agent should give rise to a single, stable derivative per An improved method for the quantitative derivatizaamino acid. The derivative should be detectable with tion of amino acids with fluorenylmethyl chlorohigh sensitivity and the reagent itself or its degradaformate (FMOC-Cl) is described. Amino acids are detion products should not interfere with the chromatorivatized in borate buffer at pH 11.4 for 40 min at graphic separation. 9-Fluorenylmethyl chloroformate ambient temperature. All amino acids resulted in sta-(FMOC-Cl) 2 meets most of these criteria. This label ble derivatives. In particular, improved derivatization was introduced for amino acid analysis by Einarsson was obtained with the troublesome amino acids His et al. . FMOC-Cl reacts rapidly and quantitatively and Tyr: exclusively monosubstituted His and disubwith both primary and secondary amino acids under stituted Tyr were formed, eluting as free peaks in the mild conditions and is relatively insensitive for salts. chromatogram. These derivatives show a higher fluo-The resulting carbamates exhibit high fluorescent rerescence response than their disubstituted and monosponse (detection at the low picomole level), are stable substituted counterparts, respectively, resulting from at room temperature for several days, and show excelother protocols. Under the new conditions, considerlent chromatographic behavior on reversed-phase coable less of the hydrolysis product of FMOC-Cl is seen lumns. in the chromatograms. Baseline noise was substan-A disadvantage of FMOC-Cl is its reactivity toward tially reduced at a higher emission wavelength (630 water; after hydrolysis and decarboxylation, the fluonm instead of 313 or 340 nm). With simple precautions, rescent alcohol, FMOC-OH, elutes in the middle of the extensive adsorption of the disubstituted derivatives chromatogram. At high concentrations, FMOC-OH (Lys, Hyl, and Tyr) on plastic or glass surfaces could overlaps with other amino acids in the chromatogram, be prevented. Calibration curves were linear over a 10 complicating the quantification of these amino acids. to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the Inasmuch as FMOC-Cl is also fluorescent, excess rederivatives are stable for more than 48 h, thus permit-agent should be removed before chromatography, for ting automated analysis of multiple samples. แญง 1996 instance by extraction with organic solvents. With such Academic Press, Inc.
extractions part of the FMOC-OH is also removed (5-15). To avoid the cumbersome extraction step, excess FMOC-Cl can be eliminated with 1-adamantylamine or hydroxylamine (19,. A disadvantage Although numerous precolumn derivatization reis that the amount of FMOC-Cl added and reaction agents have been introduced for the analysis of amino acids (1-4), all of them show various shortcomings. The ideal reagent should react rapidly and quantitatively, 2 Abbreviations used: FMOC-Cl, 9-fluorenylmethyl chloroformate; under mild conditions, with both primary and second-FMOC-OH, hydrolysis product of FMOC-Cl with water; Hyl, hydrox- ylysine; Hyp, hydroxyproline; N-FMOC-His, monosubstituted deriv-ary amino acids, without being disturbed by sample ative of histidine (a-amino); N-FMOC-Tyr, monosubstituted derivamatrix components such as salts. Furthermore, the retive of tyrosine (a-amino); N,NH-FMOC-His, disubstituted derivative of histidine (a-amino and imidazole NH); N,O-FMOC-Tyr, disubstituted derivative of tyrosine (a-amino and phenolic hydroxyl