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Avapritinib (AVP) was the first precision drug to be approved by the US Food and Drug Administration (FDA) in 2020 for patients suffering from metastatic gastrointestinal stromal tumors (GISTs) and progressive systemic mastocytosis. The analysis of AVP in pharmaceutical tablets and human plasma was then carried out using a fast, efficient, sensitive, and simple fluorimetric method using a fluorescamine reagent. The procedure is based on the interaction between fluorescamine as a fluorogenic reagent and the primary aliphatic amine moiety in AVP using borate buffer solution at pH 8.8. The produced fluorescence was measured at 465 nm (Excitation at 395 nm). The calibration graph's linearity range was discovered to be 45.00–500.0 ng mL−1. Utilizing the International Council for Harmonization (ICH) and US‐FDA recommendations, the research technique was validated and bioanalytically validated. The proposed approach was effectively employed for determining the stated pharmaceuticals in plasma with a high percentage of recovery ranging from 96.87 to 98.09 and pharmaceutical formulations with a percentage of recovery equal to 102.11% ± 1.05%. In addition, the study was extended to a pharmacokinetic study of AVP with 20 human volunteers as a step for AVP management in therapeutic cancer centers. [ABSTRACT FROM AUTHOR]

Diaryl-furanones are specific analytical reagents for the biochemical detection of primary amines by fluorescence techniques. Well-known reagents are fluorescamine (Fluram) and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), yielding fluorescent products with λem at 480–490 nm. Although the reaction products claim to be pyrrolinones, recent studies show that they are really 3-oxopyrrole (pyrrolone) derivatives. Both reagents have been used for the cytochemical demonstration of primary amines. In this work, we have applied the fluorescent products of MDPF with amines (n-butylamine, BA; glucosamine, GA; and spermine, Sp), which showed interesting fluorescence reactions with chromatin DNA. 2,4-diphenyl-3-oxopyrrole products (diPOPy) can be easily synthesized according to well-known procedures, by mixing solutions of MDPF in acetone with water at pH 9 containing the amino compounds. DiPOPy derivatives of BA, GA, and Sp were used for spectroscopic, microscopic, and molecular modeling studies, showing a bright and selective blue–green fluorescence on DNA substrates, mainly chromatin, kinetoplast DNA, and stretched chromatin fibers. The cationic diPOPy fluorophore is planar, with a high partial positive charge in the N atom, and suitable for intercalative binding to DNA. A mechanism of fluorescamine fluorescence due to an inner-salt isomeric form is proposed, and an astonishing correlation between adenine–thymine-rich centromeric heterochromatin in mouse metaphase chromosomes after reaction of the fluorescamine reagent with protein amino groups is also discussed.

Diaryl-furanones are specific analytical reagents for the biochemical detection of primary amines by fluorescence techniques. Well-known reagents are fluorescamine (Fluram) and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), yielding fluorescent products with λem at 480–490 nm. Although the reaction products claim to be pyrrolinones, recent studies show that they are really 3-oxopyrrole (pyrrolone) derivatives. Both reagents have been used for the cytochemical demonstration of primary amines. In this work, we have applied the fluorescent products of MDPF with amines (n-butylamine, BA; glucosamine, GA; and spermine, Sp), which showed interesting fluorescence reactions with chromatin DNA. 2,4-diphenyl-3-oxopyrrole products (diPOPy) can be easily synthesized according to well-known procedures, by mixing solutions of MDPF in acetone with water at pH 9 containing the amino compounds. DiPOPy derivatives of BA, GA, and Sp were used for spectroscopic, microscopic, and molecular modeling studies, showing a bright and selective blue–green fluorescence on DNA substrates, mainly chromatin, kinetoplast DNA, and stretched chromatin fibers. The cationic diPOPy fluorophore is planar, with a high partial positive charge in the N atom, and suitable for intercalative binding to DNA. A mechanism of fluorescamine fluorescence due to an inner-salt isomeric form is proposed, and an astonishing correlation between adenine–thymine-rich centromeric heterochromatin in mouse metaphase chromosomes after reaction of the fluorescamine reagent with protein amino groups is also discussed. [ABSTRACT FROM AUTHOR]

