9-Anthryldiazomethane, fluorescent

Biotin does not exhibit UV absorbance. It neither shows fluorescence nor electrochemical activity. Therefore, it needs to be derivatized. 4-Bromomethylmethoxiycoumarin (BMMC) [590], 9-anthryldiazomethane (ADAM) [591], and 1-pyrenyldiazomethane (PDAM) [592] have been used as precolumn reagents to convert biotin to fluorescent absorbing derivatives. Instead, to obtain derivatives that are UV detectable, hydrazines are used, such as 2-nitrophenylhydrazine hydrochloride... 

Nimura, N. and Kinoshita, T., Fluorescent labeling of fatty acids with 9-anthryldiazomethane (ADAM) for high performance liquid chromatography, Anal. Lett., 13, 191, 1980. 

The following fluorescent alkylating agents have been introduced as pre-chromatographic reagents 9-bromo-methylacridine [480], 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone [481], naphthacyl bromide (2-bromoacetonaphthone) [482, 483], p-(anthroyloxy)-phenacyl bromide (panacyl bromide) [484], 1-bromo-acetylpyrene [485], 9-chloromethy[anthracene [486], and 9-anthryldiazomethane [487, 488]. 

Nimura and Kinoshita [96] developed a derivatization technique for the analysis of fatty acids that is based on 9-anthryldiazomethane (ADAM). The latter is very stable in solution and reacts with fatty acids at room temperature without a catalyst to yield strongly fluorescent esters, which can be chromatographed on an ODS phase with acetonitrile/water mixtures. The derivatization of fatty acid is carried out with a 0.1% methanohc solution of 9-anthryldiazomethane, which is prepared via oxidation of 9-anthraldehyde-hydrazone following a procedure by Nakaya et al. [97]. The fluorescence and excitation spectra of fatty acid-methyl-anthracene esters exhibit maxima at 412 and 365 nm, respectively. As an example, Figure 8.62 shows the separation of ADAM derivatives, a mixture of saturated and unsaturated long-chain fatty acids, which can be detected down to the lowest picomole range. 

Biotin and its analogues can be discriminated by their structural dilference using reversed phase or anion exchange HPLC (Bowers-Komro et al. 1986 Chastain et al. 1985 Livaniou et al. 2000) with no further derivitization. However, fluorescent derivatizations with 4-bromomethyl-7-methoxycumarin (Br-Mmc), 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), thiamine, o-phthalaldehyde (OPA) or 3-mercaptopropionic acid (3-MPA) have usually been attempted to obtain better detection limit (Nojiri et al. 1998 Yokoyama and Kinoshita 1991). 

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