Construction of a New Probe Based on Copper Chaperone Protein for Detecting Cu2+ in Cells.

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 Cu2+. With the combination of Cu2+, the probe was subjected to fluorescence quenching. The research suggested that the probe FP carried out the highly sensitive detection of Cu2+ with detection limits of 1.7 nM. The fluorescence quenching of fluorescamine was induced by Cu2+ perhaps due to the PET (photoinduced electron transfer) mechanism. The FP-Cu2+ complex shows weak fluorescence, which is likely due to the PET quenching effect from Cu2+ to fluorescamine fluorophore. Moreover, the probe FP can be employed for imaging Cu2+ 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 Cu2+ in cells, and it has promising applications in the mechanism research and diagnosis of Cu2+-associated diseases.

Applicability of fluorescamine as a fluorogenic reagent for highly sensitive fluorimetric analysis of the tyrosine kinase inhibitor (avapritinib) in pharmaceuticals and biological samples.

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.

Application of quality-by-design for adopting an environmentally green fluorogenic determination of benoxinate hydrochloride in eye drops and artificial aqueous humour.

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 (24 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.

Sulfonamide drugs: Low-cost spectrofluorometric determination using a computer monitor calibrator for detection.

A possibility of the use of a common monitor calibrator as a portable and inexpensive tool for the fluorometric determination of sulfonamide drugs after their reaction with fluorescamine was examined. The luminescence measurements with a calibrator are based on irradiation of a test sample by the device lamp with a broadband spectrum in the visible and near UV regions and simultaneous registration of the secondary radiation by the device detector. Two types of cuvettes with black light absorbing sides eliminating the reflected self-radiation were tested. The commercially available Eppendorf-type black plastic microtubes ("LightSafe") were suggested as a good option for such measurements. It was shown that a monitor calibrator can be applied for optimization of the determination conditions. By the example of sulfanilamide and sulfamethazine, it was shown that the procedure should be carried out at pH 4-6 and fluorescamine concentration of 200 µmol L-1, and 40 min of the interaction. The limit of detection of sulfanilamide and sulfamethazine using a monitor calibrator is 0.9 µmol L-1 and 0.8 µmol L-1, respectively, which is comparable with their spectrophotometric determination.

Salvage parenteral antibiotics for multidrug-resistant (MDR) Gram-negative bacteria; a fluorescamine-based technique for ultrasensitive spectrofluorimetric measurement of Polymyxins; human plasma application.

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.

A feasible fluorimetric approach anchored in diaryl pyrrolone derivative for the facile analysis of milnacipran in tablets; evaluation of the method greenness.

In the present work a new, feasible, and green approach was employed for the analysis of milnacipran. A drug is used in the management of depression in addition to fibromyalgia. It inhibits of the reuptake of two essential neurotransmitters serotonin and nor-adrenaline. In slightly alkaline buffer (pH 8.5) the primary amino group of milnacipran reacted with fluorescamine to give a substituted pyrrolone derivative which exhibited high fluorescence activity. The fluorescence of produced derivative was measured at (λex 385 nm, λem 477 nm), and the experimental factors were cautiously optimized. The measured intensity of fluorescence was plotted versus the respective concentration of milnacipran to setup the calibration plot which has a linear concentration range of 50-300 ng/mL. The ICH guidelines were utilized to totally validate the presented approach. In addition the method could be efficiently incorporated in the analysis of commercial milnacipram tablets with no considerable effect on the results of the assay for milnacipran. The developed approach is characterized by its high simplicity and greenness of the procedure which make it suitable for routine analysis.

Controlling the semi-permeability of protein nanocapsules influences the cellular response to macromolecular payloads.

Nanocapsules are an excellent platform for the delivery of macromolecular payloads such as proteins, nucleic acids or polyprodrugs, since they can both protect the sensitive cargo and target its delivery to the desired site of action. However, the release of macromolecules from nanocapsules remains a challenge due to their restricted diffusion through the nanoshell compared to small molecule cargo. Here, we designed degradable protein nanocapsules with varying crosslinking densities of the nanoshell to control the release of model macromolecules. While the crosslinking did not influence the degradability of the capsules by natural proteases, it significantly affected the release profiles. Furthermore, the optimized protein nanocapsules were successfully used to deliver and effectively release a bioactive macromolecular vaccine adjuvant in vitro and, thus, can be used as an efficient platform for the design of potential nanovaccines.

Derivatization of tranexamic acid for its rapid spectrofluorimetric determination in pure form and pharmaceutical formulations: Application in human plasma.