Polymyxins (PMS), namely Colistin (CS) and polymyxin B (poly B), are antimicrobial drugs that have been recently used to treat multiresistant Gram‐negative bacteria infections and their resurgence, owing to a lack of new antibiotics. A speedy, simple, and ultrasensitive spectrofluorimetric screening of PMS in pharmaceutical formulations and biological fluids was urgently required from this point forwards. A reaction between fluorescamine and the aliphatic amino moiety found in both drugs was performed in a slightly alkaline borate buffer (pH 8.5) resulted in highly fluorescent products measured at λem 460 (after λex 390.5 nm). Linear calibration curves were constructed over the concentration range 70–1800 ng ml−1 and 100 to 1400 ng ml−1, with slope values of 0.273 and 0.286, correlation coefficients of 0.9998 and 0.9997, and determination coefficient of 0.9997 and 0.9994 for poly B and CS, respectively. The ultrasensitivity of the proposed method was demonstrated by the very low limit of quantification values of 67.56 ng ml−1 and 94.89 ng ml−1 for poly B and CS, respectively. The cited drugs were successfully determined in their intravenous market preparations by the prescribed method. Moreover, due to the high sensitivity, the suggested method was used to assay the investigated drugs in biological fluids. [ABSTRACT FROM AUTHOR]

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.

Aggregation of amyloid-β (Aβ) protein plays a central role in Alzheimer's disease. Because protein aggregation is a concentration-dependent process, rigorous investigations require accurate concentration measurements. Owing to the high aggregation propensity of Aβ protein, working solutions of Aβ are typically in the low micromolar range. Therefore, an ideal Aβ quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280 nm is frequently used to measure Aβ concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the Aβ sequence. Here we present a fluorescence method for Aβ quantification using fluorescamine, which gives high fluorescence upon reaction with primary amines. We show that, using hen egg white lysozyme as a standard, fluorescence correlates linearly with primary amine concentration across a wide range of fluorescamine concentrations, from 62.5 to 1000 µM. The maximal sensitivity of detection is achieved at a fluorescamine concentration of 250 µM or higher. The fluorescamine method is compatible with the presence of dimethyl sulfoxide, which is commonly used in the preparation of Aβ oligomers, and limits the use of absorbance at 280 nm due to its high background reading. Using aggregation kinetics, we show that the fluorescamine method gives accurate concentration measurements at low micromolar range and leads to highly consistent aggregation data. We recommend the fluorescamine assay to be used for routine and on-the-fly concentration determination in Aβ oligomerization and fibrillization experiments.

A new method for determining the composition of protein-nanoparticle conjugates is proposed in this paper that uses fluorescamine as a fluorescent marker. This method allows obtaining high accuracy results and is characterized by minimal duration and laboriousness. Conjugates of gold nanoparticles with streptococcal protein G were characterized by composition and immunoglobulin-binding properties. The optimal concentrations of protein G and gold nanoparticles with an average diameter of 25 nm were found to be 8 μg/mL and 2.5 nM (OD520 = 5), respectively. The obtained conjugates were used for the developed immunochromatographic analysis of the antibiotic sulfonylamide as a relevant contaminant that is controlled in food products. Indirect labeling of specific antibodies allowed using crude rabbit antiserum in the analysis. The visual and instrumental detection limits of sulfonylamide were 100 ng/mL and 0.3 ng/mL, respectively. The time of the assay was 10 min. The developed assay was tested for the characterization of honey samples. [ABSTRACT FROM AUTHOR]

