Ninhydrin reagent, preparing

Rao et al. [46] reported the use of a spectrophotometric method for the determination of primaquine phosphate with ninhydrin. Standard solution of 0.01% primaquine phosphate solution (3 mL) or solution prepared from the drug or its tablets was mixed with 2 mL of water, 5 mL of 2-methoxyethanol and then with 4 mL of ninhydrin reagent. The mixture was boiled for 35 min, and, after cooling and dilution to 25 mL with water, the absorbance was measured at 570 nm versus a reagent blank. Beer s law was obeyed from 4 to 20 pg/mL with recoveries of 99.4— 100.2% for 25 mg of primaquine phosphate. 

To prepare the ninhydrin reagent, ninhydrin (2 g) and hydrindantin (300 mg) are dissolved in dimethyl sulfoxide (DMSO) (75 ml) in a foil-covered 400 ml flask (to exclude light) and are adjusted to 100 ml with 4 M lithium acetate buffer. The jar is stoppered with a rubber bung with inlet and outlet tubes, and the reagent is flushed with 02-free N2 for 30 minutes. The operations must be done in a fume hood. The final solution is then stored sealed at room temperature. Ninhydrin is toxic and stains the skin, so gloves should be worn at all times. 

The separation of amino acids has become a standard laboratory method of analysing protein hydrolysates, which are routinely prepared in the study of chemistry of proteins, and amino acid analyzers based on considerations discussed above are commercially available. Figure 8.5 is a schematic diagram of an amino acid analyzer using the ninhydrin reagent for quantitative determination. 

An overpressured layer chromatographic procedure, with photodensitometric detection for the simultaneous determination of water-soluble vitamins in multivitamin pharmaceutical preparations, was developed and evaluated. HPTLC on silica gel plates with 1-butanol-pyridine-water (50 35 15 vol/vol) as mobile phase was used. The quantitation was carried out without derivatization [vitamin B2 (Rf value 0.30), vitamin Bg (Rf value 0.64), folic acid (Rf value 0.37), nicotinamide (Rf value 0.80), and vitamin C (Rf value 1.02)] or after spraying ninhydrin reagent [calcium panthothenate (Rf value 0.72)] or 4-demethylaminocin-namaldehyde [vitamin B12 (Rf value 1.84) and biotin (Rf value 0)]. 

Fig. f. Detection of panthenol and Ca pantothenate. Solvent ethanol-water (80 + 20). Plate heated, sprayed with ninhydrin reagent and again heated to 160 C. 5—10 pig of each active material 7 panthenol 2 aminopropanol 3 extract from a cosmetic preparation 4 Ca pantothenate 5 /3-alanine 6 extract from multivitamin tablets... 

Overpressure derivatization (OPD) is a relatively new method in which an absorbent polymeric pad prewetted with detection reagent is pressed onto the TLC plate. OPD was found to give more reproducible results when compared to spraying of ninhydrin reagent in the quantitative analysis of tryptophan on a cellulose layer using densitometric scanning. Use of OPD for a variety of other detection reagents and application to the analysis of a multivitamin preparation was described by Postaire et al. (1990). 

Materials consist of standard solutions of amino acids, acetate buffer (4 M, pH 5.5), ethanol (50%), methyl cellosolve (ethylene glycol monomethyl ether), and ninhydrin reagent (0.9 g of ninhydrin and 0.12 g of hydrantin dissolved in 30 ml of methyl cellosolve and 10 ml of acetate buffer, prepared fresh). 

The release of GS from the core-shell electrospun fibers was determined by colorimetric procedure [13] for gentamicin quantification. Triplicate samples of 35 mg of core-shell electrospun GS-loaded fibers eg., core-shell electrospun of sPLA-cPEG/GS, core-shell electrospun of sPLA/CAl-cPEG/GS and core-shell electrospun of sPLA/CA3-cPEG/GS were prepared. Each coreshell PEG/PLA fibrous sample was immersed in 10 ml phosphate buffer saline (PBS) in tubes. The tubes are placed in water bath at 37 C. The buffer solution was collected at 0.5, 1.0, 1.5, 2.0, 3.5, 4.5, 5.5, 9.5, 13.5, 17.5, 24, 36, 48, 60 and 72 hour [14] and replaced with an equal amount of fresh PBS each time. Aliquots (5 ml) of this buffer solution were mixed with the ninhydrin reagent 1.5 ml and pH 7.4 phosphate buffer 3.5 ml and heated in a water bath at 95°C for 30 min. After the UV-visible absorption at 400 nm, the results were presented in terms of cumulative release (%) as a function of release time [1] ... 

The ninhydrin colour reaction has proved very useful in qualitative work and is widely used in the visualization of amino acid bands after electrophoretic or chromatographic separation of mixtures. The reagent used in such circumstances is usually prepared in ethanol and, if 2,4,6-collidine is added, the variations in colour produced by different amino acids will aid their identification (Table 10.6). 

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

In order to prepare the Pauly reagent two solutions, solution I (0.4 M sodium sulfanilate in water) and solution II (0.4 M sodium nitrite in water) are made separately. Just before use, solution I with solution II, 0.25 M HC1, and 2 M NaOH (1 1 8 10) are mixed successively. This solution, which is not stable, should be applied as a spray within minutes and discarded. The ninhydrin test will not interfere with the Pauly reagent. 

A further interesting case is provided by the synthesis of ninhydrin (Expt 5.99, cognate preparation) from indane-l,3-dione (Expt 7.9) in which the methylene group is activated by two adjacent carbonyl groups. Ninhydrin is the stable monohydrate of the triketone, indane-l,2,3-trione, and is a well-known colorimetric reagent for amino acids (Section 2.31). 

Reagent Solution B. A solution of ninhydrin (2.5 g) in absolute EtOH (50 ml) is prepared and maintained in a light-proof container, preferably under inert atmosphere. 

Polyolefin laminate Drug product stored in plastic hags, SPE preparation HPTLC. Plate = 10 x 20 cm silica gel. Mobile phase acetone - chloroform - concentrated sodium hydroxide (20 80 0.2). Photodensitometric detection, at 200 and 234 nm before derivitization, 388 nm after derivitization with ninhydrin and 580 nm after derivitization with Bratton/Marshall reagent. e-Caprolactam Eganox 1010 Butylhydrox)4oluene 4,4 -methylene dianiline 7... 

Ninhydrin. Reacts with drugs that contain primary or secondary amines, such as amphetamine and other sympathomimetic amines. Dissolve 100 mg ninhydrin in 100 mL of acetone. Prepare fresh reagent for each test. 

---