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.

Analysis of Polypeptides by Amino Acid Analysis.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

A naphthyl appended ninhydrin based colorimetric chemosensor for Cu2+ ion: Detection of cysteine and ATP.

A ninhydrin-based colorimetric chemosensor (LH) was synthesized using 3-hydroxy-2-naphthoic hydrazide and 11H-indeno[1,2-b]quinoxalin-11-one. It was characterized by spectroscopic and single crystal X-ray diffraction techniques. In a semi-aqueous (MeOH/HEPES) system, LH displayed a characteristic chromogenic change from colorless to yellow upon adding Cu2+ ion, with the appearance of a new peak at λmax = 460 nm. A 1:1 binding stoichiometry between LH and Cu2+ ion has been found, with LOD = 2.3 µM (145 ppb) and LOQ = 8 µM (504 ppb). Based on experimental results the formula of [Cu(L)Cl(H2O)2] (1) was assigned and this in-situ generated 1 was found to exhibit a discoloration of upon gradual addition of cysteine (LOD = 60 nM) as well as ATP (LOD = 130 nM) having 1:2 and 1:1 stoichiometry respectively. The LH was useful for recognition of Cu2+ ion in real water samples and on filter paper strips. A two-input-two-output logic gate circuitry was also constructed by employing 1 and cysteine. The DFT/TDDFT calculations performed on LH and 1 were consistent with experimental findings. The binding affinity of LH towards HSA and BSA were determined with HSA having greater affinity than BSA, which was also supported by theoretical calculations.

Diaryl pyrrolone fluorescent probing strategy for Mirabegron determination through condensation with ninhydrin and phenylacetaldehyde: Application to dosage forms, human urine and plasma.

Mirabegron (MRB) is a ß3-adrenoceptor agonist used for managing overactive bladder syndrome. A cost-effective, environmentally friendly, and highly sensitive spectrofluorimetric method was suggested to serve the purpose of quantifying MRB in its pure state, pharmaceutical tablets, spiked human plasma and urine, and testing content uniformity. In the present study, ninhydrin and phenylacetaldehyde react with the amino group moiety of MRB in Teorell-Stenhagen buffer (pH 7.5) to generate a strongly fluorescent diaryl pyrrolone compound that emits fluorescence at a wavelength of 477 nm upon excitation at 385 nm. The obtained calibration curve showed a linear relationship with a high correlation coefficient (r = 0.9997) in the concentration range of 0.25 to 5.0 µg mL-1. Limits of detection (LOD) and quantitation (LOQ) were 0.082 and 0.248 µg mL-1 respectively. The procedure was verified in accordance with the ICH guidelines. The suggested approach could be utilized for the selective analysis of MRB in its pharmaceuticals, either containing a single drug or co-formulated with solifenacin succinate. The greenness of the suggested method was confirmed using different green analytical metrics.

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 laboratory perspective: Confirming the integrity of fingermark enhancement reagents.

Validation is particularly important in forensic science. Each process utilised by a forensic laboratory will undergo validation to ensure it is functioning as expected. Some pieces of equipment (hardware) will be simple to validate, and this will be conducted regularly. However, many commonly employed fingermark enhancement reagents, such as ninhydrin and 1,2-Indandione, do not have clearly defined and robust validation parameters. To that end, artificial fingermark solutions, particularly amino acid solutions, have previously been investigated as a controlled validation method, via deposition onto a substrate. Whilst amino acid reagents are important, there is also a wider group of latent fingermark constituents (targets) that require the use of other enhancement reagents within the forensic laboratory. The work presented herein seeks to expand on the concept of amino acid printing, by exploring the possibilities of depositing a sebaceous solution matrix, thereby facilitating the testing of reagents such as Oil-Red-O and Physical Developer. In addition, we present a method that extends capabilities beyond fingermark enhancement reagents for porous substrates, by developing a methodology that enables process validation of the cyanoacrylate fuming technique to be easily facilitated. These simple and effective solutions have the capacity to serve as a crucial process validation check within the laboratory validation workflow.