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Using two green and sensitive spectrofluorimetric methods, we quantified two cephalosporins, cefepime (CFM) and cefazolin (CFZ), in raw and pharmaceutical formulations. The first method is based on the reaction between CFM and fluorescamine (borate buffer, pH 8), which yields a highly fluorescent product. After excitation at 384 nm, the fluorescent product emits light at 484 nm. At concentration ranges from 12.0 to 120.0 ng ml−1, the relative fluorescence intensity/concentration curve was linear with a limit of quantification (LOQ) of 2.46 ng ml−1. The second method relied on measuring the CFZ quenching action on acriflavine fluorescence through formation of an ion-associate complex using Britton–Robinson buffer at pH 8. We measured acriflavine fluorescence at 505 nm after excitation at 265 nm. The decrease in acriflavine fluorescence intensity was CFZ concentration-dependent. Using this method, we quantified CFZ in concentrations ranging from 1 to 10 µg ml−1 with a LOQ of 0.48 µg ml−1. We studied and optimized the factors influencing reaction product formation. Moreover, we adapted our methods to the investigation of the mentioned drugs in raw and pharmaceutical formulations with greatly satisfying results. We statistically validated our methods according to International Council on Harmonisation Guidelines. The obtained results were consistent with those obtained with the official high-performance liquid chromatography methods.

Collagenases are important enzymes frequently used for isolation of cells. Accurate estimation of collagenase activity is an important requirement. Available techniques for assaying collagenase activity are (i) ninhydrin-based assays, in which ninhydrin reacts with freshly generated N-termini resulting from collagenase action upon collagen, with a colorimetric read-out, (ii) synthetic substrate-based assays, in which collagenase action releases either a radioactive group or a fluorescent moiety. These methods are associated with some drawbacks. Here, we have standardized a simple, cost-effective method for assaying collagenase activity, using fluorescence readouts of N-terminal-specific fluorescamine adduction with peptides and proteins at pH 6.0. Collagenase-mediated degradation of gelatin and collagen was performed at pH 7.0. Following incubation, fluorescamine was added at pH 6.0. A standard plot based on the use of BSA (bovine serum albumin) was used for determination of the moles of freshly-generated N-termini detected, based on fluorescence owing to fluorescamine adduction to N-termini. The method was also tested with two other proteases (trypsin and cathepsin-K). Specific reaction of fluorescamine with α amino groups at pH 6.0 can be used for labeling of freshly generated N-termini in collagen subjected to degradation by a collagenase. The kinetic parameters determined by the fluorescamine assay are comparable to those determined previously. Our improved method of using fluorescamine for protease activity addresses the previously associated drawbacks of this assay, thereby lifting this assay up to an unprecedented level of reliability and convenience. We found that the technique can also be extended to use with other proteases. [ABSTRACT FROM AUTHOR]

[Display omitted] • Selective derivatization of primary amine using fluorescamine: a fluorescent probe. • Spectrofluorimetric determination of benoxinate in Benox® eye drops. • First order derivative coupled with fluorescamine-benoxinate reaction product. • The proposed method was fully validated as per pharmacopeial guidelines. In this research, fluorogenic labelling followed by applying first-order derivative spectrofluorimetry for the developed fluorophore is discussed as an alternative, sensitive and selective analytical approach. Benoxinate, containing a primary amine and fluorescamine reagent were selected for the study. Then the proposed methodology relies on the reaction between the primary amine in benoxinate with fluorescamine that selectively reacts with primary amines to develop highly fluorescent products. The fluorescamine-benoxinate developed fluorophore is identified by its sharp first-order derivative peak at 465 nm following excitation at 386 nm in borate buffer, pH 8. The optimum reaction conditions were ascertained. Following ICH validation guidelines, the first order derivative of the relative fluorescence intensity for the developed fluorophore was linearly related to benoxinate concentration and ranged from 20.0 to 200.0 ng / mL with a detection limit of 3.36 ng/mL and a quantitation limit of 10.19 ng/mL, moreover, satisfying accuracy and precision values were obtained upon statistical analysis of results. The offered analytical method was successfully applied to quantify benoxinate in raw material and Benox® eye drops as a direct application to a commercial formulation. [ABSTRACT FROM AUTHOR]

Biomacromolecular probes have been extensively employed in the detection of metal ions for their prominent biocompatibility, water solubility, high selectivity, and easy modification of fluorescent groups. In this study, a fluorescent probe FP was constructed. The probe FP exhibited high specificity recognition for Cu 2+ . With the combination of Cu 2+ , the probe was subjected to fluorescence quenching. The research suggested that the probe FP carried out the highly sensitive detection of Cu 2+ with detection limits of 1.7 nM. The fluorescence quenching of fluorescamine was induced by Cu 2+ perhaps due to the PET (photoinduced electron transfer) mechanism. The FP-Cu 2+ complex shows weak fluorescence, which is likely due to the PET quenching effect from Cu 2+ to fluorescamine fluorophore. Moreover, the probe FP can be employed for imaging Cu 2+ in living cells. The new fluorescent probe developed in this study shows the advantages of good biocompatibility and low cytotoxicity. It can be adopted for the targeted detection of Cu 2+ in cells, and it has promising applications in the mechanism research and diagnosis of Cu 2+ -associated diseases.

