Development of sensitive spectrofluorometric approach based on formation of pyrrolidone derivative for determination of linagliptin through condensation reaction with ninhydrin and phenylacetaldehyde: Application to content uniformity and real plasma.

The new drug linagliptin belongs to the class of dipeptidyl peptidase-4 enzyme inhibitors. Linagliptin is used to treat type 2 diabetes and is taken orally either alone or in combination with other drugs. In this instance, a new, simple, and economical technique for analyzing linagliptin was developed by the effective use of a pyrrolidone derivative. The primary amine group of linagliptin permits its condensation with ninhydrin (0.14% w/v) to produce a fluorescent product in the presence of phenylacetaldehyde (0.02% v/v). All experimental parameters were carefully examined and adjusted in order to monitor the generation of the pyrrolidone derivative at excitation and emission wavelengths of 385 and 475 nm, respectively. The calibration graph was made by plotting the intensity of the fluorescence in relation to linagliptin concentration. A significant linearity was found for values ranging from 20 to 460 ng/mL. The process's validity has been verified by a thorough assessment of the instructions provided by the International Conference on Harmonization (ICH). The results indicate excellent uniformity with a reference method, showing that there is no substantial difference in precision and accuracy. The proposed approach was utilized for determining linagliptin in real rat plasma successfully owing to its high sensitivity. Additionally, the proposed approach was evaluated using the Eco-Scale evaluation tool and showed a high degree of eco-friendliness (86/100).

The Ninhydrin Reaction Revisited: Optimisation and Application for Quantification of Free Amino Acids.

The ninhydrin reaction is commonly used for the detection of amino acids. However, in the literature, different conditions with respect to the buffer system, its pH and concentration, type of organic solvent, incubation time, and temperature, as well as the concentrations of the reagents, are described. To identify the most suitable conditions, colour development with reagents of varying compositions and different reaction temperatures and times were investigated using asparagine as a model amino acid. Asparagine was selected since it is one of the most abundant free amino acids in many types of samples. The optimal reaction mixture consisted of 0.8 mol L-1 potassium acetate, 1.6 mol L-1 acetic acid, 20 mg mL-1 ninhydrin and 0.8 mg mL-1 hydrindantin in DMSO/acetate buffer 40/60 (v/v) (final concentrations). The best reaction condition was heating the samples in 1.5 mL reaction tubes to 90 °C for 45 min. Afterwards, the samples were diluted with 2-propanol/water 50/50 (v/v) and the absorbance was measured at 570 nm. The proteinogenic amino acids showed a similar response except for cysteine and proline. The method was highly sensitive and showed excellent linearity as well as intra-day and inter-day reproducibility.

Designing stimuli-responsive upconversion nanoparticles based on a mimetic immunoassay for potential accurate diabetic nephropathy diagnosis.

Diabetic nephropathy (DN) is the most common microvascular complication associated with incurable diabetes. The gold standard diagnostic method for DN is based on the detection of proteinuria but it overlooks cases of non-proteinuria (NP-DN). To address this limitation, urinary sialic acid (SA) has been confirmed as an effective biomarker for various DNs. Herein, we constructed an ultrasensitive non-proteinuria assay platform to accurately diagnose DN within 20 min. This platform utilized the ninhydrin reaction between acidic ninhydrin and urinary sialic acid (SA) as an effective biomarker for various DNs. A compound with a maximum absorption peak at 470 nm was produced in this reaction and contributed to the fluorescence decrease of the blue-emission core-shell upconverting nanoparticles through the inner filter effect (IFE). By integrating the inner filter effect (IFE) with a mimetic immunoassay, the imperceptible color was converted into highly sensitive fluorescence signals. This protocol shows a stable and high sensitivity with a detection limit of 20 nmol L-1 and provides 100% positive prediction for urine samples, demonstrating its potential for clinical diagnosis and long-term monitoring of DN.

Survey reaction of 2-amino-N-(aryl) benzimidamides with ninhydrin in different conditions and investigation of reaction mechanism using density functional theory (DFT).

