A new HPLC micromethod to measure total plasma homocysteine in newborn.

ABSTRACT

Total plasma homocysteine (tHcy) in children may be an useful biochemical marker for genetic risk of premature cardiovascular disease. We reported a rapid, isocratic HPLC method able to process very small amount of newborn plasma samples. A blood sample from heel capillary circulation was collected, using a heparinized capillary glass tube. Plasma sample from 1 to 10 microl was derivatized with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate after reduction with tri-n-butylphosphine and analyzed on Discovery C18 column, with a solution of acetonitrile-dihydrogenphosphate 0.1 M (892 v/v pH*2.1). This assay ensures a good recovery (95%), precision (CV 4.5%) and linearity (y=2.41x + 0.31, r=1). Due to its simplicity and reliability, our method is suitable for routine analysis of tHcy and other aminothiols (Cys, Cys-Gly, GSH) assessed for clinical and research purposes. With this HPLC method we have assayed tHcy levels in 1400 apparently healthy newborn babies (tHcy mean value=4.9+/-2.7 microM). In conclusion, this accurate and linear HPLC method allows measurement of tHcy in newborn during the routinary capillary blood collection in the fourth living day without any other invasive procedure.