An ingenious approach for determination of tranexamic acid spectrofluorimetrically has been developed. This experiment is very simple, sensitive and selective method for determination of tranexamic acid in pure form, pharmaceutical dosage forms and in spiked human plasma. All optimal conditions needed in our proposed experiment have been determined and validated precisely. This developed method based on the reaction between the primary amino group found in the chemical structure of tranexamic acid with the fluorescamine reagent in presence of borate buffer (pH 8.3) that result in the formation of fluorescence product measured at 473.5 nm after excitation at 392 nm. We notice that the linearity of the resulted calibration curve found to be (0.1-0.9 µg/mL) with LOD and LOQ results were 0.0237 and 0.0719 respectively. The validation of the developed method is according to the international council for Harmonization (ICH) guidelines indicating good accuracy and precision. Finally, the developed method has been applied for in vitro study of tranexamic acid by making spiked human plasma with a mean percentage recovery 99.430 ± 0.623 as well as in its pharmaceutical dosage forms tablets and ampoules.

LC-MS/MS quantification of asymmetric dimethyl arginine and symmetric dimethyl arginine in plasma using surrogate matrix and derivatization with fluorescamine.

Aim: A novel LC-MS/MS method using a surrogate matrix and derivatization with fluorescamine was developed and validated for simultaneous quantification of asymmetric dimethyl arginine and symmetric dimethyl arginine. Methods & results: Asymmetric dimethyl arginine, symmetric dimethyl arginine and corresponding internal standards were extracted using protein precipitation and derivatization with fluorescamine followed by SPE. Derivatives were analyzed by turbo ion spray LC-MS/MS in the positive ion mode. Methodology was successfully transferred across multiple preclinical species and utilized in the support of several investigative studies. Conclusion: A new LC-MS/MS analytical methodology that utilizes a surrogate matrix and derivatization with fluorescamine was successfully developed and validated.

Protective actions of bioactive flavonoids chrysin and luteolin on the glyoxal induced formation of advanced glycation end products and aggregation of human serum albumin: In vitro and molecular docking analysis.

The post-translational modification of proteins by nonenzymatic glycation (NEG) and the accumulation of AGEs are the two underlying factors associated with the long-term pathogenesis in diabetes. Glyoxal (GO) is a reactive intermediate which has the ability to modify proteins and generate AGEs at a faster rate. Human serum albumin (HSA) being the most abundant serum protein has a higher chance to be modified by NEG. The key objective of the present study is to investigate the potency of chrysin and luteolin as antiglycating and antifibrillating agents in the GO-mediated glycation and fibril formation of HSA. AGEs formation were confirmed from the absorption and fluorescence spectral measurements. Both the flavonoids were able to quench the AGEs fluorescence intensity in vitro indicating the antiglycating nature of the molecules. The formation of fibrils in the GO-modified HSA was confirmed by the Thioflavin T (ThT) fluorescence assay and the flavonoids were found to exihibit the antifibrillation properties in vitro. Docking results suggested that both the flavonoids interact with various amino acid residues of subdomain IIA including glycation prone lysines and arginines via non-covalent forces and further stabilized the structure of HSA, which further explains their mechanisms of action as antiglycating and antifibrillating agents.

Spectrofluorometric determination of alogliptin an antidiabetic drug in pure and tablet form using fluorescamine, a fluorogenic agent: application to content uniformity test.

Alogliptin is an antidiabetic drug that belongs to a group called dipeptidyl peptidase-4 enzyme inhibitors. As the drug contains a primary amino group in its structure, it readily reacts with fluorescamine in slightly alkaline medium (borate buffer, pH 8.8) to form a highly fluorescent product. Emission of this product was measured at 477 nm (λex = 387 nm). The linear range between the fluorescence intensity and the drug concentration was 0.1-0.5 µg ml-1 with a good correlation coefficient (0.9986). Limits of detection and quantitation were 22 and 72 ng ml-1 , respectively. Guidelines of the International Conference for Harmonisation were followed to validate the developed method with acceptable results. Alogliptin content was determined successfully in its commercial dosage form using the fluorescamine method with good recovery (98.60-101.26%). The method has excellent levels of accuracy and precision compared with the reported method as assessed using Student's t-test and Fisher's exact test. The method was applied successfully for the content uniformity test with high recovery and low relative standard deviation.

The convenient use of fluorescamine for spectrofluorimetric quantitation of pramipexole in pure form and pharmaceutical formulation; application to content uniformity testing.

Pramipexole is a selective dopamine receptor agonist which is used in the treatment of Parkinson's disease and restless legs syndrome. The present work illustrates the development and validation of a sensitive and selective spectrofluorometric method for quantitation of pramipexole (PMP) through its interaction with fluorescamine at pH 7.5 using aqueous borate buffer to produce a highly fluorescent product. The fluorescent intensity of the formed product was measured at 480 nm after excitation at 391 nm. Experimental factors that could influence the formation, stability and the fluorescence intensity of the formed product were investigated and optimized. The linearity of the proposed method was achieved in the concentration range of 0.05-2.0 µg/mL. The quantitation and detection limits were 47 and 15 ng/mL, respectively. The proposed method has been validated in respect to guidelines of ICH and pharmaceutical tablets of PMP were successfully analyzed. Moreover, the method was applied for studying the content uniformity test according to the guidelines of United States Pharmacopeia.