In this investigation, firstly, 1-(2-amino-phenyl)-N-(aryl) methane diamine derivatives were synthesized by reaction of 2-aminobenzo nitrile with aromatic amines in the presence of aluminum chloride as the catalyst. Then, the reaction of these intermediates with ninhydrin in different conditions was investigated. The reaction between ninhydrin and 2-amino-N'-(aryl) benzimidamide derivatives in water as solvent under reflux conditions resulted in the synthesis of diazepine derivatives. The same results were obtained when the reaction was done in EtOH and in the presence of a few drops of sulfuric acid at room temperature. Also, this reaction was carried out in ethanol as solvent without the presence of sulfuric acid at room temperature which resulted in the synthesis of spiro [indene-2,2'-quinazoline] derivatives. And finally, the reaction was carried out in ethanol as solvent without the presence of sulfuric acid at the reflux conditions which resulted in the synthesis of isoquinolino-quinazoline derivatives. These N-heterocycles compounds are important biologically. Mild reaction conditions, simple procedure and purification and also product diversity with changing conditions are important advantages of this method. Also, to better understanding reaction mechanism on the condensation reactions of 2-amino-N-(aryl) benzimidamides with ninhydrin in different conditions, density functional theory (DFT)-based quantum chemical methods have been applied. Calculated atomic charges suggest that the C-1 (+ 0.54 a.u.) center of ninhydrin is a better electrophile than C-2 (+ 0.42 a.u.) center.

Diastereo- and regioselective petasis aryl and allyl boration of ninhydrins towards synthesis of functionalized indene-diones and dihydrobenzoindeno-oxazin-ones.

Petasis aryl and allyl borations were accomplished using substituted ninhydrins, boronic acids or 2-allyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and 1,2-aminophenols in Hexafluoroisopropanol (HFIP) without any catalysts to synthesize different aryl and allyl derivatives of ninhydrins. The nature of substitution in the boronic acids and 1,2-amino phenols was the key factor in determining the diastereo-regioselectivity and the type of product distributions. The products were isolated and characterized by HMBC, HSQC, 1H, 13C NMR experiments and X-ray single crystallographic analysis. A probable reaction pathway involves in situ formation of acyclic and cyclic ninhydrin-amino alcohol adducts, with the positioned hydroxyl group determining the stereo-regioselective outcome via tetracoordinated boron intermediates. A metal free diastereo- and regioselective Petasis aryl and allyl boration of ninhydrins.

A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent.

An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates.

Organocatalytic Regio- and Enantioselective [3 + 2]-Annulations of Ninhydrin-Derived Morita-Baylis-Hillman Carbonates with 3-Methyleneoxindoles.

A type of Morita-Baylis-Hillman (MBH) carbonates has been developed from ninhydrin. These MBH carbonates have been successfully employed as 3C-synthons in the organocatalytic asymmetric [3 + 2]-annulations of the isatin-derived electron-deficient olefins, affording structurally diverse spirooxindoles in high yield with excellent stereoselectivity. In particular, the regioselectivity of MBH carbonates was controlled by the reaction partner, 3-methyleneoxindoles with carbonyl groups (R = ArCO), affording ß-selective products and 3-methyleneoxindoles with ester groups (R = CO2Me) furnishing γ-selective products. The representative scale-up reactions and transformation of product were examined. The reaction mechanism was expounded by control experiments.

Synthesis of 1,3,5-trisubstituted pyrazoles via 1,3-dipolar cycloaddition of nitrile imines with ninhydrin-derived Morita-Baylis-Hillman carbonates.

An effective synthetic method for 1,3,5-trisubstituted pyrazoles via 1,3-dipolar cycloaddition reaction has been developed. This reaction could smoothly proceed between ninhydrin-derived Morita-Baylis-Hillman carbonates and nitrilimines to provide a wide scope of differently substituted pyrazoles in high yields (up to 95%). In addition, the reaction mechanism was also proposed to explain its regioselectivity.

Description of Prasinibacter corallicola gen. nov., sp. nov., a zeaxanthin-producing bacterium isolated from stony coral Porites lutea.

