Applications of the Ninhydrin Reaction for Analysis of Amino Acids, Peptides, and Proteins to Agricultural and Biomedical Sciences

Abstract

The reaction of ninhydrin with primary amino groups to form the purple dye now called Ruhemann's purple (RP) was discovered by Siegfried Ruhemann in 1910. In addition, imines such as pipecolic acid and proline, the guanidino group of arginine, the amide groups of asparagine, the indole ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with ninhydrin to form various chromophores of analytical interest. Since its discovery, extensive efforts have been made to apply manual and automated ninhydrin reactions as well as ninhydrin spray reagents to the detection, isolation, and analysis of numerous compounds of interest across a broad spectrum of disciplines. These include agricultural, biochemical, clinical, environmental, food, forensic, histochemical, microbiological, medical, nutritional, plant, and protein sciences. This reaction is unique among chromogenic reactions in that at pH 5.5 it results in the formation of the same soluble chromophore by all primary amines which react, be they amines, amino acids, peptides, proteins, and even ammonia. Because the chromophore is not chemically bound to the protein or other insoluble material, it is not lost when the insoluble substrate is removed by centrifugation or filtration after the reaction is completed. The visible color of the chromophore is distinctive and is generally not affected by the yellow colors present in many food, plant, and tissue extracts. Adaptations of the classical ninhydrin reaction to specialized needs in analytical chemistry and biochemistry include the use of acid, alkaline, and fluorogenic ninhydrin reagents. To cross-fertilize information among several disciplines wherein an interest in the ninhydrin reaction has developed, and to enhance its utility, this review attempts to integrate and correlate the widely scattered literature on ninhydrin reactions of a variety of structurally different compounds. Specifically covered are the following aspects: historical perspective, chemistry and mechanisms, applications, and research needs. A better understanding of these multifaceted ninhydrin reactions provide a scientific basis for further improvements of this important analytical technique.

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

“…[1][2][3][4][5][6] For over thirty years most amino acid analyses have been performed by ion-exchange chromatography with post-column derivatization with ninhydrin. [7] Alternatively, amino acids can be analyzed by precolumn fluorescent derivatization and then separated by reverse-phase high-performance liquid chromatography (HPLC). [8] The most common agent used for the fluorescent derivatization of amino acids is o-phthalaldehyde (OPA).…”

Angiotensin-Converting Enzyme-Inhibitory Peptides Isolated from Pepsin Hydrolyzate of Apios americana Tuber and Their Hypotensive Effects in Spontaneously Hypertensive Rats

“…After cooling, 3 mL of 50% methanol solution was added and mixed, and absorption at 570 nm was measured (Friedman, 2004). Amino acid content in the sample was standardized with leucine.…”

Biochemical changes and drug residues in ascidian Halocynthia roretzi after formalin–hydrogen peroxide treatment regimen designed against soft tunic syndrome

“…Free amino acids were analyzed according to the ninhydrin post-column derivatization method (Friedman 2004) optimized for the Hitachi amino acid analyzer (Hitachi L-8900, Hitachi, Tokyo, Japan). Soft tissues (5 g) were homogenized with distilled water (5 mL) and centrifuged at 3000 × g for 10 min at 3°C.…”