The influence of storage conditions on fingermarks developed with 1,2-indanedione-ZnCl.

The most important class of reagents for the enhancement of fingermarks on porous surfaces are those that react with the amino acid constituents of fingermarks. Ninhydrin, DFO (1,8-diazafluoren-9-one), and 1,2-indanedione are the three most common techniques widely known in forensic laboratories for the visualization of latent fingermarks on porous surfaces. In 2012 the Netherlands Forensic Institute replaced DFO by 1,2-indanedione-ZnCl after an internal validation, just like an increasing numbers of laboratories. In 2003, Gardner et al. published an article showing that fingermarks treated with 1,2-indanedione (without ZnCl), that were stored in daylight only decreased by 20% in fluorescence in 28 days. However, during casework we observed that the fluorescence of fingermarks treated with 1,2-indanedione with ZnCl decreased more rapidly. In this study, the effect of various storage conditions and aging times on the fluorescence of marks after treatment with 1,2-indanedione-ZnCl were assessed. Latent printed fingermarks from the digital matrix printer (DMP) and natural fingermarks from a known donor were used. The results showed that storing fingermarks in daylight (wrapped and un-wrapped) drastically decrease (over 60% loss) in fluorescence in approximately three weeks. Storage of the marks in a dark environment (at room temperature, in the refrigerator or even in the freezer) resulted in a decrease in fluorescence of less than 40%. Our recommendation is to always store treated fingermarks with 1,2-indanedione-ZnCl in a dark environment and, if possible, photographing them directly (within 1-2 days after treatment) to minimize the decrease of fluorescence.

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.

Preliminary evaluation of Solstice® PF as a replacement carrier solvent for Australian fingermark detection.

HFE-7100 is a routine carrier solvent in amino acid-sensitive fingermark detection reagents such as ninhydrin and 1,2-indanedione/zinc chloride (IND/Zn). However, a potential EU ban on hydrofluoroethers may require reformulation of these treatments worldwide. Solstice® PF has shown promise as a replacement for HFE-7100 in the United Kingdom. However, the performance (and hence optimal formulation) of IND/Zn is impacted by differences in climate and substrate composition, necessitating assessments under local conditions for different regions. We present a series of preliminary investigations in an Australian context, using the IND/Zn formulation used by Australian forensic service providers. The general performance of Solstice® PF-based IND/Zn was comparable to that using HFE-7100 on three substrate types, three ageing periods (1, 7 and 30 days) and 5 donors. However, slight differences in colour and luminescence intensity, as well as increased ink diffusion, suggest chemical interactions with other reagent components that may affect stability. Specifically, Solstice® PF-based reagent formed a precipitate within a month of storage, though this did not affect performance over a 4 month period. HFE-7100-based IND/Zn was found to be marginally more effective than Solstice® PF when applied to incidental fingermarks. These results indicate that Solstice® PF is a satisfactory alternative carrier solvent to HFE-7100 in an Australian context, though users should be aware of possible limitations regarding compatibility with other evidence components (particularly inks) and shelf-life.

Evaluation of alizarin and purpurin dyes for their ability to visualize latent fingermark on porous surfaces.

Anthraquinones are a group of dye with many members and a wide range of industrial uses ranging from food, textiles to printing. Even though the reactions of anthraquinone dyes with amino acids are known, they have not yet been subjected to an examination regarding their ability in fingermark detection. In the presented study, the potential of alizarine and purpurin, natural anthraquinone dyes, as a fingermark reagent was examined in comparison with ninhydrin and lawsone. Alizarin and purpurin react with latent fingermark residue on copier paper and thermal paper to yield yellow-orange coloured impressions. The products formed also display photoluminescence properties when illuminated at 440 nm viewed through red filter. Both natural anthraquinone dyes exhibited some superior properties at the development of latent fingermarks on these surfaces compared to ninhydrin and lawsone.

Ninhydrin-functionalized chitosan for selective removal of Pb(II) ions: Characterization and adsorption performance.