Thermal stress is considered one of the main causes of mass scleractinian coral degradation; however, it is still unknown how corals can adapt to future global warming. In this study, 11 strains of coral-associated Flavobacteria were shown to produce zeaxanthin, a carotenoid antioxidant, which may help coral holobionts to alleviate thermal stress. In addition, a novel zeaxanthin-producing Flavobacterium, designated R38T, was identified using polyphasic taxonomy. Although strain R38T shared a maximum 16S rRNA gene sequence similarity of 93% with Mesoflavibacter aestuarii KYW614T, phylogenetic analyses based on whole genome and 16S rRNA gene sequences revealed that strain R38T forms a distinct branch in a robust cluster composed of strain R38T and Leptobacterium flavescens KCTC 22160T under the family Flavobacteriaceae. Strain R38T exhibited average nucleotide identities of 70.2% and 72.5% for M. aestuarii KYW614T and L. flavescens KCTC 22160T, respectively. The only detected respiratory quinone was menaquinone 6 (MK-6). The genomic DNA G + C content was 33.2 mol%. The major polar lipids were phosphatidylmethylethanolamine, phosphatidylethanolamine, one unidentified ninhydrin phospholipid, three unidentified ninhydrin-positive lipids, and three unidentified lipids. The major cellular fatty acids were iso - C15: 0, iso - C15: 0 ω6c, C16:2 DMA, and C13:1 ω3c. The distinct biochemical, chemotaxonomic, phylogenetic, and phylogenomic differences from validly published taxa suggest that strain R38T represents a new species of a new genus, for which Prasinibacter corallicola gen. nov., sp. nov. is proposed. The type strain R38T (= MCCC 1K03889T = KCTC 72444T).

Design and strategy for spectrofluorimetric determination of tranexamic acid in its authentic form and pharmaceutical preparations: application to spiked human plasma.

A creative, very sensitive and noncomplicated spectrofluorimetric technique was established and further validated to determine tranexamic acid in both its authentic form and its pharmaceutical preparation dosage forms. In the introduced technique, a reaction was found between the aliphatic primary amino group of tranexamic acid and ninhydrin/phenylacetaldehyde reagents in the presence of Torell and Steinhagen buffer pH 7.0, which led to the production of a highly fluorescent product; fluorescence intensity was measured at 475 nm after excitation at 391 nm. A calibration curve was drawn with a linear range of 0.3-2 µg/ml. Limit of detection and limit of quantification values were 0.051 and 0.155 µg/ml respectively. The introduced technique was validated based on the International Council for Harmonisation guidelines and agreed for determination of tranexamic acid in its pharmaceutical formulation. Finally, this simple method was also applied for determination of tranexamic acid in spiked human plasma.

A new feasible approach based on utility of ninhydrin for selective fluorimetric analysis of baclofen. Application to content uniformity evaluation.

In the current proposed analysis, a new, feasible, and selective fluorimetric approach was designed for baclofen assay. Baclofen is a medication prescribed as a therapy for muscle spasticity that originated from multiple sclerosis or a spinal cord injury, and other cases such as hiccups. The analytical approach relies on the use of ninhydrin to form a fluorescent derivative that was monitored at λex 386 nm or λem 480 nm. Under suitable reaction conditions, the primary amino moiety in baclofen was condensed with ninhydrin and phenylacetaldehyde in the presence of Teorel buffer as a buffered medium. The method exhibited linearity when baclofen concentration was plotted against response in the range 1-10 µg ml-1 . Adjustment of the reaction variables and study of validation parameters according to ICH directives were performed correctly. An interference study was implemented to ensure that no discrepancy from the excipient had occurred. Finally, the proposed method was applied successfully for baclofen assay in dosage form and extended to test Mylobac content uniformity.

In Situ Interactions of Eu(TTA)3(H2O)2 with Latent Fingermark Components-A Time-Gated Visualization of Latent Fingermarks on Paper.