A method was developed for the determination of nonvolatile amines, such as histamine, tyramine, putrescine, and cadaverine, in foods. These nonvolatile amines were extracted from a sample with 5％ trichloroacetic acid, and the extract was purified using an InertSep MC-1 cartridge column. The four amines were derivatized with fluorescamine, determined by HPLC with a fluorescence detector, and confirmed by LC-MS/MS. The average recoveries（ n＝5） and the relative standard deviations from 11 foods （pacific saury, dried mackerel, canned mackerel in brine, canned tuna in oil, fish sauce, surimi, rice-koji miso, soy sauce, gouda cheese, red wine, and beer） spiked at 100 mg/kg were 81-100％ and 0.4-3.1％, respectively. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Hydrophilic-interaction planar chromatography is an efficient technique for the separation of polar compounds. • Creative utilization of experimental design approach in optimization of chromatographic conditions. • Innovative combination between modified QuEChERS extraction technique and HILIC planar chromatography. • Economic application of pre-chromatographic derivatization with fluoresacmine to achieve the highest sensitivity. • Applicability of the developed HPTLC method to a pharmacokinetic study of cefalexin in goat milk samples. Abstract A highly sensitive, selective and precise HPTLC method coupled with fluorescence detection was developed and validated for the determination of α-aminocephalosporin antibiotics; namely cefalexin, cefadroxil and cefradine in their standard solutions. The applicability of the developed methodology was demonstrated via analysis of cefalexin in goat milk samples. Full optimization of the fluorescence derivatization reaction was carried out with regard to the standard solutions of the studied compounds or after extraction of milk samples. The separation of the studied compounds was performed on HPTLC precoated silica gel plates 60 F 254 using acetonitrile: water in a ratio 85:15 (v/v) as a mobile phase. The retention behavior of the formed derivatives was discussed in detail. It was found that hydrophilic interaction mode is the main interaction mechanism governing the retention of the formed derivatives. In addition, an experimental design approach was conducted for optimization of the chromatographic conditions. Modified QuEChERS was applied as an efficient extraction technique of cefalexin from both spiked and real goat milk samples. Optimization of QuEChERS extraction technique to achieve the highest extraction recovery was performed and the results indicate that this method provides a good extraction recovery (83–116%) for cefalexin from goat milk samples. Limit of detection (LOD) of the developed method was found to be 0.023, 0.005, and 0.023 ng band−1 for cefalexin, cefadroxil and cefradine, respectively in their standard solutions and 0.165 ng band−1 for cefalexin in goat milk samples. According to the achieved LOD values, the method sensitivity was quasi-equivalent to other methods based on expensive techniques such as HPLC-UV and HPLC-MS and it is sufficient to determine cefalexin below its MRL in milk samples. Moreover, the method was successfully applied to a pharmacokinetic study of cefalexin in goat milk after single intramuscular injection of 10 mg of cefalexin kg−1 per body weight. [ABSTRACT FROM AUTHOR]

