Determination of total homocysteine in human plasma by isocratic high-performance liquid chromatography.
A simple, sensitive and precise isocratic HPLC method for the determination of total homocysteine in human plasma is described. The thiol compounds were liberated from plasma proteins by reduction with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulphonate. The derivatives were separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column as stationary phase. By using this approach more than 200 samples a day can be assayed for total homocysteine. The method was linear up to 100 mumol/l and proved to be sensitive with a detection limit of 0.1 mumol/l and the lowest limit of reliable quantification of 0.5 mumol/l for homocysteine in buffer. Intra- and inter-assay coefficients of variation were both < 4% at a concentration of 10 mumol/l homocysteine. Similar results were obtained for homocysteine concentrations between 0.5 and 100 mumol/l. The analytical recovery for these concentrations ranged from 94.9 to 117.0%. As compared to other protocols published so far, this modified method is less complicated but equally sensitive and reproducible and allows a rapid determination of total homocysteine and cysteine in human plasma under routine conditions.
Feussner
A
A
Medizinische Poliklinik, Ludwig-Maximilians-Universität München, Klinikum Innenstadt, Germany.
Rolinski
B
B
Weiss
N
N
Deufel
T
T
Wolfram
G
G
Roscher
A A
AA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000097:blood; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction; D010720:Phosphines; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D010720:Phosphines; D006710:Homocysteine; C043255:tri-n-butylphosphine; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; D003545:Cysteine