Quantification of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355) by high-performance liquid chromatography using o-phthalaldehyde/N-acetyl-L-cysteine derivatization.
A new and rapid high-performance liquid chromatographic assay has been developed for the determination of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355,I), a novel prodrug of L-DOPA. The method involves precolumn derivatization of the drug in biological samples with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) in a triethanolamine buffer (pH 8.0), giving a fluorescent compound that is stable for 2 h at 4 degrees C. Use of an internal standard improved the assay in accuracy and reliability. A programmable injector allowed automatic derivatization of large numbers of samples. Chromatographic separation was performed on a reversed-phase column (Capcell Pak C18) in which the silica gel was coated with silicone polymer. The peaks corresponding to compound I and the internal standard were eluted within 16 min with a mobile phase of acetonitrile-phosphate buffer (pH 7.1). The reliable limit of quantification was 0.5 pmol per injection (0.05 micrograms equivalents of L-DOPA per ml in plasma). The method was successfully applied for the measurements of dog plasma concentrations after oral dosing of compound I.
Hisaka
A
A
Central Research Laboratories, Banyu Pharmaceutical Co., Ltd, Tokyo, Japan.
Kasamatsu
S
S
Takenaga
N
N
Ohtawa
M
M
D000111:Acetylcysteine; D000818:Animals; D001682:Biological Availability; D002230:Carbidopa / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D007980:Levodopa / Q000031:analogs & derivatives* / Q000097:blood; D008297:Male; D011355:Prodrugs / Q000032:analysis*; D009764:o-Phthalaldehyde
D011355:Prodrugs; C061031:L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine; D007980:Levodopa; D009764:o-Phthalaldehyde; D002230:Carbidopa; D000111:Acetylcysteine