VISIBLE DIODE LASER-INDUCED FLUORESCENCE DETECTION OF PHENYLACETIC ACID IN PLASMA DERIVATIZED WITH NILE BLUE AND USING PRECOLUMN PHASE-TRANSFER CATALYSIS

This study reports the application of Nile blue (NB), a far-red oxazin e label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma, The measurement of P AA in psychiatric populations is important because it provides a marke r for 2-phenylethylamine (PEA), which has been implicated in the patho genesis of schizophrenia and major depression, PAA was derivatized wit h NE through an amide linkage in the presence of 2-chloro-1-methylpyri dinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively, The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromat ography, reversed phase liquid chromatography, and electrospray mass s pectrometry, The formation of the NB-PAA derivative was optimized usin g a sequential single factor approach, The optimal conditions for the formation and chromatographic separation of the derivative were determ ined to be 8.0 nmol/mL NE, 390 nmol/mL CMP, 2 mu mol/mL TEA, a reactio n time of 45 min, and a reaction temperature of 25 degrees C. This der ivatization scheme was performed in a phase transfer catalysis mode th at enabled the simultaneous extraction, preconcentration, and derivati zation of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivati ve from a side reaction product. The method was used to estimate endog enous free levels of PAA in human plasma samples, The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been r eported in the literature, The limit of detection of the derivative wa s determined to be 7.33 x 10(-11) M using a laboratory-constructed HPL C-VDLIF detector.