High-performance liquid chromatographic methods with fluorescence detection for the determination of branched-chain amino acids and their alpha-keto analogues in plasma samples of healthy subjects and uraemic patients.
High-performance liquid chromatographic (HPLC) methods have been developed for the quantification of branched-chain amino acids (BCAA) and their keto analogues (BCKA). Amino acids and their keto analogues were derivatized with o-phthalaldehyde, 2-mercaptoethanol and o-phenylenediamine sulphate prior to HPLC. Both separations were performed on a reversed-phase column, using a multi-step gradient system with two solvents and a fluorescence detector. These methods are simple and sensitive and give highly reproducible results. By using an automatic system, the instability problem is avoided and the reaction kinetics are controlled. The use of a simple clean-up procedure with preparative cation-exchange chromatography before BCKA analysis concentrates the dilute plasma sample. The methods were applied to the determination of BCAA and BCKA in plasma samples of healthy volunteers and patients with chronic renal disorders. The relationship between the concentrations of BCAA and BCKA in plasma for these two groups is shown.
Qureshi
G A
GA
Department of Renal Medicine, Karolinska Institute, Huddinge University Hospital, Sweden.
D000597:Amino Acids, Branched-Chain / Q000097:blood*; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007651:Keto Acids / Q000097:blood; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014511:Uremia / Q000097:blood*
D000597:Amino Acids, Branched-Chain; D002021:Buffers; D007651:Keto Acids; D012997:Solvents