Derivatization and Fluorescence Detection of Amino Acids and Peptides with 9-Fluorenylmethyl Chloroformate on the Surface of a Solid Adsorbent

Abstract

An approach that exploits the surface of a solid adsorbent is proposed for precolumn FMOC derivatization of amino acids and peptides. Amino acids (Ser, Glu, GABA, Val, Phe, Lys) and two neuropeptides (substance P and Leuenkephalin) were adsorbed on alkaline silica gel cartridges. After drying, they were reacted with 9-fluorenyl-methyl chloroformate (FMOC-Cl) in toluene. After washing off the excess FMOC-Cl with ethyl acetate, the derivatives were eluted with aqueous eluant. The eluates were separated and detected by means of HPLC with fluorescence detection. Compared with the traditional derivatization in the liquid phase, the extent of formation of byproducts of FMOC-Cl with water was greatly decreased, and the excess FMOC-Cl was eliminated completely.

High-performance liquid chromatographic method for determination of amino acids by precolumn derivatization with 4-chloro-3,5-dinitrobenzotrifluoride

“…PITC needs to remove any excess reagent by drying, because it interferes with the separation of the amino acids derivatives [38]. Fmoc-Cl has been reported to yield multiple derivatives and significant interference due to reagent is observed unless the reagent is extracted prior to chromatographic analysis or the molar excess of reagent carefully limited [39,40]. DNFB is an ultraviolet (UV) derivatization reagent for amino acids in aqueous borate buffer.…”

Solid‐phase derivatization of tryptic peptides for rapid protein identification by matrix‐assisted laser desorption/ionization mass spectrometry

“…The requirement for sample cleanup prior to MALDI analyses suggested the possibility of simplifying the entire derivatization protocol by carrying out the N‐terminal sulfonation reaction with peptides after they are adsorbed onto C 18 ZipTips™. Solid‐phase derivatization reactions have been successfully used for years to prepare derivatives for GC, LC or CE analyses 27–31. Potential advantages of the solid‐phase approach, relative to current solution methods, include: pre‐concentration of dilute solutions to improve overall method sensitivity, reduced reaction times and convenient sample cleanup prior to analyses.…”

Techniques for neuropeptide determination

“…Pre-column derivatization resulting in detection limits in the lower pmol range has been used for lysinecontaining neuropeptides employing reagents, such as fluorescamine [25], naphtalene-2,3-dicarboxylaldehyde/cyanide [26] and 9-fluorenylmethyl chloroformate [27]. The major drawback with pre-column derivatization is that several reaction sites may be present and that can result in multiple peaks for one neuropeptide and possible quenching of the fluorescence signal.…”