg ml^?1, respectively, and indicated good sensitivity of the proposed method. Mean per cent recovery was 99.78 ± 1.78%. The proposed analytical approach was successfully applied to DGF in the quality control laboratory and would be suitable as a stability‐indicating assay.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Development of sensitive high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent for determination of bisphenol A in plasma samples

A sensitive HPLC method for determination of bisphenol A (BPA) in plasma samples using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a fluorescence labeling reagent was developed. The fluorescence labeling reaction was completed within 10 min at room temperature. DIB-Cl reacts with the phenolic hydroxyl group of BPA in the presence of triethylamine (TEA). The DIB-Cl derivative of BPA (DIB-BPA) was separated within 30 min with an ODS column using acetonitrile–water (90:10, v/v) as the isocratic eluent. Calibration graphs were linear over the range of 1.0–100 ng/ml (r=0.999). The detection limit of DIB-BPA was 0.05 ng/ml (2.5 pg) at a signal-to-noise ratio of 3. The relative standard deviations (RSDs) of the method for between-run were 1.0–5.0%. The analytical recoveries of known amounts (1.0 and 100 ng/ml) of BPA-spiked rabbit plasma were around 95%.     

First derivative synchronous spectrofluorimetric method for the simultaneous determination of propofol and cisatracurium besylate in biological fluids

Propofol and cisatracurium besylate have been simultaneously determined using a highly sensitive first derivative synchronous spectrofluorometric method. The method is based on measuring first derivative synchronous spectrofluorimetric amplitude at Δλ = 40 nm with a scanning rate of 600 nm/min. The different experimental parameters affecting the fluorescence intensity of the two drugs were carefully studied and optimized. The amplitude–concentration plots were rectilinear over the range 40.0–400.0 ng/mL and 20.0–280.0 ng/mL for propofol and cisatracurium, respectively with lower detection limits of 4.0 and 2.35 ng/mL and quantification limits of 12.1 and 7.1 ng/mL for propofol and cisatracurium, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial ampoules. The high sensitivity attained using the proposed method allowed the simultaneous determination of both drugs in spiked plasma samples. The mean % recoveries in spiked human plasma (n = 3) were 96.53 ± 0.90 and 96.20 ± 1.64 for each of propofol and cisatracurium, respectively. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.