Abstract Ceftazidime pentahydrate (CEF) and Vancomycin hydrochloride (VAN) are antimicrobial drugs they are used worldwide especially in developing countries for management several diseases caused by bacterial infections especially against Pseudomonas species. From this point, ultrasensitive, simple, rapid, cost effective method was developed for assay of CEF and VAN in real human plasma. The proposed spectrofluorimetric method was achieved using fluorescamine reagent. This method based on reaction of fluorescamine reagent with primary amine moiety (aromatic and aliphatic) in CEF and VAN respectively, with calibration graph from 30 to 300 ng mL−1 and 0.5–30 ng mL−1 was plotted under optimum conditions. The investigated method was developed and bio-analytically validated using ICH and US-FDA recommendations. The proposed method was successfully used for determination of the cited drugs in real human plasma with high percentage of recovery ranged from 95.72 ± 0.95% to 97.72 ± 2.02 and pharmaceutical formulations with percentage 101.55 ± 0.55% and 101.99 ± 0.43% for CEF and VAN respectively. The study was also utilized to examine the stability of the CEF and VAN in real human plasma. Highlights • Ultrasensitive spectrofluorimetric method for determination of Ceftazidime (CEF) and vancomycin (VAN) was developed. • The determination based on reaction of cited drugs with fluorescamine reagent. • The proposed method was validated according to ICH guidelines and FDA recommendations. • The proposed methods were applied for detection of CEF and VAN in real human plasma. [ABSTRACT FROM AUTHOR]

A method for the screening of biogenic amines in waters, whose presence at some concentration levels potentially cause adverse effects on humans, was developed for the first time. A suitable and easy to operate system, with low reagent consumption was devised. The proposed flow-based system was divided into two analytical parts, preconcentration and derivatization of the biogenic amines. Solid phase extraction, using a Chelex 100 resin, was the newly chosen strategy for preconcentration of the analyte and also removal of possible matrix interferences. Fluorescamine was used as derivatization reagent for biogenic amines followed by fluorimetric detection. The influence of different sorbent materials for preconcentration and flow system parameters such as pH of standards and buffer, composition of the eluent solution, flow-rates, standard/sample volume, were studied. The interference of ammonia was assessed, and no interference was observed. The limits of detection and quantification were 1.7 and 5.6 µmol L−1, respectively. The developed system was applied to water samples and the recovery results were 98 ± 7%. [ABSTRACT FROM AUTHOR]

A novel sensitive and simple spectrofluorimetric method was developed then validated for determination of midodrine in both its authentic pure form and its tablets. This method is based on the reaction between midodrine's aliphatic primary amine moiety with fluorescamine reagent, using borate buffer at pH 7.8 and yielding a highly fluorescent product whose fluorescence intensity was measured at 462 nm after excitation at 388 nm. This method represents the first attempt for determination of midodrine spectrofluorimetrically. A calibration curve was constructed showing that the linear range was 0.2–3.0 μg/ml. The limit of detection and limit of quantitation values were 0.06 and 0.19 μg/ml respectively. The correlation coefficient (r) and the determination coefficient (r2) values were 0.9992 and 0.9984 respectively. The proposed method was validated according to ICH guidelines and successfully applied for determination of midodrine in its tablets with an overall % recovery of 99.56 ± 0.95. Finally, the presented method was adapted to study the content uniformity test according to United States Pharmacopeia guidelines. [ABSTRACT FROM AUTHOR]

Avapritinib (AVP) was the first precision drug to be approved by the US Food and Drug Administration (FDA) in 2020 for patients suffering from metastatic gastrointestinal stromal tumors (GISTs) and progressive systemic mastocytosis. The analysis of AVP in pharmaceutical tablets and human plasma was then carried out using a fast, efficient, sensitive, and simple fluorimetric method using a fluorescamine reagent. The procedure is based on the interaction between fluorescamine as a fluorogenic reagent and the primary aliphatic amine moiety in AVP using borate buffer solution at pH 8.8. The produced fluorescence was measured at 465 nm (Excitation at 395 nm). The calibration graph's linearity range was discovered to be 45.00-500.0 ng mL -1 . Utilizing the International Council for Harmonization (ICH) and US-FDA recommendations, the research technique was validated and bioanalytically validated. The proposed approach was effectively employed for determining the stated pharmaceuticals in plasma with a high percentage of recovery ranging from 96.87 to 98.09 and pharmaceutical formulations with a percentage of recovery equal to 102.11% ± 1.05%. In addition, the study was extended to a pharmacokinetic study of AVP with 20 human volunteers as a step for AVP management in therapeutic cancer centers., (© 2023 John Wiley & Sons Ltd.)