We introduce a new latent fingermark (LFM) development method, where compounds showing long lifetime luminescence are generated in situ by the reactions of Eu(TTA)3(H2O)2 with LFM components. Until now, time-gated imaging could not be used to develop LFM on porous surfaces due to the difficulties with selective binding of the developing agents to the fingermark ridges. The nature of the interactions of Eu(TTA)3(H2O)2 with the LFM material has been investigated for three model compounds commonly found in the LFM composition-oleic acid, l-serine, and squalene. The LFMs developed with the europium ß-diketonate complex have been successfully photographed using a time-gated imaging scheme. The presented new approach has been demonstrated to give similar or better results than developing agents commonly used for paper samples (ninhydrin and 1,2-indanedione). Moreover, contrary to the methods mentioned above, the new approach allows for the development of amino acid-poor LFM on paper.

Method comparison of HPLC-ninhydrin-photometry and UHPLC-PITC-tandem mass spectrometry for serum amino acid analyses in patients with complex congenital heart disease and controls.

INTRODUCTION: Metabolomics studies are not routine when quantifying amino acids (AA) in congenital heart disease (CHD). OBJECTIVES: Comparative analysis of 24 AA in serum by traditional high-performance liquid chromatography (HPLC) based on ion exchange and ninhydrin derivatisation followed by photometry (PM) with ultra-high-performance liquid chromatography and phenylisothiocyanate derivatisation followed by tandem mass spectrometry (TMS); interpretation of findings in CHD patients and controls. METHODS: PM: Sample analysis as above (total run time, ~ 119 min). TMS: Sample analysis by AbsoluteIDQ® p180 kit assay (BIOCRATES Life Sciences AG, Innsbruck, Austria), which employs PITC derivatisation; separation of analytes on a Waters Acquity UHPLC BEH18 C18 reversed-phase column, using water and acetonitrile with 0.1% formic acid as the mobile phases; and quantification on a Triple-Stage Quadrupole tandem mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with electrospray ionisation in the presence of internal standards (total run time, ~ 8 min). Calculation of coefficients of variation (CV) (for precision), intra- and interday accuracies, limits of detection (LOD), limits of quantification (LOQ), and mean concentrations. RESULTS: Both methods yielded acceptable results with regard to precision (CV < 10% PM, < 20% TMS), accuracies (< 10% PM, < 34% TMS), LOD, and LOQ. For both Fontan patients and controls AA concentrations differed significantly between methods, but patterns yielded overall were parallel. CONCLUSION: Serum AA concentrations differ with analytical methods but both methods are suitable for AA pattern recognition. TMS is a time-saving alternative to traditional PM under physiological conditions as well as in patients with CHD. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier NCT03886935, date of registration March 27th, 2019 (retrospectively registered).

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR.

3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornene-functionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials. Graphical abstract.

Synthesis of new pyrimidine-containing compounds: 5-(2-(alkylamino)-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)-6-hydroxypyrimidine-2,4(1H,3H)-dione derivatives.

In this study, the one-pot reaction between primary amines, 1,1-bis-(methylthio)-2-nitroethene, ninhydrin, and barbituric acid as an enolizable C-H-activated compound provides a simple method for the preparation of 5-(2-(alkylamino)-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)-6-hydroxypyrimidine-2,4(1H,3H)-dione derivatives with potential synthetic and pharmacological interest. This reaction shows attractive characteristics, such as substrate availability, good yields, existence of numerous hydrogen-bonding possibilities in product, and its mild conditions in ethanol media.

Cascade process for direct synthesis of indeno[1,2-b]furans and indeno[1,2-b]pyrroles from diketene and ninhydrin.

Novel and efficient multicomponent reactions (MCRs) involving diketene, ninhydrin (indane-1,2,3-trione) and one primary amine (3CR) or two different primary amines (4CR) were achieved for the successful synthesis of dihydro-4H-indeno[1,2-b]furan-3-carboxamides or tetrahydroindeno[1,2-b]pyrrole-3-carboxamides, respectively. The merits of this method are highlighted by using either commercially available or easily accessible starting materials, operational simplicity, facile workup procedure, efficient usage of all the reactants, tolerance of a variety of functional groups and ability to conduct under un-catalyzed reaction condition. The products were also isolated by just decantation of the solvent, and for the purification column chromatography was non-required.

Applicability of ninhydrin as a fluorescent reagent for estimation of teicoplanin in human plasma using salting-out assisted liquid-liquid extraction technique.

