Matrix effects in the derivatization of amino acids with naphthalene dicarboxaldehyde, 9-fluorenylmethyl chloroformate and phenylisothiocyanate.
Pre-column derivatizations of amino acids often present two major challenges: 1) automation, due to the multi-step manipulations for pH control, reagent addition, mixing and extraction, and 2) effect of matrices in the sample such as salts, buffers and surfactants. Both issues have been addressed in a previous publication on derivatization methods using 9-fluorenylmethyl chloroformate (FMOC) and phenylisothiocyanate (PITC). In this paper, a third method of derivatization, which has recently been developed and published, was studied to address the same issues. The derivatization reagent, naphthalene-2,3-dicarboxaldehyde (NDA), is a modification from o-phthalaldehyde (OPA) and yields more stable derivatives with high fluorescence efficiencies. An autosampler was programmed to mix amino acid samples with cyanide and NDA reagent, allow a programmed reaction time and finally inject onto the HPLC. To study sample matrix effects, amino acid samples were spiked with various concentrations of Tris-HCl, phenol, citrate, sulfosalicylic acid, sodium chloride and sodium dodecyl sulfate. The recoveries of amino acids in varied sample matrices were compared to pure amino acid standards. The matrix effects using the NDA method were similar to those using the FMOC method. Comparisons of all three methods (NDA, FMOC and PITC) are discussed and tabulated.
Lai
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F
Varian Chromatography Systems, Walnut Creek, CA 94598.
Sheehan
T
T
D000596:Amino Acids / Q000032:analysis*; D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005069:Evaluation Studies as Topic; D005449:Fluorenes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D009281:Naphthalenes; D013861:Thiocyanates
D000596:Amino Acids; D005449:Fluorenes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D009281:Naphthalenes; D013861:Thiocyanates; C005441:phenylisothiocyanate; C027717:2,3-naphthalenedicarboxaldehyde; C054007:1-(9-fluorenyl)methyl chloroformate