Ninhydrin positive substances

Ninhydrin positive substances present in official amino acids... 

NINHYDRIN POSITIVE SUBSTANCES PRESENT IN OFFICIAL AMINO ACIDS... 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

Figure 2. Typical chromatogram of urine from a subject eating corn flakes. Key , ninhydrin-positive substances after hydrolysis (absorbance at 570 nm) O, zinc concentration in eluate. (chromatography conditions same as Figure 1.)...

The procedure was applied to additional dipeptides, and the course of the alkylation at 35-40°C followed by argentometric titration (Toennies and Kolb, 1945a). With over 90% formation of carbamylmethylsulfonium salts only 8% of peptide cleavage occurred at 40°C however, on brief heating at 95°C the yields of liberated amino acid increased to 54-85 % (Table XVI). Paper chromatography and paper electrophoresis of such reaction mixtures showed that in each case the hberated amino acid was the only ninhydrin-positive substance present. 

Secondary amines and amino acids react in an entirely different way. Indeed, L-proline forms a mixture of the four possible tautomers, but, after a time, ninhydrin-positive substances may be formed by breakdown of the proUne molecule. (Other ketoses do not give this reaction.) Secondary amines, such as piperidine, will not react at 0°, but, at higher temperatures (40-60°), some D-fructosylamine is formed, together with a higher proportion of D-glucose by isomerization. Morpholine or dicyclohexylamine give a 20 to 25% yield of D-n6o-hexulose, and may be used on a preparative... 

A summary of the effects of DL-isoproterenol on all ninhydrin positive substances (NFS) in the rat ventricle is shown in Table 3. 

Blood levels of many of the ninhydrin positive substances were also affected by DL-isoproterenol. However, taurine was the only substance that increased while many of the essential amino acids decreased. Utilization of the essential amino acids for protein synthesis in the damaged myocardium might account for their depletion in the circulation. The increase in taurine concentration was most likely a result of taurine loss from cardiac tissue. 

NFS ninhydrin positive substances NEM N-ethylmaleimide ODC = ornithine decarboxylase 0-MT = 0-methylthreonine P = pantothenate PAP = phosphoadenosylphosphate PAPCA = phosphoadenosylphosphocysteic acid PAPS = phosphoadenosylphosphosulfate PCA = perchloric acid PCYM = pantetheine... 

A systematic investigation of the free amino acids of the Leguminosae led to the isolation of a novel ninhydrin-positive compound from the leaves of Derris elliptica Benth. (Papilionidae) (93). This substance was analyzed as C6H,3N04 (microanalysis and high resolution mass spectrometry) and was shown to be an amino alcohol. The absence of a carbonyl in the 1R, the loss of 31 mass units in the mass spectrum, and a positive periodate cleavage reaction were best embodied into a dihydroxydihydroxymethylpyrrolidine structure. The relative simplicity of the NMR spectra (three peaks in the 13C spectrum four spin-system in the H spectrum) pointed out a symmetrical structure. Inasmuch as the material was optically active ([a]D 56.4, c = 7, H20), meso structures were ruled out, and the 2R, 3R, 4R, 5R relative configuration was retained (93). This structure (53) was further confirmed by an X-ray determination (94). 

Also Tojo and Uenoyama (T2) by means of paper chromatography detected neither 5-hydroxyanthranilic acid nor its conjugate forms in urine of senile cataract patients. They found, however, an ethanol-extractable substance giving a positive ninhydrin reaction and Rf value coinciding with that of tryptophan. The oral administration of a dose of anthra-nilic acid did not alter their findings. 

It must be remembered that with a buffer solution there is always the possibility of interaction between the substance under examination and the buffer components. The best known example of this type is the effect of borates which form complexes with e.g. a-dihydroxy or a-hydroxycarbonyl compounds and result in shifts, diminutions or complete suppressions of their polarographic waves (e.g. with benzil< > or ninhydrin< >). Similarly, the interaction of primary amines (ammonia, glycine) with carbonyl compounds may cause a change in polarographic behaviour. The ketimines formed by condensation are reduced in a new wave at potentials more positive than that of the carbonyl form. A simple wave for carbonyl compounds can be obtained with a veronal buffer or buffers prepared from tertiary amines over the pH-range 8-10. 

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