High-performance liquid chromatographic determination of total plasma homocysteine with or without internal standards

Hyperhomocysteinemia is an independent risk factor for atherosclerosis and vascular occlusive disease. Assessment of total plasma concentration of hom ocysteine (tHcys) requires accurate and reproducible measurements. The aim of this study was to test a rapid isocratic HPLC method for tHcys analysis with an internal standard (I.S.) of alpha-mercaptopropionylglycine (MPG), 2 -mercaptoethylamine (ME), or N-acetylcysteine (NAC) or without I.S., and to verify whether the use of an I.S. improves the precision. The method witho ut I.S. showed an excellent linearity (y=1.59x-0.15, r=1), recovery (100%) and a within-assay relative standard deviation (R.S.D.) of 1.2%. Instead, i n our hands, the presence of I.S,s decreased the reproducibility (within-as say R.S.D. ranged from 3.5 to 6.5%) and lengthened the chromatogram by up t o four to five times. In conclusion, HPLC measurement of plasma tHcys witho ut I.S. improves accuracy with respect to determination with I.S.; moreover , this approach allows to routinely process larger amounts of plasma sample s. (C) 1998 Published by Elsevier Science B.V. All rights reserved.