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

S Bartesaghi, R Accinni, G De Leo, C F Cursano, J Campolo, C Galluzzo, P G Vegezzi, O Parodi

Index: J. Pharm. Biomed. Anal. 24(5-6) , 1137-41, (2001)

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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 (8:92 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.