The applicability of ninhydrin, a widely used derivatizing reagent, for determination of teicoplanin (TEIC) in its pure form, pharmaceutical vials, and in human plasma was investigated. The presented spectrofluorimetric method was based on a condensation reaction between ninhydrin and the primary amine group existing in TEIC (in the presence of phenylacetaldehyde) to produce a highly fluorescent product detected at 460 nm (λex ,390 nm). Calibration plots were constructed in the concentration range 60-600 ng mL-1 with a good correlation coefficient of 0.9998 and a low detection limit of 10.84 ng mL-1 . The method was subjected to a bioanalytical validation study according to US-FDA recommendations. The proposed method was applied for analysis of TEIC in commercial vials with high recovery result 101.88 ± 1.11%. In addition, the method was utilized efficiently for detection of TEIC in human plasma using salting-out assisted liquid-liquid extraction technique (SALLE) with a recovery range from 96.71 ± 1.08% to 97.71 ± 0.86%. SALLE is an effective approach used for extraction of TEIC from human plasma without interferences using ammonium sulphate. The proposed method is highly recommended to monitor TEIC in clinical laboratory samples and therapeutic drug monitoring systems.

Silane Coupling Agent Modifies the Mechanical Properties of a Chitosan Microfiber.

Chitosan microfibers are widely used in medical applications because they have favorable inherent properties. However, their mechanical properties require further improvement. In the present study, a trimethoxysilane aldehyde (TMSA) crosslinking agent was added to chitosan microfibers to improve their tensile strength. The chitosan microfibers were prepared using a coagulation method. The tensile strength of the chitosan microfibers was improved by crosslinking them with TMSA, even when only a small amount was used (less than 1%). TMSA did not change the orientation of the chitosan molecules. Furthermore, aldehyde derived from TMSA did not remain, and siloxane units were formed in the microfibers.

Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach.

Qualitative and quantitative determination of amino acid composition using amino acid analysis (AAA) is an important quality attribute and considered an identity of therapeutic peptide drugs by the regulatory agencies. Although huge literature is available on pre- and post- column derivatization AAA methods, arriving at an appropriate hydrolysis protocol coupled with adequate separation of the derivatized/underivatized amino acids is always challenging. Towards achieving a facile and comprehensive protocol for AAA, the present work is geared towards developing a deeper understanding of the extent of hydrolysis of peptide, and the nature and stability of amino acids present in the peptide backbone. This defines the suitability of the method in meeting the end goals and the regulatory requirement. Analysis of historical data generated during the method optimization of AAA for icatibant acetate (ICT) using head space oven hydrolysis (HSOH) and microwave-assisted hydrolysis (MAH) methods helped in arriving at fast (< 1 h) and efficient hydrolysis (0.9-1.1 of theoretical residue) conditions. Better separations for the natural and unnatural amino acids were achieved using 3.45 ≤ pH ≤ 10.85, and a column oven gradient program. This approach was useful in meeting the method quality attributes [resolution (Rs) > 2.0; plate count (N) > 5600; and USP tailing factor < 1.2] with a target analytical method profile of relative amino acid mole ratios (RAAMR) in the range of 0.9-1.1 for Ser, Oic, Tic, Hyp, Ala (Thi), Gly and Pro, and between 2.7 and 3.3 for Arg. The developed method was validated as per the ICH guidelines and is precise, accurate, linear and robust.

Criteria for identification of Schistosoma mansoni eggs in faecal sediments prepared with the Helmintex method and stained by ninhydrin.

Helmintex is a sensitive method used for detecting Schistosoma mansoni eggs. Here, we describe the observed frequency of six proposed criteria associated with the identification of S. mansoni eggs prepared with the Helmintex method and stained with ninhydrin. The efficacy of these criteria in classifying S. mansoni eggs when applied in various combinations was also examined. Nine observers registered the presence or absence of 6 different criteria in 100 eggs using a microscope at 100x magnification. Ninhydrin purple, which was frequently observed, was the criterion associated with the lowest inter-observer variability. At least three criteria were associated with a significantly better performance in egg identification. In conclusion, ninhydrin staining and a combination of criteria are recommended for microscope examination of faecal sediments.