Fluorescamine-based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma.

An innovative approach to determine heptaminol spectrofluorimetrically was developed, determining the optimum conditions needed, then validated for determination of heptaminol in its pure form, its tablets and in spiked human plasma. The presented method is based on the reaction between fluorescamine reagent with the primary amine group found in heptaminol, using a borate buffer at pH 9.0 that yields a highly fluorescent product, fluorescence was measured at 471 nm after excitation at 393 nm. The linearity of the constructed calibration curve was (75-850 ng/ml) with LOD and LOQ values 23.85 and 72.29 ng/ml respectively. The method was validated following the International Council for Harmonisation (ICH) guidelines indicating good accuracy and precision. Finally, the presented approach was adapted for in vitro study of heptaminol in spiked human plasma with a mean percentage recovery 100.52 ±â€¯1.19% as well as in its tablets with a mean percentage recovery 99.47 ±â€¯1.25%.

[Determination of Nonvolatile Amines in Foods by HPLC Following Fluorescamine Derivatization].

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.

Hantzsch pre-column derivatization for simultaneous determination of alendronate sodium and its pharmacopoeial related impurity: Comparative study with synchronous fluorometry using fluorescamine.

High performance liquid chromatographic (HPLC) method with a pre-column derivatization based on Hantzsch condensation reaction was applied for simultaneous determination of alendronate sodium (ALN) and its main related impurity, 4-Aminobutanoic acid (ABA) at its pharmacopeial limit. The separation of colored condensation products of ALN and ABA were achieved on Agilent Zobrax Eclipse SB-C18 analytical column (250 × 4.6 mm, 5 µm) using a mobile phase composed of acetonitrile-0.1 M acetate buffer, pH 5.0 (15:85, v/v). The flow rate was 1 mL min-1. The detection was carried out at 340 nm using photo-diode array detector. Peak areas were used for the linear regression line in the range of 10-500 and 0.2-40 µg mL-1 for ALN and ABA, respectively. Different conditions for the optimization of the derivatization reactions as well as for the HPLC measurement were studied. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. This method was used to check the purity of ALN in the presence of ABA (related impurity) at the pharmacopeial limit (0.5%). For comparison purpose, another method was proposed which involves synchronous fluorescence measurement after ALN reaction with fluorescamine. In this method, the third derivative synchronous spectra were estimated as peak to peak measurement from 339 to 370 nm for ALN determination with LOD and LOQ of 24 and 73 ng mL-1, respectively, showing very high sensitivity. Both methods have been applied for determination of the alendronate sodium (ALN) in bulk and pharmaceutical preparations without interference of additives in tablets or oral solution.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization.

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3⁻114.0%), with repeatability and reproducibility in the range of 2.7⁻9.1% to 5.9⁻14.9%, respectively. Decision limit (CCα) and detection capability (CCß) were 197.7⁻274.6 and 263.2⁻337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5⁻79.5 and 41.3⁻89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing.

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.

In Vivo Femtosecond Laser Subsurface Cortical Microtransections Attenuate Acute Rat Focal Seizures.

Recent evidence shows that seizures propagate primarily through supragranular cortical layers. To selectively modify these circuits, we developed a new technique using tightly focused, femtosecond infrared laser pulses to make as small as ~100 µm-wide subsurface cortical incisions surrounding an epileptic focus. We use this "laser scalpel" to produce subsurface cortical incisions selectively to supragranular layers surrounding an epileptic focus in an acute rodent seizure model. Compared with sham animals, these microtransections completely blocked seizure initiation and propagation in 1/3 of all animals. In the remaining animals, seizure frequency was reduced by 2/3 and seizure propagation reduced by 1/3. In those seizures that still propagated, it was delayed and reduced in amplitude. When the recording electrode was inside the partially isolated cube and the seizure focus was on the outside, the results were even more striking. In spite of these microtransections, somatosensory responses to tail stimulation were maintained but with reduced amplitude. Our data show that just a single enclosing wall of laser cuts limited to supragranular layers led to a significant reduction in seizure initiation and propagation with preserved cortical function. Modification of this concept may be a useful treatment for human epilepsy.

Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-l-homoserine lactone acylase activity.

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.

In-capillary derivatization with fluorescamine for the rapid determination of adamantane drugs by capillary electrophoresis with UV detection.

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.