In‐capillary derivatization using fluorescamine as the labeling reagent was proposed to enhance the detectability of adamantine drugs (memantine, amantadine and rimantadine) by spectrophotometric detection. Fluorescamine and the drugs were delivered to the capillary electrophoresis instrument at a ratio of 10:1 by zone injection. The derivatization reaction occurred following the application of voltage (20 kV). The derivatized products, hydrolyzed‐ fluorescamine and excess fluorescamine were separated in 7 min using 100 mM borate buffer (pH 10.0) containing 0.1% w/v of Brij®‐35 and 20% v/v of acetonitrile. Validation data showed good linearity (r2 > 0.98), precision (%RSDs < 3.4), and accuracy (recoveries ranging from 98.0 to 102.0%). The detection and quantitation limits are in the range of 6.0–8.5 and 18–25 μM, respectively. The validation data is comparable to reported methods, however, the current method offers better precision with enhanced sensitivity (up to six times). Applications of the method show percent labeled amounts found in the studied samples within 100.6–109.3%, which complied with the United States Pharmacopeia limit (90.0–110.0%). The method was simple, rapid and, automated, which required no extra instrumentation or skillful operators. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive method for determining sulfonamides in water was developed and validated through in situ derivatization and hollow‐fiber liquid‐phase microextraction with ultra‐high performance liquid chromatography and fluorescence detection. The target sulfonamides were sulfadiazine, sulfacetamide, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole, and sulfisoxazole. Following in situ derivatization with fluorescamine, three‐phase hollow‐fiber liquid‐phase microextraction with an S 6/2 polypropylene hollow‐fiber membrane was applied automatically using a multipurpose autosampler. Experimental parameters including derivatization time, choice of organic phase, pH of donor and acceptor phase, stirring rate, extraction temperature and time were optimized. Under optimized conditions, the target sulfonamides achieved excellent linearity with correlation coefficients of 0.9924–0.9994 within the concentration range of 0.05–5 μg/L. The limits of detection of the eight sulfonamides were 3.1–11.2 ng/L, and the limits of quantification were 10.3–37.3 ng/L. Enrichment factors of 0.1 and 5 μg/L sulfonamides spiked in lake water were 14–60, and recoveries were 56–113% with relative standard derivations of 3–19%. Applied with the developed method, sulfamerazine and sulfamethoxazole were measurable in both influent and effluent water of the three sewage treatment plants in Guangzhou, China. The developed method was sensitive and provided an alternative method for simultaneously enriching and quantifying multiple sulfonamides in environmental water. [ABSTRACT FROM AUTHOR]

Synthesis and spectral study of two new cyano-substituted fluorescamine as the fluorescent probes for amino acid detection have been carried out comparing with the original fluorescamine. Of the three compounds, the derivative with a cyano group at the meta -position on the 4-phenyl group was found to be superior to the original one in the reactivity toward some amino acids as well as the fluorescence intensity of the adducts. The fluorescent amino acid adducts were also applied to the peroxyoxalate chemiluminescence system as the fluorophores, in which the derivative described above was found to be more effective also in chemiluminescence than the original one. [ABSTRACT FROM AUTHOR]

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called “edge effect”), thereby increasing usable wells to the entire plate, not just interior wells. [ABSTRACT FROM AUTHOR]

Aggregation of amyloid-β (Aβ) protein plays a central role in Alzheimer's disease. Because protein aggregation is a concentration-dependent process, rigorous investigations require accurate concentration measurements. Owing to the high aggregation propensity of Aβ protein, working solutions of Aβ are typically in the low micromolar range. Therefore, an ideal Aβ quantification method requires high sensitivity without sacrificing speed and accuracy. Absorbance at 280 nm is frequently used to measure Aβ concentration, but the sensitivity is low with only one tyrosine and no tryptophan residues in the Aβ sequence. Here we present a fluorescence method for Aβ quantification using fluorescamine, which gives high fluorescence upon reaction with primary amines. We show that, using hen egg white lysozyme as a standard, fluorescence correlates linearly with primary amine concentration across a wide range of fluorescamine concentrations, from 62.5 to 1000 µM. The maximal sensitivity of detection is achieved at a fluorescamine concentration of 250 µM or higher. The fluorescamine method is compatible with the presence of dimethyl sulfoxide, which is commonly used in the preparation of Aβ oligomers, and limits the use of absorbance at 280 nm due to its high background reading. Using aggregation kinetics, we show that the fluorescamine method gives accurate concentration measurements at low micromolar range and leads to highly consistent aggregation data. We recommend the fluorescamine assay to be used for routine and on-the-fly concentration determination in Aβ oligomerization and fibrillization experiments.

