Matrix effects in the derivatization of amino acids with 9-fluorenylmethyl chloroformate and phenylisothiocyanate.
Pre-column derivatization of amino acids is widely practiced today for the HPLC analysis of amino acids. Due to the requirement for pH control, excess reagent removal and multi-step manipulations, automation is a major challenge in the derivatization of amino acids. Another challenge for pre-column chemistries is the effect of matrices in the sample such as salts, buffers and surfactants. This paper reports on automated derivatization using phenylisothiocyanate (PITC) and compares matrix effects between this PITC method and that of derivatization using 9-fluorenylmethyl chloroformate (FMOC). An autosampler was programmed to mix amino acid samples with PITC or FMOC reagent, allow a programmed reaction time, extract excess reagent 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. Using the PITC method and the FMOC method, the recoveries of amino acids in varied sample matrices were compared to pure amino acid standards. The PITC method appears to be affected less by matrix effects than the FMOC method. However, the FMOC method has a higher sensitivity so that sample dilution (up to 30 times) can be used to eliminate matrix effects.
Lai
F
F
Varian Chromatography Systems, Walnut Creek, CA 94598.
Mayer
A
A
Sheehan
T
T
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005449:Fluorenes / Q000737:chemistry*; D007202:Indicators and Reagents / Q000737:chemistry*; D017879:Isothiocyanates; D015394:Molecular Structure; D013861:Thiocyanates / Q000737:chemistry*
D000596:Amino Acids; D005449:Fluorenes; D007202:Indicators and Reagents; D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate; C054007:1-(9-fluorenyl)methyl chloroformate