Separation-Oriented Derivatization of Native Fluorescent Compounds through Fluorous Labeling Followed by Liquid Chromatography with Fluorous-Phase

Abstract

We have developed a new and simple method based on "fluorous derivatization" for LC of native fluorescent compounds. This method involves the use of a column with a fluorous stationary phase. Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag, and the fluorous-labeled analytes are retained in the column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are not detected. In this study, biologically important carboxylic acids (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindoleacetic acid) and drugs (naproxen, felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluorous-phase column can selectively retain fluorous compounds including fluorous-labeled analytes on the basis of fluorous separation. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis.

Selective and sensitive liquid chromatographic determination method of 5-hydroxyindoles with fluorous and fluorogenic derivatization

“…The fluorescence excitation and emission maxima obtained with HFBA-derivatized 5-HIs and BA-derivatized 5-HIs are shown in Table 1. As expected, the HFBA-derivatized 5-HIs exhibited the fluorescence with longer wavelength, similar to BA-derivatized 5-HIs, than the underivatized 5-HIs themselves ( ex 280 nm, em 340 nm) [29]. However, the maximum emission wavelengths obtained with HFBA-derivatized 5-HIs were shifted to slightly shorter wavelengths around 450 nm compared to BA-derivatized 5-HIs (465 nm).…”

“…Recently, we have also developed LC analytical methods for native fluorescent compounds based on "fluorous derivatization," in which "fluorous" refers to the unique affinity between highly fluorinated compounds [29][30][31]. The principle of this method is that native fluorescent analytes are derivatized precolumn by attachment of a non-fluorescent fluorous compound to a particular functional group; thereafter, the fluorous-derivatized analytes are extremely retained on a fluorous phase LC column, thus achieving selective separation of analytes from biological sample matrices.…”

Fluorous derivatization and fluorous-phase separation for fluorometric determination of naproxen and felbinac in human plasma

“…The optimum conditions for fluorous derivatization reaction were optimized according to the previous method [16]. The concentrations of HFUA and DMT-MM were selected as 20 mM and 10 mM, respectively.…”

“…Recently, we developed a simple LC determination method of native fluorescent compounds based on "fluorous derivatization" [16]. "Fluorous" is that between highly fluorinated compounds and highly fluorinated compounds show the special property to fluorophilic, a branch of organic chemistry is dedicated to research on fluorous chemistry [17][18][19].…”

Highly sensitive and selective derivatization-LC method for biomolecules based on fluorescence interactions and fluorous separations

“…Sakaguchi et al [107] have applied fluorous derivatization to the LC-fluorescence analysis of native fluorescent compounds of biologically important metabolites (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindole-3-acetic acid) and nonsteroidal anti-inflammatory drugs (NSAIDs) (naproxen, felbinac, flurbiprofen, and etodolac). In this study, 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoron-undecylamine (HFUA) was used as a derivatization reagent for carboxylic acids with a fluorous derivatization reagent, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) [108,109] was used as an effective condensation reagent between HFUA and the analytes.…”