A new, sensitive, and selective high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of moxifloxacin (MOX) in human breast milk. MOX was precolumn derivatized with fluorescamine; the fluorescent derivative was separated on an RP C18 column using a mobile phase composed of acetonitrile-10 mM orthophosphoric acid by isocratic elution with flow rate of 0.5 mL min-1. The method was based on the measurement of the derivative using fluorescence detection at 481 nm with excitation at 351 nm. The calibration curve was linear over the range of 1-40 μg mL-1. Limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.3 and 1 μg mL-1, respectively. Intra-day and interday repeatabilities were less than 3.15%. [ABSTRACT FROM AUTHOR]

Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yieldedR2 ≥ 0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously. [ABSTRACT FROM AUTHOR]

Abstract N -acyl- l -homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format. Highlights • N -acyl- l -homoserine lactone (AHL) acylases degrade quorum sensing molecules. • A fluorescamine-based assay for the detection of homoserine lactone was optimised. • This assay can be used to screen potential acylases for activity towards AHLs. [ABSTRACT FROM AUTHOR]

Here we report the validation of a derivatization method that makes use of fluorescamine as a selective reactant for the quantitative analysis of peptide and protein drugs in the dissolution profile from depot formulations. Typical current methods require separation of the nano/microparticles and time-consuming chromatographic runs. In this study we report a method which can be conducted without the need for complete physical separation of the particles or removal of the unreacted probe. This method was used here for the analysis of the release profile of octreotide in a depot formulation, with results in excellent agreement with reported chromatographic assays.

Herein, a sensitive and selective spectrofluorimetric method has been developed for the determination of the ocular local anesthetic benoxinate hydrochloride (BEN-HCl) in eye drops and artificial aqueous humour. The proposed method is based on the interaction of fluorescamine with the primary amino group of BEN-HCl at room temperature. Following the excitation of the reaction product at 393 nm, the emitted relative fluorescence intensity (RFI) was measured at 483 nm. The key experimental parameters were carefully examined and optimized by adopting an analytical quality-by-design approach. The method used a two-level full factorial design (2 4 FFD) to obtain the optimum RFI of the reaction product. The calibration curve was linear at the range of 0.10-1.0 μg/mL of BEN-HCl with sensitivity down to 0.015 μg/mL. The method was applied for analyzing the BEN-HCl eye drops and could also assess its spiked levels in artificial aqueous humour with high % recoveries (98.74-101.37%) and low SD values (≤ 1.11). To investigate the green profile of the proposed method, a greenness assessment was performed with the aid of the Analytical Eco-Scale Assessment (ESA) and GAPI. The developed method obtained a very high ESA rating score in addition to being sensitive, affordable, and environmentally sustainable. The proposed method was validated according to ICH guidelines., (© 2023. The Author(s).)

