Determination of lipoic acid and dihydrolipoic acid in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.
A highly sensitive method for the determination of alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma and urine has been developed. Samples were acidified and extracted with organic solvent, and the free sulfhydryls of DHLA protected as the dicarboxyethylate by treatment with ethylchloroformate. The free carboxylic function of LA and the SH-protected DHLA were converted into their amide derivatives with the strong fluorophore 2-(4-aminophenyl)-6-methylbenzothiazole in the presence of a coupling agent and a base catalyst. The resulting fluorescent amides of both LA and DHLA were separated on a reversed-phase column (Ultrasphere C8) using simple isocratic elution with acetonitrile-water (80:20) and detected fluorimetrically (excitation 343, emission 423 nm). The method is highly sensitive, reproducible, and is easily applied for the simultaneous determination of LA and DHLA in biological samples.
Haj-Yehia
A I
AI
Department of Pharmaceutics, The David R. Bloom Center for Pharmacy, School of Pharmacy, Hebrew University of Jerusalem, Israel. abdullah@cc.huji.ac.il
Assaf
P
P
Nassar
T
T
Katzhendler
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000032:analysis* / Q000097:blood / Q000652:urine
D008063:Thioctic Acid; C015144:dihydrolipoic acid