A chitosan-based adsorbents (CS-Ninhydrin) was prepared by grafting ninhydrin for Pb(II) ions adsorption. SEM-EDS, XRD and FTIR analysis were used to characterize the synthesized CS-Ninhydrin. The static adsorption experiments showed that CS-Ninhydrin had a good removal rate for Pb(II) ions in a wide range of pH 3 to 7, quickly reached equilibrium (120 min) and had a higher adsorption capacity (196 mg/g). Pseudo second-order and Langmuir models showed that the adsorption process of Pb(II) by CS-Ninhydrin was a single-layer chemical adsorption. Temperature experiments showed that the reaction was a spontaneous exothermic process. In the wastewater experiment, CS-Ninhydrin showed an excellent selectivity to Pb(II) ions. The reusability of CS-Ninhydrin was perfect after five adsorption-desorption cycles. The main adsorption mechanism was the chelating and electrostatic action between N and O groups in CS-Ninhydrin and Pb(II) ions. Therefore, the new adsorbent CS-Ninhydrin was expected to promote the wide application of chitosan in Pb(II) adsorption.

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.

On the Ruhemann's Purple electronic spectrum: the role of torsion angle and coordination with Zn(II).

The Ruhemann's Purple (RP) is a chromophore formed from ninhydrin reaction with amines mainly applied to develop latent fingerprints in forensic science. Further enhancement of RP photoluminescence is achieved thought post-treatment with transition metal salt (commonly ZnCl2 alcohol solution), due to the coordination of RP with the metal ion. The literature attributes this improvement to the RP planarity increase in the complex, suggesting this characteristic as important to the post-treatment success. In the present work, the experimentally reported ZnRP complexes and the isolated RP ligand were investigated at the ωB97XD/6-311++G(d,p)/LanL2DZ/IEFPCM(MeOH) level, and their geometrical parameters, complexation energies, and spectroscopic properties (harmonic vibrations and electronic spectrum) were evaluated and agree with the experimental data. The systematic variation of the RP planarity (tilt angle) showed that the lowest energy electronic transition is quite sensitive to RP of this torsion angle. However, the RP planarity increase corresponds to approximately 30% of the total band shift achieved in the complex formation. The results reinforce the importance of the metal ion in the photoluminesce property changing and draw attention to improvements on transition metal complexes design in the post-treatment of developed latent fingermarks.

A Kinetic Investigation of Metal-Dipeptide Complex with Ninhydrin in the Absence and Presence of CTAB Micelles.

This paper presents the results of a kinetic study performed between ninhydrin and a Ni(II) dipeptide complex under various conditions. The rate of formation of the imine adduct was measured spectrophotometrically both in plain aqueous media and in aqueous micellar media in which CTAB (cetyltrimethylammonium bromide) is used as the surfactant. These studies were carried out at pH 5 and over a temperature a range of 50 to 70°C. Studies were also conducted to elucidate the effect of some organic sodium salts on the rate of this reaction. In these studies, it was found that the formation of imine adduct followed a first-order kinetics with respect to [Ni(II)-Gly-Leu]+ in both aqueous and micellar medium. A fractional-order kinetics was observed with respect to ninhydrin, again in both aqueous and micellar media. Increase in the total concentration of CTAB from 0 to 40×10-3 mol dm-3 resulted in approximately two folds increase in the pseudo-first-order rate constant (kψ). The rate constant (kΨ) in micellar medium first increased with increase in CTAB concentration, reached a maximum value, and finally, with further increase in CTAB concentration, a decreasing effect was observed. Quantitative kinetic analysis of kψ-[CTAB] data was performed on the basis of the pseudo-phase model of the micelles. The rate profile in presence of CTAB suggests a cooperative effect in the enhanced formation of the imine adduct as is commonly found in enzyme catalyzed reactions. Addition of organic sodium salts (such as benzoate, salicylate and tosylate) enhanced the rate at lower concentrations but rates start to decrease at higher concentrations. This suggests that tightly binding organic counter-anions were the most effective. Viscosity of the reaction media seems to affect the kinetic behavior in micellar media.

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.

Reductive amination of α-Ketoglutarate in metabolite extracts results in glutamate overestimation.

