Amines, acetylation detection

The conversion of several biogenic amines into their acetyl derivatives has been attempted [26] for their analysis by HPLC with UV detection. The calibration graphs are linear within the tested range and the limits of detection range from 30 to 250 ng depending on the amine. 

LIMITS OF DETECTION OF SOME AMINES IN HPLC AFTER ACETYLATION... 

The HPLC separation of a number of acetylated amines of biological interest is shown in Fig.4.11. The separation was achieved on a 30-cm column of Spherosil XOA-400 (25 °C) with methylene chloride (prepared by shaking 11 of methylene chloride with 17 ml of water and 15 ml of absolute ethanol for 10 h, then discarding the polar aqueous phase) as the eluting solvent at a flow-rate of 1.3 ml/min (1S75 p.s.i.). The limits of detection of 11 amines carried through this system are given in Table 4.2. 

The reduction of 2, either catalytically or with zinc in acetic acid, leads to 3-amino-4-hydroxy-2-quinolone 20 [72TH000], These amino compounds are rather unstable they dimerize with loss of ammonia to "bis-amines", which in turn are readily oxidized to dyes similar to those obtained from ninhydrin and primary amines [68M1205] [68M1543], The amino derivatives 20 are therefore conveniently converted into 0,N-diacetyl derivatives, the N-acetyl derivative 21, or its dehydrated form, the oxazolo derivative 22 [95MI000], The variety of biological activity of oxazolo-quinolines of type 22 has been detected only in recent years [94JHC1647],... 

TMS derivatives of biogenic amines are used in combination with acyl derivatives for electron-capture detection. Horning et al. [97] presented retention data of TMS-N-acetyl and TMS-N-HFB derivatives of a number of these substances on SE-30, OV-1 and OV-17. The derivatives were prepared by the following procedure. A 1-mg amount of the amine or amino hydrochloride was dissolved in 0.1 ml of acetonitrile and 0.2 ml of TMS-imida-zole was added. After heating for 3 h at 60°C, 5 mg of N-acetylimidazole (or 0.1 ml of HFB-imidazole) were added and the solution was heated at 80°C for 3 h (30 min at 60°C). The solution was used directly for the GC analysis. 

It appears that the NO+ group can be transferred directly only to those species which can behave as reasonably powerful nucleophiles in moderately acid solution. This is undoubtedly the reason why species such as azide ion and amines do not appear to act in this way since they are very extensively protonated in these solutions. Azide ion catalysis has been reported in one case, however (Meyer and Williams, to be published) the denitrosation of N-acetyl-N-nitrosotryptophan, which reacts very rapidly, has been examined at pH 6 where a substantial quantity of free azide ion exists. Presumably the same would be true for some amines at these very low acidities. However, there is experimental evidence from two independent groups which shows that a direct (sometimes called trans-) nitrosation by nitrosamines can occur with aromatic amines. The reactions of N-nitrosodiphenylamine in aqueous acid solution were studied in this context (Challis and Osborne, 1973). The reaction was acid-catalysed for all the substrates studied and was believed to involve the protonated form of the nitrosamine (here written as PhjNNO, H" ). Two separate pathways were detected (a) a direct reaction with N-methylaniline (without the formation of free nitrous acid or a derivative of nitrous acid) as outlined in Scheme 20 and b) an indirect reaction with... 

The first step in the Polonovski reaction of an amine N-oxide with an acid anhydride, acyl chloride or chloroformate ester is the formation of the 0-acylimonium salt (1 Scheme 1). Such species are highly unstable. However, they can be detected in situ ( H NMR), and in certain cases actually isolated, if prepared using powerful acylating agents such as acetyl perchlorate. 

Initial studies indicated that these techniques were also amenable to monitoring the acetylation and deacetylation kinetics in mice, dogs and monkeys. These studies will be published in detail at a later date. It should be noted that dogs do not acetylate amines. In dogs administered deuterium labelled N-acetyl LY201116, no non-deuterated N-acetyl LY201116 was detected, further evidence that the deuterium was not exchanged in vivo CQ. 

Prolonged heating of Bi(02CMe)3 with amines or alcohols around 150°C affords the respective acetylated derivatives in moderate yields [73JOC764]. Bi(02CMe)3 can be used successfully for the Prevost type reaction of olefins [89CC407]. The reaction is highly stereospecific no trans isomer is detectable under wet conditions, while no cis isomer is formed under dry conditions. 

Derivatization is also useful to detect volatile metabolites. Liu et al. [282] described a specific, rapid, and sensitive in situ derivatization solid-phase microextraction (SPME) method for determination of volatile trichloroethylene (TCE) metabolites, trichloroacetic acid (TCA), dichloroacetic acid (DCA), and trichloroethanol (TCOH), in rat blood. The metabolites were derivatized to their ethyl esters with acidic ethanol, extracted by SPME and then analyzed by gas chromatography/negative chemical ionization mass spectrometry (GC-NCI-MS). After validation, the method was successfully applied to investigate the toxicokinetic behavior of TCE metabolites following an oral dose of TCE. Some of the common derivatization reagents include acetyl chloride and TV-methyl-iV- ft-b u (y Idi methyl si I y I) tro (1 uoroacctam i nc (MTBSTFA) for phenols and aliphatic alcohols and amines, dansyl chloride and diazomethane for phenols, dansyl chloride for amines, acidic ethanol and diazomethane for carboxylic acids, and hydrazine for aldehydes. 

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