DETERMINATION OF L-CARNITINE, ACETYL-L-CARNITINE AND PROPIONYL-L-CARNITINE IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER PRECOLUMN DERIVATIZATION WITH 1-AMINOANTHRACENE

A new sensitive high-performance liquid chromatographic procedure for the determination of L-carnitine (LC), acetyl-L-carnitine (ALC) and pr opionyl-L-carnitine (PLC) in human plasma has been developed. Precolum n derivatization with 1-aminoanthracene (1AA), performed in phosphate buffer in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimid e hydrochloride (EDC) as catalyst, is involved. The fluorescent deriva tives were isocratically separated on a reversed-phase column (C-18). The eluate was monitored with a fluorimetric detector set at 248 nm (e xcitation wavelength) and 418 nm (emission wavelength). Because of the presence of endogenous carnitines, the validation was performed using dialyzed plasma. The identity of the derivatized compounds was assess ed by mass spectrometry and the purity of the chromatographic peaks wa s confirmed by HPLC-tandem mass spectrometry. The limits of quantitati on were 5 nmol/ml for LC, 1 nmol/ml for ALC and 0.25 nmol/ml for PLC. The recovery of the extraction procedure was in the range 82.6%-95.4% for all 3 compounds. Good linearity (R approximate to 0.99) was observ ed within the calibration ranges studied: 5-160 nmol/ml for LC, 1-32 n mol/ml for ALC and 0.25-8 nmol/ml for PLC. Precision was in the range 0.3-16.8% and accuracy was always lower than 10.6%.