Artifacts due to metabolite extraction, derivatization, and detection techniques can result in aberrant observations that are not accurate representations of actual cell metabolism. Here, we show that α-ketoglutarate (α-KG) is reductively aminated to glutamate in methanol:water metabolite extracts, which introduces an artifact into metabolomics studies. We also identify pyridoxamine and urea as amine donors for α-KG to produce glutamate in methanol:water buffer in vitro, and we demonstrate that the addition of ninhydrin to the methanol:water buffer suppresses the reductive amination of α-KG to glutamate in vitro and in metabolite extracts. Finally, we calculate that glutamate levels have been overestimated by 10-50%, depending on cell line, due to α-KG reductive amination. These findings suggest that precautions to account for α-KG reductive amination should be taken for the accurate quantification of glutamate in metabolomics studies.

Positive control tests for fingermark development reagents.

At the start of this study, no reliable, consistent, challenging and representative positive control test was commercially available for fingermark visualisation techniques. The goal of this study was to determine the lower limits of detection (LOD) of the fingermark visualisation techniques ninhydrin and 1,2-indanedione-ZnCl and developing positive control tests for these techniques. Spot tests with an amino acid solution were produced to determine the lower LOD's. Secondly, a Dimatix Materials Printer (DMP) was used to print simulated fingermarks using different concentrations of an amino acid solution, to determine the lower LOD of ninhydrin and 1,2-indanedione-ZnCl using the DMP. Concept positive control tests were developed using the lower LOD of ninhydrin and 1,2-indanedione-ZnCl. These were later on adjusted, to form the definitive positive control tests, after a testing period of two months.

Highly sensitive enclosed multilayer paper-based microfluidic sensor for quantifying proline in plants.

Free proline, termed proline, is a biomarker used for diagnosing drought stress in plants. A previously developed proline-ninhydrin reaction-based paper sensor could quickly and easily detect proline, but it was limited by low sensitivity. In this study, we developed an enclosed multilayer paper-based microfluidic sensor with high sensitivity for the quantitative detection of proline in plants. The multilayer paper-based sensor was manufactured using simple wax printing and origami methods, and contained an internal mixing channel to allow good mixing of the proline with ninhydrin, increasing the proline-ninhydrin reactivity and providing accurate and sensitive proline detection. By preloading ninhydrin onto the sample loading area, uniform coloration of the sensing window was achieved, allowing quantitative analysis of various proline concentrations using a constant reaction time. Only the sensing window and sample loading area were exposed to limit sample evaporation and contamination from the external environment. The LOD of the fabricated sensor was 23 µM, which is approximately 29-fold lower than that of the previously proposed paper sensor (657 µM). Samples were extracted from A. thaliana plants subjected to drought stress for proline detection. The proline concentrations measured using the developed paper sensor and a spectrophotometric method were not statistically significant at a confidence level of 95%. Therefore, the developed sensor can be applied to measure proline concentrations precisely in the field with a low detection limit. The developed paper-based sensor can be used to detect the early stages of drought in plants and thus improve crop productivity.

A Ninhydrin-Type Urea Sorbent for the Development of a Wearable Artificial Kidney.

The aim of this study is to develop polymeric chemisorbents with a high density of ninhydrin groups, able to covalently bind urea under physiological conditions and thus potentially suitable for use in a wearable artificial kidney. Macroporous beads are prepared by suspension polymerization of 5-vinyl-1-indanone (vinylindanone) using a 90:10 (v/v) mixture of toluene and nitrobenzene as a porogen. The indanone groups are subsequently oxidized in a one-step procedure into ninhydrin groups. Their urea absorption kinetics are evaluated under both static and dynamic conditions at 37 °C in simulated dialysate (urea in phosphate buffered saline). Under static conditions and at a 1:1 molar ratio of ninhydrin: urea the sorbent beads remove ≈0.6-0.7 mmol g-1 and under dynamic conditions and at a 2:1 molar excess of ninhydrin ≈0.6 mmol urea g-1 sorbent in 8 h at 37 °C, which is a step toward a wearable artificial kidney.

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.