O objetivo deste trabalho foi avaliar e validar um método para deteminação de resíduos de sulfatiazol (STZ), sulfametazina (SMZ) e sulfadimetoxina (SDM) em leite UHT integral. A extração foi realizada com diclorometano e coluna de extração em fase sólida de sílica. Os resíduos, após derivação com fluorescamina, foram quantificados por cromatografia líquida de alta eficiência com detector de fluorescência. O limite de detecção das três sulfas em amostra de leite integral foi 0,3 µg L-1 e o limite de quantificação foi 1 µg L-1 para STZ e SMZ e 2,5 µg L-1 para SDM, com coeficientes de variação entre 4,4 e 6,6%. Os valores de recuperação para STZ, SMZ e SDM foram 63,2, 91,2 e 63,2%, respectivamente. Considerando o limite máximo de resíduo estabelecido pela legislação brasileira de 100 µg kg-1 para a soma das concentrações totais de STZ, SMZ e SDM, o método descrito permite a determinação simultânea dos três analitos em amostras de leite UTH integral.The objective of this work was to evaluate and validate a method for analysis of sulfathiazole (STZ), sulfamethazine (SMZ) and sulfadimethoxine (SDM) residues in milk. Extraction was carried out with diclhoromethane followed by silica solid phase extraction. The extracts were derivatizated with fluorescamine and quantified by reversed-phase high performance liquid chromatography with fluorescence detection. The detection limit for the three sulfonamides was 0.3 µg L-1 and the quantification limit was 1 µg L-1 for STZ and SMZ and 2.5 µg L-1 for SDM, with coefficient of variation ranging from 4.4 to 6.6%. The recoveries for STZ, SMZ and SDM were 63.2, 91.2 and 63.2%, respectively. Considering that Brazilian regulation sets maximum residue limit in milk of 100 µg kg-1 for total sulfonamide concentrations (STZ, SMZ and SDM), the present method is adequate to quantify the residues of three sulfonamides simultaneously in UHT milk.

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine-threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R = 0.956. [ABSTRACT FROM AUTHOR]

A highly sensitive, simple and selective spectrofluorimetric method has been developed and validated for determination of doxazosin mesylate in pure form, pharmaceutical formulations and human plasma. The method is based on the reaction between doxazosin mesylate and fluorescamine in Teorell buffer solution (pH 3) to give highly fluorescent derivative that can be measured at 489 nm using excitation wavelength of 385 nm. Different experimental parameters affecting the reaction were carefully studied and optimized. The calibration plot was constructed over the concentration range of 16–400 ng mL − 1 with quantitation limit of 14.3 ng mL − 1 . The developed procedure was validated according to ICH guidelines and the results were satisfactory. The proposed method has been successfully applied to the analysis of the cited drug in its pharmaceutical preparations as well as for content uniformity testing. The results showed excellent agreement with the reported method with respect to precision and accuracy. In addition, the drug concentration was determined in the spiked human plasma by the suggested method with % recovery in the range of 96.2–98.3% (SD; 0.76–0.93, n = 5). [ABSTRACT FROM AUTHOR]

Angiotensin-(1-7) is an important active component in the renin-angiotensin-system. Due to its cardio protective effects it is now under investigation in combination with antioxidants as a reperfusion solution. The combination showed impressive effects on isolated hearts of male Wistar rats after induced ischemia. In this work a high performance liquid chromatography method with fluorescence detection was developed for the first time for in-process measurements as well as for stability tests of the peptide in the novel antioxidant-containing Karal® solution. For fluorescence detection of angiotensin-(1-7) fluorescamine as derivatization dye was applied. Under optimized conditions the method showed linearity over the range of 50 to 5000 ng/mL with R of 0.9988 and an overall precision better than 5.0 %. LOD and LOQ were determined to be in the femtomol range on column. It was found that stability of angiotensin-(1-7) could be significantly improved in the antioxidant containing preparation compared to aqueous solutions. [ABSTRACT FROM AUTHOR]

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions. [ABSTRACT FROM AUTHOR]

Deposition of amyloid-β (Aβ) aggregates in the form of amyloid plaques is a central feature of Alzheimer's disease. The end products of Aβ aggregation are amyloid fibrils. Soluble Aβ aggregates called oligomers are also formed either on or off the pathway of fibril formation. The amyloid fibrils from different clinical subtypes of Alzheimer's disease have been found to adopt different structures, a phenomenon called fibril polymorphism. Meanwhile, different types of Aβ oligomers have also been found. Recently, it has been shown that different types of Aβ42 oligomers may form fibrils of different structures, linking oligomer heterogeneity to fibril polymorphism. In this chapter, we describe methods to prepare heterogeneous Aβ42 oligomers and to quantify the concentration of these oligomers at a low micromolar range using a fluorescamine method. Fractionation of these oligomers by size using ultrafiltration filters allows for the formation of Aβ42 fibrils with different structural properties., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)